Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 215283 13 pageshttpdxdoiorg1011552013215283

Research ArticlePPAR Activation Induces M1 Macrophage Polarization viacPLA2-COX-2 Inhibition Activating ROS Production againstLeishmania mexicana

J A Diacuteaz-Gandarilla C Osorio-TrujilloV I Hernaacutendez-Ramiacuterez and P Talamaacutes-Rohana

Departamento de Infectomica y Patogenesis Molecular Centro de Investigacion y de Estudios Avanzados del IPN Avenida InstitutoPolitecnico Nacional No 2508 Colonia San Pedro Zacatenco Delegacion Gustavo A Madero 07360 Mexico DF Mexico

Correspondence should be addressed to P Talamas-Rohana ptrcinvestavmx

Received 11 August 2012 Revised 6 December 2012 Accepted 11 December 2012

Academic Editor Jorge Morales-Montor

Copyright copy 2013 J A Dıaz-Gandarilla et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Defence against Leishmania depends upon Th1 inflammatory response and a major problem in susceptible models is the turnoffof the leishmanicidal activity of macrophages with IL-10 IL-4 and COX-2 upregulation as well as immunosuppressive PGE

2

all together inhibiting the respiratory burst Peroxisome proliferator-activated receptors (PPAR) activation is responsible formacrophages polarization on Leishmania susceptible models where microbicide functions are deactivated In this paper wedemonstrated that at least for L mexicana PPAR activation mainly PPAR120574 induced macrophage activation through theirpolarization towardsM1 profilewith the increase ofmicrobicide activity against intracellular pathogenLmexicana PPARactivationinduced IL-10 downregulation whereas the production of proinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 remainedhigh Moreover PPAR agonists treatment induced the deactivation of cPLA

2-COX-2-prostaglandins pathway together with an

increase in TLR4 expression all of whose criteriameet theM1macrophage profile Finally parasite burden in treatedmacrophageswas lower than that in infected nontreated macrophages most probably associated with the increase of respiratory burst in thesetreated cells Based on the above data we conclude that PPAR agonists used in this work induces M1 macrophages polarization viainhibition of cPLA

2and the increase of aggressive microbicidal activity via reactive oxygen species (ROS) production

1 Introduction

Leishmaniasis is a collection of parasitic diseases caused bytwo dozens species of protozoa belonging to the genus Leish-mania and spread by the bite of a sandfly Two main clinicalforms are known cutaneous leishmaniasis affecting the skincausing scars and eventually disfiguration and systemic orvisceral leishmaniasis that can lead to fatal complications ifuntreated [1]

In Mexico Leishmania mexicana is the causative agent oftwo forms of cutaneous leishmaniasis Localized cutaneousleishmaniasis (LCL) is characterized by ulcerative skin lesionsthat develop at the site of the bite of the sandfly diffusecutaneous leishmaniasis (DCL) which consists of nonulcer-ative nodules that spread throughout the skin leads to severe

mutilation because of the invasion of naso- and oropha-ryngeal mucosa [2 3] Peroxisome proliferator-activatedreceptors (PPARs) are ligand-activated transcription factorsexpressed in macrophages where they control the inflamma-tory response there are three isoforms PPAR120572 PPAR120573120575and PPAR120574 that exhibit different tissue distribution aswell as different ligand specificities [4] PPAR120574 promotesthe differentiation of monocytes into anti-inflammatory M2macrophages in humans and mice while the role of PPAR120573120575in this process has been reported only in mice and no dataare available for PPAR120572 [5]

Differential cytokine production is a key feature of pola-rized macrophages while Th1 cytokines promote proin-flammatory M1 macrophages Th2 cytokines support anldquoalternativerdquo anti-inflammatory M2 macrophage phenotype

2 BioMed Research International

Modulation of proinflammatory cytokines by Leishmaniaspecies in vitro and in vivo is reported elsewhere [6] Ingeneral Leishmania infections induce tumor necrosis factor(TNF-120572) production interleukin-1120573 (IL-1120573) generation isabrogated by L donovani infection in vitro and in vivowhereas it is induced by L major infection These obser-vations indicate that different species of Leishmania candifferentially modulate the proinflammatory cytokines Inaddition it is now well documented that these cytokinesplay a decisive role in the modulation of chemokines whichare recognized for their function in cell recruitment andpromotion of the inflammatory reaction [6]

Regarding anti-inflammatory cytokines recent studieshave demonstrated the critical role of IL-10 in susceptibilityto cutaneous and visceral leishmaniasis caused by differentLeishmania species such asLmajor L donovaniLmexicanaand L amazonensis IL-10 suppresses IFN-120574 synthesis byinhibiting accessory cell functions and also can reduce theproduction of Nitric Oxide (NO) by activated macrophagesIL-10 also downregulates the expression of MHC class I andclass II molecules as well as costimulatory B7 moleculeson macrophages Moreover a recent study has shown thatIL-10-deficient BALBc mice can control infection with Lmajor suggesting that IL-10 plays a key role in mediating thesusceptibility and pathogenesis of cutaneous leishmaniasis[7 8]

Prostaglandins are often associated with anti-infla-mmatory activities such as inhibition of effector functionsof inflammatory cells These include inhibition of mediatorrelease frommacrophages neutrophils mast cells basophilsand lymphocytes they can also downregulate macrophagefunctions particularly prostaglandin E

2(PGE2) [9] which

is synthesized throughout the duration of the inflammatoryresponse largely via the sequential activities of cytosolicphospholipase A

2(cPLA

2) cyclooxygenase-2 (COX-2 rate-

limiting enzyme) andmicrosomal PGE synthase-1 (mPGES-1)This pleiotropic prostanoid serves as an underlyingmodu-lator of inflammation bymediating andmodulating cytokine-target gene expression at transcriptional and posttranscrip-tionaltranslational levels [10ndash13]

The cPLA2and COX-2 promoters contain a PPAR

response element (PPRE) thus PPAR120574 agonists includinganti-inflammatory drugs may affect COX-2 and cPLA

2tran-

scription and expression Perez-Santos and Talamas-Rohana[14] demonstrated that indomethacin (INDO) administra-tion induced the intracellular killing of L mexicana par-asites in infected BALBc mice these results suggest thatsuppression of PGs by INDO promotes the developmentof a protective Th1 type response in susceptible mice byenhancement of IL-12 IFN-120574 and NO production

Classically activated macrophages have a high capac-ity to present antigens and to produce IL-6 IL-1120573 TNF-120572 and toxic intermediates (NO and ROS) consequentlyorienting the immune system to a polarized type I responseThe various life-cycle stages of Leishmania have differentsensitivities to ROS and elicit different oxidative responsesof the macrophage Leishmania protects itself against themacrophagersquos oxidative burst through the expression ofantioxidant enzymes and proteins as well as actively by the

inhibition of NO and ROS production in the macrophage[15]

In the present work we analyzed the effect of PPARrsquos ago-nists during the early-time infection of J774A1 macrophageswith Lmexicana and addressed the issue of whether the addi-tion of PPAR agonists to J774A1 macrophages infected withL mexicana could increase ROS production by polarizationof M2 towards M1 macrophages inhibiting cPLA

2and COX-

2 enzymes

2 Material and Methods

21 Antibodies and Reagents PPAR120573120575 and PPAR120574 antibod-ies were purchased from Cayman Chemical (Ann ArborMI USA) cPLA

2 p-cPLA

2(Ser505) COX-2 MRCD206

and p4442 MAP kinase (ERK12) antibodies were fromSanta Cruz Biotechnology Inc (Santa Cruz CA USA)TLR4CD284 antibody was from IMGENEX (San DiegoCA USA) Anti-mouse IgG-conjugated horseradish perox-idase was from Pierce Biotechnology Inc (Rockford ILUSA) and anti-rabbit IgG-conjugated horseradish perox-idase was from Zymed Laboratories (San Francisco CAUSA) PPAR120573120575 agonist (GW501516) and PPAR120574 agonist(GW1929) were obtained from Alexis Biochemicals (ENZOLife Sciences Inc Ann Arbor MI USA) cPLA

2inhibitor

(arachidonyl trifluromethyl ketone (ATK)) was obtainedfrom Cayman Chemical All D-MEM and RPMI-1640 mediawere purchased from Gibco-BRL Life Technologies (GrandIsland NY USA) Fetal bovine serum (FBS) was fromPAA Laboratories (GE Healthcare UK) All materials forSDS-PAGE were purchased from Bio-Rad Lipopolysac-charide (LPS Escherichia coli serotype 0111B4) and allthe other chemicals and biochemicals were from Sigma-Aldrich (St Louis MO USA) Prostaglandin E

1(PGE1)

prostaglandin F1120572

(PGF1120572) 6-ketoprostaglandin F

1120572(6-

keto-PGF1120572) prostaglandin E

2(PGE2) prostaglandin F

2120572

(PGF2120572) and deuterated prostaglandins (2H)were purchased

from Cayman Chemical HPLC grade solvents glacial aceticacid acetonitrile methanol chloroform and all the otherchemicals were from Sigma-Aldrich Solid-phase extraction(SPE) cartridges (C18) were purchased from Millipore (Mil-ford MA USA)

22 Parasites Leishmania mexicana (MHOMMX92UAY-68) promastigoteswere grown at 26∘C inRPMI-1640mediumsupplemented with 10 heat-inactivated FBS 2mM L-glutamine 100 IUmL penicillin 100 120583gmL streptomycinand 10mMHEPES Promastigotes were used at the stationaryphase of growth

23 Cell Culture Murinemacrophage cell line J774A1 (Ame-rican Type Culture Collection Rockville MD USA) wascultured at 37∘C in humidified 5 CO

295 air in DMEM

containing 10 heat-inactivated FBS 2mM L-glutamine100 IUmL penicillin and 100 120583gmL streptomycin Cellswere incubated for 24 h before being used for the requiredassays For all experiments cells were grown up to 80ndash90confluence and then the medium was replaced with a fresh

BioMed Research International 3

medium and cells were incubated with L mexicana Cellswere not subjected to more than 20 cell passages

24 Infection of Macrophages J774A1 macrophages (1 times106well) were cultured in 24-well culture plates Cells wereincubated with promastigotes of L mexicana at a ratio of 20parasites per macrophage or treated with LPS (1120583gmL) forthe indicated periods after which noningested promastigoteswere washed off with warm D-MEM Where indicated cellswere also pretreated for 24 h with pharmacological agonistsGW501516 (100 nM) GW1929 (600 nM) and for 1 hwithATK(75 120583M) prepared inDMSOor ethanol Vehicle controls wereincluded in each experiment

25 Real-Time PCR Assays The total mRNA from non-infected L mexicana-infected or LPS-stimulated J774A1macrophages was extracted with TRIzol reagent (Life Tech-nologies Corporation USA) according to the manufacturerrsquosinstructions The retrotranscription reaction was performedwith the First Strand cDNA Synthesis Kit according to themanufacturerrsquos instructions (Fermentas Life Sciences USA)in an iCycler Thermal Cycler (Bio-Rad) RT-PCR amplifica-tions were performed as described by Estrada-Figueroa et al[16] Reactions were done in a real-time PCR 7500 apparatus(Applied Biosystems) in a final volume of 20 120583L using 100 ngof cDNA and 10 120583L of TaqMan Universal Master Mix II(Applied Biosystems Foster City CA USA) Primers werefrom Applied Biosystems (TaqMan Gene Expression Assay)TNF-120572 (Mm00443258 m1) IL-1120573 (Mm01336189 m1) IL-6(Mm00446190 m1) COX-2 (PTGS2 Mm00478374 m1) IL-10 (Mm00439614 m1) and 120573-Actin (Mm00607939 s1) withthe following conditions 50∘C for 2min then 95∘C for10min followed by 40 cycles at 95∘C for 15 sec and 60∘Cfor 1min To verify results each sample was analyzed inquadruplicate Levels of transcription were normalized tothose of 120573-actin (internal standard) to determine the vari-ability in the amount of cDNA in each sample With the CTvalues obtained the 2minusΔΔCT method was followed to calculatethe level of expression of each cytokine or mediator genein treated macrophages in comparison with the expressionlevel of the same cytokines or mediators in the nontreatedmacrophages according to the formula [17]

ΔΔCT = (CT target minus CT120573 actin) treated

minus (CT target minus CT120573 actin) nontreated(1)

26 Preparation of Cell Extracts and Western Blot AnalysisMacrophages were pretreated for 24 h with the PPAR120573120575 andPPAR120574 agonists or for 1 h with cPLA

2inhibitor ATK while

some (basal control) were not After incubation conditions(noninfected L mexicana-infected or 2 h LPS-stimulatedmacrophages where indicated) cells were quickly washedtwice with icecold PBS and lysed by scraping in RIPAbuffer (50mM Tris-HCl pH 74 150mM NaCl 1 NonidetP-40 1mM PMSF 1 120583gmL aprotinin 1120583gmL leupeptin1mM EDTA 1mM NaF and 1mM Na

3VO4) Lysates were

centrifuged at 10000timesg for 15min at 4∘C to yield the wholecell extract Supernatants were transferred to fresh tubes and

stored at minus70∘C until required Protein concentration wasdetermined using a BCA protein assay with bovine serumalbumin as standard Equal amounts of total cell lysates (60120583gprotein) were solubilized in sample buffer by boiling for5min separated on 10 SDS-PAGE and then transferredonto a nitrocellulose membrane using a Trans blot system(Bio-Rad) Nitrocellulose membranes were then incubatedsuccessively in TBST blocking buffer (50mM Tris-HCl pH74 150mMNaCl (TBS)) containing 5 skimmed dried milkand 005 Tween 20 for 1 h at room temperature to blocknonspecific protein binding Membranes were incubatedovernight at 4∘C with a specific anti-PPAR120573120575 (1 500) anti-PPAR120574 (1 500) anti-phospho-cPLA

2(1 500) total cPLA

2

(1 500) or anti-COX-2 (1 500) antibodies in TBST Mem-branes were washed with TBST five times for 5min eachand incubated with the appropriate Horseradish peroxidase-conjugated secondary antibody (1 1000) for 1 h at roomtemperature Blots initially probed with an antibody werestripped by incubation in 50mMTris-HCl pH 67 100mM120573-mercaptoethanol and 2 SDS for 30min at 50∘C Followingextensive washing blots were reprobed with an anti-ERK12antibody (1 5000) as a loading control Immunoreactiveproteins were visualized by enhanced chemiluminescencedetecting system Densitometry analyses of immunoblotswere performed using Syngene GeneGenius scanning densit-ometer and software

27 Phagocytic Assays A flow cytometry-based method wasused to study the phagocytic activity of macrophages J774A1macrophages were seeded at 1 times 106 cellsmL per well in24-well tissue culture plates and incubated at 37∘C 5CO2for 24 h Macrophages were treated or not with PPAR

agonists (24 h) or cPLA2antagonist (1 h) and incubated

with (FITC)-conjugated zymosan A BioParticles (Molec-ular Probes Europe BV Leiden The Netherlands) at 50particlescell ratio for 2 h unless indicated otherwise or10 120583M CFSE-labeled promastigotes (1 10 ratio) for 60 or120min at 37∘C except control wells After incubation excessnonphagocytized promastigotes or particles were removed bywashing Cells were collected in tubes and phagocytosis wasdetermined by two criteria (1) the number of phagocytizingcells and (2) the mean fluorescent intensity (MFI) in aFACSCalibur

28 Expression of Mannose Receptor and Toll-Like Receptor4 (TLR4) Macrophages were pretreated or not for 24 hwith the PPAR120573120575 and PPAR120574 agonists or for 1 h withcPLA2inhibitor ATK After incubation conditions non-

infected L mexicana-infected LPS or zymosan-stimulatedmacrophages where it is indicated cells were quickly washedtwice with icecold PBS containing 2 of FBS (FACS buffer)and scraped on FACS buffer then cells were spin downand the supernatant was removed After that cells wereresuspended in 1mL of fixer solution and incubated for1 h at 37∘C then cells were centrifuged and washed twicewith FACS buffer Nonspecific staining was blocked with10 PBS-goat serum for 1 h at 37∘C (Fc block) After twowashes with FACS buffer the appropriate antibodies were

4 BioMed Research International

added rabbit anti-human MR (1 100) and mouse anti-human TLR4 (1 100) and incubated for 1 h at 37∘C in FACSbuffer After washing the samples twice they were incubatedwith the appropriate antibody goat anti-rabbit IgG (H + L)rhodamine-conjugated antibody (1 100 Millipore) donkeyanti-mouse IgG (H + L) and Pacific blue-conjugated anti-body (1 100 Sigma-Aldrich) for 1 h at 37∘C Finally sampleswere washed twice and read in a FACSCalibur

29 Oxidative Metabolism The oxidative metabolism ofJ774A1 macrophages was measured by their ability to reduceyellow-colored nitroblue tetrazolium (NBT) to blue for-mazan through the production of superoxide anions asdescribed by Nessa et al [18] Macrophages were simultane-ously incubated with promastigotes and NBT (1mgmL inPBS) for the indicated times To determine if PPAR agonistswere able to increase oxidative metabolism macrophages(2mL 1 times 106mL) were allowed to adhere to coverslips inplastic Petri dishes (35 times 10mm Nunclon Denmark) byincubation at 37∘C for 24 h with PPAR agonists or cPLA

2

antagonist for 1 h Then macrophages were infected with Lmexicana promastigotes (1 10 ratio) After that 1mL of NBTsolution was added to the reaction mixture and incubatedat 37∘C for the indicated times The reaction was stopped byadding 1mL of 05 HCl and cells were further stained withfuccina for 30 sec Then they were washed three times withPBS and positive-oxidative burst cells were counted in a lightmicroscope (100 cellfield and six fieldscondition) Quan-titative production of formazan was determined in 96-wellplates [19] macrophages (1 times 105) were stimulated with PPARagonists as described above and then they were infected withpromastigotes for the indicated periods and NBT added Atthe end of each time macrophages were washed with 70methanol in order to remove nonreduced NBT the producedformazan was dissolved in DMSO and the optical densityof the solution was measured in a spectrophotometer (Bio-Rad) at 630 nm wavelength A Petri dish or a 96-well platewith noninfected macrophages was incubated with NBT andserved as control for each type of experiment

210 Prostaglandins Extraction The following procedure wasdeveloped for the separation of eicosanoids from 24-well cellculture plates containing 2mL of media Media was collectedand centrifuged for 5min at 10000 xg to remove cellulardebris Produced eicosanoids were isolated via solid-phaseextraction using SPE cartridge C

18from Millipore Columns

were prewashed with 2mL of MeOH followed by 2mL ofH2O After applying the sample to the columns they were

washed with 1mL of 10 MeOH and prostaglandins wereeluted with 1mL of MeOH The eluate was dried undervacuum and redissolved in 100120583L of chloroform-MeOH(2 1)

211 Mass Spectrometry (MS) All MS analyses were per-formed using an Applied Biosystems 3200 QTRAP hybridtriple-quadrupole linear ion trap mass spectrometerequipped with a Turbo V ion source and operated in MRMmode For all experiments the Turbo V ion source wasoperated in a negative electrospray mode with N

2gas and

the QTRAP parameters DP EP CE and CXP were set andmaximized for each eicosanoid and all the samples wereloaded by direct infusion at 10 120583Lmin

212 Statistical Analysis To take into account all values of thekinetics of macrophages infection a statistical analysis wasperformed using two-way ANOVA and Bonferronirsquos multiplecomparison tests using GraphPad Prism 40 (GraphPadSoftware San Diego CA) Differences with 119875 lt 005 wereconsidered significant

3 Results

31 PPAR Agonists Downregulate cPLA2and COX-2 Expres-

sion in J774A1 Macrophages Infected with Leishmania mex-icana Promastigotes In L mexicana-infected macrophagesthe activation of cPLA

2by phosphorylation and the expres-

sion of COX-2 are triggered The activation of theseenzymes is considered necessary within the proinflamma-tory response whereas PPAR activation is considered asan important part of the anti-inflammatory process bothin vivo and in vitro [20ndash22] To examine the possibilitythat PPAR agonists could inhibit cPLA

2phosphorylation

and COX-2 expression J774A1 macrophages were incu-bated with different PPAR agonists and then cells wereinfected with L mexicana promastigotes The effects of Lmexicana promastigotes on the expression of PPAR COX-2 and cPLA

2phosphorylation in J774A1 macrophages were

examined (Figure 1) Results showed that there were nochanges in the expression of PPARs after infection with Lmexicana promastigotes of macrophages whether treatedor not with PPAR agonists however cPLA

