research article carrageenan-induced colonic...

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 397642 13 pageshttpdxdoiorg1011552013397642

Research ArticleCarrageenan-Induced Colonic Inflammation Is Reduced inBcl10 Null Mice and Increased in IL-10-Deficient Mice

Sumit Bhattacharyya12 Liquan Xue3 Suzanne Devkota4 Eugene Chang5

Stephan Morris3 and Joanne K Tobacman12

1 Department of Medicine University of Illinois at Chicago 840 S Wood Street CSN 440 MC 718 Chicago IL 60612 USA2 Jesse Brown VA Medical Center Chicago IL 60612 USA3Department of Pathology St Jude Childrenrsquos Research Hospital Memphis TN 38105 USA4Department of Medicine University of Chicago Chicago IL 02215 USA5 Joslin Diabetes Center Boston MA 60637 USA

Correspondence should be addressed to Joanne K Tobacman jktuicedu

Received 25 February 2013 Revised 29 April 2013 Accepted 8 May 2013

Academic Editor Assaf Rudich

Copyright copy 2013 Sumit Bhattacharyya 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

The common food additive carrageenan is a known activator of inflammation in mammalian tissues and stimulates both thecanonical and noncanonical pathways of NF-120581B activation Exposure to low concentrations of carrageenan (10 120583gmL in thewater supply) has produced glucose intolerance insulin resistance and impaired insulin signaling in C57BL6 mice B-cellleukemialymphoma 10 (Bcl10) is amediator of inflammatory signals fromToll-like receptor (TLR) 4 inmyeloid and epithelial cellsSince the TLR4 signaling pathway is activated in diabetes and by carrageenan we addressed systemic and intestinal inflammatoryresponses following carrageenan exposure in Bcl10 wild type heterozygous and null mice Fecal calprotectin and circulatingkeratinocyte chemokine (KC) nuclear RelA and RelB phospho(Thr559)-NF-120581B-inducing kinase (NIK) and phospho(Ser36)-I120581B120572in the colonic epithelial cells were significantly less (119875 lt 0001) in the carrageenan-treated Bcl10 null mice than in controls IL-10-deficient mice exposed to carrageenan in a germ-free environment showed an increase in activation of the canonical pathwayof NF-120581B (RelA) activation but without increase in RelB or phospho-Bcl10 and exogenous IL-10 inhibited only the canonicalpathway of NF-120581B activation in cultured colonic cells These findings demonstrate a Bcl10 requirement for maximum developmentof carrageenan-induced inflammation and lack of complete suppression by IL-10 of carrageenan-induced inflammation

1 Introduction

Carrageenans are sulfated polygalactans and are obtainedfrom several species of red seaweed (Rhodophyceae) Chemi-cally they are composed of repeating disaccharide units andconsist of sulfated or unsulfated D-galactose residues thatare linked in alternating 120573-14 and 120572-13 bonds They resem-ble the endogenous human galactose-containing sulfatedglycosaminoglycans chondroitin sulfate and keratan sulfatebut differ by the presence of the unusual 120572-13 linkages [1 2]Carrageenans predictably induce an inflammatory responseand can activate immune responses that are mediated byB-cell leukemialymphoma (Bcl) 10 and Toll-like receptor

(TLR) 4 [3 4] Due to their marked chemical reactivitycarrageenans are widely used in processed foods to improvethe texture of food products although their harmful effectsare well recognized [5 6]

Animal models have demonstrated that ingestion of car-rageenan induces colonic inflammation with development ofinflammatory infiltrates ulcerations and clinical evidence ofcolitis [5 6] In studies of the effects of carrageenan on humancolonic epithelial cells in cell culture carrageenan stimulatedNF-120581B activation and increased secretion of IL-8 by TLR4-Bcl10-dependent and Bcl10-independent pathways [3 4 78] Specific phosphorylations of Bcl10 were required for theNF-120581B responses [9] and prolongation of the effects of

2 Mediators of Inflammation

carrageenan in colonic epithelial cells on both noncanonicaland canonical inflammatory pathways occurred due to thepresence of an NF-120581B binding site in the Bcl10 promoter [10]

The carrageenan-induced activation of the innateimmune response in the colonic epithelial cells was attributa-ble to its distinctive chemical structure and in particularits unusual 120572-D-Gal (1rarr 3) D-Gal link [11] Carrageenanexposure was shown to produce NF-120581B activation by threedistinct mechanisms including (1) TLR4-Bcl10-IKK120574-IKK120573-inhibitory factor (I120581B)120572-NF-120581B (RelA) pathway (2) TLR4-Bcl10-NF-120581B-inducing kinase (NIK)-IKK120572-NF-120581B (RelB)pathway and (3) a reactive-oxygen-species-(ROS-) mediatedpathway that involves Hsp27-IKK120573-I120581B120572-NF-120581B (RelA) thatdoes not require Bcl10 [3 4 7 8] These pathways indicatethat Bcl10 is a key signaling molecule in the carrageenan-initiated inflammation in vitro in colonic epithelial cellsRecently the impact of carrageenan on glucose toleranceinsulin resistance and impaired insulin signaling in a mousemodel was reported [12] consistent with the evidence thatTLR4-mediated inflammation is involved in diabetes [13ndash15]Inhibition of inflammation by Bcl10 silencing or TLR4blocking antibody reduced the carrageenan-induced impair-ment of insulin signaling [12]

Many recent investigations involve carrageenan includ-ing almost 500 published reports in 2012 Most of these pub-lications address the effects of anti-inflammatory treatmentsto alleviate carrageenan-induced inflammation includingeffects of lactobacilli [16] but without specific attention to themechanistic pathways by which carrageenan causes inflam-mation Other reports consider a variety of topics includingthe chemical characteristics of carrageenan as a gel in foodproducts [17] the use of carrageenan hydrogels as therapeuticdelivery vehicles [18] carrageenan as a therapy including forthe common cold [19] and thrombotic effects of carrageenan[20]

This is the first report to address the requirement for Bcl10as a mediator of carrageenan-induced inflammation in thein vivo animal model and extends previous findings aboutthe mechanisms by which carrageenan produces inflamma-tion Prior work has shown the role of Bcl10 in normaldevelopment and function of lymphocyte populations [21]Overexpression of Bcl10 by the translocation t(1 14)(p22 q32)was associated with development of the mucosa-associatedlymphoid tissue (MALT) lymphomas and with the consti-tutive overexpression of NF-120581B [22] by both non-canonicaland canonical pathways of NF-120581B activation [23] Bcl10 hasbeen reported to mediate inflammatory cascades in colonicepithelial cells mouse embryonic fibroblasts hepatocytesand human embryonic kidney 293T cells as well as incells of myeloid origin [3 24ndash26] Other recent work hasidentified Bcl10 as a link between fat (palmitate) intakeand hepatic NF-120581B activation and insulin resistance [27]and Bcl10 was reported to coordinate the NF-120581B-mediatedresponse associated with endosomal trafficking and F-actinremodeling in human macrophages [28] Recognition ofthe participation of the CARMA-Bcl10-Malt1 (CBM) sig-nalosome in vital processes in cells continues to emergeincluding distinct roles of CARMA1 in immune cells andCARMA3 in epithelialendothelial cells The CBM complex

appears to be a signaling platform enabling the integration ofeffects from receptors with downstream cascades includingeffects from G-protein coupled receptors [29] The CBMcomplex was shown to promote angiotensin II-dependentvascular inflammation and atherogenesis and these effectswere reported to be reduced in Bcl10-deficient mice [30]The protein A20 also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3) acts as a negative regulatorof the Bcl10-CARMA interactions in both the lymphoid andnonlymphoid cells due to its effects as a ubiquitin ligase anddeubiquitinase [31]

In this report we evaluate the effect of carrageenan inBcl10 null mice and the pathways of carrageenan-inducedinflammation mediated by activation of canonical and non-canonical cascades Also since the microbiome and IL-10 areboth widely recognized to impact intestinal inflammationthe effects of the germ-free environment and of exogenousIL-10 on carrageenan-induced inflammation are evaluated inorder to provide additional insights into the mechanisms andmediators by which carrageenan provokes inflammation

2 Materials and Methods

21 Animal Care and Carrageenan Exposure Eight-week-old male C57BL6J mice were purchased (Jackson Labora-tories Bar Harbor ME USA) and housed in the VeterinaryMedicine Unit (VMU) at the Jesse Brown VAMedical Center(JBVAMC Chicago IL USA) Principles of laboratory ani-mal care were followed and all procedures were approved bythe Animal Care Committee Adult mice were fed a standarddiet and maintained in individual cages with routine light-dark cycles After acclimatizing to the environment the watersupply was changed to ddH

2Owith undegraded carrageenan

(120582-120581 carrageenan 10mgL Sigma Chemical Co St LouisMO USA 119899 = 6) or without carrageenan (119899 = 6) forsim12 weeks Weight and water consumption were measuredregularly Bcl10 null (119899 = 8) heterozygote (119899 = 3)and wild type (119899 = 6) mice were bred and genotyped atSt Jude Childrenrsquos Research Hospital by study investigators(Stephen Morris and Liquan Xue) [21ndash23] Adult mice wereshipped to the JBVAMC VMU Following quarantine adultmice were treated with carrageenan in their water supplyStool was collected and mice were euthanized after receivingcarrageenan forsim14weeks Total carrageenan intake averagedsim115mg30 g mouse Blood was collected by cardiac punc-ture at the time of euthanasia and organs were immediatelyharvested and frozen Carrageenan (10mgL times 19 days +100mgL times 9 days) was also given in the water supply to adultC57BL6 mice (119899 = 5) and IL-10-deficient mice (119899 = 4)in the germ-free facility at the University of Chicago underan approved protocol and direction of study investigators(Eugene Chang and Suzanne Devkota) [32] Control litter-mates were not given carrageenan Mice were euthanizedby carbon dioxide and exsanguination by cardiac punctureblood was collected and stool and organs were harvestedand frozen pending further studies Germ-free status wasassessed by detection of 16S rRNA by PCR from stool andcecal contents

Mediators of Inflammation 3

22 NCM460 Cells in Tissue Culture NCM460 cells(INCELL San Antonio TX USA) were grown in M310Amedia under the established conditions as previouslydescribed [3 4 33] Bcl10 siRNA was used as previouslydescribed [3 4] Cells were exposed to 120582-CGN (1mgL times24 h) in the presence or absence of bioactive exogenousIL-10 (Sigma-Aldrich Chemical Co St Louis MO USA 10or 20120583gL times 24 h) At the end of the treatment spent mediawere collected from control and treated wells and stored atminus80∘C Total cell protein was measured by the BCA proteinassay kit (Pierce Rockford IL USA) using BSA as thestandard

23 Measurement of Bcl10 and Phospho-Bcl10 Bcl10 proteinin the colonic epithelium of control and carrageenan-fedmice was determined by a solid-phase ELISA reported pre-viously [34] To determine phospho-Bcl10 in mouse colonicepithelium and in control and CGN-treated NCM460 cellsBcl10 in the samples was captured in a 96-well ELISA platethat was precoated with Bcl10 antibody sandwiched by aphospho(Ser138)-Bcl10 second antibody and then detectedby a specific anti-rabbit IgG and hydrogen peroxide-TMBchromogenic substrate [8 9] Intensity of the developedcolor was proportional to the quantity of phospho-Bcl10in the sample and optical density values were determined(FLUOstar BMG Cary NC USA) and normalized using thetotal cell protein determined by a protein assay kit (Pierce)Results were expressed as percent of total Bcl10 in theuntreated control

24 ELISA for Phospho-I120581B120572 in Colon of Carrageenan-Exposed Mice and in NCM460 Cells Commercial sandwichELISA for phospho(Ser32)-I120581B120572 (Cell Signaling Technol-ogy Inc Danvers MA USA) was used to determine thephosphorylation of I120581B120572 that was produced following car-rageenan exposure in the mouse colonic epithelial tissueand in NCM460 cells [3 4] Briefly the I120581B120572 in the cellextracts was captured in a 96-well ELISA plate that was pre-coated with mouse monoclonal antibody against I120581B120572Phospho-I120581B120572was determined by a specific phospho(Ser32)-I120581B120572 antibody and detected by horseradish peroxidase-(HRP-) conjugated secondary antibody and hydrogen per-oxide-tetramethylbenzidine (TMB) chromogenic substratePhospho-I120581B120572 was expressed as percent of total I120581B120572 in theuntreated control

25 Oligonucleotide-Based ELISA for Measurement of NuclearRelA and RelB Nuclear extracts were prepared from eitherthe colonic epithelial cells of control and carrageenan-fedmice or carrageenan-treated and control NCM460 cells bya nuclear extraction kit (Active Motif Carlsbad CA USA)ActivatedNF-120581Bs includingRelA andRelBwere determinedby oligonucleotide-based ELISA (ActiveMotif) as previouslydescribed [8] Treated and control samples were incubatedin 96-well microtiter plates that were coated with the NF-120581Bconsensus nucleotide sequence (51015840-GGGACTTTCC-31015840) NF-120581B from the samples attached to thewells andwas captured byspecific antibody to either RelA or RelBThe extent of binding

of the antibody was detected by anti-rabbit-HRP-conjugatedIgG and color was developed with hydrogen peroxide-TMBchromogenic substrate Intensity of the developed color wasproportional to the quantity of either RelA or RelB in thesample Specificity of the NF-120581B binding to the nucleotidesequence was determined by the premixing of either freeconsensus nucleotide or mutated consensus nucleotide to thenuclear extract sample before adding the sample to the wellThe optical density values were normalized using the total cellprotein determined by a protein assay kit (Pierce) and resultswere expressed as percent of the unexposed control

26 ELISA for Measurement of KC and Other Cytokinesin Mouse Serum Serum keratinocyte chemokine (KC)the mouse homolog of IL-8 was detected in control andcarrageenan-exposed Bcl10 wild type heterozygotic and nullmice and untreated control C57BL6J mice (total 119899 = 12)by DuoSet ELISA (RampD Systems Minneapolis MN USA)as previously described [4] In addition to the KC ELISAa custom cytokine ELISA array that included TNF-120572 IL-6 IFN120574 IL-1120573 IL-10 IL-12 MCP- (monocyte chemotacticprotein-) 1 IL-12 and IL-23 was prepared (Signosis Sun-nyvale CA USA) and serum of carrageenan-treated Bcl10wild type heterozygotic null mice and untreated controlC57BL6Jmice (total 119899 = 12) was tested Hydrogen peroxide-TMB chromogenic substrate was used to develop the colorcolor development was stopped with a stop solution and theconcentrations of the cytokines were directly proportionalto the intensity of color measured spectrophotometricallyat 450 nm in an ELISA plate reader (FLUOstar BMG) KCconcentrations were extrapolated from a standard curveOther chemokines and cytokines were expressed as percentof control based on determinations from mice unexposedto carrageenan Technical duplicates were performed for allmeasurements and the mean of the two readings was used insubsequent comparisons

27Measurement of Fecal Calprotectin Fecal calprotectinwasmeasured in stools of control and carrageenan-treated miceby ELISA (Alpco Diagnostics Salem NH USA) [35] Feceswere collected from control and treated animals at the timeof euthanasia and recommended procedures were followedStool samples were extracted in the supplied extractionbuffer centrifuged for 5 minutes at 13000 g then used inthe sandwich ELISA TMB was used for the development ofcolor which was read at an OD of 450 nm in a plate reader(FLUOstar) and compared to the standard curve

28 Western Blots for Phospho(Thr559)-NIK and Phos-pho(Thr184)-Tak1 Tissue homogenates were prepared fromcolonic epithelial tissue of control and carrageenan-exposedmice in lysis buffer (Cell Signaling Technology Inc Dan-vers MA USA) with protease and phosphatase inhibitors(Halt Protease and Phosphatase Inhibitor Cocktail ThermoScientific Pittsburgh PA USA) Western blots were per-formed on 10 SDS gels with commercial antibodiesto phospho(Thr559)-NIK phospho(Thr184)-TAK and 120573-actin (Santa Cruz Biotechnology Santa Cruz CA USA)

4 Mediators of Inflammation

Immunoreactive bands were visualized using enhancedchemiluminescence (Amersham GE Healthcare PiscatawayNJ USA) ImageJ software (NIH Bethesda MD USA) wasused for densitometry The density of the protein of interestwas normalized with the density of 120573-actin or tubulin fromthe same specimen and density of treated and control sam-ples was compared

29 Histopathology of Colon from Bcl10 Wild Type and NullMice Following Carrageenan Intestine and other organsfrom control and carrageenan-fed mice were sampled pro-cessed in 10 neutral-buffered formalin and paraffin blockswere made by standard procedures The paraffin blocks weresectioned and tissues were stained with hematoxylin andeosin by the Veterinary Diagnostic Laboratory of the Uni-versity of Illinois (Urbana IL USA) Slides of duodenumjejunum ileum cecum colon and rectum from three Bcl10wild type three Bcl10 heterozygotic and three Bcl10 nullmiceexposed to carrageenanwere analyzed by a veterinary pathol-ogist (Susan Ball-Kell Global Path Imaging GermantownHills IL USA) who examined blindly the tissue sectionsfrom the gastrointestinal sites and recorded on a standard-ized scale ratings of inflammation corresponding to theAmeho criteria with six gradations [36] These grades wereoccasional = 0ndash3 inflammatory cellshpf grade 0 of normalnormal to trace inflammatory infiltrate = 4ndash10 inflammatorycellshpf grade ldquo1rdquo or small mild inflammatory infiltrate =11ndash20 inflammatory cellshpf grade ldquo2rdquo or small-mediummild-moderate inflammatory infiltrate = 21ndash30 inflammatorycellshpf grade ldquo3rdquo ormediummoderate inflammatory infil-trate = 31ndash40 inflammatory cellshpf grade ldquo4rdquo or medium-large moderate to severe inflammatory infiltrate = 41ndash50cellshpf grade ldquo5rdquo or large severe inflammatory infiltrate=gt50 inflammatory cellshpf grade ldquo6rdquo or marked Photomi-crographs were taken with 20x objective

210 Statistical Analysis Data were analyzed using InStat3software (GraphPad La Jolla CA USA) Mean values plusmnstandard deviation (SD) were calculated and differencesbetween carrageenan-treated and control results were com-pared by one-way ANOVA with Tukey-Kramer post-testfor multiple comparisons or by unpaired t-tests two-tailedUnless stated otherwise the one-way ANOVA test was usedfor the reported comparisons All measurements are theresult of independent experiments with at least three biolog-ical samples and two technical replicates with the exceptionof the control IL-10-deficient mice in which the 119899 was 2 Inthe figures lowast is for 119875 le 005 lowastlowast is for 119875 le 001 and lowastlowastlowast is for119875 le 0001

3 Results

31 Histopathology of Colonic Tissue Following CarrageenanExposure Following exposure to sim18mg of carrageenan inthewater supply over 14weeks Bcl10wild type heterozygoticand null mice and unexposed C57BL6J mice were eutha-nized and the intestine and other organs were excised No

gross differences in the length of the intestine or in macro-scopic lesions were evident The intestine was immersed in10 neutral-buffered formalin embedded in paraffin sec-tioned and stained with hematoxylin and eosin Disruptionof the mucosal surface with hemorrhage and inflammatoryinfiltrate was seen in the cecal tissue of one Bcl10 wildtype mouse (Figure 1(a)) but not in any of the other mice(Figure 1(b)) No macroscopic abnormalities were observedand the intestine did not appear shortened

The intestines were evaluated for microscopic changesusing a six-point scale based on the extent of leukocyteinfiltration as described in Section 2 Throughout the smalland large intestines the wild-type mice had more leukocyteinfiltration including granulocytes plasma cells and lym-phocytes than the Bcl10 null mice consistent with increasedinflammation when Bcl10 was present (Figure 1(c)) Thedifferences in the scores among the Bcl10 WT heterozygousand null mice were not statistically significant Leukocyteinfiltrationwas greater in the small intestine than in the colonand rectum for all of the mice (119875 lt 001) The Bcl10 WTmice hadmore cecal infiltration than the null or heterozygousmice No edema was seen in the null mice in contrast toedema of the lamina propria and submucosa in the cecumand rectum of the heterozygous andWTmiceThe initial andfinal weights of the mice indicated no significant differencesamong the groups (Figure 1(d))

32 KC and Fecal Calprotectin Increased Less in Bcl10Null Mice Following Carrageenan The serum keratinocytechemokine (KC) the mouse homolog of IL-8 increased tosim20 times the no carrageenan baseline value in the wild typeand heterozygous Bcl10 mice (119875 lt 0001) The increase inserum KC following carrageenan was less (to sim15 times thebaseline) in the Bcl10 null mice than in the Bcl10 wild typeor heterozygous mice (119875 lt 0001) (Figure 2(a)) Similarlythe increase in fecal calprotectin following carrageenan wasless in the Bcl10 null mice than in the Bcl10 wild type or het-erozygous mice (119875 lt 0001) (Figure 2(b)) These findings areconsistent with reduced intestinal and systemic inflammationin the absence of Bcl10

33 Increased Serum IL-6 and MCP-1 Following CarrageenanExposure in Bcl10 Mice Serum was collected from theBcl10 WT heterozygous and null mice after exposure tocarrageenan for 14 weeks Cytokine ELISA was performedand indicated significant and similar increases following car-rageenan in MCP-1 and IL-6 in the Bcl10 WT heterozygousand controlmice compared to controls that were not exposedto carrageenan (119875 lt 0001) (Figure 3) Serum TNF120572 IFN120574IL-1120573 IL-10 IL-12 and IL-23 were unchanged followingcarrageenan

34 Activation of NF-120581B RelA Reduced and of RelB Inhibitedin Bcl10 Null Mice Following Carrageenan Measurements ofactivated nuclear RelA and RelB in the colonic epitheliumof carrageenan-exposed mice and controls revealed thatactivation of RelA was significantly increased in wild typeheterozygous and Bcl10 null mice after carrageenan expo-sure but the increase was significantly less in the Bcl10 null

Mediators of Inflammation 5

Bcl10 WT

(a)

Bcl10 null

(b)

005

115

225

335

Leuk

ocyt

e infi

ltrat

ion

mea

n sc

ore

WTHetNull

Duodenum Jejunum Ileum Cecum Colon Rectum

(c)

048

12162024283236

Mea

n w

eigh

t (gm

s)

InitialFinal

WT no CGN WT + CGN Het + CGN Null + CGN

(d)

Figure 1 Histopathology of intestinal tissue in Bcl10wild type and nullmice following carrageenan exposure (a) (b) Bcl10WT heterozygousand null mice were sacrificed after ingestion of carrageenan (10120583gmL) added in their water for 30 days Histopathology of HampE sectionsof cecum demonstrated disruption of the mucosa in a WT mouse with no similar findings in the Bcl10 null mice consistent with reducedinflammation in the absence of Bcl10 [WT wild type] (c) Throughout the mouse intestine the extent of inflammatory infiltrate includinggranulocytes lymphocytes and plasma cells was greater in the Bcl10WTmice than in the Bcl10 null miceThe histopathology was scored for3WT 3 heterozygous and 3 null mice and the mean scores for leukocyte infiltration for each site are compared The scores for the WTmiceare higher at each site than for the null mice although the differences are not statistically significant The extent of inflammatory infiltratewas significantly greater in the small intestine than in the colon and rectum for each of the groups (119875 lt 001) Cecal inflammation includingleukocyte infiltration and edema was greater in theWTmice than in the heterozygous or null mice (d)Themouse weights at the onset of thecarrageenan exposure and at termination are presented and indicate no significant differences in weight and slight weight gain in all groups(n for no CGN control=3 n for WT with CGN = 6 n for het with CGN = 3 n for null with CGN = 8) (WT wild type het heterozygousCGN carrageenan)

mice (119875 lt 0001) (Figure 4(a)) Activated RelB was increasedsim75 in wild type and heterozygous mice but this increasewas completely inhibited in the Bcl10 null mice (119875 lt 0001)(Figure 4(b))

35 Phosphorylations of Bcl10 and of I120581B120572 Phosphorylationsof Bcl10 and I120581B120572 were measured by ELISAs in colonicepithelial tissue following exposure to carrageenan in theBcl10 transgenic mice and in control untreated C57BL6mice Four fold increases in phospho(Ser138)-Bcl10 weremeasured in the carrageenan-exposed Bcl10 wild type andheterozygous mice (119875 lt 0001) but not in the Bcl10 null

mice as anticipated (Figure 5(a)) Phospho(Ser32)-I120581B120572 levelwas measured by ELISA in the colonic epithelial tissue ofthe carrageenan-treated Bcl10 wild type heterozygous andhomozygous null mice and control untreated C57BL6 miceFollowing carrageenan phospho-I120581B120572 increased to almosttwice the level of the untreated control value in the wild typeand heterozygousmice but in the Bcl10 nullmice the increasewas nearly 50 less (119875 lt 0001) (Figure 5(b))

36 Phosphorylations of NIK and of Tak1 To compare theactivation of the canonical and non-canonical pathways inthe Bcl10 transgenic mice phospho-NIK which is required

6 Mediators of Inflammation

250

0

50

100

150

200

KC (n

gL)

No CGN WT + CGN Het + CGN Null + CGN

lowastlowastlowast lowastlowastlowast

(a)

0

40

80

120

160

200

No CGN WT + CGN Het + CGN Null + CGN

lowastlowastlowast lowastlowastlowast

Feca

l cal

prot

ectin

(120583g

kg)

(b)

Figure 2 Carrageenan-induced increases in KC and calprotectinare less in Bcl10 null mice (a) KC the mouse homolog of IL-8increased less in the Bcl10 null mice (119899 = 8) than in the wild type(119899 = 6) or heterozygous (119899 = 3)mice following carrageenan tosim77of the higher value (1422 plusmn 90 ngL versus 1885 plusmn 64 ngL 119875 lt0001) (b) Fecal calprotectin an indicator of colonic inflammationalso increased less following carrageenan in the Bcl10 null micethan in the wild type or heterozygous mice to 1151 plusmn 80 120583gkgversus 1827 plusmn 143 120583gkg in the wild type mice (119875 lt 0001) CGNcarrageenan KC keratinocyte chemokine WT wild type hetheterozygous

for RelB activation by the non-canonical pathway andphospho-Tak which is involved in the canonical pathwaywere determined Western blots of colonic epithelial tis-sue from untreated C57BL6 mice and carrageenan-treatedBcl10 wild type and null mice indicated no increase inphospho(Thr559)-NIK in the Bcl10 null mice in contrastto the wild type mice (Figure 6(a)) Densitometry confirmsthe visual impression of the greater increase in phospho-NIK in the Bcl10 wild type than in the Bcl10 null mice(119875 = 001) (Figure 6(b)) The carrageenan-induced increasein phospho(Thr184)-Tak in the mouse colonic epitheliumwas significantly greater in the Bcl10 wild type mice than inthe Bcl10 null mice (Figure 6(c)) compared to the untreatedcontrol (119875 lt 005 by densitometry) (Figure 6(d)) Absenceof phospho(Thr559)-NIK in the Bcl10 null mice is consistentwith the requirement for Bcl10 in the non-canonical RelBpathway ofNF-120581B activation by carrageenan In the Bcl10 nullmice the increase in phospho(Thr184)-Tak was less than inthe Bcl10 wild type mice consistent with the presence of bothBcl10-dependent and Bcl10-independent pathways by whichcarrageenan increases phospho(Thr184) Tak1

0

50

100

150

200

250

WT

cont

rol (

)

WT WT + CGNNull + CGN Het + CGN

TNF-120572 IL-6 IFN-120574 IL-1120573 IL-10 MCP-1 IL-12 IL-23

lowastlowastlowast lowastlowastlowast

Figure 3 Serum IL-6 and MCP-1 increase post-carrageenan expo-sure and increases are similar in Bcl10 WT heterozygous andnull mice Carrageenan exposure produced significant increases inserum levels of IL-6 and MCP-1 in the WT heterozygous and nullmice (119875 lt 0001) compared to the unexposed controls WT heter-ozygotes and null mice serum levels of MCP-1 and IL-6 bothincreased sim85 Serum levels of TNF-120572 IFN120574 IL-1120573 IL-10 IL-12and IL-23 did not change

37 Effects of Carrageenan in the Germ-Free Environmentand in IL-10 Null Mice C57BL6 mice were exposedto carrageenan in the germ-free environment Thegerm-free environment did not affect the responses ofKC (Figure 7(a)) fecal calprotectin (Figure 7(b)) Bcl10(Figure 7(c)) RelA (Figure 7(d)) RelB (Figure 7(e)) orphospho-Bcl10 (Figure 7(f)) These findings enabled thecomparison between the C57BL6 mice and the IL-10 micewhich require the germ-free environment due to theircompromised immunity

In the IL-10 null mice in the germ-free environmentthe baseline values without carrageenan of KC (119875 lt 005)(Figure 7(a)) fecal calprotectin (119875 lt 005) (Figure 7(b))Bcl10 (119875 lt 0001) (Figure 7(c)) and RelA (119875 lt 005)(Figure 7(d)) were all significantly greater than in the C57BL6 mice Carrageenan exposure produced larger increases inthese measures in the IL-10 null mice than in the C57BL6 mice (119875 lt 0001) In contrast the baseline values andcarrageenan-stimulated increases in RelB (Figure 7(e)) andphospho-Bcl10 (Figure 7(f)) were similar in the IL-10-deficient mice in the C57BL6 mice and in the germ-freeenvironment indicating that IL-10 deficiency did not affectactivation of the non-canonical NF-120581B pathway

38 Inhibition of the Carrageenan-Activated Canonical butNot the Non-Canonical NF-120581B Pathway by Exogenous IL-10 When human colonic NCM460 cells were exposed tocarrageenan and exogenous IL-10 the RelA (Figure 8(a))and phospho-I120581B120572 (Figure 8(b)) responses to carrageenandeclined but the RelB (Figure 8(c)) and phospho-Bcl10(Figure 8(d)) responses were unaffected These results areconsistent with the in vivo findings above and indicate thatIL-10 acts only on the canonical pathway of NF-120581B activationthat involves phospho-I120581B120572 and RelA Phospho-Bcl10 andRelB which participate in the non-canonical pathway are not

Mediators of Inflammation 7

0

50

RelA

( co

ntro

l)

100

150

200

250

No CGN WT + CGN Het + CGN Null + CGN

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

RelB

( co

ntro

l)

ND

lowastlowastlowast lowastlowastlowast

No CGN WT + CGN Het + CGN Null + CGN

(b)

Figure 4 Carrageenan-induced increase in RelA is reduced andis absent in RelB in Bcl10 null mice (a) Nuclear RelA measuredby oligonucleotide assay increased by sim89 in the wild type andheterozygous mice following carrageenan but only by about 40 inthe Bcl10 null mice (119875 lt 0001) (b) In contrast the increase in RelBwas completely inhibited in the Bcl10 null mice but increased bysim75 in the heterozygous and wild typemice following carrageenan(119875 lt 0001) CGN carrageenan WT wild-type Het heterozygousND no difference

inhibited since the anti-inflammatory cytokine IL-10 inhibitsthe activation of the phospho-I120581B120572-mediated pathway

4 Discussion

BCL10 has emerged as a key molecule in the activation ofthe inflammatory cascade in epithelial cells as well as inimmune cells [3 24ndash26] Recent investigations in colonicepithelial cells demonstrated the requirement for Bcl10 Ser138phosphorylation for the activation of the non-canonicalpathway of NF-120581B activation [9] The identification of anNF-120581B-Bcl10 loop by which an NF-120581B binding site in theBcl10 promoter can lead to transcriptional effects on Bcl10expression presents a mechanism by which carrageenan andother Bcl10 stimulators can lead to prolonged stimulation ofinflammatory responses [10]

The studies in this report demonstrate a profound impactof Bcl10-mediated inflammation on the mammalian intes-tine In Bcl10 null mice histopathology following exposure to

050

100150200250300350400450

No CGN WT + CGN Het + CGN Null + CGN

Phos

pho(

Ser1

38)-

Bcl1

0(

no

CGN

cont

rol)

lowastlowastlowast lowastlowastlowast

(a)

0

25

50

75

100

125

150

175

200

225

No CGN WT + CGN Het + CGN Null + CGN

Phos

pho-

I120581B

( n

o CG

N co

ntro

l)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Figure 5 Phosphorylation of Bcl10 is absent and of I120581B120572 is reducedin Bcl10 nullmice following carrageenan (a) Phospho(Ser138)-Bcl10was increased to sim400 of the no carrageenan baseline when theBcl10 wild type and heterozygousmice were exposed to carrageenan(119875 lt 0001) but not in the Bcl10 null mice (b) The increase inphospho(Ser32)-I120581B120572 is less in the Bcl10 null mice compared to thewild type and heterozygous mice (increase of sim37 versus sim91)but the increase from the baseline no carrageenan value is alsosignificant (119875 lt 0001) CGN carrageenan WT wild type Hetheterozygous

carrageenan showed less intense inflammatory response thanin the wild type mice Decline in activation of both canonicaland non-canonical NF-120581B activation occurred when Bcl10was absent as indicated by reductions in RelA and RelBIn contrast studies in IL-10-deficient mice treated with car-rageenan demonstrated effects on the canonical but not thenon-canonical pathway ofNF-120581B activation Complementarystudies in NCM460 cells with exogenous IL-10 followingexposure to carrageenan showed effects of IL-10 on RelAbut not on RelB activation IL-10 gene-knockout mice areknown to develop a colitis that resembles IBD [37] and IL-10gene and IL-10 receptor mutations have been associated withclinical IBD [38] However IL-10 therapy has not becomeas useful clinically as anticipated [39] By the demonstrationof Bcl10-mediated non-canonical inflammation in the IL-10 deficient mouse model the study findings suggest thatspecific therapeutic targeting of the non-canonical pathway

8 Mediators of Inflammation

Phospho-NIK

Control Bcl10 nullBCL10 WTNo CGN + CGN + CGN

120573-Actin

(a)

0

05

1

15

2

25

3

35

4

Control WT + CGN Null + CGN

Den

sitom

etry

Phos

pho-

NIK

(fol

d-ch

ange

)

lowastlowast

(b)

Control Bcl10 null + CGN + CGN

Phospho(Thr184)-Tak1

Tubulin

BCL10 WTNo CGN

(c)

0123456789

101112

Control WT + CGN Null + CGN

Den

sitom

etry

Phos

pho-

Tak1

(fol

d-ch

ange

)

lowast

(d)

Figure 6 Different responses of phospho(Thr559)-NIK and of phospho(Thr184)-Tak1 following carrageenan in Bcl10 null mice (a) Theimmunoblot shows that phospho-NIK does not increase following exposure to carrageenan in the Bcl10 null mice in contrast to the findingin the wild type mice (b) Densitometry confirms the increase in phospho-NIK in the Bcl10 WT mice treated with carrageenan compared tothe Bcl10 null mice (119875 = 001 unpaired t-test two-tailed) (c) In contrast phospho-Tak1(Thr184) was somewhat increased in the Bcl10 nullmicersquos intestinal tissue although less than in the wild type mouse tissue (d) Densitometry confirms the visual impression that the phospho-Tak1 is significantly reduced in the Bcl10 null versus Bcl10 WTmouse following CGN exposure (119875 lt 005 unpaired t-test two-tailed) CGNcarrageenan WT wild type NIK NF-120581B inducing kinase Tak TGF-120573 activated kinase

of NF-120581B activation as well as targeting of the canonicalpathway by IL-10 may lead to improved clinical outcomesRecent findings that oral Lactobacillus significantly reducedcarrageenan-induced paw edema and downregulated TNF-120572 and upregulated IL-10 [16] are suggestive that combinedinterventions which impact upon both canonical and non-canonical NF-120581B signaling pathways may lead to improvedresponses

The study findings include data that indicate that car-ageenan-induced inflammation was not suppressed in thegerm-free environment This result suggests that the fecal

microflora as constituted in the C57BL6 mice under stan-dard conditions either do not modify carrageenan or do notmodify carrageenan in any way that enhances inflammationThe response to Lactobacillus noted above suggests that thespecificmicroflora can affect the response to carrageenan butthe effect appears to be indirect through changes in cytokinesor circulating immune cells

Carrageenan has been widely used in the laboratory fordecades to stimulate inflammation in animal models andin cell-based experiments [5 6] The animal models haveincluded injection of carrageenan directly into rat hind paw

Mediators of Inflammation 9

KC (n

gL)

0

50

100

150

200

250

300

350

400

450

No CGN CGN

ND

NDlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

300

350

400

No CGN CGN

Feca

l cal

prot

ectin

(

C57

BL6

no

CGN

)

ND

ND

lowastlowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

400

No CGN CGN

Bcl1

0 (

C57

BL6

no

CGN

)

ND

ND

lowastlowastlowast

lowastlowastlowast

(c)

0

50

100

150

200

250

300

350

No CGN CGN

RelA

(

C57

BL6

no

CGN

)ND

ND

lowast

lowastlowastlowast

(d)

0

40

80

120

160

200

240

No CGN CGN

RelB

( in

C57

BL6

cont

rol

no C

GN

)

ND

ND

C57BL6Germ-freeIL-10 null

(e)

05

1015202530354045

No CGN CGN

Phos

pho-

Bcl1

0 (

cont

rol)

ND

ND

C57BL6Germ-freeIL-10 null

(f)

Figure 7 Effects of carrageenan exposure in IL-10-deficient mice compared to C57BL6 control in germ-free environment and in standardhousing (a) KC was not different in the C57BL6 germ-free mice versus mice with standard housing at baseline or following carrageenanexposure In the IL-10-deficient mice KC was greater at baseline (119875 lt 005) and following carrageenan (119875 lt 0001) than in the control miceand the increase in KC was greater in the IL-10-deficient mice than in the controls (sim271 ngL versus sim87 ngL) (b) Fecal calprotectin wasnot different in the germ-free mice versus the standard housing control mice with or without carrageenan exposure Fecal calprotectin wasgreater in the IL-10-deficient mice at baseline (119875 lt 005) and following CGN (119875 lt 0001) (c) Bcl10 was similar in the C57BL6 mice in thegerm-free environment as in standard housing Baseline Bcl10 was significantly greater in the IL-10 null mice than in the controls (119875 lt 0001)and increased more in the IL-10-deficient mice (sim229 versus sim144) following carrageenan (119875 lt 0001) than in the control mice (d) Thegerm-free environment did not affect the RelA results in the C57BL6mice Baseline RelA was significantly greater in the IL-10 null mice thanin the control C57Bl6mice (119875 lt 005) and increasedmore following carrageenan in the IL-10-deficient mice than in the controls (119875 lt 0001)(e) Carrageenan-induced increase in RelB was unaffected by IL-10 deficiency (f) Carrageenan-induced increase in phospho(Ser138)-Bcl10was unaffected by IL-10 deficiency or the germ-free environment CGN carrageenan ND no difference

10 Mediators of Inflammation

0

25

50

75

100

125

150

175

200

225

250

Control Control si Bcl10 si

RelA

( co

ntro

l)lowastlowastlowast lowastlowastlowast

lowastlowastlowast

(a)

Control Control si Bcl10 si0

25

50

75

100

125

150

175

200

225

( co

ntro

l)Ph

osph

o(Se

r32)

-I120581B

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

175

RelB

( co

ntro

l)

NDND

Control Control si Bcl10 si

ND

UntreatedIL-10 + CGNIL-10 (20120583gL)

CGN (1mgL)

(c)

0

50

100

150

200

250

300

350

400

450

500

Phos

pho(

Ser1

38)-

Bcl1

0(

unt

reat

ed co

ntro

l)

NDND

ND

Control Control si Bcl10 si

UntreatedIL-10 + CGNIL-10 (20120583gL)

CGN (1mgL)

(d)

Figure 8 InNCM460 cells exogenous IL-10 inhibits the canonical but not the non-canonical NF-120581B pathway that is activated by carrageenanand mediated by Bcl10 (a) Increase in RelA following exposure to carrageenan was partially inhibited by exogenous IL-10 in the NCM460cells When Bcl10 was silenced by siRNA the increase in RelA was reduced and completely inhibited by IL-10 (20120583gL times 24 h) (b) Theincrease in phospho(Ser32)-I120581B120572 was partially inhibited by exogenous IL-10 in the control and control siRNA cells The increase was lesswhen Bcl10 was silenced and was almost completely inhibited by exogenous IL-10 (c) In contrast to the above findings the increase in RelBwas unaffected by exogenous IL-10 (d) Phospho(Ser138)-Bcl10 increased to over four times the baseline in the control and control siRNAcells and the increases were not inhibited by exogenous IL-10 ND no difference IL-10 interleukin-10 si small interfering siRNA CGNcarrageenan

pleura peritoneum bursa subcutaneous air bleb or largejoints and oral ingestion to induce colonic inflammationThe signaling mechanisms by which carrageenan inducesinflammation have been investigated [3 4 7 8] and themediators by which carrageenan exposure can lead to sys-temic effects have been reported [3 4 7 8 40ndash42]The tissueand systemic effects of carrageenan are also mediated byeffects on immune cells including neutrophils macrophages

and lymphocytes Mediation of carrageenan-induced inflam-mation by an inflammatory cell infiltrate was recognizedin early experiments in which macrophages in the laminapropria spleen and liver were observed to contain fibrillarinclusions andor havemetachromatic staining following oralexposure to carrageenan [43 44] Extracolonic manifesta-tions of carrageenan exposure leading to inflammation andimpaired insulin signaling in the mouse liver were associated

Mediators of Inflammation 11

with glucose intolerance and insulin resistance in recentexperiments [12]

In B and T cells and in epithelial cells Bcl10 is involvedin NF-120581B signal transduction involving TLR4 and TAK1[3 4 24ndash26 45ndash47] The assembly of the lipid raft-asso-ciated CARMA-Bcl10-MALT1 (CBM) signalosome involvesCARMA1 in the immune cells and CARMA3 in nonimmunecells [23 48 49] The CBM complex promotes the activationand ubiquitination of IKK120574 (NEMO) the regulatory compo-nent of the IKK signalosome leading to RelA nuclear translo-cation Additional experiments that better characterize thecomponents of the inflammatory infiltrate in the intestineby FACS analysis and that study chimeric mice in which theeffects of Bcl10 deficiency in the colonic epithelial cells versusthe bone marrow can be differentiated will clarify specificsubsets that mediate the response to carrageenan Immuneeffects of carrageenan have been recognized for decades[43] and increased attention to the role of Bcl10 providesan opportunity to further differentiate the specific signalingmediators and mechanisms involved in innate and adaptiveimmunity

In the experiments in this report the Bcl10 transgenicmice weighing about 30 grams received carrageenanin their water supply at a concentration of 10 120583gmLand drank about 5 mLday for a dose of 50120583g30 gday(=sim17 120583ggday = sim17mgkgday) The reported averagedaily intake of carrageenan in the typical Western dietis reported to be 250mgday [50] corresponding to250mg60 kgday (=sim42mgkgday) considerably morethan the amount ingested by themice Greater understandingof the mediators by which carrageenan exerts inflammatoryeffects in vivo and consideration of interventions that mightinhibit carrageenan-induced inflammation are relevant tohuman diseases such as diabetes and atherosclerosis inwhich inflammation is involved and may yield new insightsinto the role of inflammation on initiation and progressionof human diseases

Conflict of Interests

The authors have no conflict of interests to report

Acknowledgments

Funding was by Merit Review from the Veteranrsquos Adminis-tration to J K Tobacman and DDRCC (DK42086) and UH3DK083993 to E Chang for the support of the gnotobioticfacility The authors thank Dr Susan Ball-Kell for her eval-uation of the gastrointestinal pathology in the mice

References

[1] B A Macher and U Galili ldquoThe Gal12057213Gal12057314GlcNAc-R (120572-Gal) epitope a carbohydrate of unique evolution and clinicalrelevancerdquo Biochimica et Biophysica Acta vol 1780 no 2 pp75ndash88 2008

[2] W R Blakemore and A R Harpell ldquoCarrageenanrdquo in Food Sta-bilisersThickeners and Gelling Agents Ullmanrsquos Encyclopedia ofIndustrial Chemistry chapter 5 pp 73ndash94 John Wiley amp SonsNew York NY USA 2009

[3] S Bhattacharyya A Borthakur P K Dudeja and J K Tobac-man ldquoCarrageenan induces interleukin-8 production throughdistinct Bcl10 pathway in normal human colonic epithelialcellsrdquo The American Journal of Physiology vol 292 no 3 ppG829ndashG838 2007

[4] S Bhattacharyya R Gill L C Mei et al ldquoToll-like receptor4 mediates induction of the Bcl10-NF120581B-interleukin-8 inflam-matory pathway by carrageenan in human intestinal epithelialcellsrdquo The Journal of Biological Chemistry vol 283 no 16 pp10550ndash10558 2008

[5] J K Tobacman ldquoReview of harmful gastrointestinal effects ofcarrageenan in animal experimentsrdquo Environmental Health Per-spectives vol 109 no 10 pp 983ndash994 2001

[6] IARC Working Group on the Evaluation of the CarcinogenicRisk of Chemicals to Humans ldquoCarrageenanrdquo IARC Mono-graphs on the Evaluation of Carcinogenic Risks to Humans vol31 pp 79ndash94 1983

[7] S Bhattacharyya P K Dudeja and J K Tobacman ldquoCar-rageenan-induced NF120581B activation depends on distinct path-ways mediated by reactive oxygen species and Hsp27 or byBcl10rdquo Biochimica et Biophysica Acta vol 1780 no 7-8 pp 973ndash982 2008

[8] S Bhattacharyya A Borthakur S Tyagi et al ldquoB-cellCLLlymphoma 10 (BCL10) is required for NF-120581B productionby both canonical and noncanonical pathways and for NF-120581B-inducing kinase (NIK) phosphorylationrdquo The Journal ofBiological Chemistry vol 285 no 1 pp 522ndash530 2010

[9] S Bhattacharyya A Borthakur A N Anbazhagan S Katyal PK Dudeja and J K Tobacman ldquoSpecific effects of BCL10 serinemutations on phosphorylations in canonical and noncanonicalpathways of NF-120581B activation following carrageenanrdquo TheAmerican Journal of Physiology vol 301 no 3 pp G475ndashG4862011

[10] A Borthakur S Bhattacharyya ANAnbazhagan A Kumar PK Dudeja and J K Tobacman ldquoProlongation of carrageenan-induced inflammation in human colonic epithelial cells byactivation of an NF120581B-BCL10 looprdquo Biochimica et BiophysicaActa vol 1822 no 8 pp 1300ndash1307 2012

[11] S Bhattacharyya H Liu Z Zhang et al ldquoCarrageenan-inducedinnate immune response is modified by enzymes that hydrolyzedistinct galactosidic bondsrdquo Journal of Nutritional Biochemistryvol 21 no 10 pp 906ndash913 2010

[12] S Bhattacharyya I O-Sullivan S Katyal T Unterman and JK Tobacman ldquoExposure to common food additive carrageenanleads to glucose intolerance insulin resistance and inhibitionof insulin signaling in HepG2 cells and C57BL6J micerdquoDiabetologia vol 55 no 1 pp 194ndash208 2012

[13] H Shi M V Kokoeva K Inouye I Tzameli H Yin and JS Flier ldquoTLR4 links innate immunity and fatty acid-inducedinsulin resistancerdquo Journal of Clinical Investigation vol 116 no11 pp 3015ndash3025 2006

[14] M J Song K H Kim J M Yoon and J B Kim ldquoActivationof toll-like receptor 4 is associated with insulin resistance inadipocytesrdquo Biochemical and Biophysical Research Communica-tions vol 346 no 3 pp 739ndash745 2006

[15] M K Mohammad M Morran B Slotterbeck et al ldquoDys-regulated toll-like receptor expression and signaling in bonemarrow-derived macrophages at the onset of diabetes in thenon-obese diabetic mouserdquo International Immunology vol 18no 7 pp 1101ndash1113 2006

12 Mediators of Inflammation

[16] S Amdekar P Roy V Singh A Kumar R Singh and P SharmaldquoAnti-inflammatory activity of lactobacillus on carrageenan-induced paw edema in male wistar ratsrdquo International Journalof Inflammation vol 2012 Article ID 752015 6 pages 2012

[17] F Ma C Chen L Zheng C Zhou K Cai and Z Han ldquoEffectof high pressure processing on the gel properties of salt-solublemeat protein contain CaCl

2and 120581-carrageenanrdquo Meat Science

vol 95 no 1 pp 22ndash26 2013[18] E Popa R Reis and M Gomes ldquoChondroigenic phenotype

of different cells encapsulated in 120581-carrageenan hydrogels forcartilage regeneration strategiesrdquo Biotechnology and AppliedBiochemistry vol 59 no 2 pp 132ndash141 2012

[19] R Eccles C Meier M Jawad R Weinmullner A Grassauerand E Prieschl-Grassauer ldquoEfficacy and safety of an antiviralIota-Carrageenan nasal spray a randomized double-blindplacebo-controlled exploratory study in volunteers with earlysymptoms of the common coldrdquo Respiratory Research vol 11article 108 2010

[20] J R Simkhada S S Cho P Mander Y H Choi and J C YooldquoPurification biochemical properties and antithrombotic effectof a novel Streptomyces enzyme on carrageenan-induced micetail thrombosis modelrdquoThrombosis Research vol 129 no 2 pp176ndash182 2012

[21] L Xue S WMorris C Orihuela et al ldquoDefective developmen-tal and function of Bcl10-deficient follicular marginal zone andB1 B cellsrdquo Nature Immunology vol 4 no 9 pp 857ndash865 2003

[22] Q Zhang R Siebert M Yan et al ldquoInactivating mutationsand overexpression of BCL 10 a caspase recruitment domain-containing gene in MALT lymphoma with t(114)(p22q32)rdquoNature Genetics vol 22 no 1 pp 63ndash68 1999

[23] Z Li H Wang L Xue et al ldquoE120583-BCL10 mice exhibit consti-tutive activation of both canonical and noncanonical NF-120581Bpathways generating marginal zone (MZ) B-cell expansion asa precursor to splenic MZ lymphomardquo Blood vol 114 no 19pp 4158ndash4168 2009

[24] D Wang Y You P C Lin et al ldquoBcl10 plays a critical rolein NF-120581B activation induced by G protein-couple receptorsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 1 pp 145ndash150 2007

[25] S Klemm S Zimmermann C Peschel T W Mak and JRuland ldquoBcl10 and Malt1 control lysophosphatidic acid-in-duced NF-120581B activation and cytokine productionrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 1 pp 134ndash138 2007

[26] L M McAllister-Lucas J Ruland K Siu et al ldquoCARMA3Bcl10MALT1-dependent NF-120581B activation mediates angiot-ensin II-responsive inflammatory signaling in nonimmunecellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 104 no 1 pp 139ndash144 2007

[27] M Van Beek K I Oravecz-Wilson P C Delekta et al ldquoBcl10links saturated fat overnutrition with hepatocellular NF-120581Bactivation and insulin resistancerdquo Cell Reports vol 1 no 5 pp444ndash452 2012

[28] S Marion J Mazzolini F Herit et al ldquoThe NF-120581B signalingprotein Bcl10 regulates actin dynamics by controlling AP1 andOCRL-bearing vesiclesrdquo Developmental Cell vol 23 no 5 pp954ndash967 2012

[29] S Rosebeck A O Rehman P C Lucas and L M McAllister-Lucas ldquoFromMALT lymphoma to the CBM signalosome threedecades of discoveryrdquo Cell Cycle vol 10 no 15 pp 2485ndash24962011

[30] L M McAllister-Lucas X Jin S Gu et al ldquoThe CARMA3-Bcl10-MALT1 signalosome promotes angiotensin II-dependentvascular inflammation and atherogenesisrdquo The Journal of Bio-logical Chemistry vol 285 no 34 pp 25880ndash25884 2010

[31] R Stilo E Varricchio D Liguoro A Leonardi and P VitoldquoA20 is a negative regulator of BCL10- and CARMA3-mediatedactivation of NF-120581Brdquo Journal of Cell Science vol 121 no 8 pp1165ndash1171 2008

[32] S Devkota Y Wang M W Musch et al ldquoDietary fat inducedtaurocholic acid promotes pathobiont expansion and colitis inIL10-- micerdquo Nature vol 487 no 7405 pp 104ndash108 2012

[33] M PMoyer L AManzano R LMerriman J S Stauffer and LR Tanzer ldquoNCM460 a normal human colonmucosal epithelialcell linerdquo In vitro Cellular ampDevelopmental Biology Animal vol32 no 6 pp 315ndash317 1996

[34] S Bhattacharyya N Pant P K Dudeja and J K TobacmanldquoDevelopment evaluation and application of a highly sensitivemicrotiter plate ELISA for human Bcl10 proteinrdquo Journal ofImmunoassay and Immunochemistry vol 28 no 3 pp 173ndash1882007

[35] F Costa M G Mumolo M Bellini et al ldquoRole of faecalcalprotectin as non-invasivemarker of intestinal inflammationrdquoDigestive and Liver Disease vol 35 no 9 pp 642ndash647 2003

[36] C K Ameho A A Adjei E K Harrison et al ldquoProphylacticeffect of dietary glutamine supplementation on interleukin 8and tumour necrosis factor 120572 production in trinitrobenzenesulphonic acid induced colitisrdquo Gut vol 41 no 4 pp 487ndash4931997

[37] R Kuhn J Lohler D Rennick K Rajewsky and W MullerldquoInterleukin-10-deficient mice develop chronic enterocolitisrdquoCell vol 75 no 2 pp 263ndash274 1993

[38] E O Glocker D Kotlarz C Klein N Shah and B GrimbacherldquoIL-10 and IL-10 receptor defects in humansrdquoAnnals of the NewYork Academy of Sciences vol 1246 pp 102ndash107 2011

[39] M C Li and S H He ldquoIL-10 and its related cytokines fortreatment of inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 10 no 5 pp 620ndash625 2004

[40] S Cuzzocrea EMazzon LDugo et al ldquoAbsence of endogenousinterleukin-10 enhances the evolution of acute lung injuryrdquoEuropean Cytokine Network vol 13 no 3 pp 285ndash297 2002

[41] F DrsquoAcquisto A Ianaro A Ialenti T Iuvone V Colantuoniand R Carnuccio ldquoActivation of nuclear transcription factor120581B in rat carrageenin-induced pleurisyrdquo European Journal ofPharmacology vol 369 no 2 pp 233ndash236 1999

[42] L Ellis V Gilston C C Y Soo C J Morris B L Kidd and PGWinyard ldquoActivation of the transcription factorNF-120581B in therat air pouch model of inflammationrdquo Annals of the RheumaticDiseases vol 59 no 4 pp 303ndash307 2000

[43] A W Thomson and E F Fowler ldquoCarrageenan a review of itseffects on the immune systemrdquo Agents and Actions vol 11 no3 pp 265ndash273 1981

[44] R Mankes and R Abraham ldquoLysosomal dysfunction incolonic submucosal macrophages of rhesus monkeys causedby degraded iota carrageenanrdquo Proceedings of the Society forExperimental Biology and Medicine vol 150 no 1 pp 166ndash1701975

[45] H Zhou I Wertz K OrsquoRourke et al ldquoBcl10 activates the NF-120581B pathway through ubiquitination of NEMOrdquoNature vol 427no 6970 pp 167ndash171 2004

Mediators of Inflammation 13

[46] W Dong Y Liu J Peng et al ldquoThe IRAK-1-BCL10-MALT1-TRAF6-TAK1 cascade mediates signaling to NF-120581B from toll-like receptor 4rdquoThe Journal of Biological Chemistry vol 281 no36 pp 26029ndash26040 2006

[47] G Takaesu R M Surabhi K J Park J Ninomiya-Tsuji KMatsumoto and R B Gaynor ldquoTAK1 is critical for I120581B kinase-mediated activation of theNF-120581Bpathwayrdquo Journal ofMolecularBiology vol 326 no 1 pp 105ndash115 2003

[48] O Gaide F Martinon O Micheau D Bonnet M Thome andJ Tschopp ldquoCarma1 a CARD-containing binding partner ofBcl10 induces Bcl10 phosphorylation and NF-120581B activationrdquoFEBS Letters vol 496 no 2-3 pp 121ndash127 2001

[49] A Borthakur S Bhattacharyya W A Alrefai J K TobacmanK Ramaswamy and P K Dudeja ldquoPlatelet-activating factor-induced NF-120581B activation and IL-8 production in intestinalepithelial cells are Bcl10-dependentrdquo Inflammatory Bowel Dis-eases vol 16 no 4 pp 593ndash603 2010

[50] Encyclopaeligdia Britannica ldquoalgaerdquo Encyclopaeligdia BritannicaEncyclopaeligdia Britannica Online 2010 httpsearchebcomebarticle-31714

Submit your manuscripts athttpwwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom