Effect of the Leptin Receptor Q223R Polymorphism on the HostTranscriptome following Infection with Entamoeba histolytica

Nicole M. Mackey-Lawrence,a Xiaoti Guo,a,b Daniel E. Sturdevant,c Kimmo Virtaneva,c Matthew M. Hernandez,c Eric Houpt,a

Alan Sher,d Stephen F. Porcella,c William A. Petri, Jr.a

Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia School of Medicine, Charlottesville, Virginia, USAa; North ShoreLong Island Health System, Manhasset, New York, USAb; Rocky Mountain Laboratory Research Technologies Section, Genomics Unit, National Institute of Allergy andInfectious Diseases, NIH, Hamilton, Montana, USAc; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USAd

Resistance to amebiasis is associated with a polymorphism in the leptin receptor. Previous studies demonstrated that humans with theancestral Q223 leptin receptor allele were nearly four times less likely to be infected with Entamoeba histolytica than those carrying themutant R223 allele. We hypothesized that the Q223 allele protected against E. histolytica via STAT3-mediated transcription of genesrequired for mucosal immunity. To test this, mice containing the humanized LEPR Q or R allele at codon 223 were intracecally infectedwith E. histolytica. Susceptibility to amebiasis was assessed, and cecal tissues were analyzed for changes in gene expression. By 72 hpostchallenge, all Q223 mice had cleared E. histolytica, whereas 39% of 223R mice were infected. Thirty-seven genes were differentiallyexpressed in response to infection at 72 h, including proinflammatory genes (CXCL2, S100A8/9, PLA2G7, ITBG2, and MMP9) andfunctions pertaining to the movement and activity of immune cells. A comparison at 12 h postchallenge of infected Q223 versus R223mice identified a subset of differentially expressed genes, many of which were closely linked to leptin signaling. Further analyses indi-cated that the Q223 gene expression pattern was consistent with a suppressed apoptotic response to infection, while 223R showed in-creased cellular proliferation and recruitment. These studies are the first to illuminate the downstream effects of leptin receptor poly-morphisms on intestinal infection by E. histolytica. As such, they are important for the insight that they provide into this previouslyuncharacterized mechanism of mucosal immunity.

The association of leptin signaling with infectious diseases haslong been suspected; however, only recently was the signifi-

cant association between leptin and human disease described (1–4). It was determined that a common genetic mutation of theleptin receptor (LEPR), Q223R, was associated with an approxi-mately 4-fold increase in likelihood of infection with Entamoebahistolytica in a Bangladeshi cohort of children (4). The murinemodel of amebiasis recapitulated the human findings, demon-strating that 223R (homozygous for arginine) mice were signifi-cantly more susceptible to infection with E. histolytica than Q223(homozygous for glutamine) mice. In addition, both ob/ob (lep-tin�/�) and db/db (LEPR�/�) mice had increased rates of E. his-tolytica infection and worse tissue destruction and mortality. Fi-nally, the use of tissue-specific LEPR knockouts demonstratedthat the site of action for LEPR was located within the intestinalepithelial cell (IEC) population (5).

LEPR belongs to the gp130 family of cytokine receptors. Uponleptin binding to the homodimeric LEPR, Janus kinase 2 (JAK2) isactivated, leading to the phosphorylation of tyrosines 985, 1077,and 1138 on LEPR, which in turn are recognized by Src homology2 domain-containing tyrosine phosphatase (SHP2) and suppres-sor of cytokine signaling 3 (SOCS3), STAT5a/b, and STAT3 (re-spectively) (6–8). Murine studies demonstrated that signaling atboth Tyr985 and Tyr1138 was essential for protection against E.histolytica, implicating SHP2 and/or SOCS3 (Tyr985) and STAT3(Tyr1138) as important downstream effectors of LEPR in amebi-asis (5). In support of the role of STAT3 downstream of LEPR, itwas previously found that HEK cell lines transfected with LEPRunable to signal at Tyr1138 had decreased resistance to amebiccytotoxicity (9). The effects downstream of the Q223R polymor-phism are currently unknown; however, HEK cells transfectedwith the 223R receptor exhibited decreased STAT3 activation in

both the presence and absence of leptin, indicating that the Q223Rpolymorphism may attenuate LEPR signaling through STAT3.

LEPR signaling via STAT3 can have multiple effects, includinginduction of cytokines, repression of apoptosis, and IEC prolifer-ation, all of which conceivably could be protective (3, 9–14). Thus,we hypothesized that the 223R mutation in LEPR modulatesSTAT3 signaling in IECs leading to increased susceptibility toameba-induced apoptosis and attenuated restitution of the co-lonic epithelium. To assess the differences in the transcriptionalresponse to E. histolytica, here we infected mice harboring eitherthe Q223 or 223R LEPR allele (Q223infected and 223Rinfected mice)and measured transcriptional responses at 12 and 72 h postchal-lenge.

MATERIALS AND METHODSAnimals. Mice containing the humanized LEPR allele at codon 223 were agift from Rudy Leibel (15). Briefly, homologous recombination in129P3/J ES embryonic stem cells was used to replace mouse leptin recep-tor exon 4 with an exon that was identical except for the Q223R substitu-tion. An embryonic stem cell clone was injected into C57BL/6J blastocysts,and the founders were crossed to 129 mice and F1 progeny interrogated

Received 7 December 2012 Returned for modification 7 January 2013Accepted 12 February 2013

Published ahead of print 19 February 2013

Editor: J. F. Urban, Jr.

Address correspondence to William A. Petri, Jr., wap3g@virginia.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01383-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01383-12

1460 iai.asm.org Infection and Immunity p. 1460–1470 May 2013 Volume 81 Number 5

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

for the targeted LEPR allele. The 129P3/J mice were maintained byheterozygous breeding. Only those homozygous for Q223 and 223R wereevaluated. Animals were maintained under specific-pathogen-free condi-tions at the University of Virginia. All experiments were done under pro-tocols approved by the Institutional Animal Care and Use Committee.

E. histolytica culture and intracecal injection. Animal-passagedHM1:IMSS E. histolytica trophozoites were cultured from cecal contentsof infected mice in complete trypsin-yeast-iron (TYI-33) medium supple-mented with Diamond Vitamin (JRH Biosciences), 100 U/ml of penicillinand streptomycin, and bovine serum (Sigma-Aldrich). Prior to injection,trophozoites were grown to log phase, and 2 � 106 parasites were sus-pended in 150 �l and injected intracecally (16). To reduce extraneousvariation, Q223 and 223R mice, 5 to 12 weeks of age, were challengedusing a randomized block design (see Table S1 in the supplemental ma-terial). Q223 and 223R mice were sacrificed at 12 and 72 h followingchallenge or when moribund, and ceca were harvested and contents eval-uated for infection using ex vivo culture of E. histolytica trophozoites fromharvested cecal contents.

RNA and DNA extraction. The sample harvest order was maintainedthroughout the study during both nucleic acid extraction and target prep-aration. Tissues were homogenized in 600 �l of RLT lysis buffer (Qiagen)and 0.145 M �-mercaptoethanol in a FastPrep Green lysing matrix vial for20 s at setting 6 in a FastPrep-24 instrument (MP Biomedicals). The lysatewas passed through a Qiashredder column (Qiagen) at 21,000 � g for 2min to homogenize genomic DNA in order to increase the final RNAyield. RNA was extracted from a 10-mg aliquot of homogenized cecaltissue. Both RNA and genomic DNA were extracted using the AllPrepDNA/RNA 96 kit (Qiagen) following the manufacturer’s recommenda-tions, except that RNA samples received an additional on-column DNaseI treatment during extraction. Cecal RNAs were extracted to study mousemRNA expression profiles during infection. Extracted DNA was used toestimate the E. histolytica copy number in the infected tissues. RNA andDNA concentrations were measured by spectrophotometry at 260 and280 nm in a 384-well format (Molecular Devices). RNA purity (A260/A280

ratio) and quality were verified on an Agilent 2100 Bioanalyzer using thePico analysis kit (Agilent Technologies).

cDNA amplification, synthesis, and labeling. Five nanograms of eachRNA sample was concentrated to 5 �l and processed according to themanufacturer’s instructions using the WT-Ovation Pico system (version1) RNA amplification system (Nugen Inc.). Each RNA sample was alsospiked with four Bacillus subtilis poly(A)-tailed mRNAs (dapB, lys, thr,and trp) to monitor cDNA synthesis and amplification during target prep-aration. The resulting amplified single-stranded cDNAs were purifiedwith the QIAquick 96-well protocol (Qiagen) with a modified centrifuga-tion protocol (17). Single-stranded single-primer isothermal amplifica-tion (SPIA) cDNA quality was determined by running an aliquot in theAgilent 2100 Bioanalyzer.

The standard cDNA (ST-cDNA) generation protocol was followed bysynthesis of second-strand cDNA. Three micrograms of SPIA single-stranded cDNA was concentrated to 20 �l in a vacuum concentrator(Eppendorf). The double-stranded cDNA was generated according to themanufacturer’s instructions using the WT-Ovation Exon Module (ver-sion 1; Nugen Inc.). Double-stranded cDNA was purified with the QiagenQIAquick 96-well system as described before (17).

The fragmentation and labeling of 2.5 �g of double-stranded cDNAwas performed according to the manufacturer’s protocol using the FL-Ovation cDNA Biotin Module V2 (Nugen Inc.). All samples were frag-mented to a size of 25 to 85 bp. Fragmented double-stranded cDNAs werethen end labeled with biotin using terminal transferase (Nugen Inc.).Aliquots of unlabeled and labeled fragmented double-stranded cDNAswere diluted to 5 ng/�l in 10 �l H2O. Each sample was incubated at roomtemperature with 2.5 ng streptavidin protein (ImmunoPure NeutrAvidin;Thermo Scientific) for 10 min. Unlabeled and labeled fragmented cDNAsamples were subjected to “mobility shift” analysis using a 2100 Bioana-

lyzer. This method allowed a qualitative labeling efficiency estimate forfragmented double-stranded cDNAs.

Microarray chip processing and analysis. Hybridization, fluidics andscanning were performed according to standard Affymetrix protocols(Affymetrix, Santa Clara, CA). Command Console (CC v3.1; Affymetrix)software was used to convert the image files to cell intensity data (cel files).The cel files were input into Partek Genomics Suite software (v6.66.12.0420; Partek, Inc.) and quantile normalized to produce the principal-component analysis (PCA) graph (see Fig. S1 in the supplemental mate-rial). An analysis of variance (ANOVA) was performed within Partek toobtain multiple-test-corrected P values (MTCP) using the false-discov-ery-rate method (18). Rankings were assigned from P values and foldchange then combined using custom Excel templates for each comparisonof interest to view any trends from a more consistently performing subset.Separation between comparisons was defined as the distance between theindividual comparison’s resulting fold difference for the same probe setanchored to 1-fold. This distance was assigned P values by using the indi-vidual replicate fold comparisons as a metric for following trends in sep-aration not as an absolute measure of significance.

TaqMan Q-RT-PCR. Candidate quantitative real-time PCR (Q-RT-PCR) probe and primer sets were designed using Primer Express version3.0 (Life Technologies). The succinate dehydrogenase complex subunit C(SDHC), sulfide quinone reductase-like (SQRDL), and tyrosine 3-mono-oxygenase/tryptophan 5-monooxygenase activation protein epsilon poly-peptide (YWHAE) genes were used as housekeeping genes.

Prior to synthesis of Q-RT-PCR template cDNA, RNAs were tested forcontaminating genomic DNA. The mouse hypoxanthine guanine phos-phoribosyltransferase gene (HPRT) was used to quantitate genomic DNAusing reference mouse genomic DNA for the Q-RT-PCR standard curve(Clontech). Contaminating genomic DNA was undetectable in all 58RNA samples (data not shown). The SuperScript VILO cDNA synthesiskit (Life Technologies) was used to generate cDNA from the test samplesaccording to the manufacturer’s protocol. An aliquot of 2.5 �g of RNAwas taken and vacuum centrifuged to 14 �l. cDNA synthesis reactions forall 57 samples and two controls without reverse transcriptase were run for2 h at 42°C to increase cDNA yields. The resulting cDNAs were purifiedaccording to the QIAquick 96-well protocol (Qiagen) with a modifiedcentrifugation protocol (17).

TaqMan assays were performed on all 58 samples and negative con-trols. To ensure precision and consistency in volumes of template andmaster mix, the Biomek NXP automated lab assistant (Beckman CoulterInc.) was used to prepare the plates for Q-RT-PCR analyses. Plates in96-well format were loaded with 60 �l of master mix for each multiplexreaction. From these plates, 15 �l of master mix was loaded onto a 384-well format in triplicate. With the same automated format, 5 �l of cDNAtemplate was added to each replicate well. The prepared plates were storedat �80°C. Data were analyzed according to comparative threshold cycle(CT) method (Life Technologies). The Spearman rank correlation (seeTable S2 in the supplemental material) between the overall Q-RT-PCRresults and the quantile normalized Affymetrix GeneChip results was cal-culated using GraphPad Prism 5.0 software (GraphPad Software). All

TABLE 1 Q223 mice are more resistant to amebiasis than 223R mice at72 ha

Genotype Time point (h)Infection rate, %(no. infected/total)

QQ 12 55.6 (5/9)RR 12 76.9 (10/13)QQ 72 0.0 (0/5)RR 72 38.9 (7/18)a Q223 and 223R mice were sacrificed at either 12 or 72 h postchallenge. Infectionstatus was determined by ex vivo culture of E. histolytica trophozoites from collectedcecal contents. No significant differences were observed using Fisher’s exact t test.

Leptin Receptor Polymorphism and Response to Amebiasis

May 2013 Volume 81 Number 5 iai.asm.org 1461

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

Q-RT-PCR values showed a highly significant correlation to the Af-fymetrix GeneChip probe set signals.

Ingenuity pathway analysis (IPA). Functional analysis identified thebiological functions that were most significant to the data set. Genes witha P value of �0.1 and associated with biological functions in the IngenuityKnowledge Base were considered for the analysis. A right-tailed Fisher

exact test was used to calculate a P value determining the probability thateach biological function assigned to that data set is due to chance alone.

My Pathways is a graphical representation of the molecular relation-ships between genes. Genes are represented as nodes, and the biologicalrelationship between two nodes is represented as an edge (line). All edgesare supported by at least one reference from the literature, from a text-

FIG 1 Q223infected and 223Rinfected mice have similar parasite burdens. E. histolytica genomic equivalents are indicated for individual animals. Controls are shownas circles, while experimental mice are shown as triangles. Closed symbols represent homozygous Q223 mice and open symbols homozygous 223R mice.Genomic equivalents were determined by Q-RT-PCR. Differences between Q223 and 223R genomic burdens were not significant. Control animals were mockinfected with spent amebic culture medium and thus are likely contaminated with E. histolytica DNA.

FIG 2 223Rinfected mice show significant regulation of genes at 12 h. 223Rinfected mice significantly regulated 27 genes in response to infection with E. histolytica(223Rinfected versus 223Rmock) at 12 h postchallenge (MTCP � 0.00008). While the results were not significant, Q223infected mice displayed similar trends inexpression (Q223infected versus Q223mock). — indicates an unannotated transcript.

Mackey-Lawrence et al.

1462 iai.asm.org Infection and Immunity

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

book, or from canonical information stored in the Ingenuity KnowledgeBase. Human, mouse, and rat orthologs of a gene are stored as separateobjects in the Ingenuity Knowledge Base but are represented as a singlenode in the network. The intensity of the node color indicates the degreeof upregulation (red) or downregulation (green). Nodes are displayedusing various shapes that represent the functional class of the gene prod-uct. Only pertinent and biologically relevant relationships (lines) areshown (Ingenuity Systems, Redwood City, CA).

Microarray data accession number. All microarray data discussed inthis paper were deposited into NCBI’s Gene Expression Omnibus (19)and are accessible through GEO Series accession number GSE43372 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc�GSE43372).

RESULTS223R mice display increased susceptibility to infection with E.histolytica. It has been shown that Q223 mice were significantlymore resistant to infection by E. histolytica than 223R mice (4). Toobserve the rate of clearance by Q223 mice and the establishmentof infection in 223R mice, two time points, 12 and 72 h postchal-lenge, were chosen for analysis. Resistant Q223 mice and suscep-tible 223R mice were randomly distributed into experimental andcontrol groups using a randomized block method (see Table S1 inthe supplemental material). Experimental animals were chal-lenged with 2 � 106 E. histolytica trophozoites using the previouslypublished intracecal injection model (16). Due to the xenic cul-ture conditions used to grow and maintain virulence of the ani-mal-passaged E. histolytica, control animals underwent laparot-omy but were injected with an equal volume of spent amebicculture medium to control for the bacteria that are cocultivatedwith the ameba. Mice were sacrificed at 12 and 72 h postchallenge,and infection status was determined by ex vivo culture of cecalcontents to observe E. histolytica trophozoites. As shown in Table1, 56% of Q223 mice and 77% of 223R mice were classified asinfected at 12 h postchallenge. Furthermore, at 12 h, Q223 and223R infected mice showed similar parasite burden (Fig. 1). By 72h however, all Q223 mice had cleared E. histolytica, while 39% of223R mice were infected. These findings were consistent with thepreviously reported observation that 223R mice were more sus-ceptible to infection with E. histolytica.

E. histolytica infection elicits a significant transcriptomic re-sponse in infected 223R mice. LEPR signaling in intestinal epi-thelial cells (IECs) is a mediator of resistance against E. histolytica;thus, it was predicted that the site of action for the Q223R poly-morphism would also be within the colon, specifically in IECs (5).The use of total cecal RNA allowed for the capture of the entirelocal transcriptional response to infection, including both IECresponses and those downstream (i.e., infiltrating cells). Analysiswas performed on animals that were infected at the indicated timepoints postchallenge; those which had cleared E. histolytica werenot analyzed. As shown in Fig. 2 and Table 2, microarray analysisof whole ceca from 223Rinfected animals at 12 h postchallengefound 27 genes to be significantly differentially regulated in re-sponse to infection (223Rinfected versus 223Rmock). Analysis ofQ223infected mice found only one gene, that for hydroxysteroid(17-beta)dehydrogenase (HSBD17B13), to be significantly regu-lated at 12 h (not shown). Although not reaching statistical signif-icance, the trends in expression for the majority of the significantlyregulated genes observed in 223Rinfected mice were similar inQ223infected mice at 12 h.

The 27 genes significantly regulated in 223Rinfected mice at 12 hdemonstrated that the host transcriptional response at 12 h of

infection was predominantly proinflammatory; for example, am-phiregulin (AREG) (20–22), a potent activator of the epidermalgrowth factor receptor (EGFR) which is transactivated by LEPR,and CD47 (23) and CD38 (24) were induced (25). Ingenuity path-way analysis (IPA) indicated enrichment of gene functions in-volved in cell-to-cell signaling and interaction and in cellularfunction and maintenance, with immune cells being especiallytargeted (Table 3). Thus, a proinflammatory transcriptional re-sponse in 223R mice was initiated during infection with E. histo-lytica at 12 h.

In order to observe how the host transcriptional responsechanges over time during infection, mice that were infected at 72 hwere analyzed. As shown in Fig. 3 and Table 4, at 72 h a total of 37genes were significantly regulated by 223R mice in response toinfection. Like the host response at 12 h, the transcriptional re-sponse at 72 h appeared to be proinflammatory, as 10 proinflam-matory genes, including CXCL2/KC (26) and the genes for calpro-tectin (S100A8/9) (27–29), platelet-activating factor acetylhydrolase(PLA2G7) (25, 30, 31), CD18 or integrin �2 (ITBG2) (32, 33), F4/80or EGF-like module containing, mucin-like, hormone receptor-like 1 (EMR1) (34), and matrix metallopeptidase 9 (MMP9) (35,36), were significantly induced. Furthermore, pathway analysis

TABLE 2 Genes and pathways differentially expressed in the cecumduring E. histolytica colitis: microarray results for comparison of223Rinfected versus 223Rmock mice at 12 ha

Gene P valueExpression change,b

Rinfected vs Rmock Fold change

Anxa3c,d,f 2.07E�06 � 3.71Aregc,d,e,f,g 8.86E�06 � 2.60Adra2ac,f,g 1.16E�05 � �1.67Ak3l1g 1.31E�05 � 5.37Gm5068 1.39E�05 � 2.06Anxa1c,d,e,f,g 1.50E�05 � 2.32Ska1 1.80E�05 � 1.74Ugdh 1.82E�05 � �1.49Krtap1-3 1.93E�05 � �1.27Wdr12e,g 2.53E�05 � 2.09Cebpde,g 2.75E�05 � 1.57Ugt1a9 2.89E�05 � �1.28Cd47c,d,e,f,g 3.09E�05 � 1.57Ly6c2c,d,e,g 3.47E�05 � 3.87Irf6e,g 3.56E�05 � �1.56Prpsap1 4.08E�05 � �1.78Zfp664 4.08E�05 � �1.26Fam134a 4.43E�05 � �1.50Abp1 4.62E�05 � �2.48Tmem171 4.66E�05 � �1.89Ino80c 4.68E�05 � 1.80Gna14 5.55E�05 � 2.89Ccdc94 7.27E�05 � �1.20Abcg2c,e,g 7.42E�05 � �1.36Cd38c,d,e,f,g 7.83E�05 � 2.23Idi1 8.43E�05 � 2.53a See Fig. 1. Twenty-seven genes were significantly differentially regulated (MTCP �

0.00008). Gene functional categories are indicated.b �, downregulated; �, upregulated.c Cell-to-cell signaling and interaction.d Cellular function and maintenance.e Cellular development.f Cellular movement.g Cellular growth and proliferation.

Leptin Receptor Polymorphism and Response to Amebiasis

May 2013 Volume 81 Number 5 iai.asm.org 1463

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

found functions pertaining to the movement and activity of im-mune cells significantly enriched (Table 3). While no overlappinggenes were significantly regulated at both time points, similar genefunctions were enriched at both the early and late time points in223R mice (Table 3; Fig. 4). As shown in Fig. 4, it appeared thatfunctions involved in inflammation and cellular compromisewere augmented after 72 h of infection. Along with proinflamma-tory genes, infected 223R mice induced calprotectin, a damage-associated molecular pattern (DAMP) expressed in response totissue damage and cellular stress by IECs as well as monocytes(27–29). Based on directionality of expression changes in both

MMP9 (37) and sirtuin 3 (SIRT3) (38), interleukin-10 (IL-10)synthesis was significantly predicted to occur via IPA (Fig. 4, Pro-tein Synthesis), implying that at 72 h postchallenge the host at-tempts to limit inflammation, likely in order to curtail tissue dam-age (39, 40). While being proinflammatory, MMP9 also activatesEGFR, suggesting that EGFR was activated following 12 and 72 hof infection (40). Thus, although no significantly regulated genesoverlapped at 12 and 72 h, a continuation of the inflammatoryresponse and activation of a similar pathway(s) were observed(Fig. 3; Tables 2 and 4). For microarray validation, nine signifi-cantly regulated genes were chosen for analysis by Q-RT-PCR. All

TABLE 3 Molecular and cellular functions of significantly regulated genes in 223Rinfected micea

Timepoint (h) Function P value

No. ofmolecules Direction

12 Cell-to-cell signaling and interaction 7.12E�05 to 4.36E�02 8 �Cellular function and maintenance 7.12E�05 to 4.90E�02 6 �Cellular development 1.78E�04 to 4.55E�02 9 �Cellular movement 4.86E�04 to 4.19E�02 6 �/�Cellular growth and proliferation 5.37E�04 to 4.31E�02 11 �/�

72 Cellular movement 5.24E�10 to 1.70E�02 12 ��Cell-to-cell signaling and interaction 5.36E�08 to 1.70E�02 10 ��Cellular compromise 6.45E�07 to 1.55E�02 8 �/�Cellular function and maintenance 6.45E�07 to 1.40E�02 10 �/�Lipid metabolism 6.21E�05 to 1.66E�02 7 �/�

a Molecular and cellular functions predicted to be significantly enriched at either 12 or 72 h postchallenge by Ingenuity pathway analysis (IPA). Predictions are based on genes thatwere significantly regulated between 223Rinfected and 223Rmock mice at each time point.

FIG 3 223Rinfected mice show significant regulation of genes at 72 h. Microarray analysis of 223Rinfected mice showed significant regulation of 37 genes in responseto infection with E. histolytica (223Rinfected versus 223Rmock) at 72 h postchallenge (MTCP � 0.0001). There were no Q223infected mice available for comparison.— indicates an unannotated transcript.

Mackey-Lawrence et al.

1464 iai.asm.org Infection and Immunity

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

genes analyzed demonstrated significant correlation with the mi-croarray results (see Table S2 in the supplemental material).

Genes differentially expressed due to the Q223R polymor-phism. As discussed above, infected Q223 and 223R mice hadcomparable burdens of E. histolytica and displayed similar geneexpression patterns for the majority of genes analyzed at 12 h ofinfection. In order to observe whether there was a subset of genesdifferentially regulated in response to infection with E. histolytica,the Q223infected/Q223mock versus 223Rinfected/223Rmock ratios werecompared. The trends of separation were filtered at an arbitrarycutoff of P � 0.05 to produce a list of exploratory genes deemed tobe differentially regulated in response to infection. The same anal-ysis could not be performed at 72 h postchallenge since no Q223mice remained infected at that time. As shown in Table 5, 42differentially regulated genes were identified. Pathway analysis in-

dicated that the regulated genes were significantly enriched infunctions involved in cellular compromise and DNA replication,recombination, and repair (Table 6). Eighteen of the 42 geneswere closely associated with leptin signaling (separated fromLEPR by no more than one gene product), suggesting that theobserved differential regulation may be directly downstream ofthe 223R polymorphism (Fig. 5).

The majority of leptin-linked differentially regulated geneswere involved in apoptosis, cellular proliferation, or recruitmentof hematopoietic cells. Antiapoptotic genes such as chitinase3-like 1 (CHI3L1) (41–43), RELA/NF-B (44, 45), and AKT3 (46–48) were decreased in infected 223R mice. These findings suggestthat 223R mice are more susceptible to cellular damage and deathcaused by E. histolytica, and they support our previous observa-tions that 223R mice displayed significantly more caspase 3 activ-ity and reduced AKT activation than Q223 mice (4). While apop-tosis appeared to be increased in infected 223R mice, cellularproliferation and recruitment of hematopoietic cells were also in-creased with the expression changes in genes, including DEC1 orstimulated by retinoic acid 13 homolog (STRA13) (49–51), insu-lin-like growth factor binding protein 7 (IGFBP7) (52), CCL2/13(53, 54), vimentin (VIM) (55), and sprouty-related EVH1 domaincontaining 2 (SPRED2) (56, 57).

The implication that 223R mice were more susceptible to dam-age and ameba-induced apoptosis than infected Q223 mice, inconjunction with the previous findings that STAT3 signaling atTyr1138 was essential for resistance to in vitro killing by E. histo-lytica, led to the hypothesis that STAT3 signaling downstream ofthe Q223R polymorphism was attenuated (5, 9). This hypothesiswas supported with the observation that of the 11 genes connectedto one of the downstream signaling molecules of LEPR, 82% (9/11) were predicted to be regulated by STAT3, while 100% wereregulated by STAT3 and/or SOCS3 (see Fig. S2 in the supplemen-tal material). Analysis of direct relationships with STAT3 sug-gested that STAT3 activity was decreased in 223R mice.

DISCUSSION

The most important finding of this study was the impact of the223R leptin receptor mutation on host gene expression duringexperimental amebiasis. Comparison of cecal gene expression ininfected Q223 versus 223R mice at 12 h was consistent with amutation-induced attenuation of the antiapoptotic effect of leptinand augmentation of recruitment of immune cells, presumably inresponse to intestinal damage. Analysis of gene expression in micewith the 223R LEPR mutation demonstrated that the host re-sponse to infection continues to evolve over time, as expressionchanges at 72 h did not overlap those observed at 12 h and con-sisted of regulation of proinflammatory and immune cell recruit-ment genes. A picture therefore emerges of a more robust immuneresponse in susceptible mice with the leptin receptor mutation.Whether the immune response is ultimately protective or delete-rious will be an important issue for future study.

Previously the 223R LEPR mutation was shown to be associ-ated with increased susceptibility to amebiasis in humans (bothamebic colitis and amebic liver abscess) (4). While it is one of themost common LEPR polymorphisms, the impact of the 223R mu-tation on in vivo receptor function had not been described (3, 4).A decrease in STAT3 activation by the mutant receptor observedin vitro in transfected cells had, however, led to the plausible hy-pothesis that the mutation rendered mice susceptible to amebiasis

TABLE 4 Genes and pathways differentially expressed in the cecumduring E. histolytica colitis: microarray results for comparison of223Rinfected versus 223Rmock mice at 72 ha

Gene P valueExpression change,b

Rinfected versus Rmock Fold change

Upk3b 2.51E�07 � �2.17Pla2g7c,d 3.34E�07 � 2.91Hmox1c,d,e 1.28E�06 � 2.86Sirt3d,e 3.07E�06 � �1.39Cxcl2c,e,f,g 6.58E�06 � 11.12Mpeg1 8.32E�06 � 3.86Emr1g 8.94E�06 � 3.50Fpr3c 9.73E�06 � 1.95Mir425 1.02E�05 � �1.55Cfp 1.16E�05 � 1.84S100a9c,f 2.07E�05 � 14.96Ms4a6d 2.08E�05 � 2.50Csf1rc,f,g 2.13E�05 � 1.72S100a8c,f 2.35E�05 � 15.81Arhgap20 2.39E�05 � �1.41Clec4a2 2.67E�05 � 1.95Tmem180 3.07E�05 � �1.37Mmp9c,d,e,g 3.27E�05 � 1.57Ptgesc,d,f 3.36E�05 � 1.58Clec4eg 3.87E�05 � 6.91Fpr1c,d,e,f,g 3.99E�05 � 4.43Skap2 4.05E�05 � 1.64Fam108a 4.25E�05 � �1.30Gm4942 4.98E�05 � �1.36BstIg 6.30E�05 � 2.52Ifitm1 6.31E�05 � 3.13Krt222 6.36E�05 � �1.62EG214403 6.83E�05 � �1.32Szt2 7.34E�05 � �1.47Itgb2c,e,f,g 9.27E�05 � 2.03Luzp4 9.69E�05 � �1.22Abca1d,e,f,g 1.07E�04 � 1.44Pf4c,e,f,g 1.14E�04 � 3.78Tmem82 1.26E�04 � �1.71a See Fig. 2. Thirty-seven genes were significantly differentially regulated (MTCP �

0.0001). Gene functional categories are indicated.b �, downregulated; �, upregulated.c Cellular movement.d Lipid metabolism.e Cellular compromise.f Cell-to-cell signaling and interaction.g Cellular function and maintenance.

Leptin Receptor Polymorphism and Response to Amebiasis

May 2013 Volume 81 Number 5 iai.asm.org 1465

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

by decreasing transcription mediated via STAT3 signaling (9). At72 h postchallenge, no Q223 mice were infected, while 40% of223R mice were. These data suggested that the mechanism(s) ofresistance in Q223 mice occurred early following challenge, im-plying an impact of the mutation on innate immunity.

Whether a more robust innate immune response was protec-tive or deleterious is an important question from this work. On theone hand, a proinflammatory response that involves cell-medi-ated immunity and gamma interferon (IFN-�) has been shown tobe protective against E. histolytica (58–61). On the other hand, it ispostulated that too robust an immune response augments diseaseby exacerbation of tissue damage, allowing further invasion of E.histolytica into the colonic epithelium (58, 61). For example, in-creased levels of tumor necrosis factor alpha (TNF-�), a proin-flammatory cytokine, are associated with symptomatic and recur-rent E. histolytica infections (62). The current results demonstratethat infected 223R mice significantly regulate expression of nu-merous proinflammatory genes at both time points, whereasQ223 mice infected at 12 h do not (no Q223 mice were infected at72 h), supporting the idea that inflammation was ineffective anddetrimental during infection with E. histolytica. Indeed, induction

of calprotectin (S100A8/9) provided evidence of increased tissuedamage and stress in infected 223R mice (9, 27–29, 63–66).

Comparison of significantly regulated genes in infected mice at12 and 72 h postchallenge demonstrated that a robust proinflam-matory response occurs. Interestingly, none of the significantlyregulated genes at 12 h and 72 h (223Rinfected only) overlapped,suggesting a continuously evolving transcriptional response to E.histolytica. The cause of this phenomenon is likely complex andmultifactorial; however, it is possible that the differences in regu-lation over time are due to the kinetics of E. histolytica infection. Ithas been previously suggested that E. histolytica establishes infec-tion sequentially, beginning with attachment to and degradationof the mucus layer, followed by invasion and destruction of theepithelial layer, and finally invasion into the lamina propria anddamage of the enteric nervous system (67–69). Thus, it is likelythat at early time points during infection (i.e., at 12 h) transcrip-tional changes are due to superficial invasion of the mucosa, whilelater responses represent deeper invasion and more tissue damage.Along with amebic invasion, immune infiltration occurs in dis-tinct phases, with neutrophils recruited early and macrophage re-cruitment and granuloma formation occurring later (68, 70).

FIG 4 Comparison of transcriptional responses (significantly enriched functions) in 223Rinfected mice at 12 and 72 h. Infected mice at 72 h were enriched in genefunctions for cell-to-cell signaling and interaction, cellular function and maintenance, cellular movement, cellular compromise, and protein synthesis. Thenegative log of the P value is indicated on the x axis. Results for the 12-h and 72-h time points are shown as black and gray bars, respectively.

Mackey-Lawrence et al.

1466 iai.asm.org Infection and Immunity

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

Thus, the data support the dynamic nature of both E. histolyticainvasion and also the adapting host inflammatory response.

In order to determine how the Q223 and 223R transcriptionalresponses differ during infection, a separation statistic was used toidentify genes differentially regulated between the two in responseto infection. Of the 42 genes identified, 18 were closely linked toleptin signaling and as such may illuminate how the Q223R poly-morphism modulates the effects of leptin during infection. Anal-ysis of these genes suggested that apoptotic pathways were in-

duced in infected 223R mice compared to Q223 mice. Thesetrends are not unexpected. E. histolytica is known to kill host cellsby inducing a rapid form of apoptosis, thus supporting the hy-pothesis that leptin protects against E. histolytica via its antiapop-totic activity, increasing the host cell’s intrinsic resistance (9, 63–66). Furthermore, differential regulation of these genes suggeststhat the Q223R polymorphism attenuates the ability of LEPRstimulation to protect cells against amebic killing and/or apopto-sis. As we have previously observed greater caspase 3 activationamong infected 223R mice, it is likely that this pathway is involvedin the mechanism of increased apoptosis in these animals (4).

Along with the increase in apoptotic genes, 223R mice alsoregulated genes involved in cellular proliferation, differentiation,and chemotaxis. Most notably, CCL2 (CCL13 or MCP-1), encod-ing a potent macrophage chemoattractant, was observed in in-fected 223R mice compared to Q223 (53, 54). Like the significantregulation of proinflammatory genes in infected 223R mice at 12and 72 h, differential regulation of CCL2 at the 12-h time point,when both Q223 and 223R mice were infected, suggests that en-hanced recruitment of myeloid-derived cells could have beenharmful and potentiated infection with E. histolytica.

Through in vitro assays, we have previously found that LEPR sig-naling through STAT3 is critical for protection against E. histolytica,presumably via its antiapoptotic activity (9). In support of the in vitrofindings, most leptin-linked genes were also linked to STAT3 and/orSOCS3, implicating the STAT3 pathway in the phenotype of the 223Rreceptor. These findings are in apparent contrast with those reportedby Stratigopoulos et al., where no significant differences in STAT3expression or activity were observed between cell line transfected withQ223 or 223R LEPR (15). However, the results reported here areconsistent with those observed by Marie et al. (9), where Q223 LEPRhad significantly greater STAT3 activity than the 223R LEPR in vitro,and further support the hypothesis that the Q223R polymorphismsignificantly affects LEPR signaling via STAT3. Therefore, the mech-anism(s) by which the polymorphism impacts the STAT3 signalingpathway will be a subject of further study.

A caveat to these studies was the use of whole tissue and the inabil-ity to assign transcriptional responses specifically to IECs, whereLEPR has been shown to play a critical role. Thus, other differencesdownstream of the Q223R polymorphism in these cells may havebeen diluted to below our level of detection. However, the methodsused allowed us to visualize the complete transcriptional environ-ment in the colon during infection and to observe effects downstreamof IECs.

It has been previously reported that both leptin and LEPR con-

TABLE 5 Genes and pathways differentially expressed in the cecumduring E. histolytica colitis: microarray results for comparison of Q223versus 223R mice in response to infection at 12 ha

Gene Rratio vs Qratiob P valuec

Acta2 � 0.04Atf7ip � 0.04Alpi � 0.02Cd81d,e,f,g � 0.03Ccl2/13d,e,f,g � 0.05Chi3l1d,f,g � 0.01Commd6 � 0.04AU042671 � 0.03Fmo2g,h � 0.04Gfpt2 � 0.03Igfbp7 � 0.01Jph2 � 0.00Kif3c � 0.03Lilrb4e,g � 0.03Ldb2 � 0.03Laptm5 � 0.03Mfsd8 � 0.04Mnd1 � 0.02Npm3 � 0.00Nup43 � 0.05Osbpl9 � 0.02Ppp2r5c � 0.04Ptpn9e � 0.03Qtrt1h � 0.01Rabif � 0.02Rad51h � 0.022310045A20R � 0.024932438A13R � 0.014932438A13R � 0.03Rnf121 � 0.01Slc17a4 � 0.03Spred2 � 0.03Stra13e � 0.01Tex12h � 0.02Akt3 � 0.02Timp2d,f � 0.01Tmem159 � 0.01Tmem195 � 0.01Vimd,e,f,h � 0.04Relad,f,g � 0.03Xrcc4h � 0.00a See Fig. 3. Gene functional categories are indicated.b �, downregulated; �, upregulated.c P value of the separation statistic (distance between Q223infected/Q223mock and223Rinfected/223Rmock ratio).d Cellular movement.e Cellular compromise.f Cell morphology.g Protein synthesis.h DNA replication, recombination, and repair.

TABLE 6 Molecular and cellular functions of genes differentiallyregulated in 223Rinfected and Q223infected micea

Function P valueNo. ofmolecules

Cellular compromise 1.01E�04 to 1.91E�02 6DNA replication, recombination,

and repair2.17E�04 to 4.17E�02 8

Cell morphology 6.20E�04 to 4.82E�02 8Protein synthesis 7.14E�04 to 3.74E�02 6Cellular movement 1.34�03 to 4.82E�02 6a Molecular and cellular functions predicted to be significantly enriched for genesdifferentially regulated in 223Rinfected compared to Q223infected mice at 12 hpostchallenge by Ingenuity pathway analysis (IPA).

Leptin Receptor Polymorphism and Response to Amebiasis

May 2013 Volume 81 Number 5 iai.asm.org 1467

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

centrations/expression differ by sex, with females having largeramounts than males (71–73). Humans homozygous for 223R havebeen found to have significantly lower levels of LEPR expression;however, differences in expression due to sex were not reported (4).Due to the small sample size for infected mice, we are unable to assesswhether sex-dependent differences in leptin/LEPR expression im-pacted the transcriptional responses identified here. Thus, the possi-bility that the phenotype of the Q223R polymorphism may be morepronounced depending on sex cannot be ruled out; however, differ-ences in concentration and expression of leptin or LEPR by sex inQ223R mice and humans have not been described (4, 15).

In summary, we found that infected 223R mice responded toinfection with E. histolytica by significantly regulating numerousgenes, many of which are involved in augmentation of an inflam-

matory response. Genes that were differentially regulated betweenQ223 and 223R mice during infection point to the 223R mutationleading to an attenuation of the antiapoptotic effect of leptin andaugmentation of the ability to recruit immune cells. These studiesare the first to propose a mechanism by which the Q223R poly-morphism affects leptin signaling and leptin-mediated effects inthe gut. The implications of the current work are of broad interestfor the insight provided into a novel mechanism of innate mucosaldefense of the intestinal mucosa.

ACKNOWLEDGMENTS

This work was supported by NIH grant 5RO1 AI026649 toW.A.P. N.M.M.-L was supported by BioDefense Research Trainingand Career Development NIH grant T32AI07046-32.

FIG 5 Differentially regulated 223Rinfected mouse genes linked to leptin. Genes differentially regulated between 223R and Q223 mice in response toinfection were analyzed for linkage to leptin/LEPR signaling. Using IPA software, target genes were connected to leptin and/or LEPR. To be listed, thegenes must have had one intervening gene between leptin/LEPR whose interaction made biological sense and the relationship resulted in the observedexpression changes. Eighteen of 42 genes met these criteria and were defined as leptin linked. Differentially regulated genes are shown in red (increased)or green (decreased); genes not present in the data set have no fill. Gene expression is indicated by bar chart, where expression in Q223 mice is first,followed by expression in 223R mice.

Mackey-Lawrence et al.

1468 iai.asm.org Infection and Immunity

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

REFERENCES1. Ozata M, Ozdemir IC, Licinio J. 1999. Human leptin deficiency caused

by a missense mutation: multiple endocrine defects, decreased sympa-thetic tone, and immune system dysfunction indicate new targets for lep-tin action, greater central than peripheral resistance to the effects of leptin,and spontaneous correction of leptin-mediated defects. J. Clin. Endocri-nol. Metab. 84:3686 –3695.

2. Farooqi IS, Wangensteen T, Collins S, Kimber W, Matarese G, KeoghJM, Lank E, Bottomley B, Lopez-Fernandez J, Ferraz-Amaro I, DattaniMT, Ercan O, Myhre AG, Retterstol L, Stanhope R, Edge JA, McKenzieS, Lessan N, Ghodsi M, De Rosa V, Perna F, Fontana S, Barroso I,Undlien DE, O’Rahilly S. 2007. Clinical and molecular genetic spectrumof congenital deficiency of the leptin receptor. N. Engl. J. Med. 356:237–247.

3. Mackey-Lawrence NM, Petri WA, Jr. 2012. Leptin and mucosal immu-nity. Mucosal Immunol. 5:472– 479.

4. Duggal P, Guo X, Haque R, Peterson KM, Ricklefs S, Mondal D, AlamF, Noor Z, Verkerke HP, Marie C, Leduc CA, Chua SC, Jr, Myers MG,Jr, Leibel RL, Houpt E, Gilchrist CA, Sher A, Porcella SF, Petri WA, Jr.2011. A mutation in the leptin receptor is associated with Entamoebahistolytica infection in children. J. Clin. Invest. 121:1191–1198.

5. Guo X, Roberts MR, Becker SM, Podd B, Zhang Y, Chua SC, Jr, MyersMG, Jr, Duggal P, Houpt ER, Petri WA, Jr. 2011. Leptin signaling inintestinal epithelium mediates resistance to enteric infection by Entam-oeba histolytica. Mucosal Immunol. 4:294 –303.

6. Fruhbeck G. 2006. Intracellular signalling pathways activated by leptin.Biochem. J. 393:7–20.

7. Leinninger GM, Myers MG, Jr. 2008. LRb signals act within a distributednetwork of leptin-responsive neurones to mediate leptin action. ActaPhysiol. 192:49 –59.

8. Myers MG, Jr. 2004. Leptin receptor signaling and the regulation ofmammalian physiology. Recent Prog. Horm. Res. 59:287–304.

9. Marie CS, Verkerke HP, Paul SN, Mackey AJ, Petri WA, Jr. 2012. Leptinprotects host cells from Entamoeba histolytica cytotoxicity by a STAT3-dependent mechanism. Infect. Immun. 80:1934 –1943.

10. Gove ME, Pini M, Fayad R, Cabay RJ, Fantuzzi G. 2009. Adiponectindeficiency modulates adhesion molecules expression and cytokine pro-duction but does not affect disease severity in the transfer model of colitis.Cytokine 47:119 –125.

11. Hruz P, Dann SM, Eckmann L. 2010. STAT3 and its activators in intes-tinal defense and mucosal homeostasis. Curr. Opin. Gastroenterol. 26:109 –115.

12. Li Y, de Haar C, Chen M, Deuring J, Gerrits MM, Smits R, Xia B,Kuipers EJ, van der Woude CJ. 2010. Disease-related expression of theIL6/STAT3/SOCS3 signalling pathway in ulcerative colitis and ulcerativecolitis-related carcinogenesis. Gut 59:227–235.

13. Pickert G, Neufert C, Leppkes M, Zheng Y, Wittkopf N, Warntjen M,Lehr HA, Hirth S, Weigmann B, Wirtz S, Ouyang W, Neurath MF,Becker C. 2009. STAT3 links IL-22 signaling in intestinal epithelial cells tomucosal wound healing. J. Exp. Med. 206:1465–1472.

14. Agrawal S, Gollapudi S, Su H, Gupta S. 2011. Leptin activates human Bcells to secrete TNF-alpha, IL-6, and IL-10 via JAK2/STAT3 andp38MAPK/ERK1/2 signaling pathway. J. Clin. Immunol. 31:472– 478.

15. Stratigopoulos G, LeDuc CA, Matsuoka N, Gutman R, Rausch R,Robertson SA, Myers MG, Jr, Chung WK, Chua SC, Jr, Leibel RL. 2009.Functional consequences of the human leptin receptor (LEPR) Q223Rtransversion. Obesity (Silver Spring) 17:126 –135.

16. Houpt ER, Glembocki DJ, Obrig TG, Moskaluk CA, Lockhart LA,Wright RL, Seaner RM, Keepers TR, Wilkins TD, Petri WA, Jr. 2002.The mouse model of amebic colitis reveals mouse strain susceptibility toinfection and exacerbation of disease by CD4� T cells. J. Immunol. 169:4496 – 4503.

17. Graham MR, Virtaneva K, Porcella SF, Barry WT, Gowen BB, JohnsonCR, Wright FA, Musser JM. 2005. Group A Streptococcus transcriptomedynamics during growth in human blood reveals bacterial adaptive andsurvival strategies. Am. J. Pathol. 166:455– 465.

18. Klipper-Aurbach Y, Wasserman M, Braunspiegel-Weintrob N, BorsteinD, Peleg S, Assa S, Karp M, Benjamini Y, Hochberg Y, Laron Z. 1995.Mathematical formulae for the prediction of the residual beta cell functionduring the first two years of disease in children and adolescents with insu-lin-dependent diabetes mellitus. Med. Hypotheses 45:486 – 490.

19. Edgar R, Domrachev M, Lash AE. 2002. Gene Expression Omnibus:

NCBI gene expression and hybridization array data repository. NucleicAcids Res. 30:207–210.

20. Val S, Belade E, George I, Boczkowski J, Baeza-Squiban A. 2012. FinePM induce airway MUC5AC expression through the autocrine effect ofamphiregulin. Arch. Toxicol. 86:1851–1859.

21. Wilson KJ, Mill C, Lambert S, Buchman J, Wilson TR, Hernandez-Gordillo V, Gallo RM, Ades LM, Settleman J, Riese DJ, II. 2012. EGFRligands exhibit functional differences in models of paracrine and auto-crine signaling. Growth Factors 30:107–116.

22. Scheller J, Chalaris A, Garbers C, Rose-John S. 2011. ADAM17: amolecular switch to control inflammation and tissue regeneration. TrendsImmunol. 32:380 –387.

23. Westlund J, Livingston M, Fahlen-Yrlid L, Oldenborg PA, Yrlid U.2012. CD47-deficient mice have decreased production of intestinal IgAfollowing oral immunization but a maintained capacity to induce oraltolerance. Immunology 135:236 –244.

24. Grahnert A, Grahnert A, Klein C, Schilling E, Wehrhahn J, HauschildtS. 2011. NAD�: a modulator of immune functions. Innate Immun. 17:212–233.

25. Slomiany BL, Slomiany A. 2009. Leptin-induced cytosolic phospholipaseA2 activation in gastric mucosal protection against ethanol cytotoxicityinvolves epidermal growth factor receptor transactivation. Inflammop-harmacology 17:6 –14.

26. Shang K, Bai YP, Wang C, Wang Z, Gu HY, Du X, Zhou XY, Zheng CL,Chi YY, Mukaida N, Li YY. 2012. Crucial involvement of tumor-associated neutrophils in the regulation of chronic colitis-associated car-cinogenesis in mice. PLoS One 7:e51848. doi:10.1371/journal.pone.0051848.

27. Ehrchen JM, Sunderkotter C, Foell D, Vogl T, Roth J. 2009. Theendogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) asinnate amplifier of infection, autoimmunity, and cancer. J. Leukoc. Biol.86:557–566.

28. Foell D, Wittkowski H, Ren Z, Turton J, Pang G, Daebritz J, EhrchenJ, Heidemann J, Borody T, Roth J, Clancy R. 2008. Phagocyte-specificS100 proteins are released from affected mucosa and promote immuneresponses during inflammatory bowel disease. J. Pathol. 216:183–192.

29. Foell D, Wittkowski H, Vogl T, Roth J. 2007. S100 proteins expressed inphagocytes: a novel group of damage-associated molecular pattern mole-cules. J. Leukoc. Biol. 81:28 –37.

30. Tew DG, Southan C, Rice SQ, Lawrence MP, Li H, Boyd HF, MooresK, Gloger IS, Macphee CH. 1996. Purification, properties, sequencing,and cloning of a lipoprotein-associated, serine-dependent phospholipaseinvolved in the oxidative modification of low-density lipoproteins. Arte-rioscler. Thromb. Vasc. Biol. 16:591–599.

31. Tjoelker LW, Eberhardt C, Unger J, Trong HL, Zimmerman GA,McIntyre TM, Stafforini DM, Prescott SM, Gray PW. 1995. Plasmaplatelet-activating factor acetylhydrolase is a secreted phospholipase A2with a catalytic triad. J. Biol. Chem. 270:25481–25487.

32. Merched A, Tollefson K, Chan L. 2010. Beta2 integrins modulate theinitiation and progression of atherosclerosis in low-density lipoproteinreceptor knockout mice. Cardiovasc. Res. 85:853– 863.

33. Simon AJ, Lev A, Wolach B, Gavrieli R, Amariglio N, Rosenthal E,Gazit E, Eyal E, Rechavi G, Somech R. 2010. The effect of gentamicin-induced readthrough on a novel premature termination codon of CD18leukocyte adhesion deficiency patients. PLoS One 5:e13659. doi:10.1371/journal.pone.0013659.

34. Gordon S, Hamann J, Lin HH, Stacey M. 2011. F4/80 and the relatedadhesion-GPCRs. Eur. J. Immunol. 41:2472–2476.

35. Van Lint P, Libert C. 2007. Chemokine and cytokine processing bymatrix metalloproteinases and its effect on leukocyte migration and in-flammation. J. Leukoc. Biol. 82:1375–1381.

36. Price NM, Gilman RH, Uddin J, Recavarren S, Friedland JS. 2003.Unopposed matrix metalloproteinase-9 expression in human tuberculousgranuloma and the role of TNF-alpha-dependent monocyte networks. J.Immunol. 171:5579 –5586.

37. Wang M, Qin X, Mudgett JS, Ferguson TA, Senior RM, Welgus HG.1999. Matrix metalloproteinase deficiencies affect contact hypersensitiv-ity: stromelysin-1 deficiency prevents the response and gelatinase B defi-ciency prolongs the response. Proc. Natl. Acad. Sci. U. S. A. 96:6885– 6889.

38. Hirschey MD, Shimazu T, Jing E, Grueter CA, Collins AM, AouizeratB, Stancakova A, Goetzman E, Lam MM, Schwer B, Stevens RD,Muehlbauer MJ, Kakar S, Bass NM, Kuusisto J, Laakso M, Alt FW,Newgard CB, Farese RV, Jr, Kahn CR, Verdin E. 2011. SIRT3 deficiency

Leptin Receptor Polymorphism and Response to Amebiasis

May 2013 Volume 81 Number 5 iai.asm.org 1469

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

and mitochondrial protein hyperacetylation accelerate the developmentof the metabolic syndrome. Mol. Cell 44:177–190.

39. Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. 2011.Regulation and functions of the IL-10 family of cytokines in inflammationand disease. Annu. Rev. Immunol. 29:71–109.

40. Roelle S, Grosse R, Aigner A, Krell HW, Czubayko F, Gudermann T.2003. Matrix metalloproteinases 2 and 9 mediate epidermal growth factorreceptor transactivation by gonadotropin-releasing hormone. J. Biol.Chem. 278:47307– 47318.

41. Lee CG, Da Silva CA, Dela Cruz CS, Ahangari F, Ma B, Kang MJ, HeCH, Takyar S, Elias JA. 2011. Role of chitin and chitinase/chitinase-likeproteins in inflammation, tissue remodeling, and injury. Annu. Rev.Physiol. 73:479 –501.

42. Chen CC, Pekow J, Llado V, Kanneganti M, Lau CW, Mizoguchi A,Mino-Kenudson M, Bissonnette M, Mizoguchi E. 2011. Chitinase3-like-1 expression in colonic epithelial cells as a potentially novel markerfor colitis-associated neoplasia. Am. J. Pathol. 179:1494 –1503.

43. Dela Cruz CS, Liu W, He CH, Jacoby A, Gornitzky A, Ma B, Flavell R,Lee CG, Elias JA. 2012. Chitinase 3-like-1 promotes Streptococcus pneu-moniae killing and augments host tolerance to lung antibacterial re-sponses. Cell Host Microbe 12:34 – 46.

44. Janssens S, Tschopp J. 2006. Signals from within: the DNA-damage-induced NF-kappaB response. Cell Death Differ. 13:773–784.

45. Campbell KJ, Perkins ND. 2004. Reprogramming RelA. Cell Cycle3:869 – 872.

46. Shao Y, Aplin AE. 2010. Akt3-mediated resistance to apoptosis in B-RAF-targeted melanoma cells. Cancer Res. 70:6670 – 6681.

47. Yang ZZ, Tschopp O, Di-Poi N, Bruder E, Baudry A, Dummler B,Wahli W, Hemmings BA. 2005. Dosage-dependent effects of Akt1/protein kinase Balpha (PKBalpha) and Akt3/PKBgamma on thymus, skin,and cardiovascular and nervous system development in mice. Mol. Cell.Biol. 25:10407–10418.

48. Ding L, Biswas S, Morton RE, Smith JD, Hay N, Byzova TV, FebbraioM, Podrez EA. 2012. Akt3 deficiency in macrophages promotes foam cellformation and atherosclerosis in mice. Cell Metab. 15:861– 872.

49. Wang Y, Rao VK, Kok WK, Roy DN, Sethi S, Ling BM, Lee MB, TanejaR. 2012. SUMO modification of Stra13 is required for repression of cyclinD1 expression and cellular growth arrest. PLoS One 7:e43137. doi:10.1371/journal.pone.0043137.

50. Yun Z, Maecker HL, Johnson RS, Giaccia AJ. 2002. Inhibition of PPARgamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: amechanism for regulation of adipogenesis by hypoxia. Dev. Cell 2:331–341.

51. Shen M, Yoshida E, Yan W, Kawamoto T, Suardita K, Koyano Y,Fujimoto K, Noshiro M, Kato Y. 2002. Basic helix-loop-helix proteinDEC1 promotes chondrocyte differentiation at the early and terminalstages. J. Biol. Chem. 277:50112–50120.

52. Nousbeck J, Sarig O, Avidan N, Indelman M, Bergman R, Ramon M,Enk CD, Sprecher E. 2010. Insulin-like growth factor-binding protein 7regulates keratinocyte proliferation, differentiation and apoptosis. J. In-vestig. Dermatol. 130:378 –387.

53. Kolattukudy PE, Niu J. 2012. Inflammation, endoplasmic reticulumstress, autophagy, and the monocyte chemoattractant protein-1/CCR2pathway. Circ.Res. 110:174 –189.

54. Menezes-Souza D, Guerra-Sa R, Carneiro CM, Vitoriano-Souza J, Gi-unchetti RC, Teixeira-Carvalho A, Silveira-Lemos D, Oliveira GC,Correa-Oliveira R, Reis AB. 2012. Higher expression of CCL2, CCL4,CCL5, CCL21, and CXCL8 chemokines in the skin associated with para-site density in canine visceral leishmaniasis. PLoS Negl. Trop. Dis. 6:e1566.doi:10.1371/journal.pntd.0001566.

55. Satelli A, Li S. 2011. Vimentin in cancer and its potential as a moleculartarget for cancer therapy. Cell. Mol. Life Sci. 68:3033–3046.

56. Nobuhisa I, Kato R, Inoue H, Takizawa M, Okita K, Yoshimura A, TagaT. 2004. Spred-2 suppresses aorta-gonad-mesonephros hematopoiesis byinhibiting MAP kinase activation. J. Exp. Med. 199:737–742.

57. Taniguchi K, Kohno R, Ayada T, Kato R, Ichiyama K, Morisada T, OikeY, Yonemitsu Y, Maehara Y, Yoshimura A. 2007. Spreds are essential forembryonic lymphangiogenesis by regulating vascular endothelial growthfactor receptor 3 signaling. Mol. Cell. Biol. 27:4541– 4550.

58. Rojas-Lopez AE, Soldevila G, Meza-Perez S, Dupont G, Ostoa-SalomaP, Wurbel MA, Ventura-Juarez J, Flores-Romo L, Garcia-Zepeda EA.2012. CCR9� T cells contribute to the resolution of the inflammatoryresponse in a mouse model of intestinal amoebiasis. Immunobiology 217:795– 807.

59. Guo X, Barroso L, Lyerly DM, Petri WA, Jr, Houpt ER. 2011. CD4�and CD8� T cell- and IL-17-mediated protection against Entamoeba his-tolytica induced by a recombinant vaccine. Vaccine 29:772–777.

60. Guo X, Stroup SE, Houpt ER. 2008. Persistence of Entamoeba histolyticainfection in CBA mice owes to intestinal IL-4 production and inhibition ofprotective IFN-gamma. Mucosal Immunol. 1:139 –146.

61. Mortimer L, Chadee K. 2010. The immunopathogenesis of Entamoebahistolytica. Exp. Parasitol. 126:366 –380.

62. Peterson KM, Shu J, Duggal P, Haque R, Mondal D, Petri WA, Jr. 2010.Association between TNF-alpha and Entamoeba histolytica diarrhea. Am.J. Trop. Med. Hyg. 82:620 – 625.

63. Ralston KS, Petri WA. 2011. The ways of a killer: how does Entamoebahistolytica elicit host cell death? Essays Biochem. 51:193–210.

64. Ralston KS, Petri WA, Jr. 2011. Tissue destruction and invasion byEntamoeba histolytica. Trends Parasitol. 27:254 –263.

65. Boettner DR, Petri WA. 2005. Entamoeba histolytica activates host cellcaspases during contact-dependent cell killing. Curr. Top. Microbiol. Im-munol. 289:175–184.

66. Becker SM, Cho KN, Guo X, Fendig K, Oosman MN, Whitehead R,Cohn SM, Houpt ER. 2010. Epithelial cell apoptosis facilitates Entam-oeba histolytica infection in the gut. Am. J. Pathol. 176:1316 –1322.

67. Lourenssen S, Houpt ER, Chadee K, Blennerhassett MG. 2010. Entam-oeba histolytica infection and secreted proteins proteolytically damageenteric neurons. Infect. Immun. 78:5332–5340.

68. Guo X, Houpt E, Petri WA, Jr. 2007. Crosstalk at the initial encounter:interplay between host defense and ameba survival strategies. Curr. Opin.Immunol. 19:376 –384.

69. Bansal D, Ave P, Kerneis S, Frileux P, Boche O, Baglin AC, Dubost G,Leguern AS, Prevost MC, Bracha R, Mirelman D, Guillen N, LabruyereE. 2009. An ex-vivo human intestinal model to study Entamoeba histo-lytica pathogenesis. PLoS Negl. Trop. Dis. 3:e551. doi:10.1371/journal.pntd.0000551.

70. Baxt LA, Singh U. 2008. New insights into Entamoeba histolytica patho-genesis. Curr. Opin. Infect. Dis. 21:489 – 494.

71. Chan JL, Bluher S, Yiannakouris N, Suchard MA, Kratzsch J, Mantzo-ros CS. 2002. Regulation of circulating soluble leptin receptor levels bygender, adiposity, sex steroids, and leptin: observational and interven-tional studies in humans. Diabetes 51:2105–2112.

72. Smith JT, Waddell BJ. 2003. Developmental changes in plasma leptin andhypothalamic leptin receptor expression in the rat: peripubertal changesand the emergence of sex differences. J. Endocrinol. 176:313–319.

73. Hickey MS, Israel RG, Gardiner SN, Considine RV, McCammon MR,Tyndall GL, Houmard JA, Marks RH, Caro JF. 1996. Gender differencesin serum leptin levels in humans. Biochem. Mol. Med. 59:1– 6.

Mackey-Lawrence et al.

1470 iai.asm.org Infection and Immunity

on May 22, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

Effect of the Leptin Receptor Q223R Polymorphism on the HostTranscriptome following Infection with Entamoeba histolytica

Nicole M. Mackey-Lawrence, Xiaoti Guo, Daniel E. Sturdevant, Kimmo Virtaneva, Matthew M. Hernandez, Eric Houpt, Alan Sher,Stephen F. Porcella, William A. Petri, Jr.

Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia School of Medicine, Charlottesville, Virginia, USA; North ShoreLong Island Health System, Manhasset, New York, USA; Rocky Mountain Laboratory Research Technologies Section, Genomics Unit, National Institute of Allergy andInfectious Diseases, NIH, Hamilton, Montana, USA; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA

Volume 81, no. 5, p. 1460 –1470, 2013. Page 1468, Acknowledgments: The following sentence should be added.

This research was supported in part by the Intramural Research Program of the NIH, NIAID.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00689-13

AUTHOR’S CORRECTION

4322 iai.asm.org Infection and Immunity p. 4322 November 2013 Volume 81 Number 11