2phosphoryla-

tion diminished significantly through the infection whenmacrophages were treated with PPAR agonists (Figures1(a) and 1(b)) in addition COX-2 protein also diminishedsignificantly after infection (Figures 1(a) and 1(c)) COX-2mRNA expressionwas strongly upregulated after Lmexicanainfection (see Figure 1 available in Supplementary Materialonline at httpdxdoiorg1011552013215283) but it wasdownregulated in a time-dependent manner (Figure 1(d))when J774A1 macrophages were treated with PPAR agonistsand infected with L mexicana promastigotes

32 PPAR Agonists Downregulate IL-10 and Sustain Proin-flammatory Cytokines Expression after Infection Cytokinesand microbial products profoundly and differentially affectthe function of mononuclear phagocytes It is well estab-lished that different species of Leishmania can differentiallymodulate important inflammatory response mediators [23]In order to see how PPAR agonists could modulate theinflammatory response on J774A1 macrophages infectedwith L mexicana we evaluated the transcripts of someinflammatory cytokines and anti-inflammatory IL-10 Thecapacity of thesemacrophages to produceTNF-120572 IL-1120573 IL-6and IL-10 cytokines in response to L mexicana promastigoteswas tested via qRT-PCR (Figure 2) The infection of J774A1macrophages resulted in an increase in TNF-120572 IL-1120573 andIL-6 expression IL-10 was downregulated during Leishmaniainfection although its expression recovered compared to

BioMed Research International 5

ERK12

COX-2

p-cPLA

cPLA

LPS LPS LPS

2

2

Time post infection Time post infection Time post infection

(-) GW501516 120573120575 GW1929 120574

PPAR120574

PPAR120573120575

MΦ MΦ MΦ5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400

(a)

0

50

100

150

Den

sitom

etric

anal

ysis

of p

-cPL

A(

)

LPS

wTX

2

Time after infection

(b)

0

755025

150125100

225200175175

Den

sitom

etric

anal

ysis

of C

OX-

2 (

)

LPS

wTX

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowast

Time after infection

(c)

0001

1

01

001

1000

100

10

Rela

tive e

xpre

ssio

n le

vel C

OX-

2

LPS 30 60 120

ATK

PPARβδPPARγ

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowast

Mφ

Time after infection

(d)

Figure 1 PPAR agonists inhibit cPLA2phosphorylation and COX-2 expression in L mexicana-infected macrophages (a) Protein expression

for PPARs and COX-2 and cPLA2phosphorylation levels were evaluated by Western blotting (b) Densitometry analyses of cPLA

2

phosphorylation and (c) COX-2 expression were performed in basal conditions as well as in macrophages treated or not with PPAR agonists(d) COX-2 mRNA expression was evaluated by qRT-PCR and analyzed by 2minusΔΔCT method Total ERK12 was probed to normalize proteinloading Results are representative of three independent experiments Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast)119875 lt 005 (lowastlowast)119875 lt 001 and (lowastlowastlowast)119875 lt 0001

noninfected macrophages (Supplementary Figure 2) How-ever when noninfected macrophages were previously treatedwith PPAR120573120575 agonist (white bars) proinflammatory IL-6was upregulated and its expressionwas held during infectionTNF-120572 and IL-1120573 were overexpressed but they were down-regulated at 30 to 60min (after infection) and its expres-sion recovered at 120min (after infection) Moreover whennoninfected macrophages were treated with PPAR120574 agonist(black bars) TNF-120572 was upregulated but its expression wasdownregulated after infection overregulation of IL-6 dimin-ished after infection but it was held equivalent to nontreatedmacrophages IL-1120573 was upregulated and its expression washeld during infection Anti-inflammatory IL-10 cytokine wasdownregulated with both PPAR agonists after infection On

the other hand when cPLA2was blocked 1 h before the

infection by treatment ofmacrophageswith cPLA2antagonist

ATK (gray bars) proinflammatory cytokines TNF-120572 IL-1120573and IL-6were upregulated furthermore IL-10 expressionwassignificantly affectedwhen cPLA

2was inhibited In summary

PPAR agonists and cPLA2antagonist set down the levels of

IL-10 at the same time they upregulated TNF-120572 IL-1120573 andIL-6 cytokines or at least they were held overexpressed afterinfection as occurred with nontreated infected macrophages(Supplementary Figure 2) all together are evidence of apossible M2 to M1 polarization

33 PPAR Activation and cPLA2Inhibition Induce TLR4

Expression in L mexicana-Infected Macrophages Polarized

6 BioMed Research International

lowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0001

001

01

1

10

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-10

Time after infection

(a)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n le

vel

LPS

Time after infection

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-6

ATK

Time after infection

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowastlowast

lowastlowast

lowast

lowast lowast

0001

001

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

ATK

Time after infection

(d)

Figure 2 Cytokine determination in L mexicana-infected macrophages Levels of gene expression for each sample were normalized with120573-actin RNA as internal control Modulation was expressed relative to the untreated control using the 2minusΔΔCT method The 119909-axis interceptsthe 119910-axis at ldquo1rdquo to show the increase and the decrease of each cytokine compared to nontreated infected macrophages Relative expressionlevel for each cytokine was calculated according to ΔΔCT = (CT test minus CT 120573-actin) treated minus (CT test minus CT 120573-actin) untreated formula [17]Graph bars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versusnontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages differ in terms of receptor expression cytokineand chemokine repertoires and effector function M1macrophages exposed to the classic activation signals expressreceptors such as CD16 CD32 CD64 TLR2 and TLR4whereas M2 macrophages are characterized by abundantlevels of nonopsonic receptors such as the mannose receptor(MR) [24] In order to investigate if PPAR agonists andcPLA2antagonist induced macrophage polarization during

Lmexicana infection we evaluatedMR andTLR4 expressionby flow cytometry Since BALBcmice macrophages can fullysupport maturation of alternatively activated macrophages

J774A1 macrophages were incubated with PPAR agonistsand cPLA

2antagonist and then infected or stimulated with

LPS and zymosan neither infection nor treatments inducedMR expression an M2 receptor classified as an alternativeactivation marker (Supplementary Figure 3) however PPARagonists and cPLA

2antagonist induced an increase of TLR4

expression at 60 and 120min after infection (Figure 3) anM1 receptor classified as classical activation marker Theseresults show that PPAR120573120575 GW501516 and PPAR120574 GW1929agonists and cPLA

2antagonist do not help to keep the

M2 polarization profile instead PPAR agonists and cPLA2

BioMed Research International 7

0

50

100

150

TLR4+

(MFI

)

LPS

wTXATK

lowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

Time after infection

Figure 3 TLR4 expression in L mexicana-infected macrophagesCells were treated or not with PPAR agonists for 24 h and cPLA

2

antagonist for 1 h before infection TLR4 expression was analyzed byflow cytometry LPS (2 h) was used as a positive control of inductionGraph bars are mean plusmn SEM of three independent experiments andstatistical analysis was performed comparing for each time treatedversus non-treated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and(lowastlowastlowast) 119875 lt 0001

antagonist promote the polarization of macrophages towardan M1 profile

34 PPAR120574Activation byAgonist and cPLA2Inhibition Reduce

Parasite Burden PPAR120574 expression is strongly associatedwith maturation of M2 macrophages Gallardo-Soler et alandAdapala andChan [25 26] have demonstrated that PPARagonists increased intracellular growth of L major in bone-marrow-derived macrophages moreover PPAR120574 agonistCurcumine induced PPAR120574 expression in residential liverand spleen macrophages of BALBc mice In addition oraladministration of Curcumin further increases PPAR120572 andPPAR120574 expression and this increase was associated with aheavier parasite burden To analyze how PPAR120573120575GW501516and PPAR120574 GW1929 agonists and cPLA

2inhibition affected

parasite burden in treated macrophages their phagocyticactivity was evaluated through two parameters the phago-cytizing cell percentage and the number of phagocytizedzymosan particles or parasitescell treated or nontreatedmacrophages were incubated with FITC-labeled zymosanparticles by 2 h or infected with CFSE-labeled promastigotesby 1-2 h (Figure 4) PPAR120574GW1929 agonist does not increasethe number of zymosan-phagocytizing macrophages butPPAR120573120575 GW501516 agonist and cPLA

2ATK antagonist

treatments decreased the number of zymosan-phagocytizingmacrophages (119875 lt 005 and 119875 lt 0001 resp Figure 4(a)(zymosan)) on the contrary zymosan particlescell (meanfluorescence intensity (MFI)) increased significantly withall treatments GW501516 and GW1929 (119875 lt 0001) andATK (119875 lt 005) (Figure 4(b) (zymosan)) This result shows

that phagocytic activity per se was not affected by treat-ments The number of CFSE-labeled parasites-phagocytizingmacrophages did not increase by treatments either insteadparasites-phagocytizing macrophages diminished signifi-cantly with GW1929 (119875 lt 001) and ATK (119875 lt 0001)at 60min after infection and 2h after infection GW501516(119875 lt 001) GW1929 (119875 lt 0001) and ATK (119875 lt 0001)(Figure 4(a)) Only cPLA

2antagonist (Figure 4(b)) slightly

increased the number of parasitescell (119875 lt 005) at 60minafter infection but neither PPAR120573120575 nor PPAR120574 agonistsincreased it however at 120min after infection PPAR120573120575agonist slightly increased parasite burden (119875 lt 005)but PPAR120574 agonist and cPLA

2inhibition decreased parasite

burden significantly (119875 lt 0001) (Figure 4(b)) These resultstogether demonstrate that PPAR activation by these agonistsand cPLA

2inhibition did not increase parasite load

35 PPAR Activation by Agonists Selectively Regulates Pros-taglandin Production in L mexicana-Infected MacrophagesProstaglandins are potent ligands of the intracellular PPARreceptors in macrophages and their binding to PPAR120572and PPAR120574 causes macrophage deactivation Perez-Santosand Talamas-Rohana [14] have demonstrated that COX-2 inhibition induced leishmanicidal activity by splenocytes[14] Thus one possible mechanism of intracellular sur-vival of Leishmania is the deactivation of macrophages byprostaglandins produced [27] In order to investigate ifPPAR agonists couldmodulate inflammatory prostaglandinsmacrophages were treated with PPAR agonists or cPLA

2

antagonist before the infection to look for PGrsquos metabolitesin the conditioned media (Figure 5) PPAR activation andcPLA2inhibition decreased significantly 6k-PGF

1120572 PGE

1

and PGF1120572

production (Supplementary Figure 4) howeverPPAR120574 agonist was not able to reduce PGE

2production

which increased significantly after infection (119875 lt 0001) at60 and 120min respectively (Figure 5) PPAR120573120575 agonist andcPLA2antagonist did not increase PGE

2production even its

production diminished (119875 lt 005 and 119875 lt 0001) at 120minafter infection respectively moreover PPAR agonists andcPLA2antagonist significantly increased PGF

2120572production

(119875 lt 0001) at 60 and 120min after infection respectively Insummary PPAR agonists and cPLA

2antagonist diminished

6k-PGF1120572 PGF

1120572 and PGE

1 and neither Leishmania infec-

tion nor LPS was able to recover their production howeverPGF2120572

production was increased after infection and onlyPPAR120574 activation increased PGE

2production

36 PPARActivation and cPLA2Inhibition Increase theOxida-

tive Burst during J774A1 Macrophages Infection with Lmexicana Promastigotes Several studies have demonstratedthat ROS modulate arachidonic acid metabolism and pro-duction of eicosanoids in activated macrophages [28 29]In murine and human macrophages it has been establishedthat the respiratory burst of the cell with production ofROS such as H

2O2and O

2

minus is primarily responsible forparasite control as these molecules have been reportedto be fatal for Leishmania promastigotes We next ana-lyzed the effect of PPAR agonists and cPLA

2antagonist

on the oxidative burst induced by L mexicana on J774A1

8 BioMed Research International

0

10

20

30

40

50

60

70Ph

agoc

ytiz

ing

cells

()

Zy

wTXATK

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

Time after infection

(a)

0

200

400

600

800

Part

icle

s or p

aras

ite lo

adc

ell (

MFI

)

Zy

wTXATK

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 4 Phagocytic activity of J774A1 macrophages was determined for zymosan and L mexicana promastigotes uptake treated ornontreated macrophages were incubated with zymosan-FITC for 2 h or infected with CFSE-promastigotes for 1-2 h (a) Phagocytizingmacrophage percentage (b) Zymosan particles or parasitescell (MFI) Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 (lowastlowastlowast) 119875 lt 0001

0

200

400

600

800

1000

LPS

wTXATK

PGE 2

prod

uctio

n (p

gm

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

Time after infection

(a)

0

10

20

30

40

LPS

wTXATK

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 5 Prostaglandin production by L mexicana-infectedmacrophages Prostaglandins were analyzed byMSMS assay product scanningexperiments were conducted using nitrogen as collision gas and the collision energy was optimized for individual compounds to generatethe most abundant product ions These product ion spectra were then used to select the precursor-product ion pairs for the developmentof MRM assays Deuterium-labeled prostaglandins were used as internal standards for quantitation Graph bars are mean plusmn SEM of threeindependent experiments and statistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005(lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages (Figure 6)We found that positivemacrophagesto L mexicana-induced oxidative burst increased from sim341to 586 (119875 lt 0001) by PPAR120574 agonist at 120min afterinfection compared to non-treated macrophages whereastreatment with PPAR120573120575 agonist increased from sim341 to4032 (119875 lt 005) (Figures 6(a) and 6(b)) PPAR120573120575agonist slightly increased the number of positive oxidativeburst macrophages at 120min after infection and when

the oxidative burst was quantified it increased sim13-foldat 120min after infection too (119875 lt 005) (Figure 6(c)) Onthe other hand cPLA

2inhibition increased sim251-fold the

oxidative burst at 120min after infection (119875 lt 0001)compared to nontreated macrophages (Figure 6(c)) FinallyPPAR120574 agonist increased the oxidative burst sim252-(119875 lt0001) and sim355-fold (119875 lt 0001) at 60 and 120minafter infection respectively indicating that PPAR120574 activation

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Molecular Biology International

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Marine BiologyJournal of

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology

2 BioMed Research International

Modulation of proinflammatory cytokines by Leishmaniaspecies in vitro and in vivo is reported elsewhere [6] Ingeneral Leishmania infections induce tumor necrosis factor(TNF-120572) production interleukin-1120573 (IL-1120573) generation isabrogated by L donovani infection in vitro and in vivowhereas it is induced by L major infection These obser-vations indicate that different species of Leishmania candifferentially modulate the proinflammatory cytokines Inaddition it is now well documented that these cytokinesplay a decisive role in the modulation of chemokines whichare recognized for their function in cell recruitment andpromotion of the inflammatory reaction [6]

Regarding anti-inflammatory cytokines recent studieshave demonstrated the critical role of IL-10 in susceptibilityto cutaneous and visceral leishmaniasis caused by differentLeishmania species such asLmajor L donovaniLmexicanaand L amazonensis IL-10 suppresses IFN-120574 synthesis byinhibiting accessory cell functions and also can reduce theproduction of Nitric Oxide (NO) by activated macrophagesIL-10 also downregulates the expression of MHC class I andclass II molecules as well as costimulatory B7 moleculeson macrophages Moreover a recent study has shown thatIL-10-deficient BALBc mice can control infection with Lmajor suggesting that IL-10 plays a key role in mediating thesusceptibility and pathogenesis of cutaneous leishmaniasis[7 8]

Prostaglandins are often associated with anti-infla-mmatory activities such as inhibition of effector functionsof inflammatory cells These include inhibition of mediatorrelease frommacrophages neutrophils mast cells basophilsand lymphocytes they can also downregulate macrophagefunctions particularly prostaglandin E

2(PGE2) [9] which

is synthesized throughout the duration of the inflammatoryresponse largely via the sequential activities of cytosolicphospholipase A

2(cPLA

2) cyclooxygenase-2 (COX-2 rate-

limiting enzyme) andmicrosomal PGE synthase-1 (mPGES-1)This pleiotropic prostanoid serves as an underlyingmodu-lator of inflammation bymediating andmodulating cytokine-target gene expression at transcriptional and posttranscrip-tionaltranslational levels [10ndash13]

The cPLA2and COX-2 promoters contain a PPAR

response element (PPRE) thus PPAR120574 agonists includinganti-inflammatory drugs may affect COX-2 and cPLA

2tran-

scription and expression Perez-Santos and Talamas-Rohana[14] demonstrated that indomethacin (INDO) administra-tion induced the intracellular killing of L mexicana par-asites in infected BALBc mice these results suggest thatsuppression of PGs by INDO promotes the developmentof a protective Th1 type response in susceptible mice byenhancement of IL-12 IFN-120574 and NO production

Classically activated macrophages have a high capac-ity to present antigens and to produce IL-6 IL-1120573 TNF-120572 and toxic intermediates (NO and ROS) consequentlyorienting the immune system to a polarized type I responseThe various life-cycle stages of Leishmania have differentsensitivities to ROS and elicit different oxidative responsesof the macrophage Leishmania protects itself against themacrophagersquos oxidative burst through the expression ofantioxidant enzymes and proteins as well as actively by the

inhibition of NO and ROS production in the macrophage[15]

In the present work we analyzed the effect of PPARrsquos ago-nists during the early-time infection of J774A1 macrophageswith Lmexicana and addressed the issue of whether the addi-tion of PPAR agonists to J774A1 macrophages infected withL mexicana could increase ROS production by polarizationof M2 towards M1 macrophages inhibiting cPLA

2and COX-

2 enzymes

2 Material and Methods

21 Antibodies and Reagents PPAR120573120575 and PPAR120574 antibod-ies were purchased from Cayman Chemical (Ann ArborMI USA) cPLA

2 p-cPLA

2(Ser505) COX-2 MRCD206

and p4442 MAP kinase (ERK12) antibodies were fromSanta Cruz Biotechnology Inc (Santa Cruz CA USA)TLR4CD284 antibody was from IMGENEX (San DiegoCA USA) Anti-mouse IgG-conjugated horseradish perox-idase was from Pierce Biotechnology Inc (Rockford ILUSA) and anti-rabbit IgG-conjugated horseradish perox-idase was from Zymed Laboratories (San Francisco CAUSA) PPAR120573120575 agonist (GW501516) and PPAR120574 agonist(GW1929) were obtained from Alexis Biochemicals (ENZOLife Sciences Inc Ann Arbor MI USA) cPLA

2inhibitor

(arachidonyl trifluromethyl ketone (ATK)) was obtainedfrom Cayman Chemical All D-MEM and RPMI-1640 mediawere purchased from Gibco-BRL Life Technologies (GrandIsland NY USA) Fetal bovine serum (FBS) was fromPAA Laboratories (GE Healthcare UK) All materials forSDS-PAGE were purchased from Bio-Rad Lipopolysac-charide (LPS Escherichia coli serotype 0111B4) and allthe other chemicals and biochemicals were from Sigma-Aldrich (St Louis MO USA) Prostaglandin E

1(PGE1)

prostaglandin F1120572

(PGF1120572) 6-ketoprostaglandin F

1120572(6-

keto-PGF1120572) prostaglandin E

2(PGE2) prostaglandin F

2120572

(PGF2120572) and deuterated prostaglandins (2H)were purchased

from Cayman Chemical HPLC grade solvents glacial aceticacid acetonitrile methanol chloroform and all the otherchemicals were from Sigma-Aldrich Solid-phase extraction(SPE) cartridges (C18) were purchased from Millipore (Mil-ford MA USA)

22 Parasites Leishmania mexicana (MHOMMX92UAY-68) promastigoteswere grown at 26∘C inRPMI-1640mediumsupplemented with 10 heat-inactivated FBS 2mM L-glutamine 100 IUmL penicillin 100 120583gmL streptomycinand 10mMHEPES Promastigotes were used at the stationaryphase of growth

23 Cell Culture Murinemacrophage cell line J774A1 (Ame-rican Type Culture Collection Rockville MD USA) wascultured at 37∘C in humidified 5 CO

295 air in DMEM

containing 10 heat-inactivated FBS 2mM L-glutamine100 IUmL penicillin and 100 120583gmL streptomycin Cellswere incubated for 24 h before being used for the requiredassays For all experiments cells were grown up to 80ndash90confluence and then the medium was replaced with a fresh

BioMed Research International 3

medium and cells were incubated with L mexicana Cellswere not subjected to more than 20 cell passages

24 Infection of Macrophages J774A1 macrophages (1 times106well) were cultured in 24-well culture plates Cells wereincubated with promastigotes of L mexicana at a ratio of 20parasites per macrophage or treated with LPS (1120583gmL) forthe indicated periods after which noningested promastigoteswere washed off with warm D-MEM Where indicated cellswere also pretreated for 24 h with pharmacological agonistsGW501516 (100 nM) GW1929 (600 nM) and for 1 hwithATK(75 120583M) prepared inDMSOor ethanol Vehicle controls wereincluded in each experiment

25 Real-Time PCR Assays The total mRNA from non-infected L mexicana-infected or LPS-stimulated J774A1macrophages was extracted with TRIzol reagent (Life Tech-nologies Corporation USA) according to the manufacturerrsquosinstructions The retrotranscription reaction was performedwith the First Strand cDNA Synthesis Kit according to themanufacturerrsquos instructions (Fermentas Life Sciences USA)in an iCycler Thermal Cycler (Bio-Rad) RT-PCR amplifica-tions were performed as described by Estrada-Figueroa et al[16] Reactions were done in a real-time PCR 7500 apparatus(Applied Biosystems) in a final volume of 20 120583L using 100 ngof cDNA and 10 120583L of TaqMan Universal Master Mix II(Applied Biosystems Foster City CA USA) Primers werefrom Applied Biosystems (TaqMan Gene Expression Assay)TNF-120572 (Mm00443258 m1) IL-1120573 (Mm01336189 m1) IL-6(Mm00446190 m1) COX-2 (PTGS2 Mm00478374 m1) IL-10 (Mm00439614 m1) and 120573-Actin (Mm00607939 s1) withthe following conditions 50∘C for 2min then 95∘C for10min followed by 40 cycles at 95∘C for 15 sec and 60∘Cfor 1min To verify results each sample was analyzed inquadruplicate Levels of transcription were normalized tothose of 120573-actin (internal standard) to determine the vari-ability in the amount of cDNA in each sample With the CTvalues obtained the 2minusΔΔCT method was followed to calculatethe level of expression of each cytokine or mediator genein treated macrophages in comparison with the expressionlevel of the same cytokines or mediators in the nontreatedmacrophages according to the formula [17]

ΔΔCT = (CT target minus CT120573 actin) treated

minus (CT target minus CT120573 actin) nontreated(1)

26 Preparation of Cell Extracts and Western Blot AnalysisMacrophages were pretreated for 24 h with the PPAR120573120575 andPPAR120574 agonists or for 1 h with cPLA

2inhibitor ATK while

some (basal control) were not After incubation conditions(noninfected L mexicana-infected or 2 h LPS-stimulatedmacrophages where indicated) cells were quickly washedtwice with icecold PBS and lysed by scraping in RIPAbuffer (50mM Tris-HCl pH 74 150mM NaCl 1 NonidetP-40 1mM PMSF 1 120583gmL aprotinin 1120583gmL leupeptin1mM EDTA 1mM NaF and 1mM Na

3VO4) Lysates were

centrifuged at 10000timesg for 15min at 4∘C to yield the wholecell extract Supernatants were transferred to fresh tubes and

stored at minus70∘C until required Protein concentration wasdetermined using a BCA protein assay with bovine serumalbumin as standard Equal amounts of total cell lysates (60120583gprotein) were solubilized in sample buffer by boiling for5min separated on 10 SDS-PAGE and then transferredonto a nitrocellulose membrane using a Trans blot system(Bio-Rad) Nitrocellulose membranes were then incubatedsuccessively in TBST blocking buffer (50mM Tris-HCl pH74 150mMNaCl (TBS)) containing 5 skimmed dried milkand 005 Tween 20 for 1 h at room temperature to blocknonspecific protein binding Membranes were incubatedovernight at 4∘C with a specific anti-PPAR120573120575 (1 500) anti-PPAR120574 (1 500) anti-phospho-cPLA

2(1 500) total cPLA

2

(1 500) or anti-COX-2 (1 500) antibodies in TBST Mem-branes were washed with TBST five times for 5min eachand incubated with the appropriate Horseradish peroxidase-conjugated secondary antibody (1 1000) for 1 h at roomtemperature Blots initially probed with an antibody werestripped by incubation in 50mMTris-HCl pH 67 100mM120573-mercaptoethanol and 2 SDS for 30min at 50∘C Followingextensive washing blots were reprobed with an anti-ERK12antibody (1 5000) as a loading control Immunoreactiveproteins were visualized by enhanced chemiluminescencedetecting system Densitometry analyses of immunoblotswere performed using Syngene GeneGenius scanning densit-ometer and software

27 Phagocytic Assays A flow cytometry-based method wasused to study the phagocytic activity of macrophages J774A1macrophages were seeded at 1 times 106 cellsmL per well in24-well tissue culture plates and incubated at 37∘C 5CO2for 24 h Macrophages were treated or not with PPAR

agonists (24 h) or cPLA2antagonist (1 h) and incubated

with (FITC)-conjugated zymosan A BioParticles (Molec-ular Probes Europe BV Leiden The Netherlands) at 50particlescell ratio for 2 h unless indicated otherwise or10 120583M CFSE-labeled promastigotes (1 10 ratio) for 60 or120min at 37∘C except control wells After incubation excessnonphagocytized promastigotes or particles were removed bywashing Cells were collected in tubes and phagocytosis wasdetermined by two criteria (1) the number of phagocytizingcells and (2) the mean fluorescent intensity (MFI) in aFACSCalibur

28 Expression of Mannose Receptor and Toll-Like Receptor4 (TLR4) Macrophages were pretreated or not for 24 hwith the PPAR120573120575 and PPAR120574 agonists or for 1 h withcPLA2inhibitor ATK After incubation conditions non-

infected L mexicana-infected LPS or zymosan-stimulatedmacrophages where it is indicated cells were quickly washedtwice with icecold PBS containing 2 of FBS (FACS buffer)and scraped on FACS buffer then cells were spin downand the supernatant was removed After that cells wereresuspended in 1mL of fixer solution and incubated for1 h at 37∘C then cells were centrifuged and washed twicewith FACS buffer Nonspecific staining was blocked with10 PBS-goat serum for 1 h at 37∘C (Fc block) After twowashes with FACS buffer the appropriate antibodies were

4 BioMed Research International

added rabbit anti-human MR (1 100) and mouse anti-human TLR4 (1 100) and incubated for 1 h at 37∘C in FACSbuffer After washing the samples twice they were incubatedwith the appropriate antibody goat anti-rabbit IgG (H + L)rhodamine-conjugated antibody (1 100 Millipore) donkeyanti-mouse IgG (H + L) and Pacific blue-conjugated anti-body (1 100 Sigma-Aldrich) for 1 h at 37∘C Finally sampleswere washed twice and read in a FACSCalibur

29 Oxidative Metabolism The oxidative metabolism ofJ774A1 macrophages was measured by their ability to reduceyellow-colored nitroblue tetrazolium (NBT) to blue for-mazan through the production of superoxide anions asdescribed by Nessa et al [18] Macrophages were simultane-ously incubated with promastigotes and NBT (1mgmL inPBS) for the indicated times To determine if PPAR agonistswere able to increase oxidative metabolism macrophages(2mL 1 times 106mL) were allowed to adhere to coverslips inplastic Petri dishes (35 times 10mm Nunclon Denmark) byincubation at 37∘C for 24 h with PPAR agonists or cPLA

2

antagonist for 1 h Then macrophages were infected with Lmexicana promastigotes (1 10 ratio) After that 1mL of NBTsolution was added to the reaction mixture and incubatedat 37∘C for the indicated times The reaction was stopped byadding 1mL of 05 HCl and cells were further stained withfuccina for 30 sec Then they were washed three times withPBS and positive-oxidative burst cells were counted in a lightmicroscope (100 cellfield and six fieldscondition) Quan-titative production of formazan was determined in 96-wellplates [19] macrophages (1 times 105) were stimulated with PPARagonists as described above and then they were infected withpromastigotes for the indicated periods and NBT added Atthe end of each time macrophages were washed with 70methanol in order to remove nonreduced NBT the producedformazan was dissolved in DMSO and the optical densityof the solution was measured in a spectrophotometer (Bio-Rad) at 630 nm wavelength A Petri dish or a 96-well platewith noninfected macrophages was incubated with NBT andserved as control for each type of experiment

210 Prostaglandins Extraction The following procedure wasdeveloped for the separation of eicosanoids from 24-well cellculture plates containing 2mL of media Media was collectedand centrifuged for 5min at 10000 xg to remove cellulardebris Produced eicosanoids were isolated via solid-phaseextraction using SPE cartridge C

18from Millipore Columns

were prewashed with 2mL of MeOH followed by 2mL ofH2O After applying the sample to the columns they were

washed with 1mL of 10 MeOH and prostaglandins wereeluted with 1mL of MeOH The eluate was dried undervacuum and redissolved in 100120583L of chloroform-MeOH(2 1)

211 Mass Spectrometry (MS) All MS analyses were per-formed using an Applied Biosystems 3200 QTRAP hybridtriple-quadrupole linear ion trap mass spectrometerequipped with a Turbo V ion source and operated in MRMmode For all experiments the Turbo V ion source wasoperated in a negative electrospray mode with N

2gas and

the QTRAP parameters DP EP CE and CXP were set andmaximized for each eicosanoid and all the samples wereloaded by direct infusion at 10 120583Lmin

212 Statistical Analysis To take into account all values of thekinetics of macrophages infection a statistical analysis wasperformed using two-way ANOVA and Bonferronirsquos multiplecomparison tests using GraphPad Prism 40 (GraphPadSoftware San Diego CA) Differences with 119875 lt 005 wereconsidered significant

3 Results

31 PPAR Agonists Downregulate cPLA2and COX-2 Expres-

sion in J774A1 Macrophages Infected with Leishmania mex-icana Promastigotes In L mexicana-infected macrophagesthe activation of cPLA

2by phosphorylation and the expres-

sion of COX-2 are triggered The activation of theseenzymes is considered necessary within the proinflamma-tory response whereas PPAR activation is considered asan important part of the anti-inflammatory process bothin vivo and in vitro [20ndash22] To examine the possibilitythat PPAR agonists could inhibit cPLA

2phosphorylation

and COX-2 expression J774A1 macrophages were incu-bated with different PPAR agonists and then cells wereinfected with L mexicana promastigotes The effects of Lmexicana promastigotes on the expression of PPAR COX-2 and cPLA

2phosphorylation in J774A1 macrophages were

examined (Figure 1) Results showed that there were nochanges in the expression of PPARs after infection with Lmexicana promastigotes of macrophages whether treatedor not with PPAR agonists however cPLA

2phosphoryla-

tion diminished significantly through the infection whenmacrophages were treated with PPAR agonists (Figures1(a) and 1(b)) in addition COX-2 protein also diminishedsignificantly after infection (Figures 1(a) and 1(c)) COX-2mRNA expressionwas strongly upregulated after Lmexicanainfection (see Figure 1 available in Supplementary Materialonline at httpdxdoiorg1011552013215283) but it wasdownregulated in a time-dependent manner (Figure 1(d))when J774A1 macrophages were treated with PPAR agonistsand infected with L mexicana promastigotes

32 PPAR Agonists Downregulate IL-10 and Sustain Proin-flammatory Cytokines Expression after Infection Cytokinesand microbial products profoundly and differentially affectthe function of mononuclear phagocytes It is well estab-lished that different species of Leishmania can differentiallymodulate important inflammatory response mediators [23]In order to see how PPAR agonists could modulate theinflammatory response on J774A1 macrophages infectedwith L mexicana we evaluated the transcripts of someinflammatory cytokines and anti-inflammatory IL-10 Thecapacity of thesemacrophages to produceTNF-120572 IL-1120573 IL-6and IL-10 cytokines in response to L mexicana promastigoteswas tested via qRT-PCR (Figure 2) The infection of J774A1macrophages resulted in an increase in TNF-120572 IL-1120573 andIL-6 expression IL-10 was downregulated during Leishmaniainfection although its expression recovered compared to

BioMed Research International 5

ERK12

COX-2

p-cPLA

cPLA

LPS LPS LPS

2

2

Time post infection Time post infection Time post infection

(-) GW501516 120573120575 GW1929 120574

PPAR120574

PPAR120573120575

MΦ MΦ MΦ5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400

(a)

0

50

100

150

Den

sitom

etric

anal

ysis

of p

-cPL

A(

)

LPS

wTX

2

Time after infection

(b)

0

755025

150125100

225200175175

Den

sitom

etric

anal

ysis

of C

OX-

2 (

)

LPS

wTX

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowast

Time after infection

(c)

0001

1

01

001

1000

100

10

Rela

tive e

xpre

ssio

n le

vel C

OX-

2

LPS 30 60 120

ATK

PPARβδPPARγ

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowast

Mφ

Time after infection

(d)

Figure 1 PPAR agonists inhibit cPLA2phosphorylation and COX-2 expression in L mexicana-infected macrophages (a) Protein expression

for PPARs and COX-2 and cPLA2phosphorylation levels were evaluated by Western blotting (b) Densitometry analyses of cPLA

2

phosphorylation and (c) COX-2 expression were performed in basal conditions as well as in macrophages treated or not with PPAR agonists(d) COX-2 mRNA expression was evaluated by qRT-PCR and analyzed by 2minusΔΔCT method Total ERK12 was probed to normalize proteinloading Results are representative of three independent experiments Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast)119875 lt 005 (lowastlowast)119875 lt 001 and (lowastlowastlowast)119875 lt 0001

noninfected macrophages (Supplementary Figure 2) How-ever when noninfected macrophages were previously treatedwith PPAR120573120575 agonist (white bars) proinflammatory IL-6was upregulated and its expressionwas held during infectionTNF-120572 and IL-1120573 were overexpressed but they were down-regulated at 30 to 60min (after infection) and its expres-sion recovered at 120min (after infection) Moreover whennoninfected macrophages were treated with PPAR120574 agonist(black bars) TNF-120572 was upregulated but its expression wasdownregulated after infection overregulation of IL-6 dimin-ished after infection but it was held equivalent to nontreatedmacrophages IL-1120573 was upregulated and its expression washeld during infection Anti-inflammatory IL-10 cytokine wasdownregulated with both PPAR agonists after infection On

the other hand when cPLA2was blocked 1 h before the

infection by treatment ofmacrophageswith cPLA2antagonist

ATK (gray bars) proinflammatory cytokines TNF-120572 IL-1120573and IL-6were upregulated furthermore IL-10 expressionwassignificantly affectedwhen cPLA

2was inhibited In summary

PPAR agonists and cPLA2antagonist set down the levels of

IL-10 at the same time they upregulated TNF-120572 IL-1120573 andIL-6 cytokines or at least they were held overexpressed afterinfection as occurred with nontreated infected macrophages(Supplementary Figure 2) all together are evidence of apossible M2 to M1 polarization

33 PPAR Activation and cPLA2Inhibition Induce TLR4

Expression in L mexicana-Infected Macrophages Polarized

6 BioMed Research International

lowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

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lowastlowastlowast

lowastlowast

0001

001

01

1

10

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-10

Time after infection

(a)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

01

1

10

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10000

Rela

tive e

xpre

ssio

n le

vel

LPS

Time after infection

(b)

lowastlowastlowast

lowastlowastlowast

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lowastlowastlowast lowastlowastlowast

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01

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tive e

xpre

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vel

LPS

IL-6

ATK

Time after infection

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowastlowast

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lowast

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0001

001

01

1

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Rela

tive e

xpre

ssio

n le

vel

LPS

ATK

Time after infection

(d)

Figure 2 Cytokine determination in L mexicana-infected macrophages Levels of gene expression for each sample were normalized with120573-actin RNA as internal control Modulation was expressed relative to the untreated control using the 2minusΔΔCT method The 119909-axis interceptsthe 119910-axis at ldquo1rdquo to show the increase and the decrease of each cytokine compared to nontreated infected macrophages Relative expressionlevel for each cytokine was calculated according to ΔΔCT = (CT test minus CT 120573-actin) treated minus (CT test minus CT 120573-actin) untreated formula [17]Graph bars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versusnontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages differ in terms of receptor expression cytokineand chemokine repertoires and effector function M1macrophages exposed to the classic activation signals expressreceptors such as CD16 CD32 CD64 TLR2 and TLR4whereas M2 macrophages are characterized by abundantlevels of nonopsonic receptors such as the mannose receptor(MR) [24] In order to investigate if PPAR agonists andcPLA2antagonist induced macrophage polarization during

Lmexicana infection we evaluatedMR andTLR4 expressionby flow cytometry Since BALBcmice macrophages can fullysupport maturation of alternatively activated macrophages

J774A1 macrophages were incubated with PPAR agonistsand cPLA

2antagonist and then infected or stimulated with

LPS and zymosan neither infection nor treatments inducedMR expression an M2 receptor classified as an alternativeactivation marker (Supplementary Figure 3) however PPARagonists and cPLA

2antagonist induced an increase of TLR4

expression at 60 and 120min after infection (Figure 3) anM1 receptor classified as classical activation marker Theseresults show that PPAR120573120575 GW501516 and PPAR120574 GW1929agonists and cPLA

2antagonist do not help to keep the

M2 polarization profile instead PPAR agonists and cPLA2

BioMed Research International 7

0

50

100

150

TLR4+

(MFI

)

LPS

wTXATK

lowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

Time after infection

Figure 3 TLR4 expression in L mexicana-infected macrophagesCells were treated or not with PPAR agonists for 24 h and cPLA

2

antagonist for 1 h before infection TLR4 expression was analyzed byflow cytometry LPS (2 h) was used as a positive control of inductionGraph bars are mean plusmn SEM of three independent experiments andstatistical analysis was performed comparing for each time treatedversus non-treated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and(lowastlowastlowast) 119875 lt 0001

antagonist promote the polarization of macrophages towardan M1 profile

34 PPAR120574Activation byAgonist and cPLA2Inhibition Reduce

Parasite Burden PPAR120574 expression is strongly associatedwith maturation of M2 macrophages Gallardo-Soler et alandAdapala andChan [25 26] have demonstrated that PPARagonists increased intracellular growth of L major in bone-marrow-derived macrophages moreover PPAR120574 agonistCurcumine induced PPAR120574 expression in residential liverand spleen macrophages of BALBc mice In addition oraladministration of Curcumin further increases PPAR120572 andPPAR120574 expression and this increase was associated with aheavier parasite burden To analyze how PPAR120573120575GW501516and PPAR120574 GW1929 agonists and cPLA

2inhibition affected

parasite burden in treated macrophages their phagocyticactivity was evaluated through two parameters the phago-cytizing cell percentage and the number of phagocytizedzymosan particles or parasitescell treated or nontreatedmacrophages were incubated with FITC-labeled zymosanparticles by 2 h or infected with CFSE-labeled promastigotesby 1-2 h (Figure 4) PPAR120574GW1929 agonist does not increasethe number of zymosan-phagocytizing macrophages butPPAR120573120575 GW501516 agonist and cPLA

2ATK antagonist

treatments decreased the number of zymosan-phagocytizingmacrophages (119875 lt 005 and 119875 lt 0001 resp Figure 4(a)(zymosan)) on the contrary zymosan particlescell (meanfluorescence intensity (MFI)) increased significantly withall treatments GW501516 and GW1929 (119875 lt 0001) andATK (119875 lt 005) (Figure 4(b) (zymosan)) This result shows

that phagocytic activity per se was not affected by treat-ments The number of CFSE-labeled parasites-phagocytizingmacrophages did not increase by treatments either insteadparasites-phagocytizing macrophages diminished signifi-cantly with GW1929 (119875 lt 001) and ATK (119875 lt 0001)at 60min after infection and 2h after infection GW501516(119875 lt 001) GW1929 (119875 lt 0001) and ATK (119875 lt 0001)(Figure 4(a)) Only cPLA

2antagonist (Figure 4(b)) slightly

increased the number of parasitescell (119875 lt 005) at 60minafter infection but neither PPAR120573120575 nor PPAR120574 agonistsincreased it however at 120min after infection PPAR120573120575agonist slightly increased parasite burden (119875 lt 005)but PPAR120574 agonist and cPLA

2inhibition decreased parasite

burden significantly (119875 lt 0001) (Figure 4(b)) These resultstogether demonstrate that PPAR activation by these agonistsand cPLA

2inhibition did not increase parasite load

35 PPAR Activation by Agonists Selectively Regulates Pros-taglandin Production in L mexicana-Infected MacrophagesProstaglandins are potent ligands of the intracellular PPARreceptors in macrophages and their binding to PPAR120572and PPAR120574 causes macrophage deactivation Perez-Santosand Talamas-Rohana [14] have demonstrated that COX-2 inhibition induced leishmanicidal activity by splenocytes[14] Thus one possible mechanism of intracellular sur-vival of Leishmania is the deactivation of macrophages byprostaglandins produced [27] In order to investigate ifPPAR agonists couldmodulate inflammatory prostaglandinsmacrophages were treated with PPAR agonists or cPLA

2

antagonist before the infection to look for PGrsquos metabolitesin the conditioned media (Figure 5) PPAR activation andcPLA2inhibition decreased significantly 6k-PGF

1120572 PGE

1

and PGF1120572

production (Supplementary Figure 4) howeverPPAR120574 agonist was not able to reduce PGE

2production

which increased significantly after infection (119875 lt 0001) at60 and 120min respectively (Figure 5) PPAR120573120575 agonist andcPLA2antagonist did not increase PGE

2production even its

production diminished (119875 lt 005 and 119875 lt 0001) at 120minafter infection respectively moreover PPAR agonists andcPLA2antagonist significantly increased PGF

2120572production

(119875 lt 0001) at 60 and 120min after infection respectively Insummary PPAR agonists and cPLA

2antagonist diminished

6k-PGF1120572 PGF

1120572 and PGE

1 and neither Leishmania infec-

tion nor LPS was able to recover their production howeverPGF2120572

production was increased after infection and onlyPPAR120574 activation increased PGE

2production

36 PPARActivation and cPLA2Inhibition Increase theOxida-

tive Burst during J774A1 Macrophages Infection with Lmexicana Promastigotes Several studies have demonstratedthat ROS modulate arachidonic acid metabolism and pro-duction of eicosanoids in activated macrophages [28 29]In murine and human macrophages it has been establishedthat the respiratory burst of the cell with production ofROS such as H

2O2and O

2

minus is primarily responsible forparasite control as these molecules have been reportedto be fatal for Leishmania promastigotes We next ana-lyzed the effect of PPAR agonists and cPLA

2antagonist

on the oxidative burst induced by L mexicana on J774A1

8 BioMed Research International

0

10

20

30

40

50

60

70Ph

agoc

ytiz

ing

cells

()

Zy

wTXATK

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

Time after infection

(a)

0

200

400

600

800

Part

icle

s or p

aras

ite lo

adc

ell (

MFI

)

Zy

wTXATK

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 4 Phagocytic activity of J774A1 macrophages was determined for zymosan and L mexicana promastigotes uptake treated ornontreated macrophages were incubated with zymosan-FITC for 2 h or infected with CFSE-promastigotes for 1-2 h (a) Phagocytizingmacrophage percentage (b) Zymosan particles or parasitescell (MFI) Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 (lowastlowastlowast) 119875 lt 0001

0

200

400

600

800

1000

LPS

wTXATK

PGE 2

prod

uctio

n (p

gm

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

Time after infection

(a)

0

10

20

30

40

LPS

wTXATK

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 5 Prostaglandin production by L mexicana-infectedmacrophages Prostaglandins were analyzed byMSMS assay product scanningexperiments were conducted using nitrogen as collision gas and the collision energy was optimized for individual compounds to generatethe most abundant product ions These product ion spectra were then used to select the precursor-product ion pairs for the developmentof MRM assays Deuterium-labeled prostaglandins were used as internal standards for quantitation Graph bars are mean plusmn SEM of threeindependent experiments and statistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005(lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages (Figure 6)We found that positivemacrophagesto L mexicana-induced oxidative burst increased from sim341to 586 (119875 lt 0001) by PPAR120574 agonist at 120min afterinfection compared to non-treated macrophages whereastreatment with PPAR120573120575 agonist increased from sim341 to4032 (119875 lt 005) (Figures 6(a) and 6(b)) PPAR120573120575agonist slightly increased the number of positive oxidativeburst macrophages at 120min after infection and when

the oxidative burst was quantified it increased sim13-foldat 120min after infection too (119875 lt 005) (Figure 6(c)) Onthe other hand cPLA

2inhibition increased sim251-fold the

oxidative burst at 120min after infection (119875 lt 0001)compared to nontreated macrophages (Figure 6(c)) FinallyPPAR120574 agonist increased the oxidative burst sim252-(119875 lt0001) and sim355-fold (119875 lt 0001) at 60 and 120minafter infection respectively indicating that PPAR120574 activation

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

medium and cells were incubated with L mexicana Cellswere not subjected to more than 20 cell passages

24 Infection of Macrophages J774A1 macrophages (1 times106well) were cultured in 24-well culture plates Cells wereincubated with promastigotes of L mexicana at a ratio of 20parasites per macrophage or treated with LPS (1120583gmL) forthe indicated periods after which noningested promastigoteswere washed off with warm D-MEM Where indicated cellswere also pretreated for 24 h with pharmacological agonistsGW501516 (100 nM) GW1929 (600 nM) and for 1 hwithATK(75 120583M) prepared inDMSOor ethanol Vehicle controls wereincluded in each experiment

25 Real-Time PCR Assays The total mRNA from non-infected L mexicana-infected or LPS-stimulated J774A1macrophages was extracted with TRIzol reagent (Life Tech-nologies Corporation USA) according to the manufacturerrsquosinstructions The retrotranscription reaction was performedwith the First Strand cDNA Synthesis Kit according to themanufacturerrsquos instructions (Fermentas Life Sciences USA)in an iCycler Thermal Cycler (Bio-Rad) RT-PCR amplifica-tions were performed as described by Estrada-Figueroa et al[16] Reactions were done in a real-time PCR 7500 apparatus(Applied Biosystems) in a final volume of 20 120583L using 100 ngof cDNA and 10 120583L of TaqMan Universal Master Mix II(Applied Biosystems Foster City CA USA) Primers werefrom Applied Biosystems (TaqMan Gene Expression Assay)TNF-120572 (Mm00443258 m1) IL-1120573 (Mm01336189 m1) IL-6(Mm00446190 m1) COX-2 (PTGS2 Mm00478374 m1) IL-10 (Mm00439614 m1) and 120573-Actin (Mm00607939 s1) withthe following conditions 50∘C for 2min then 95∘C for10min followed by 40 cycles at 95∘C for 15 sec and 60∘Cfor 1min To verify results each sample was analyzed inquadruplicate Levels of transcription were normalized tothose of 120573-actin (internal standard) to determine the vari-ability in the amount of cDNA in each sample With the CTvalues obtained the 2minusΔΔCT method was followed to calculatethe level of expression of each cytokine or mediator genein treated macrophages in comparison with the expressionlevel of the same cytokines or mediators in the nontreatedmacrophages according to the formula [17]

ΔΔCT = (CT target minus CT120573 actin) treated

minus (CT target minus CT120573 actin) nontreated(1)

26 Preparation of Cell Extracts and Western Blot AnalysisMacrophages were pretreated for 24 h with the PPAR120573120575 andPPAR120574 agonists or for 1 h with cPLA

2inhibitor ATK while

some (basal control) were not After incubation conditions(noninfected L mexicana-infected or 2 h LPS-stimulatedmacrophages where indicated) cells were quickly washedtwice with icecold PBS and lysed by scraping in RIPAbuffer (50mM Tris-HCl pH 74 150mM NaCl 1 NonidetP-40 1mM PMSF 1 120583gmL aprotinin 1120583gmL leupeptin1mM EDTA 1mM NaF and 1mM Na

3VO4) Lysates were

centrifuged at 10000timesg for 15min at 4∘C to yield the wholecell extract Supernatants were transferred to fresh tubes and

stored at minus70∘C until required Protein concentration wasdetermined using a BCA protein assay with bovine serumalbumin as standard Equal amounts of total cell lysates (60120583gprotein) were solubilized in sample buffer by boiling for5min separated on 10 SDS-PAGE and then transferredonto a nitrocellulose membrane using a Trans blot system(Bio-Rad) Nitrocellulose membranes were then incubatedsuccessively in TBST blocking buffer (50mM Tris-HCl pH74 150mMNaCl (TBS)) containing 5 skimmed dried milkand 005 Tween 20 for 1 h at room temperature to blocknonspecific protein binding Membranes were incubatedovernight at 4∘C with a specific anti-PPAR120573120575 (1 500) anti-PPAR120574 (1 500) anti-phospho-cPLA

2(1 500) total cPLA

2

(1 500) or anti-COX-2 (1 500) antibodies in TBST Mem-branes were washed with TBST five times for 5min eachand incubated with the appropriate Horseradish peroxidase-conjugated secondary antibody (1 1000) for 1 h at roomtemperature Blots initially probed with an antibody werestripped by incubation in 50mMTris-HCl pH 67 100mM120573-mercaptoethanol and 2 SDS for 30min at 50∘C Followingextensive washing blots were reprobed with an anti-ERK12antibody (1 5000) as a loading control Immunoreactiveproteins were visualized by enhanced chemiluminescencedetecting system Densitometry analyses of immunoblotswere performed using Syngene GeneGenius scanning densit-ometer and software

27 Phagocytic Assays A flow cytometry-based method wasused to study the phagocytic activity of macrophages J774A1macrophages were seeded at 1 times 106 cellsmL per well in24-well tissue culture plates and incubated at 37∘C 5CO2for 24 h Macrophages were treated or not with PPAR

agonists (24 h) or cPLA2antagonist (1 h) and incubated

with (FITC)-conjugated zymosan A BioParticles (Molec-ular Probes Europe BV Leiden The Netherlands) at 50particlescell ratio for 2 h unless indicated otherwise or10 120583M CFSE-labeled promastigotes (1 10 ratio) for 60 or120min at 37∘C except control wells After incubation excessnonphagocytized promastigotes or particles were removed bywashing Cells were collected in tubes and phagocytosis wasdetermined by two criteria (1) the number of phagocytizingcells and (2) the mean fluorescent intensity (MFI) in aFACSCalibur

28 Expression of Mannose Receptor and Toll-Like Receptor4 (TLR4) Macrophages were pretreated or not for 24 hwith the PPAR120573120575 and PPAR120574 agonists or for 1 h withcPLA2inhibitor ATK After incubation conditions non-

infected L mexicana-infected LPS or zymosan-stimulatedmacrophages where it is indicated cells were quickly washedtwice with icecold PBS containing 2 of FBS (FACS buffer)and scraped on FACS buffer then cells were spin downand the supernatant was removed After that cells wereresuspended in 1mL of fixer solution and incubated for1 h at 37∘C then cells were centrifuged and washed twicewith FACS buffer Nonspecific staining was blocked with10 PBS-goat serum for 1 h at 37∘C (Fc block) After twowashes with FACS buffer the appropriate antibodies were

4 BioMed Research International

added rabbit anti-human MR (1 100) and mouse anti-human TLR4 (1 100) and incubated for 1 h at 37∘C in FACSbuffer After washing the samples twice they were incubatedwith the appropriate antibody goat anti-rabbit IgG (H + L)rhodamine-conjugated antibody (1 100 Millipore) donkeyanti-mouse IgG (H + L) and Pacific blue-conjugated anti-body (1 100 Sigma-Aldrich) for 1 h at 37∘C Finally sampleswere washed twice and read in a FACSCalibur

29 Oxidative Metabolism The oxidative metabolism ofJ774A1 macrophages was measured by their ability to reduceyellow-colored nitroblue tetrazolium (NBT) to blue for-mazan through the production of superoxide anions asdescribed by Nessa et al [18] Macrophages were simultane-ously incubated with promastigotes and NBT (1mgmL inPBS) for the indicated times To determine if PPAR agonistswere able to increase oxidative metabolism macrophages(2mL 1 times 106mL) were allowed to adhere to coverslips inplastic Petri dishes (35 times 10mm Nunclon Denmark) byincubation at 37∘C for 24 h with PPAR agonists or cPLA

2

antagonist for 1 h Then macrophages were infected with Lmexicana promastigotes (1 10 ratio) After that 1mL of NBTsolution was added to the reaction mixture and incubatedat 37∘C for the indicated times The reaction was stopped byadding 1mL of 05 HCl and cells were further stained withfuccina for 30 sec Then they were washed three times withPBS and positive-oxidative burst cells were counted in a lightmicroscope (100 cellfield and six fieldscondition) Quan-titative production of formazan was determined in 96-wellplates [19] macrophages (1 times 105) were stimulated with PPARagonists as described above and then they were infected withpromastigotes for the indicated periods and NBT added Atthe end of each time macrophages were washed with 70methanol in order to remove nonreduced NBT the producedformazan was dissolved in DMSO and the optical densityof the solution was measured in a spectrophotometer (Bio-Rad) at 630 nm wavelength A Petri dish or a 96-well platewith noninfected macrophages was incubated with NBT andserved as control for each type of experiment

210 Prostaglandins Extraction The following procedure wasdeveloped for the separation of eicosanoids from 24-well cellculture plates containing 2mL of media Media was collectedand centrifuged for 5min at 10000 xg to remove cellulardebris Produced eicosanoids were isolated via solid-phaseextraction using SPE cartridge C

18from Millipore Columns

were prewashed with 2mL of MeOH followed by 2mL ofH2O After applying the sample to the columns they were

washed with 1mL of 10 MeOH and prostaglandins wereeluted with 1mL of MeOH The eluate was dried undervacuum and redissolved in 100120583L of chloroform-MeOH(2 1)

211 Mass Spectrometry (MS) All MS analyses were per-formed using an Applied Biosystems 3200 QTRAP hybridtriple-quadrupole linear ion trap mass spectrometerequipped with a Turbo V ion source and operated in MRMmode For all experiments the Turbo V ion source wasoperated in a negative electrospray mode with N

2gas and

the QTRAP parameters DP EP CE and CXP were set andmaximized for each eicosanoid and all the samples wereloaded by direct infusion at 10 120583Lmin

212 Statistical Analysis To take into account all values of thekinetics of macrophages infection a statistical analysis wasperformed using two-way ANOVA and Bonferronirsquos multiplecomparison tests using GraphPad Prism 40 (GraphPadSoftware San Diego CA) Differences with 119875 lt 005 wereconsidered significant

3 Results

31 PPAR Agonists Downregulate cPLA2and COX-2 Expres-

sion in J774A1 Macrophages Infected with Leishmania mex-icana Promastigotes In L mexicana-infected macrophagesthe activation of cPLA

2by phosphorylation and the expres-

sion of COX-2 are triggered The activation of theseenzymes is considered necessary within the proinflamma-tory response whereas PPAR activation is considered asan important part of the anti-inflammatory process bothin vivo and in vitro [20ndash22] To examine the possibilitythat PPAR agonists could inhibit cPLA

2phosphorylation

and COX-2 expression J774A1 macrophages were incu-bated with different PPAR agonists and then cells wereinfected with L mexicana promastigotes The effects of Lmexicana promastigotes on the expression of PPAR COX-2 and cPLA

2phosphorylation in J774A1 macrophages were

examined (Figure 1) Results showed that there were nochanges in the expression of PPARs after infection with Lmexicana promastigotes of macrophages whether treatedor not with PPAR agonists however cPLA

2phosphoryla-

tion diminished significantly through the infection whenmacrophages were treated with PPAR agonists (Figures1(a) and 1(b)) in addition COX-2 protein also diminishedsignificantly after infection (Figures 1(a) and 1(c)) COX-2mRNA expressionwas strongly upregulated after Lmexicanainfection (see Figure 1 available in Supplementary Materialonline at httpdxdoiorg1011552013215283) but it wasdownregulated in a time-dependent manner (Figure 1(d))when J774A1 macrophages were treated with PPAR agonistsand infected with L mexicana promastigotes

32 PPAR Agonists Downregulate IL-10 and Sustain Proin-flammatory Cytokines Expression after Infection Cytokinesand microbial products profoundly and differentially affectthe function of mononuclear phagocytes It is well estab-lished that different species of Leishmania can differentiallymodulate important inflammatory response mediators [23]In order to see how PPAR agonists could modulate theinflammatory response on J774A1 macrophages infectedwith L mexicana we evaluated the transcripts of someinflammatory cytokines and anti-inflammatory IL-10 Thecapacity of thesemacrophages to produceTNF-120572 IL-1120573 IL-6and IL-10 cytokines in response to L mexicana promastigoteswas tested via qRT-PCR (Figure 2) The infection of J774A1macrophages resulted in an increase in TNF-120572 IL-1120573 andIL-6 expression IL-10 was downregulated during Leishmaniainfection although its expression recovered compared to

BioMed Research International 5

ERK12

COX-2

p-cPLA

cPLA

LPS LPS LPS

2

2

Time post infection Time post infection Time post infection

(-) GW501516 120573120575 GW1929 120574

PPAR120574

PPAR120573120575

MΦ MΦ MΦ5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400

(a)

0

50

100

150

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sitom

etric

anal

ysis

of p

-cPL

A(

)

LPS

wTX

2

Time after infection

(b)

0

755025

150125100

225200175175

Den

sitom

etric

anal

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of C

OX-

2 (

)

LPS

wTX

lowastlowastlowast

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lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowast

Time after infection

(c)

0001

1

01

001

1000

100

10

Rela

tive e

xpre

ssio

n le

vel C

OX-

2

LPS 30 60 120

ATK

PPARβδPPARγ

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowast

Mφ

Time after infection

(d)

Figure 1 PPAR agonists inhibit cPLA2phosphorylation and COX-2 expression in L mexicana-infected macrophages (a) Protein expression

for PPARs and COX-2 and cPLA2phosphorylation levels were evaluated by Western blotting (b) Densitometry analyses of cPLA

2

phosphorylation and (c) COX-2 expression were performed in basal conditions as well as in macrophages treated or not with PPAR agonists(d) COX-2 mRNA expression was evaluated by qRT-PCR and analyzed by 2minusΔΔCT method Total ERK12 was probed to normalize proteinloading Results are representative of three independent experiments Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast)119875 lt 005 (lowastlowast)119875 lt 001 and (lowastlowastlowast)119875 lt 0001

noninfected macrophages (Supplementary Figure 2) How-ever when noninfected macrophages were previously treatedwith PPAR120573120575 agonist (white bars) proinflammatory IL-6was upregulated and its expressionwas held during infectionTNF-120572 and IL-1120573 were overexpressed but they were down-regulated at 30 to 60min (after infection) and its expres-sion recovered at 120min (after infection) Moreover whennoninfected macrophages were treated with PPAR120574 agonist(black bars) TNF-120572 was upregulated but its expression wasdownregulated after infection overregulation of IL-6 dimin-ished after infection but it was held equivalent to nontreatedmacrophages IL-1120573 was upregulated and its expression washeld during infection Anti-inflammatory IL-10 cytokine wasdownregulated with both PPAR agonists after infection On

the other hand when cPLA2was blocked 1 h before the

infection by treatment ofmacrophageswith cPLA2antagonist

ATK (gray bars) proinflammatory cytokines TNF-120572 IL-1120573and IL-6were upregulated furthermore IL-10 expressionwassignificantly affectedwhen cPLA

2was inhibited In summary

PPAR agonists and cPLA2antagonist set down the levels of

IL-10 at the same time they upregulated TNF-120572 IL-1120573 andIL-6 cytokines or at least they were held overexpressed afterinfection as occurred with nontreated infected macrophages(Supplementary Figure 2) all together are evidence of apossible M2 to M1 polarization

33 PPAR Activation and cPLA2Inhibition Induce TLR4

Expression in L mexicana-Infected Macrophages Polarized

6 BioMed Research International

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lowastlowastlowast lowastlowastlowast

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01

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10

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-10

Time after infection

(a)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

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01

1

10

100

1000

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xpre

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n le

vel

LPS

Time after infection

(b)

lowastlowastlowast

lowastlowastlowast

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lowastlowastlowast lowastlowastlowast

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01

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tive e

xpre

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vel

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IL-6

ATK

Time after infection

(c)

lowastlowastlowast

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lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowastlowast

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lowast

lowast lowast

0001

001

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

ATK

Time after infection

(d)

Figure 2 Cytokine determination in L mexicana-infected macrophages Levels of gene expression for each sample were normalized with120573-actin RNA as internal control Modulation was expressed relative to the untreated control using the 2minusΔΔCT method The 119909-axis interceptsthe 119910-axis at ldquo1rdquo to show the increase and the decrease of each cytokine compared to nontreated infected macrophages Relative expressionlevel for each cytokine was calculated according to ΔΔCT = (CT test minus CT 120573-actin) treated minus (CT test minus CT 120573-actin) untreated formula [17]Graph bars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versusnontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages differ in terms of receptor expression cytokineand chemokine repertoires and effector function M1macrophages exposed to the classic activation signals expressreceptors such as CD16 CD32 CD64 TLR2 and TLR4whereas M2 macrophages are characterized by abundantlevels of nonopsonic receptors such as the mannose receptor(MR) [24] In order to investigate if PPAR agonists andcPLA2antagonist induced macrophage polarization during

Lmexicana infection we evaluatedMR andTLR4 expressionby flow cytometry Since BALBcmice macrophages can fullysupport maturation of alternatively activated macrophages

J774A1 macrophages were incubated with PPAR agonistsand cPLA

2antagonist and then infected or stimulated with

LPS and zymosan neither infection nor treatments inducedMR expression an M2 receptor classified as an alternativeactivation marker (Supplementary Figure 3) however PPARagonists and cPLA

2antagonist induced an increase of TLR4

expression at 60 and 120min after infection (Figure 3) anM1 receptor classified as classical activation marker Theseresults show that PPAR120573120575 GW501516 and PPAR120574 GW1929agonists and cPLA

2antagonist do not help to keep the

M2 polarization profile instead PPAR agonists and cPLA2

BioMed Research International 7

0

50

100

150

TLR4+

(MFI

)

LPS

wTXATK

lowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

Time after infection

Figure 3 TLR4 expression in L mexicana-infected macrophagesCells were treated or not with PPAR agonists for 24 h and cPLA

2

antagonist for 1 h before infection TLR4 expression was analyzed byflow cytometry LPS (2 h) was used as a positive control of inductionGraph bars are mean plusmn SEM of three independent experiments andstatistical analysis was performed comparing for each time treatedversus non-treated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and(lowastlowastlowast) 119875 lt 0001

antagonist promote the polarization of macrophages towardan M1 profile

34 PPAR120574Activation byAgonist and cPLA2Inhibition Reduce

Parasite Burden PPAR120574 expression is strongly associatedwith maturation of M2 macrophages Gallardo-Soler et alandAdapala andChan [25 26] have demonstrated that PPARagonists increased intracellular growth of L major in bone-marrow-derived macrophages moreover PPAR120574 agonistCurcumine induced PPAR120574 expression in residential liverand spleen macrophages of BALBc mice In addition oraladministration of Curcumin further increases PPAR120572 andPPAR120574 expression and this increase was associated with aheavier parasite burden To analyze how PPAR120573120575GW501516and PPAR120574 GW1929 agonists and cPLA

2inhibition affected

parasite burden in treated macrophages their phagocyticactivity was evaluated through two parameters the phago-cytizing cell percentage and the number of phagocytizedzymosan particles or parasitescell treated or nontreatedmacrophages were incubated with FITC-labeled zymosanparticles by 2 h or infected with CFSE-labeled promastigotesby 1-2 h (Figure 4) PPAR120574GW1929 agonist does not increasethe number of zymosan-phagocytizing macrophages butPPAR120573120575 GW501516 agonist and cPLA

2ATK antagonist

treatments decreased the number of zymosan-phagocytizingmacrophages (119875 lt 005 and 119875 lt 0001 resp Figure 4(a)(zymosan)) on the contrary zymosan particlescell (meanfluorescence intensity (MFI)) increased significantly withall treatments GW501516 and GW1929 (119875 lt 0001) andATK (119875 lt 005) (Figure 4(b) (zymosan)) This result shows

that phagocytic activity per se was not affected by treat-ments The number of CFSE-labeled parasites-phagocytizingmacrophages did not increase by treatments either insteadparasites-phagocytizing macrophages diminished signifi-cantly with GW1929 (119875 lt 001) and ATK (119875 lt 0001)at 60min after infection and 2h after infection GW501516(119875 lt 001) GW1929 (119875 lt 0001) and ATK (119875 lt 0001)(Figure 4(a)) Only cPLA

2antagonist (Figure 4(b)) slightly

increased the number of parasitescell (119875 lt 005) at 60minafter infection but neither PPAR120573120575 nor PPAR120574 agonistsincreased it however at 120min after infection PPAR120573120575agonist slightly increased parasite burden (119875 lt 005)but PPAR120574 agonist and cPLA

2inhibition decreased parasite

burden significantly (119875 lt 0001) (Figure 4(b)) These resultstogether demonstrate that PPAR activation by these agonistsand cPLA

2inhibition did not increase parasite load

35 PPAR Activation by Agonists Selectively Regulates Pros-taglandin Production in L mexicana-Infected MacrophagesProstaglandins are potent ligands of the intracellular PPARreceptors in macrophages and their binding to PPAR120572and PPAR120574 causes macrophage deactivation Perez-Santosand Talamas-Rohana [14] have demonstrated that COX-2 inhibition induced leishmanicidal activity by splenocytes[14] Thus one possible mechanism of intracellular sur-vival of Leishmania is the deactivation of macrophages byprostaglandins produced [27] In order to investigate ifPPAR agonists couldmodulate inflammatory prostaglandinsmacrophages were treated with PPAR agonists or cPLA

2

antagonist before the infection to look for PGrsquos metabolitesin the conditioned media (Figure 5) PPAR activation andcPLA2inhibition decreased significantly 6k-PGF

1120572 PGE

1

and PGF1120572

production (Supplementary Figure 4) howeverPPAR120574 agonist was not able to reduce PGE

2production

which increased significantly after infection (119875 lt 0001) at60 and 120min respectively (Figure 5) PPAR120573120575 agonist andcPLA2antagonist did not increase PGE

2production even its

production diminished (119875 lt 005 and 119875 lt 0001) at 120minafter infection respectively moreover PPAR agonists andcPLA2antagonist significantly increased PGF

2120572production

(119875 lt 0001) at 60 and 120min after infection respectively Insummary PPAR agonists and cPLA

2antagonist diminished

6k-PGF1120572 PGF

1120572 and PGE

1 and neither Leishmania infec-

tion nor LPS was able to recover their production howeverPGF2120572

production was increased after infection and onlyPPAR120574 activation increased PGE

2production

36 PPARActivation and cPLA2Inhibition Increase theOxida-

tive Burst during J774A1 Macrophages Infection with Lmexicana Promastigotes Several studies have demonstratedthat ROS modulate arachidonic acid metabolism and pro-duction of eicosanoids in activated macrophages [28 29]In murine and human macrophages it has been establishedthat the respiratory burst of the cell with production ofROS such as H

2O2and O

2

minus is primarily responsible forparasite control as these molecules have been reportedto be fatal for Leishmania promastigotes We next ana-lyzed the effect of PPAR agonists and cPLA

2antagonist

on the oxidative burst induced by L mexicana on J774A1

8 BioMed Research International

0

10

20

30

40

50

60

70Ph

agoc

ytiz

ing

cells

()

Zy

wTXATK

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

Time after infection

(a)

0

200

400

600

800

Part

icle

s or p

aras

ite lo

adc

ell (

MFI

)

Zy

wTXATK

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 4 Phagocytic activity of J774A1 macrophages was determined for zymosan and L mexicana promastigotes uptake treated ornontreated macrophages were incubated with zymosan-FITC for 2 h or infected with CFSE-promastigotes for 1-2 h (a) Phagocytizingmacrophage percentage (b) Zymosan particles or parasitescell (MFI) Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 (lowastlowastlowast) 119875 lt 0001

0

200

400

600

800

1000

LPS

wTXATK

PGE 2

prod

uctio

n (p

gm

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

Time after infection

(a)

0

10

20

30

40

LPS

wTXATK

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 5 Prostaglandin production by L mexicana-infectedmacrophages Prostaglandins were analyzed byMSMS assay product scanningexperiments were conducted using nitrogen as collision gas and the collision energy was optimized for individual compounds to generatethe most abundant product ions These product ion spectra were then used to select the precursor-product ion pairs for the developmentof MRM assays Deuterium-labeled prostaglandins were used as internal standards for quantitation Graph bars are mean plusmn SEM of threeindependent experiments and statistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005(lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages (Figure 6)We found that positivemacrophagesto L mexicana-induced oxidative burst increased from sim341to 586 (119875 lt 0001) by PPAR120574 agonist at 120min afterinfection compared to non-treated macrophages whereastreatment with PPAR120573120575 agonist increased from sim341 to4032 (119875 lt 005) (Figures 6(a) and 6(b)) PPAR120573120575agonist slightly increased the number of positive oxidativeburst macrophages at 120min after infection and when

the oxidative burst was quantified it increased sim13-foldat 120min after infection too (119875 lt 005) (Figure 6(c)) Onthe other hand cPLA

2inhibition increased sim251-fold the

oxidative burst at 120min after infection (119875 lt 0001)compared to nontreated macrophages (Figure 6(c)) FinallyPPAR120574 agonist increased the oxidative burst sim252-(119875 lt0001) and sim355-fold (119875 lt 0001) at 60 and 120minafter infection respectively indicating that PPAR120574 activation

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

added rabbit anti-human MR (1 100) and mouse anti-human TLR4 (1 100) and incubated for 1 h at 37∘C in FACSbuffer After washing the samples twice they were incubatedwith the appropriate antibody goat anti-rabbit IgG (H + L)rhodamine-conjugated antibody (1 100 Millipore) donkeyanti-mouse IgG (H + L) and Pacific blue-conjugated anti-body (1 100 Sigma-Aldrich) for 1 h at 37∘C Finally sampleswere washed twice and read in a FACSCalibur

29 Oxidative Metabolism The oxidative metabolism ofJ774A1 macrophages was measured by their ability to reduceyellow-colored nitroblue tetrazolium (NBT) to blue for-mazan through the production of superoxide anions asdescribed by Nessa et al [18] Macrophages were simultane-ously incubated with promastigotes and NBT (1mgmL inPBS) for the indicated times To determine if PPAR agonistswere able to increase oxidative metabolism macrophages(2mL 1 times 106mL) were allowed to adhere to coverslips inplastic Petri dishes (35 times 10mm Nunclon Denmark) byincubation at 37∘C for 24 h with PPAR agonists or cPLA

2

antagonist for 1 h Then macrophages were infected with Lmexicana promastigotes (1 10 ratio) After that 1mL of NBTsolution was added to the reaction mixture and incubatedat 37∘C for the indicated times The reaction was stopped byadding 1mL of 05 HCl and cells were further stained withfuccina for 30 sec Then they were washed three times withPBS and positive-oxidative burst cells were counted in a lightmicroscope (100 cellfield and six fieldscondition) Quan-titative production of formazan was determined in 96-wellplates [19] macrophages (1 times 105) were stimulated with PPARagonists as described above and then they were infected withpromastigotes for the indicated periods and NBT added Atthe end of each time macrophages were washed with 70methanol in order to remove nonreduced NBT the producedformazan was dissolved in DMSO and the optical densityof the solution was measured in a spectrophotometer (Bio-Rad) at 630 nm wavelength A Petri dish or a 96-well platewith noninfected macrophages was incubated with NBT andserved as control for each type of experiment

210 Prostaglandins Extraction The following procedure wasdeveloped for the separation of eicosanoids from 24-well cellculture plates containing 2mL of media Media was collectedand centrifuged for 5min at 10000 xg to remove cellulardebris Produced eicosanoids were isolated via solid-phaseextraction using SPE cartridge C

18from Millipore Columns

were prewashed with 2mL of MeOH followed by 2mL ofH2O After applying the sample to the columns they were

washed with 1mL of 10 MeOH and prostaglandins wereeluted with 1mL of MeOH The eluate was dried undervacuum and redissolved in 100120583L of chloroform-MeOH(2 1)

211 Mass Spectrometry (MS) All MS analyses were per-formed using an Applied Biosystems 3200 QTRAP hybridtriple-quadrupole linear ion trap mass spectrometerequipped with a Turbo V ion source and operated in MRMmode For all experiments the Turbo V ion source wasoperated in a negative electrospray mode with N

2gas and

the QTRAP parameters DP EP CE and CXP were set andmaximized for each eicosanoid and all the samples wereloaded by direct infusion at 10 120583Lmin

212 Statistical Analysis To take into account all values of thekinetics of macrophages infection a statistical analysis wasperformed using two-way ANOVA and Bonferronirsquos multiplecomparison tests using GraphPad Prism 40 (GraphPadSoftware San Diego CA) Differences with 119875 lt 005 wereconsidered significant

3 Results

31 PPAR Agonists Downregulate cPLA2and COX-2 Expres-

sion in J774A1 Macrophages Infected with Leishmania mex-icana Promastigotes In L mexicana-infected macrophagesthe activation of cPLA

2by phosphorylation and the expres-

sion of COX-2 are triggered The activation of theseenzymes is considered necessary within the proinflamma-tory response whereas PPAR activation is considered asan important part of the anti-inflammatory process bothin vivo and in vitro [20ndash22] To examine the possibilitythat PPAR agonists could inhibit cPLA

2phosphorylation

and COX-2 expression J774A1 macrophages were incu-bated with different PPAR agonists and then cells wereinfected with L mexicana promastigotes The effects of Lmexicana promastigotes on the expression of PPAR COX-2 and cPLA

2phosphorylation in J774A1 macrophages were

examined (Figure 1) Results showed that there were nochanges in the expression of PPARs after infection with Lmexicana promastigotes of macrophages whether treatedor not with PPAR agonists however cPLA

2phosphoryla-

tion diminished significantly through the infection whenmacrophages were treated with PPAR agonists (Figures1(a) and 1(b)) in addition COX-2 protein also diminishedsignificantly after infection (Figures 1(a) and 1(c)) COX-2mRNA expressionwas strongly upregulated after Lmexicanainfection (see Figure 1 available in Supplementary Materialonline at httpdxdoiorg1011552013215283) but it wasdownregulated in a time-dependent manner (Figure 1(d))when J774A1 macrophages were treated with PPAR agonistsand infected with L mexicana promastigotes

32 PPAR Agonists Downregulate IL-10 and Sustain Proin-flammatory Cytokines Expression after Infection Cytokinesand microbial products profoundly and differentially affectthe function of mononuclear phagocytes It is well estab-lished that different species of Leishmania can differentiallymodulate important inflammatory response mediators [23]In order to see how PPAR agonists could modulate theinflammatory response on J774A1 macrophages infectedwith L mexicana we evaluated the transcripts of someinflammatory cytokines and anti-inflammatory IL-10 Thecapacity of thesemacrophages to produceTNF-120572 IL-1120573 IL-6and IL-10 cytokines in response to L mexicana promastigoteswas tested via qRT-PCR (Figure 2) The infection of J774A1macrophages resulted in an increase in TNF-120572 IL-1120573 andIL-6 expression IL-10 was downregulated during Leishmaniainfection although its expression recovered compared to

BioMed Research International 5

ERK12

COX-2

p-cPLA

cPLA

LPS LPS LPS

2

2

Time post infection Time post infection Time post infection

(-) GW501516 120573120575 GW1929 120574

PPAR120574

PPAR120573120575

MΦ MΦ MΦ5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400

(a)

0

50

100

150

Den

sitom

etric

anal

ysis

of p

-cPL

A(

)

LPS

wTX

2

Time after infection

(b)

0

755025

150125100

225200175175

Den

sitom

etric

anal

ysis

of C

OX-

2 (

)

LPS

wTX

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowast

Time after infection

(c)

0001

1

01

001

1000

100

10

Rela

tive e

xpre

ssio

n le

vel C

OX-

2

LPS 30 60 120

ATK

PPARβδPPARγ

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowast

Mφ

Time after infection

(d)

Figure 1 PPAR agonists inhibit cPLA2phosphorylation and COX-2 expression in L mexicana-infected macrophages (a) Protein expression

for PPARs and COX-2 and cPLA2phosphorylation levels were evaluated by Western blotting (b) Densitometry analyses of cPLA

2

phosphorylation and (c) COX-2 expression were performed in basal conditions as well as in macrophages treated or not with PPAR agonists(d) COX-2 mRNA expression was evaluated by qRT-PCR and analyzed by 2minusΔΔCT method Total ERK12 was probed to normalize proteinloading Results are representative of three independent experiments Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast)119875 lt 005 (lowastlowast)119875 lt 001 and (lowastlowastlowast)119875 lt 0001

noninfected macrophages (Supplementary Figure 2) How-ever when noninfected macrophages were previously treatedwith PPAR120573120575 agonist (white bars) proinflammatory IL-6was upregulated and its expressionwas held during infectionTNF-120572 and IL-1120573 were overexpressed but they were down-regulated at 30 to 60min (after infection) and its expres-sion recovered at 120min (after infection) Moreover whennoninfected macrophages were treated with PPAR120574 agonist(black bars) TNF-120572 was upregulated but its expression wasdownregulated after infection overregulation of IL-6 dimin-ished after infection but it was held equivalent to nontreatedmacrophages IL-1120573 was upregulated and its expression washeld during infection Anti-inflammatory IL-10 cytokine wasdownregulated with both PPAR agonists after infection On

the other hand when cPLA2was blocked 1 h before the

infection by treatment ofmacrophageswith cPLA2antagonist

ATK (gray bars) proinflammatory cytokines TNF-120572 IL-1120573and IL-6were upregulated furthermore IL-10 expressionwassignificantly affectedwhen cPLA

2was inhibited In summary

PPAR agonists and cPLA2antagonist set down the levels of

IL-10 at the same time they upregulated TNF-120572 IL-1120573 andIL-6 cytokines or at least they were held overexpressed afterinfection as occurred with nontreated infected macrophages(Supplementary Figure 2) all together are evidence of apossible M2 to M1 polarization

33 PPAR Activation and cPLA2Inhibition Induce TLR4

Expression in L mexicana-Infected Macrophages Polarized

6 BioMed Research International

lowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0001

001

01

1

10

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-10

Time after infection

(a)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n le

vel

LPS

Time after infection

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-6

ATK

Time after infection

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowastlowast

lowastlowast

lowast

lowast lowast

0001

001

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

ATK

Time after infection

(d)

Figure 2 Cytokine determination in L mexicana-infected macrophages Levels of gene expression for each sample were normalized with120573-actin RNA as internal control Modulation was expressed relative to the untreated control using the 2minusΔΔCT method The 119909-axis interceptsthe 119910-axis at ldquo1rdquo to show the increase and the decrease of each cytokine compared to nontreated infected macrophages Relative expressionlevel for each cytokine was calculated according to ΔΔCT = (CT test minus CT 120573-actin) treated minus (CT test minus CT 120573-actin) untreated formula [17]Graph bars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versusnontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages differ in terms of receptor expression cytokineand chemokine repertoires and effector function M1macrophages exposed to the classic activation signals expressreceptors such as CD16 CD32 CD64 TLR2 and TLR4whereas M2 macrophages are characterized by abundantlevels of nonopsonic receptors such as the mannose receptor(MR) [24] In order to investigate if PPAR agonists andcPLA2antagonist induced macrophage polarization during

Lmexicana infection we evaluatedMR andTLR4 expressionby flow cytometry Since BALBcmice macrophages can fullysupport maturation of alternatively activated macrophages

J774A1 macrophages were incubated with PPAR agonistsand cPLA

2antagonist and then infected or stimulated with

LPS and zymosan neither infection nor treatments inducedMR expression an M2 receptor classified as an alternativeactivation marker (Supplementary Figure 3) however PPARagonists and cPLA

2antagonist induced an increase of TLR4

expression at 60 and 120min after infection (Figure 3) anM1 receptor classified as classical activation marker Theseresults show that PPAR120573120575 GW501516 and PPAR120574 GW1929agonists and cPLA

2antagonist do not help to keep the

M2 polarization profile instead PPAR agonists and cPLA2

BioMed Research International 7

0

50

100

150

TLR4+

(MFI

)

LPS

wTXATK

lowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

Time after infection

Figure 3 TLR4 expression in L mexicana-infected macrophagesCells were treated or not with PPAR agonists for 24 h and cPLA

2

antagonist for 1 h before infection TLR4 expression was analyzed byflow cytometry LPS (2 h) was used as a positive control of inductionGraph bars are mean plusmn SEM of three independent experiments andstatistical analysis was performed comparing for each time treatedversus non-treated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and(lowastlowastlowast) 119875 lt 0001

antagonist promote the polarization of macrophages towardan M1 profile

34 PPAR120574Activation byAgonist and cPLA2Inhibition Reduce

Parasite Burden PPAR120574 expression is strongly associatedwith maturation of M2 macrophages Gallardo-Soler et alandAdapala andChan [25 26] have demonstrated that PPARagonists increased intracellular growth of L major in bone-marrow-derived macrophages moreover PPAR120574 agonistCurcumine induced PPAR120574 expression in residential liverand spleen macrophages of BALBc mice In addition oraladministration of Curcumin further increases PPAR120572 andPPAR120574 expression and this increase was associated with aheavier parasite burden To analyze how PPAR120573120575GW501516and PPAR120574 GW1929 agonists and cPLA

2inhibition affected

parasite burden in treated macrophages their phagocyticactivity was evaluated through two parameters the phago-cytizing cell percentage and the number of phagocytizedzymosan particles or parasitescell treated or nontreatedmacrophages were incubated with FITC-labeled zymosanparticles by 2 h or infected with CFSE-labeled promastigotesby 1-2 h (Figure 4) PPAR120574GW1929 agonist does not increasethe number of zymosan-phagocytizing macrophages butPPAR120573120575 GW501516 agonist and cPLA

2ATK antagonist

treatments decreased the number of zymosan-phagocytizingmacrophages (119875 lt 005 and 119875 lt 0001 resp Figure 4(a)(zymosan)) on the contrary zymosan particlescell (meanfluorescence intensity (MFI)) increased significantly withall treatments GW501516 and GW1929 (119875 lt 0001) andATK (119875 lt 005) (Figure 4(b) (zymosan)) This result shows

that phagocytic activity per se was not affected by treat-ments The number of CFSE-labeled parasites-phagocytizingmacrophages did not increase by treatments either insteadparasites-phagocytizing macrophages diminished signifi-cantly with GW1929 (119875 lt 001) and ATK (119875 lt 0001)at 60min after infection and 2h after infection GW501516(119875 lt 001) GW1929 (119875 lt 0001) and ATK (119875 lt 0001)(Figure 4(a)) Only cPLA

2antagonist (Figure 4(b)) slightly

increased the number of parasitescell (119875 lt 005) at 60minafter infection but neither PPAR120573120575 nor PPAR120574 agonistsincreased it however at 120min after infection PPAR120573120575agonist slightly increased parasite burden (119875 lt 005)but PPAR120574 agonist and cPLA

2inhibition decreased parasite

burden significantly (119875 lt 0001) (Figure 4(b)) These resultstogether demonstrate that PPAR activation by these agonistsand cPLA

2inhibition did not increase parasite load

35 PPAR Activation by Agonists Selectively Regulates Pros-taglandin Production in L mexicana-Infected MacrophagesProstaglandins are potent ligands of the intracellular PPARreceptors in macrophages and their binding to PPAR120572and PPAR120574 causes macrophage deactivation Perez-Santosand Talamas-Rohana [14] have demonstrated that COX-2 inhibition induced leishmanicidal activity by splenocytes[14] Thus one possible mechanism of intracellular sur-vival of Leishmania is the deactivation of macrophages byprostaglandins produced [27] In order to investigate ifPPAR agonists couldmodulate inflammatory prostaglandinsmacrophages were treated with PPAR agonists or cPLA

2

antagonist before the infection to look for PGrsquos metabolitesin the conditioned media (Figure 5) PPAR activation andcPLA2inhibition decreased significantly 6k-PGF

1120572 PGE

1

and PGF1120572

production (Supplementary Figure 4) howeverPPAR120574 agonist was not able to reduce PGE

2production

which increased significantly after infection (119875 lt 0001) at60 and 120min respectively (Figure 5) PPAR120573120575 agonist andcPLA2antagonist did not increase PGE

2production even its

production diminished (119875 lt 005 and 119875 lt 0001) at 120minafter infection respectively moreover PPAR agonists andcPLA2antagonist significantly increased PGF

2120572production

(119875 lt 0001) at 60 and 120min after infection respectively Insummary PPAR agonists and cPLA

2antagonist diminished

6k-PGF1120572 PGF

1120572 and PGE

1 and neither Leishmania infec-

tion nor LPS was able to recover their production howeverPGF2120572

production was increased after infection and onlyPPAR120574 activation increased PGE

2production

36 PPARActivation and cPLA2Inhibition Increase theOxida-

tive Burst during J774A1 Macrophages Infection with Lmexicana Promastigotes Several studies have demonstratedthat ROS modulate arachidonic acid metabolism and pro-duction of eicosanoids in activated macrophages [28 29]In murine and human macrophages it has been establishedthat the respiratory burst of the cell with production ofROS such as H

2O2and O

2

minus is primarily responsible forparasite control as these molecules have been reportedto be fatal for Leishmania promastigotes We next ana-lyzed the effect of PPAR agonists and cPLA

2antagonist

on the oxidative burst induced by L mexicana on J774A1

8 BioMed Research International

0

10

20

30

40

50

60

70Ph

agoc

ytiz

ing

cells

()

Zy

wTXATK

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

Time after infection

(a)

0

200

400

600

800

Part

icle

s or p

aras

ite lo

adc

ell (

MFI

)

Zy

wTXATK

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 4 Phagocytic activity of J774A1 macrophages was determined for zymosan and L mexicana promastigotes uptake treated ornontreated macrophages were incubated with zymosan-FITC for 2 h or infected with CFSE-promastigotes for 1-2 h (a) Phagocytizingmacrophage percentage (b) Zymosan particles or parasitescell (MFI) Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 (lowastlowastlowast) 119875 lt 0001

0

200

400

600

800

1000

LPS

wTXATK

PGE 2

prod

uctio

n (p

gm

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

Time after infection

(a)

0

10

20

30

40

LPS

wTXATK

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 5 Prostaglandin production by L mexicana-infectedmacrophages Prostaglandins were analyzed byMSMS assay product scanningexperiments were conducted using nitrogen as collision gas and the collision energy was optimized for individual compounds to generatethe most abundant product ions These product ion spectra were then used to select the precursor-product ion pairs for the developmentof MRM assays Deuterium-labeled prostaglandins were used as internal standards for quantitation Graph bars are mean plusmn SEM of threeindependent experiments and statistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005(lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages (Figure 6)We found that positivemacrophagesto L mexicana-induced oxidative burst increased from sim341to 586 (119875 lt 0001) by PPAR120574 agonist at 120min afterinfection compared to non-treated macrophages whereastreatment with PPAR120573120575 agonist increased from sim341 to4032 (119875 lt 005) (Figures 6(a) and 6(b)) PPAR120573120575agonist slightly increased the number of positive oxidativeburst macrophages at 120min after infection and when

the oxidative burst was quantified it increased sim13-foldat 120min after infection too (119875 lt 005) (Figure 6(c)) Onthe other hand cPLA

2inhibition increased sim251-fold the

oxidative burst at 120min after infection (119875 lt 0001)compared to nontreated macrophages (Figure 6(c)) FinallyPPAR120574 agonist increased the oxidative burst sim252-(119875 lt0001) and sim355-fold (119875 lt 0001) at 60 and 120minafter infection respectively indicating that PPAR120574 activation

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

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International Journal of

Volume 2014

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Molecular Biology International

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International Journal of

Microbiology

BioMed Research International 5

ERK12

COX-2

p-cPLA

cPLA

LPS LPS LPS

2

2

Time post infection Time post infection Time post infection

(-) GW501516 120573120575 GW1929 120574

PPAR120574

PPAR120573120575

MΦ MΦ MΦ5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400 5998400 10998400 15998400 3099840060 998400120998400

(a)

0

50

100

150

Den

sitom

etric

anal

ysis

of p

-cPL

A(

)

LPS

wTX

2

Time after infection

(b)

0

755025

150125100

225200175175

Den

sitom

etric

anal

ysis

of C

OX-

2 (

)

LPS

wTX

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowast

Time after infection

(c)

0001

1

01

001

1000

100

10

Rela

tive e

xpre

ssio

n le

vel C

OX-

2

LPS 30 60 120

ATK

PPARβδPPARγ

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowast

Mφ

Time after infection

(d)

Figure 1 PPAR agonists inhibit cPLA2phosphorylation and COX-2 expression in L mexicana-infected macrophages (a) Protein expression

for PPARs and COX-2 and cPLA2phosphorylation levels were evaluated by Western blotting (b) Densitometry analyses of cPLA

2

phosphorylation and (c) COX-2 expression were performed in basal conditions as well as in macrophages treated or not with PPAR agonists(d) COX-2 mRNA expression was evaluated by qRT-PCR and analyzed by 2minusΔΔCT method Total ERK12 was probed to normalize proteinloading Results are representative of three independent experiments Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast)119875 lt 005 (lowastlowast)119875 lt 001 and (lowastlowastlowast)119875 lt 0001

noninfected macrophages (Supplementary Figure 2) How-ever when noninfected macrophages were previously treatedwith PPAR120573120575 agonist (white bars) proinflammatory IL-6was upregulated and its expressionwas held during infectionTNF-120572 and IL-1120573 were overexpressed but they were down-regulated at 30 to 60min (after infection) and its expres-sion recovered at 120min (after infection) Moreover whennoninfected macrophages were treated with PPAR120574 agonist(black bars) TNF-120572 was upregulated but its expression wasdownregulated after infection overregulation of IL-6 dimin-ished after infection but it was held equivalent to nontreatedmacrophages IL-1120573 was upregulated and its expression washeld during infection Anti-inflammatory IL-10 cytokine wasdownregulated with both PPAR agonists after infection On

the other hand when cPLA2was blocked 1 h before the

infection by treatment ofmacrophageswith cPLA2antagonist

ATK (gray bars) proinflammatory cytokines TNF-120572 IL-1120573and IL-6were upregulated furthermore IL-10 expressionwassignificantly affectedwhen cPLA

2was inhibited In summary

PPAR agonists and cPLA2antagonist set down the levels of

IL-10 at the same time they upregulated TNF-120572 IL-1120573 andIL-6 cytokines or at least they were held overexpressed afterinfection as occurred with nontreated infected macrophages(Supplementary Figure 2) all together are evidence of apossible M2 to M1 polarization

33 PPAR Activation and cPLA2Inhibition Induce TLR4

Expression in L mexicana-Infected Macrophages Polarized

6 BioMed Research International

lowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0001

001

01

1

10

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-10

Time after infection

(a)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n le

vel

LPS

Time after infection

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-6

ATK

Time after infection

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowastlowast

lowastlowast

lowast

lowast lowast

0001

001

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

ATK

Time after infection

(d)

Figure 2 Cytokine determination in L mexicana-infected macrophages Levels of gene expression for each sample were normalized with120573-actin RNA as internal control Modulation was expressed relative to the untreated control using the 2minusΔΔCT method The 119909-axis interceptsthe 119910-axis at ldquo1rdquo to show the increase and the decrease of each cytokine compared to nontreated infected macrophages Relative expressionlevel for each cytokine was calculated according to ΔΔCT = (CT test minus CT 120573-actin) treated minus (CT test minus CT 120573-actin) untreated formula [17]Graph bars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versusnontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages differ in terms of receptor expression cytokineand chemokine repertoires and effector function M1macrophages exposed to the classic activation signals expressreceptors such as CD16 CD32 CD64 TLR2 and TLR4whereas M2 macrophages are characterized by abundantlevels of nonopsonic receptors such as the mannose receptor(MR) [24] In order to investigate if PPAR agonists andcPLA2antagonist induced macrophage polarization during

Lmexicana infection we evaluatedMR andTLR4 expressionby flow cytometry Since BALBcmice macrophages can fullysupport maturation of alternatively activated macrophages

J774A1 macrophages were incubated with PPAR agonistsand cPLA

2antagonist and then infected or stimulated with

LPS and zymosan neither infection nor treatments inducedMR expression an M2 receptor classified as an alternativeactivation marker (Supplementary Figure 3) however PPARagonists and cPLA

2antagonist induced an increase of TLR4

expression at 60 and 120min after infection (Figure 3) anM1 receptor classified as classical activation marker Theseresults show that PPAR120573120575 GW501516 and PPAR120574 GW1929agonists and cPLA

2antagonist do not help to keep the

M2 polarization profile instead PPAR agonists and cPLA2

BioMed Research International 7

0

50

100

150

TLR4+

(MFI

)

LPS

wTXATK

lowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

Time after infection

Figure 3 TLR4 expression in L mexicana-infected macrophagesCells were treated or not with PPAR agonists for 24 h and cPLA

2

antagonist for 1 h before infection TLR4 expression was analyzed byflow cytometry LPS (2 h) was used as a positive control of inductionGraph bars are mean plusmn SEM of three independent experiments andstatistical analysis was performed comparing for each time treatedversus non-treated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and(lowastlowastlowast) 119875 lt 0001

antagonist promote the polarization of macrophages towardan M1 profile

34 PPAR120574Activation byAgonist and cPLA2Inhibition Reduce

Parasite Burden PPAR120574 expression is strongly associatedwith maturation of M2 macrophages Gallardo-Soler et alandAdapala andChan [25 26] have demonstrated that PPARagonists increased intracellular growth of L major in bone-marrow-derived macrophages moreover PPAR120574 agonistCurcumine induced PPAR120574 expression in residential liverand spleen macrophages of BALBc mice In addition oraladministration of Curcumin further increases PPAR120572 andPPAR120574 expression and this increase was associated with aheavier parasite burden To analyze how PPAR120573120575GW501516and PPAR120574 GW1929 agonists and cPLA

2inhibition affected

parasite burden in treated macrophages their phagocyticactivity was evaluated through two parameters the phago-cytizing cell percentage and the number of phagocytizedzymosan particles or parasitescell treated or nontreatedmacrophages were incubated with FITC-labeled zymosanparticles by 2 h or infected with CFSE-labeled promastigotesby 1-2 h (Figure 4) PPAR120574GW1929 agonist does not increasethe number of zymosan-phagocytizing macrophages butPPAR120573120575 GW501516 agonist and cPLA

2ATK antagonist

treatments decreased the number of zymosan-phagocytizingmacrophages (119875 lt 005 and 119875 lt 0001 resp Figure 4(a)(zymosan)) on the contrary zymosan particlescell (meanfluorescence intensity (MFI)) increased significantly withall treatments GW501516 and GW1929 (119875 lt 0001) andATK (119875 lt 005) (Figure 4(b) (zymosan)) This result shows

that phagocytic activity per se was not affected by treat-ments The number of CFSE-labeled parasites-phagocytizingmacrophages did not increase by treatments either insteadparasites-phagocytizing macrophages diminished signifi-cantly with GW1929 (119875 lt 001) and ATK (119875 lt 0001)at 60min after infection and 2h after infection GW501516(119875 lt 001) GW1929 (119875 lt 0001) and ATK (119875 lt 0001)(Figure 4(a)) Only cPLA

2antagonist (Figure 4(b)) slightly

increased the number of parasitescell (119875 lt 005) at 60minafter infection but neither PPAR120573120575 nor PPAR120574 agonistsincreased it however at 120min after infection PPAR120573120575agonist slightly increased parasite burden (119875 lt 005)but PPAR120574 agonist and cPLA

2inhibition decreased parasite

burden significantly (119875 lt 0001) (Figure 4(b)) These resultstogether demonstrate that PPAR activation by these agonistsand cPLA

2inhibition did not increase parasite load

35 PPAR Activation by Agonists Selectively Regulates Pros-taglandin Production in L mexicana-Infected MacrophagesProstaglandins are potent ligands of the intracellular PPARreceptors in macrophages and their binding to PPAR120572and PPAR120574 causes macrophage deactivation Perez-Santosand Talamas-Rohana [14] have demonstrated that COX-2 inhibition induced leishmanicidal activity by splenocytes[14] Thus one possible mechanism of intracellular sur-vival of Leishmania is the deactivation of macrophages byprostaglandins produced [27] In order to investigate ifPPAR agonists couldmodulate inflammatory prostaglandinsmacrophages were treated with PPAR agonists or cPLA

2

antagonist before the infection to look for PGrsquos metabolitesin the conditioned media (Figure 5) PPAR activation andcPLA2inhibition decreased significantly 6k-PGF

1120572 PGE

1

and PGF1120572

production (Supplementary Figure 4) howeverPPAR120574 agonist was not able to reduce PGE

2production

which increased significantly after infection (119875 lt 0001) at60 and 120min respectively (Figure 5) PPAR120573120575 agonist andcPLA2antagonist did not increase PGE

2production even its

production diminished (119875 lt 005 and 119875 lt 0001) at 120minafter infection respectively moreover PPAR agonists andcPLA2antagonist significantly increased PGF

2120572production

(119875 lt 0001) at 60 and 120min after infection respectively Insummary PPAR agonists and cPLA

2antagonist diminished

6k-PGF1120572 PGF

1120572 and PGE

1 and neither Leishmania infec-

tion nor LPS was able to recover their production howeverPGF2120572

production was increased after infection and onlyPPAR120574 activation increased PGE

2production

36 PPARActivation and cPLA2Inhibition Increase theOxida-

tive Burst during J774A1 Macrophages Infection with Lmexicana Promastigotes Several studies have demonstratedthat ROS modulate arachidonic acid metabolism and pro-duction of eicosanoids in activated macrophages [28 29]In murine and human macrophages it has been establishedthat the respiratory burst of the cell with production ofROS such as H

2O2and O

2

minus is primarily responsible forparasite control as these molecules have been reportedto be fatal for Leishmania promastigotes We next ana-lyzed the effect of PPAR agonists and cPLA

2antagonist

on the oxidative burst induced by L mexicana on J774A1

8 BioMed Research International

0

10

20

30

40

50

60

70Ph

agoc

ytiz

ing

cells

()

Zy

wTXATK

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

Time after infection

(a)

0

200

400

600

800

Part

icle

s or p

aras

ite lo

adc

ell (

MFI

)

Zy

wTXATK

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 4 Phagocytic activity of J774A1 macrophages was determined for zymosan and L mexicana promastigotes uptake treated ornontreated macrophages were incubated with zymosan-FITC for 2 h or infected with CFSE-promastigotes for 1-2 h (a) Phagocytizingmacrophage percentage (b) Zymosan particles or parasitescell (MFI) Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 (lowastlowastlowast) 119875 lt 0001

0

200

400

600

800

1000

LPS

wTXATK

PGE 2

prod

uctio

n (p

gm

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

Time after infection

(a)

0

10

20

30

40

LPS

wTXATK

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 5 Prostaglandin production by L mexicana-infectedmacrophages Prostaglandins were analyzed byMSMS assay product scanningexperiments were conducted using nitrogen as collision gas and the collision energy was optimized for individual compounds to generatethe most abundant product ions These product ion spectra were then used to select the precursor-product ion pairs for the developmentof MRM assays Deuterium-labeled prostaglandins were used as internal standards for quantitation Graph bars are mean plusmn SEM of threeindependent experiments and statistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005(lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages (Figure 6)We found that positivemacrophagesto L mexicana-induced oxidative burst increased from sim341to 586 (119875 lt 0001) by PPAR120574 agonist at 120min afterinfection compared to non-treated macrophages whereastreatment with PPAR120573120575 agonist increased from sim341 to4032 (119875 lt 005) (Figures 6(a) and 6(b)) PPAR120573120575agonist slightly increased the number of positive oxidativeburst macrophages at 120min after infection and when

the oxidative burst was quantified it increased sim13-foldat 120min after infection too (119875 lt 005) (Figure 6(c)) Onthe other hand cPLA

2inhibition increased sim251-fold the

oxidative burst at 120min after infection (119875 lt 0001)compared to nontreated macrophages (Figure 6(c)) FinallyPPAR120574 agonist increased the oxidative burst sim252-(119875 lt0001) and sim355-fold (119875 lt 0001) at 60 and 120minafter infection respectively indicating that PPAR120574 activation

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

lowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0001

001

01

1

10

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-10

Time after infection

(a)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n le

vel

LPS

Time after infection

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

IL-6

ATK

Time after infection

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowastlowastlowast

lowastlowast

lowast

lowast lowast

0001

001

01

1

10

100

1000

Rela

tive e

xpre

ssio

n le

vel

LPS

ATK

Time after infection

(d)

Figure 2 Cytokine determination in L mexicana-infected macrophages Levels of gene expression for each sample were normalized with120573-actin RNA as internal control Modulation was expressed relative to the untreated control using the 2minusΔΔCT method The 119909-axis interceptsthe 119910-axis at ldquo1rdquo to show the increase and the decrease of each cytokine compared to nontreated infected macrophages Relative expressionlevel for each cytokine was calculated according to ΔΔCT = (CT test minus CT 120573-actin) treated minus (CT test minus CT 120573-actin) untreated formula [17]Graph bars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versusnontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages differ in terms of receptor expression cytokineand chemokine repertoires and effector function M1macrophages exposed to the classic activation signals expressreceptors such as CD16 CD32 CD64 TLR2 and TLR4whereas M2 macrophages are characterized by abundantlevels of nonopsonic receptors such as the mannose receptor(MR) [24] In order to investigate if PPAR agonists andcPLA2antagonist induced macrophage polarization during

Lmexicana infection we evaluatedMR andTLR4 expressionby flow cytometry Since BALBcmice macrophages can fullysupport maturation of alternatively activated macrophages

J774A1 macrophages were incubated with PPAR agonistsand cPLA

2antagonist and then infected or stimulated with

LPS and zymosan neither infection nor treatments inducedMR expression an M2 receptor classified as an alternativeactivation marker (Supplementary Figure 3) however PPARagonists and cPLA

2antagonist induced an increase of TLR4

expression at 60 and 120min after infection (Figure 3) anM1 receptor classified as classical activation marker Theseresults show that PPAR120573120575 GW501516 and PPAR120574 GW1929agonists and cPLA

2antagonist do not help to keep the

M2 polarization profile instead PPAR agonists and cPLA2

BioMed Research International 7

0

50

100

150

TLR4+

(MFI

)

LPS

wTXATK

lowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

Time after infection

Figure 3 TLR4 expression in L mexicana-infected macrophagesCells were treated or not with PPAR agonists for 24 h and cPLA

2

antagonist for 1 h before infection TLR4 expression was analyzed byflow cytometry LPS (2 h) was used as a positive control of inductionGraph bars are mean plusmn SEM of three independent experiments andstatistical analysis was performed comparing for each time treatedversus non-treated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and(lowastlowastlowast) 119875 lt 0001

antagonist promote the polarization of macrophages towardan M1 profile

34 PPAR120574Activation byAgonist and cPLA2Inhibition Reduce

Parasite Burden PPAR120574 expression is strongly associatedwith maturation of M2 macrophages Gallardo-Soler et alandAdapala andChan [25 26] have demonstrated that PPARagonists increased intracellular growth of L major in bone-marrow-derived macrophages moreover PPAR120574 agonistCurcumine induced PPAR120574 expression in residential liverand spleen macrophages of BALBc mice In addition oraladministration of Curcumin further increases PPAR120572 andPPAR120574 expression and this increase was associated with aheavier parasite burden To analyze how PPAR120573120575GW501516and PPAR120574 GW1929 agonists and cPLA

2inhibition affected

parasite burden in treated macrophages their phagocyticactivity was evaluated through two parameters the phago-cytizing cell percentage and the number of phagocytizedzymosan particles or parasitescell treated or nontreatedmacrophages were incubated with FITC-labeled zymosanparticles by 2 h or infected with CFSE-labeled promastigotesby 1-2 h (Figure 4) PPAR120574GW1929 agonist does not increasethe number of zymosan-phagocytizing macrophages butPPAR120573120575 GW501516 agonist and cPLA

2ATK antagonist

treatments decreased the number of zymosan-phagocytizingmacrophages (119875 lt 005 and 119875 lt 0001 resp Figure 4(a)(zymosan)) on the contrary zymosan particlescell (meanfluorescence intensity (MFI)) increased significantly withall treatments GW501516 and GW1929 (119875 lt 0001) andATK (119875 lt 005) (Figure 4(b) (zymosan)) This result shows

that phagocytic activity per se was not affected by treat-ments The number of CFSE-labeled parasites-phagocytizingmacrophages did not increase by treatments either insteadparasites-phagocytizing macrophages diminished signifi-cantly with GW1929 (119875 lt 001) and ATK (119875 lt 0001)at 60min after infection and 2h after infection GW501516(119875 lt 001) GW1929 (119875 lt 0001) and ATK (119875 lt 0001)(Figure 4(a)) Only cPLA

2antagonist (Figure 4(b)) slightly

increased the number of parasitescell (119875 lt 005) at 60minafter infection but neither PPAR120573120575 nor PPAR120574 agonistsincreased it however at 120min after infection PPAR120573120575agonist slightly increased parasite burden (119875 lt 005)but PPAR120574 agonist and cPLA

2inhibition decreased parasite

burden significantly (119875 lt 0001) (Figure 4(b)) These resultstogether demonstrate that PPAR activation by these agonistsand cPLA

2inhibition did not increase parasite load

35 PPAR Activation by Agonists Selectively Regulates Pros-taglandin Production in L mexicana-Infected MacrophagesProstaglandins are potent ligands of the intracellular PPARreceptors in macrophages and their binding to PPAR120572and PPAR120574 causes macrophage deactivation Perez-Santosand Talamas-Rohana [14] have demonstrated that COX-2 inhibition induced leishmanicidal activity by splenocytes[14] Thus one possible mechanism of intracellular sur-vival of Leishmania is the deactivation of macrophages byprostaglandins produced [27] In order to investigate ifPPAR agonists couldmodulate inflammatory prostaglandinsmacrophages were treated with PPAR agonists or cPLA

2

antagonist before the infection to look for PGrsquos metabolitesin the conditioned media (Figure 5) PPAR activation andcPLA2inhibition decreased significantly 6k-PGF

1120572 PGE

1

and PGF1120572

production (Supplementary Figure 4) howeverPPAR120574 agonist was not able to reduce PGE

2production

which increased significantly after infection (119875 lt 0001) at60 and 120min respectively (Figure 5) PPAR120573120575 agonist andcPLA2antagonist did not increase PGE

2production even its

production diminished (119875 lt 005 and 119875 lt 0001) at 120minafter infection respectively moreover PPAR agonists andcPLA2antagonist significantly increased PGF

2120572production

(119875 lt 0001) at 60 and 120min after infection respectively Insummary PPAR agonists and cPLA

2antagonist diminished

6k-PGF1120572 PGF

1120572 and PGE

1 and neither Leishmania infec-

tion nor LPS was able to recover their production howeverPGF2120572

production was increased after infection and onlyPPAR120574 activation increased PGE

2production

36 PPARActivation and cPLA2Inhibition Increase theOxida-

tive Burst during J774A1 Macrophages Infection with Lmexicana Promastigotes Several studies have demonstratedthat ROS modulate arachidonic acid metabolism and pro-duction of eicosanoids in activated macrophages [28 29]In murine and human macrophages it has been establishedthat the respiratory burst of the cell with production ofROS such as H

2O2and O

2

minus is primarily responsible forparasite control as these molecules have been reportedto be fatal for Leishmania promastigotes We next ana-lyzed the effect of PPAR agonists and cPLA

2antagonist

on the oxidative burst induced by L mexicana on J774A1

8 BioMed Research International

0

10

20

30

40

50

60

70Ph

agoc

ytiz

ing

cells

()

Zy

wTXATK

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

Time after infection

(a)

0

200

400

600

800

Part

icle

s or p

aras

ite lo

adc

ell (

MFI

)

Zy

wTXATK

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 4 Phagocytic activity of J774A1 macrophages was determined for zymosan and L mexicana promastigotes uptake treated ornontreated macrophages were incubated with zymosan-FITC for 2 h or infected with CFSE-promastigotes for 1-2 h (a) Phagocytizingmacrophage percentage (b) Zymosan particles or parasitescell (MFI) Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 (lowastlowastlowast) 119875 lt 0001

0

200

400

600

800

1000

LPS

wTXATK

PGE 2

prod

uctio

n (p

gm

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

Time after infection

(a)

0

10

20

30

40

LPS

wTXATK

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 5 Prostaglandin production by L mexicana-infectedmacrophages Prostaglandins were analyzed byMSMS assay product scanningexperiments were conducted using nitrogen as collision gas and the collision energy was optimized for individual compounds to generatethe most abundant product ions These product ion spectra were then used to select the precursor-product ion pairs for the developmentof MRM assays Deuterium-labeled prostaglandins were used as internal standards for quantitation Graph bars are mean plusmn SEM of threeindependent experiments and statistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005(lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages (Figure 6)We found that positivemacrophagesto L mexicana-induced oxidative burst increased from sim341to 586 (119875 lt 0001) by PPAR120574 agonist at 120min afterinfection compared to non-treated macrophages whereastreatment with PPAR120573120575 agonist increased from sim341 to4032 (119875 lt 005) (Figures 6(a) and 6(b)) PPAR120573120575agonist slightly increased the number of positive oxidativeburst macrophages at 120min after infection and when

the oxidative burst was quantified it increased sim13-foldat 120min after infection too (119875 lt 005) (Figure 6(c)) Onthe other hand cPLA

2inhibition increased sim251-fold the

oxidative burst at 120min after infection (119875 lt 0001)compared to nontreated macrophages (Figure 6(c)) FinallyPPAR120574 agonist increased the oxidative burst sim252-(119875 lt0001) and sim355-fold (119875 lt 0001) at 60 and 120minafter infection respectively indicating that PPAR120574 activation

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

0

50

100

150

TLR4+

(MFI

)

LPS

wTXATK

lowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

Time after infection

Figure 3 TLR4 expression in L mexicana-infected macrophagesCells were treated or not with PPAR agonists for 24 h and cPLA

2

antagonist for 1 h before infection TLR4 expression was analyzed byflow cytometry LPS (2 h) was used as a positive control of inductionGraph bars are mean plusmn SEM of three independent experiments andstatistical analysis was performed comparing for each time treatedversus non-treated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and(lowastlowastlowast) 119875 lt 0001

antagonist promote the polarization of macrophages towardan M1 profile

34 PPAR120574Activation byAgonist and cPLA2Inhibition Reduce

Parasite Burden PPAR120574 expression is strongly associatedwith maturation of M2 macrophages Gallardo-Soler et alandAdapala andChan [25 26] have demonstrated that PPARagonists increased intracellular growth of L major in bone-marrow-derived macrophages moreover PPAR120574 agonistCurcumine induced PPAR120574 expression in residential liverand spleen macrophages of BALBc mice In addition oraladministration of Curcumin further increases PPAR120572 andPPAR120574 expression and this increase was associated with aheavier parasite burden To analyze how PPAR120573120575GW501516and PPAR120574 GW1929 agonists and cPLA

2inhibition affected

parasite burden in treated macrophages their phagocyticactivity was evaluated through two parameters the phago-cytizing cell percentage and the number of phagocytizedzymosan particles or parasitescell treated or nontreatedmacrophages were incubated with FITC-labeled zymosanparticles by 2 h or infected with CFSE-labeled promastigotesby 1-2 h (Figure 4) PPAR120574GW1929 agonist does not increasethe number of zymosan-phagocytizing macrophages butPPAR120573120575 GW501516 agonist and cPLA

2ATK antagonist

treatments decreased the number of zymosan-phagocytizingmacrophages (119875 lt 005 and 119875 lt 0001 resp Figure 4(a)(zymosan)) on the contrary zymosan particlescell (meanfluorescence intensity (MFI)) increased significantly withall treatments GW501516 and GW1929 (119875 lt 0001) andATK (119875 lt 005) (Figure 4(b) (zymosan)) This result shows

that phagocytic activity per se was not affected by treat-ments The number of CFSE-labeled parasites-phagocytizingmacrophages did not increase by treatments either insteadparasites-phagocytizing macrophages diminished signifi-cantly with GW1929 (119875 lt 001) and ATK (119875 lt 0001)at 60min after infection and 2h after infection GW501516(119875 lt 001) GW1929 (119875 lt 0001) and ATK (119875 lt 0001)(Figure 4(a)) Only cPLA

2antagonist (Figure 4(b)) slightly

increased the number of parasitescell (119875 lt 005) at 60minafter infection but neither PPAR120573120575 nor PPAR120574 agonistsincreased it however at 120min after infection PPAR120573120575agonist slightly increased parasite burden (119875 lt 005)but PPAR120574 agonist and cPLA

2inhibition decreased parasite

burden significantly (119875 lt 0001) (Figure 4(b)) These resultstogether demonstrate that PPAR activation by these agonistsand cPLA

2inhibition did not increase parasite load

35 PPAR Activation by Agonists Selectively Regulates Pros-taglandin Production in L mexicana-Infected MacrophagesProstaglandins are potent ligands of the intracellular PPARreceptors in macrophages and their binding to PPAR120572and PPAR120574 causes macrophage deactivation Perez-Santosand Talamas-Rohana [14] have demonstrated that COX-2 inhibition induced leishmanicidal activity by splenocytes[14] Thus one possible mechanism of intracellular sur-vival of Leishmania is the deactivation of macrophages byprostaglandins produced [27] In order to investigate ifPPAR agonists couldmodulate inflammatory prostaglandinsmacrophages were treated with PPAR agonists or cPLA

2

antagonist before the infection to look for PGrsquos metabolitesin the conditioned media (Figure 5) PPAR activation andcPLA2inhibition decreased significantly 6k-PGF

1120572 PGE

1

and PGF1120572

production (Supplementary Figure 4) howeverPPAR120574 agonist was not able to reduce PGE

2production

which increased significantly after infection (119875 lt 0001) at60 and 120min respectively (Figure 5) PPAR120573120575 agonist andcPLA2antagonist did not increase PGE

2production even its

production diminished (119875 lt 005 and 119875 lt 0001) at 120minafter infection respectively moreover PPAR agonists andcPLA2antagonist significantly increased PGF

2120572production

(119875 lt 0001) at 60 and 120min after infection respectively Insummary PPAR agonists and cPLA

2antagonist diminished

6k-PGF1120572 PGF

1120572 and PGE

1 and neither Leishmania infec-

tion nor LPS was able to recover their production howeverPGF2120572

production was increased after infection and onlyPPAR120574 activation increased PGE

2production

36 PPARActivation and cPLA2Inhibition Increase theOxida-

tive Burst during J774A1 Macrophages Infection with Lmexicana Promastigotes Several studies have demonstratedthat ROS modulate arachidonic acid metabolism and pro-duction of eicosanoids in activated macrophages [28 29]In murine and human macrophages it has been establishedthat the respiratory burst of the cell with production ofROS such as H

2O2and O

2

minus is primarily responsible forparasite control as these molecules have been reportedto be fatal for Leishmania promastigotes We next ana-lyzed the effect of PPAR agonists and cPLA

2antagonist

on the oxidative burst induced by L mexicana on J774A1

8 BioMed Research International

0

10

20

30

40

50

60

70Ph

agoc

ytiz

ing

cells

()

Zy

wTXATK

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

Time after infection

(a)

0

200

400

600

800

Part

icle

s or p

aras

ite lo

adc

ell (

MFI

)

Zy

wTXATK

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 4 Phagocytic activity of J774A1 macrophages was determined for zymosan and L mexicana promastigotes uptake treated ornontreated macrophages were incubated with zymosan-FITC for 2 h or infected with CFSE-promastigotes for 1-2 h (a) Phagocytizingmacrophage percentage (b) Zymosan particles or parasitescell (MFI) Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 (lowastlowastlowast) 119875 lt 0001

0

200

400

600

800

1000

LPS

wTXATK

PGE 2

prod

uctio

n (p

gm

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

Time after infection

(a)

0

10

20

30

40

LPS

wTXATK

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 5 Prostaglandin production by L mexicana-infectedmacrophages Prostaglandins were analyzed byMSMS assay product scanningexperiments were conducted using nitrogen as collision gas and the collision energy was optimized for individual compounds to generatethe most abundant product ions These product ion spectra were then used to select the precursor-product ion pairs for the developmentof MRM assays Deuterium-labeled prostaglandins were used as internal standards for quantitation Graph bars are mean plusmn SEM of threeindependent experiments and statistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005(lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages (Figure 6)We found that positivemacrophagesto L mexicana-induced oxidative burst increased from sim341to 586 (119875 lt 0001) by PPAR120574 agonist at 120min afterinfection compared to non-treated macrophages whereastreatment with PPAR120573120575 agonist increased from sim341 to4032 (119875 lt 005) (Figures 6(a) and 6(b)) PPAR120573120575agonist slightly increased the number of positive oxidativeburst macrophages at 120min after infection and when

the oxidative burst was quantified it increased sim13-foldat 120min after infection too (119875 lt 005) (Figure 6(c)) Onthe other hand cPLA

2inhibition increased sim251-fold the

oxidative burst at 120min after infection (119875 lt 0001)compared to nontreated macrophages (Figure 6(c)) FinallyPPAR120574 agonist increased the oxidative burst sim252-(119875 lt0001) and sim355-fold (119875 lt 0001) at 60 and 120minafter infection respectively indicating that PPAR120574 activation

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

0

10

20

30

40

50

60

70Ph

agoc

ytiz

ing

cells

()

Zy

wTXATK

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

Time after infection

(a)

0

200

400

600

800

Part

icle

s or p

aras

ite lo

adc

ell (

MFI

)

Zy

wTXATK

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 4 Phagocytic activity of J774A1 macrophages was determined for zymosan and L mexicana promastigotes uptake treated ornontreated macrophages were incubated with zymosan-FITC for 2 h or infected with CFSE-promastigotes for 1-2 h (a) Phagocytizingmacrophage percentage (b) Zymosan particles or parasitescell (MFI) Graph bars are mean plusmn SEM of three independent experiments andstatistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 (lowastlowastlowast) 119875 lt 0001

0

200

400

600

800

1000

LPS

wTXATK

PGE 2

prod

uctio

n (p

gm

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

Time after infection

(a)

0

10

20

30

40

LPS

wTXATK

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Time after infection

(b)

Figure 5 Prostaglandin production by L mexicana-infectedmacrophages Prostaglandins were analyzed byMSMS assay product scanningexperiments were conducted using nitrogen as collision gas and the collision energy was optimized for individual compounds to generatethe most abundant product ions These product ion spectra were then used to select the precursor-product ion pairs for the developmentof MRM assays Deuterium-labeled prostaglandins were used as internal standards for quantitation Graph bars are mean plusmn SEM of threeindependent experiments and statistical analysis was done comparing for each time treated versus nontreated macrophages (lowast) 119875 lt 005(lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

macrophages (Figure 6)We found that positivemacrophagesto L mexicana-induced oxidative burst increased from sim341to 586 (119875 lt 0001) by PPAR120574 agonist at 120min afterinfection compared to non-treated macrophages whereastreatment with PPAR120573120575 agonist increased from sim341 to4032 (119875 lt 005) (Figures 6(a) and 6(b)) PPAR120573120575agonist slightly increased the number of positive oxidativeburst macrophages at 120min after infection and when

the oxidative burst was quantified it increased sim13-foldat 120min after infection too (119875 lt 005) (Figure 6(c)) Onthe other hand cPLA

2inhibition increased sim251-fold the

oxidative burst at 120min after infection (119875 lt 0001)compared to nontreated macrophages (Figure 6(c)) FinallyPPAR120574 agonist increased the oxidative burst sim252-(119875 lt0001) and sim355-fold (119875 lt 0001) at 60 and 120minafter infection respectively indicating that PPAR120574 activation

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

induces an aggressive oxidative response to intracellularparasites (Figure 6(c))

4 Discussion

ThecPLA2activation COX-2 expression and PG production

are positioned at the core of a common regulatory circuitcontrolling the initiation magnitude duration and resolu-tion of the inflammatory response During the inflammatoryphase proinflammatory genes expression is controlled attranscriptional posttranscriptional and translational levelsAccording to several reports in this work we have confirmedthat phosphorylated cPLA

2and COX-2 are key enzymes

during Leishmania infection [14 23 30] in addition wehave shown that PPAR activation by agonists preventscPLA2phosphorylation and COX-2 either protein or mRNA

expression during macrophages infection with L mexicanaPreviously Perez-Santos and Talamas-Rohana [14] showedthat COX-2 inhibition increased IL-12 and IFN120574 productionand induced NO production and parasite killing Moreoverit has been demonstrated previously that p-cPLA

2activates

COX-2 and proinflammatory cytokine genes expressionthrough PPAR120574 response elements [31] thus inhibition ofcPLA2phosphorylation suppresses those genes [32] In this

context inhibition of cPLA2phosphorylation with ATK

antagonist significantly reduced the mRNA expression ofCOX-2

We have demonstrated in this work that PPAR activationby agonists and cPLA

2inhibition by antagonist ATK are able

to downregulate IL-10 expression throughout the course ofinfection with L mexicana It has been demonstrated thatcells from IL-10minusminusmice producedmore NO IFN120574 and IL-12compared with cells from BALBcmice [8] and IL-10minusminusmicewhich become resistant to infection [7] suggesting that IL-10 increases susceptibility to L mexicana or L amazonensisinfection by inhibiting effector cell functions required forparasite killing IL-10 inhibition after treatments has severalconsequences On one hand it has been demonstrated thatit induces the cPLA

2-COX-2 pathway however results in

this work show its downregulation On the other hand IL-10 alone or in concert with other molecules activates distincttranscriptional programs that promote the alignment ofadaptive responses in a type I or type II direction as well as byexpressing specialized and polarized effector functions [24]In this case after treatments infected macrophages did notinduce IL-10 expression and remained as classically activatedmacrophages this activation program is characterized byTNF-120572 IL-1120573 and IL-6 expression these cytokines beingresponsible of the oxidative burst [15]

Although TNF-120572 and IL-1120573 have been shown as detri-mental in various pathologies in this work they are requiredto sustain classical macrophage activation combined with asmall IL-10 production several reports have demonstratedthat after Leishmania infection TNF-120572 and IL-1120573 inducethe phagocytesrsquo NADPH oxidase whereas IL-10 productioninhibits the oxidative stress [15]

The heterogeneity in macrophage phenotypes has givenplace to its classification as M1 and M2 phenotypes corres-ponding to classically and alternatively activated

macrophages respectively [33 34] M1 macrophages pro-duce high levels of proinflammatory cytokines TNF-120572IL-1 IL-6 IL-23 and ROS M2 macrophages upregulatescavenger mannose and galactose receptors and IL-1receptor antagonist and downregulate IL-1120573 and otherproinflammatory cytokines [35] Available informationsuggests that classically activated M1 macrophages arepotent effector cells integrated in Th1 responses whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines In this work wedemonstrated that PPAR activation modulates selectivelydifferent molecules suggesting a macrophage polarizationfrom M2 to M1 profile among these molecules weemphasize IL-10 down regulation and upregulation ofIL-6 IL-1120573 and TNF-120572 PPAR activation also diminishedcPLA2phosphorylation and COX-2 expression This is in

agreement with reports showing that the differentiationinto classically activated M1 macrophage increases incPLA2knockdown cells whereas the differentiation into

alternatively activated M2 macrophage was suppressed bycPLA2-knockdown [36] These findings suggest that cPLA

2

is involved in regulation of macrophage differentiation andmacrophage polarization Polarized macrophages differin terms of receptor expression M2 macrophages arecharacterized by MR (CD206) expression [37] whereasTLR4 expression is associated with M1 macrophages[24 38] Our results show that PPAR activation and cPLA

2

inhibition significantly increased the TLR4 expression afterinfection compared to nontreated macrophages indicatingmacrophage polarization to M1 profile

Recent evidence suggests that PPAR120574 activation mayincrease the replication of parasites as well as maintain thesurvival of the host In particular PPAR activation has beenassociated with parasite survival and increase of parasiteburden [25 26 39 40] Flow cytometry analysis revealedthat phagocytic activity was not affected by treatments asindicated by zymosan particles assay and neither PPARactivation nor cPLA

2inhibition increased significantly the

percentage of infected macrophages or the parasite burdenas other agonists do

Previous studies have reported that Leishmania infectionboth in vitro and in vivo conducts to PGE

2production and

it has been postulated that this may favor Leishmania persis-tence and progression [14 30] Among prostaglandins ana-lyzed 6k-PGF

1120572 PGE

1 and PGF

1120572production was increased

after infection and PPAR agonists and cPLA2antagonist

diminished their production after infection however bothPGE2and PGF

2120572production was diminished after infection

and increased in treated and infected macrophages Thisresult may seem in conflict with previous data reporting anincrease in PGE

2after infection This could be explained

by the fact that COX-2 enzyme and its principal catalyticproduct PGE

2are often equated with inflammation and

pathology a notion fueled primarily by a strong inductionof COX-2 expression at sites of inflammation and tissueinjury [41] however at a later phase COX-2 promotedresolution by generating an alternate set of reportedlyanti-inflammatory prostaglandins through a process nowregarded as ldquoeicosanoid class switchingrdquo In addition it has

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 BioMed Research International

(a)

0

10

20

30

40

50

60

70

wTX

lowast

lowastlowastlowast

Time after infection

(b)

0

250

500

750

1000

Redu

ced

NBT

(ng)

wTXATK

lowastlowastlowast

lowast

lowastlowastlowast

Time after infection

(c)

Figure 6Oxidative burst of Lmexicana-infectedmacrophages Cells were treatedwith PPAR agonists 24 h before infection and the oxidativeburst was determined by NBT reduction NBT was added simultaneously with promastigotes (a) After the indicated times after infectionslides with infected macrophages were washed and stained for 30min with Fuccina Microphotographs show positive cells to NBT reductionin comparison with control cells which were treated or not with agonists in the presence of NBT (b) The graph shows percentage of cellspositive to NBT reduction (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists Graphbars are mean plusmn SEM of three independent experiments and statistical analysis was done comparing for each time treated versus nontreatedmacrophages (lowast) 119875 lt 005 (lowastlowast) 119875 lt 001 and (lowastlowastlowast) 119875 lt 0001

been demonstrated that PGE2can modulate various steps of

inflammation at the beginning it can induce the expressionof COX-2 however as the inflammation progresses andrecovering initiates PGE

2can also inhibit the expression

of this enzyme Therefore PGE2can exert both proinflam-

matory and anti-inflammatory effects Akarasereenont et al[42] demonstrated that in HUVEC cells treated with IL-1120573 PGE

2can inhibit COX-2 but not COX-1 protein expres-

sion Therefore results suggested that PGE2can initiate

a negative feedback regulation in the induction of COX-2elicited by IL-1120573 in endothelial cells [42] Based on these

results we propose that the expression of IL-1120573 and TNF-120572maintains COX-2 expression in untreated macrophages andas stated by Akarasereenont et al PGE

2is able to inhibit

COX-2 expression in the presence of these proinflammatorycytokines together with these results cPLA

2inhibition also

inhibits COX-2 expression via PPAR120574 [29] and authorshave proposed that cPLA

2inhibition can be reverted dur-

ing M2 to M1 polarization of macrophages [36] All theseresults may explain why during PPAR120574 activation PGE

2

production increased during macrophages infection withL mexicana

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 11

In murine and human macrophages it has been estab-lished that the respiratory burst of the cell with the pro-duction of ROS such as H

2O2and O

2 is largely responsible

for parasite control as these molecules have been reportedto be fatal for Leishmania promastigotes [43 44] It hasbeen demonstrated that L donovani inhibits the respiratoryburst in macrophages [45] In this work we have shownthat PPAR activation as well as cPLA

2inhibition increased

ROS production by 1-2 folds It has been demonstrated thatlong-chain fatty acids increase intercellular ROS synthesis viaPPAR120572 and its inhibitors reduced ROS concentration [46]

The FDA has approved several synthetic PPAR ligandsas therapeutic drugs [47] These PPAR ligands could have apotential use in parasitic diseases Recently Serghides et al[48] have shown that rosiglitazone a PPAR120574 agonist is usefulin alleviating cerebral malaria in a murine model [48]

It has been demonstrated that different Leishmania spe-cies can induce a different profile of cytokines [6 49] andthe enzymes responsible for ROS production are regulatedby those cytokines therefore treatment against Leishmaniainfection would depend on the infecting species Thus cuta-neous leishmaniasis caused by L major could be alleviatedwith PPAR120572 and PPAR120574 ligands in murine models [26 39]In thiswork we have demonstrated that PPAR120573120575 andmainlyPPAR120574 activation induced macrophage activation throughtheir polarization to M1 profile with an increase of microbi-cidal activity against an intracellular pathogen L mexicanaBased on the above reasons macrophage polarization fromM2 toM1 throughPPARactivation in the presence of agonistscould be considered as a potential signaling pathway for drugdesign and eventually to be used as a strategy to controlintracellular parasitosis

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This work was supported by CONACyT Mexico (Grant104108) and ICyTDF Mexico (PIFUTP08-157) The authorsthank Belem de Luna Vergara and Ma Elena CisnerosAlbavera for their technical support J A Dıaz-Gandarillawas a recipient of a PhD fellowship from CONACyT andICyTDF Mexico (Grants 173691 and BI11-240)

References

[1] Who ldquoLeishmaniasisrdquo 2012 httpwwwwhointtopicsleish-maniasisen

[2] F A Torrentera M A Lambot J D Laman et al ldquoParasiticload and histopathology of cutaneous lesions lymph nodespleen and liver from BALBc and C57BL6 mice infected withLeishmania mexicanardquo American Journal of Tropical Medicineand Hygiene vol 66 no 3 pp 273ndash279 2002

[3] L E Rosas T Keiser J Barbi et al ldquoGenetic background influ-ences immune responses and disease outcome of cutaneous Lmexicana infection in micerdquo International Immunology vol 17no 10 pp 1347ndash1357 2005

[4] O Braissant F Foufelle C Scotto M Dauca and W WahlildquoDifferential expression of peroxisome proliferator-activatedreceptors (PPARs) tissue distribution of PPAR-120572 -120573 and -120574 inthe adult ratrdquo Endocrinology vol 137 no 1 pp 354ndash366 1996

[5] M M Chan K W Evans A R Moore and D FongldquoPeroxisome proliferator-activated receptor (PPAR) balancefor survival in parasitic infectionsrdquo Journal of Biomedicine ampBiotechnology vol 2010 Article ID 828951 9 pages 2010

[6] U Ritter J Mattner J S Rocha C Bogdan andH Korner ldquoThecontrol of Leishmania (Leishmania) major by TNF in vivo isdependent on the parasite strainrdquo Microbes and Infection vol6 no 6 pp 559ndash565 2004

[7] R Chatelain S Mauze and R L Coffman ldquoExperimentalLeishmania major infection in mice role of IL-10rdquo ParasiteImmunology vol 21 no 4 pp 211ndash218 1999

[8] U M Padigel J Alexander and J P Farrell ldquoThe role ofinterleukin-10 in susceptibility of BALBcmice to infectionwithLeishmania mexicana and Leishmania amazonensisrdquo Journal ofImmunology vol 171 no 7 pp 3705ndash3710 2003

[9] T Krakauer ldquoMolecular therapeutic targets in inflammationcyclooxygenase and NF-120581Brdquo Current Drug Targets vol 3 no3 pp 317ndash324 2004

[10] Q He B Zhai A Mancini and J A Di Battista ldquo438Modulation of the inflammatory and catabolic response byprostaglandin E

2(PGE

2) is partially dependent on the induc-

tion of dual specificity phosphatase 1 (DUSP-1) in vitro and invivordquo Osteoarthritis and Cartilage vol 16 supplement 4 ppS190ndashS191 2008

[11] W H Faour N Alaaeddine A Mancini W H Qing DJovanovic and J A Di Battista ldquoEarly growth response factor-1 mediates prostaglandin E

2-dependent transcriptional sup-

pression of cytokine-induced tumor necrosis factor-120572 geneexpression in human macrophages and rheumatoid arthritis-affected synovial fibroblastsrdquo Journal of Biological Chemistryvol 280 no 10 pp 9536ndash9546 2005

[12] F Giuliano and T D Warner ldquoOrigins of prostaglandin E2

involvements of cyclooxygenase (COX)-1 and COX-2 in humanand rat systemsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 303 no 3 pp 1001ndash1006 2002

[13] W He J P Pelletier J Martel-Pelletier S Laufer and J A DiBattista ldquoSynthesis of interleukin 1120573 tumor necrosis factor-120572and interstitial collagenase (MMP-1) is eicosanoid dependent inhuman osteoarthritis synovial membrane explants interactionswith antiinflammatory cytokinesrdquo Journal of Rheumatology vol29 no 3 pp 546ndash553 2002

[14] J L M Perez-Santos and P Talamas-Rohana ldquoIn vitroindomethacin administration upregulates interleukin-12 pro-duction and polarizes the immune response towards a Th1type in susceptible BALBc mice infected with Leishmaniamexicanardquo Parasite Immunology vol 23 no 11 pp 599ndash6062001

[15] T Van Assche M Deschacht R A I Da Luz L Maes andP Cos ldquoLeishmania-macrophage interactions insights into theredox biologyrdquo Free Radical Biology and Medicine vol 51 no 2pp 337ndash351 2011

[16] L A Estrada-Figueroa Y Ramırez-Jimenez C Osorio-Trujilloet al ldquoAbsence of CD38 delays arrival of neutrophils to theliver and innate immune response development during hepaticamoebiasis by Entamoeba histolyticardquo Parasite Immunology vol33 no 12 pp 661ndash668 2011

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 BioMed Research International

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] KNessa L Palmberg U Johard PMalmberg C Jarstrand andP Camner ldquoReaction of human alveolar macrophages to expo-sure toAspergillus fumigatus and inert particlesrdquo EnvironmentalResearch vol 75 no 2 pp 141ndash148 1997

[19] R Munoz and W Alfaro ldquoEstandarizacion de la tecnica dereduccion del NBTmediante lectura en placas de microELISArdquoRevista Medica del Hospital Nacional de Ninos Dr Carlos SaenzHerrera vol 35 pp 29ndash39 2000

[20] D S Straus and C K Glass ldquoAnti-inflammatory actions ofPPAR ligands new insights on cellular and molecular mecha-nismsrdquo Trends in Immunology vol 28 no 12 pp 551ndash558 2007

[21] J J Bright S Kanakasabai W Chearwae and S ChakrabortyldquoPPAR regulation of inflammatory signaling in CNS diseasesrdquoPPAR Research vol 2008 Article ID 658520 12 pages 2008

[22] R Ringseis N Schulz D Saal and K Eder ldquoTroglitazonebut not conjugated linoleic acid reduces gene expressionand activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophagesrdquo Journal of Nutritional Bio-chemistry vol 19 no 9 pp 594ndash603 2008

[23] C Matte and M Olivier ldquoLeishmania-induced cellular recruit-ment during the early inflammatory response modulation ofproinflammatory mediatorsrdquo Journal of Infectious Diseases vol185 no 5 pp 673ndash681 2002

[24] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[25] A Gallardo-Soler C Gomez-Nieto M L Campo et alldquoArginase I induction bymodified lipoproteins inmacrophagesa peroxisome proliferator-activated receptor-120574120575-mediatedeffect that links lipid metabolism and immunityrdquo MolecularEndocrinology vol 22 no 6 pp 1394ndash1402 2008

[26] N Adapala and M M Chan ldquoLong-term use of an anti-inflammatory curcumin suppressed type 1 immunity andexacerbated visceral Leishmaniasis in a chronic experimentalmodelrdquo Laboratory Investigation vol 88 no 12 pp 1329ndash13392008

[27] M C Noverr J R Erb-Downward and G B HuffnagleldquoProduction of eicosanoids and other oxylipins by pathogeniceukaryotic microbesrdquo Clinical Microbiology Reviews vol 16 no3 pp 517ndash533 2003

[28] C C Leslie ldquoProperties and regulation of cytosolic phospho-lipase A

2rdquo Journal of Biological Chemistry vol 272 no 27 pp

16709ndash16712 1997[29] F Okamoto K Saeki H Sumimoto S Yamasaki and T

Yokomizo ldquoLeukotriene B4 augments and restores Fc120574Rs-dependent phagocytosis in macrophagesrdquo Journal of BiologicalChemistry vol 285 no 52 pp 41113ndash41121 2010

[30] C Matte G Maion W Mourad and M Olivier ldquoLeish-mania donovani-induced macrophages cyclooxygenase-2 andprostaglandin E

2synthesisrdquo Parasite Immunology vol 23 no

4 pp 177ndash184 2001[31] R Pawliczak C Han X L Huang A Jake Demetris J H

Shelhamer and T Wu ldquo85-kDa cytosolic phospholipase A2

mediates peroxisome proliferator-activated receptor 120574 acti-vation in human lung epithelial cellsrdquo Journal of BiologicalChemistry vol 277 no 36 pp 33153ndash33163 2002

[32] R Pawliczak C Logun P Madara et al ldquoCytosolic phos-pholipase A

2Group IV120572 but not secreted phospholipase A

2

Group IIA V or X induces interleukin-8 and cyclooxygenase-2gene and protein expression through peroxisome proliferator-activated receptors 120574 1 and 2 in human lung cellsrdquo Journal ofBiological Chemistry vol 279 no 47 pp 48550ndash48561 2004

[33] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[34] S Gordon and P R Taylor ldquoMonocyte andmacrophage hetero-geneityrdquoNature Reviews Immunology vol 5 no 12 pp 953ndash9642005

[35] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[36] K Ishihara A Kuroda K Sugihara S Kanai andTNabe ldquoReg-ulation ofmacrophage differentiation and polarization by groupIVC phospholipase A

2rdquo Biochemical and Biophysical Research

Communications vol 416 no 3-4 pp 325ndash330 2011[37] M L Rodriguez S Rodriguez A E Gonzalez et al ldquoCan

taenia solium latent post-oncospheral stages be found inmuscletissue of cysticercosis-infected pigs (Sus scrofa)rdquo Journal ofParasitology vol 92 no 1 pp 199ndash201 2006

[38] D Bosisio N Polentarutti M Sironi et al ldquoStimulation of toll-like receptor 4 expression in human mononuclear phagocytesby interferon-120574 a molecular basis for priming and synergismwith bacterial lipopolysacchariderdquo Blood vol 99 no 9 pp3427ndash3431 2002

[39] J I Odegaard R R Ricardo-Gonzalez M H Goforth etal ldquoMacrophage-specific PPAR120574 controls alternative activationand improves insulin resistancerdquo Nature vol 447 no 7148 pp1116ndash1120 2007

[40] M M Chan N Adapala and C Chen ldquoPeroxisome prol-iferator-activated receptor-120574-mediated polarization of macro-phages in Leishmania infectionrdquo PPAR Research vol 2012Article ID 796235 11 pages 2012

[41] A D Mancini and J A Di Battista ldquoThe cardinal role of thephospholipase A

2cyclooxygenase-2prostaglandin e synthase

prostaglandin E2(PCPP) axis in inflammostasisrdquo Inflammation

Research vol 60 no 12 pp 1083ndash1092 2011[42] P Akarasereenont K Techatrisak S Chotewuttakorn and A

Thaworn ldquoThe induction of cyclooxygenase-2 in IL-1120573-treatedendothelial cells is inhibited by prostaglandin E

2through

cAMPrdquo Mediators of Inflammation vol 8 no 6 pp 287ndash2941999

[43] H W Murray ldquoCell-mediated immune response in experi-mental visceral Leishmaniasis II Oxygen-dependent killingof intracellular Leishmania donovani amastigotesrdquo Journal ofImmunology vol 129 no 1 pp 351ndash357 1982

[44] J H Zarley B E Britigan and M E Wilson ldquoHydrogenperoxide-mediated toxicity for Leishmania donovani chagasipromastigotes role of hydroxyl radical and protection by heatshockrdquo Journal of Clinical Investigation vol 88 no 5 pp 1511ndash1521 1991

[45] J F Dermine G Goyette M Houde S J Turco and MDesjardins ldquoLeishmania donovani lipophosphoglycan disruptsphagosome microdomains in J774 macrophagesrdquo CellularMicrobiology vol 7 no 9 pp 1263ndash1270 2005

[46] E Stachowska M Baskiewicz-Masiuk V Dziedziejko et alldquoConjugated linoleic acid increases intracellular ROS synthesisand oxygenation of arachidonic acid in macrophagesrdquo Nutri-tion vol 24 no 2 pp 187ndash199 2008

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 13

[47] P Gervois J C Fruchart and B Staels ldquoDrug insight mech-anisms of action and therapeutic applications for agonists ofperoxisome proliferator-activated receptorsrdquo Nature ClinicalPractice Endocrinology and Metabolism vol 3 no 2 pp 145ndash156 2007

[48] L Serghides S N Patel K Ayi et al ldquoRosiglitazone modulatesthe innate immune response to plasmodium falciparum infec-tion and improves outcome in experimental cerebral malariardquoJournal of Infectious Diseases vol 199 no 10 pp 1536ndash15452009

[49] M J Teixeira J Dumet Fernandes C Romero Teixeira et alldquoDistinct Leishmania braziliensis isolates induce different pacesof chemokine expression patternsrdquo Infection and Immunity vol73 no 2 pp 1191ndash1195 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology