RESEARCH ARTICLE

Environmental factors regulate Paneth cell phenotype and hostsusceptibility to intestinal inflammation in Irgm1-deficient miceAllison R. Rogala1,2, Alexi A. Schoenborn1,2, Brian E. Fee3, Viviana A. Cantillana4, Maria J. Joyce5,Raad Z. Gharaibeh6, Sayanty Roy1,2, Anthony A. Fodor1,6, R. Balfour Sartor1,7,8, Gregory A. Taylor3,4,*and Ajay S. Gulati1,2,9,*,‡

ABSTRACTCrohn’s disease (CD) represents a chronic inflammatory disorder of theintestinal tract. Several susceptibility genes have been linked to CD,though their precise role in the pathogenesis of this disorder remainsunclear. Immunity-related GTPase M (IRGM) is an established riskallele in CD. We have shown previously that conventionally raised (CV)mice lacking the IRGM ortholog, Irgm1 exhibit abnormal Paneth cells(PCs) and increased susceptibility to intestinal injury. In the presentstudy, we sought to utilize this model system to determine ifenvironmental conditions impact these phenotypes, as is thought tobe the case in human CD. To accomplish this, wild-type and Irgm1−/−

mice were rederived into specific pathogen-free (SPF) and germ-free(GF) conditions. We next assessed how these differential housingenvironments influenced intestinal injury patterns, and epithelial cellmorphology and function inwild-type and Irgm1−/−mice. Remarkably, incontrast to CV mice, SPF Irgm1−/− mice showed only a slight increasein susceptibility to dextran sodium sulfate-induced inflammation. SPFIrgm1−/− mice also displayed minimal abnormalities in PC number andmorphology, and in antimicrobial peptide expression. Goblet cellnumbers and epithelial proliferation were also unaffected by Irgm1in SPF conditions. No microbial differences were observed betweenwild-type and Irgm1−/− mice, but gut bacterial communities differedprofoundly between CV and SPF mice. Specifically, Helicobactersequences were significantly increased in CV mice; however,inoculating SPF Irgm1−/− mice with Helicobacter hepaticus was notsufficient to transmit a pro-inflammatory phenotype. In summary, ourfindings suggest the impact of Irgm1-deficiency on susceptibility tointestinal inflammation and epithelial function is critically dependent on

environmental influences. This work establishes the importance ofIrgm1−/−miceas amodel to elucidate host-environment interactions thatregulate mucosal homeostasis and intestinal inflammatory responses.Defining such interactions will be essential for developing novelpreventative and therapeutic strategies for human CD.

KEY WORDS: Immunity-related GTPases, Experimental colitis,Inflammatory bowel diseases

INTRODUCTIONCrohn’s disease (CD) is a chronic inflammatory disorder of thegastrointestinal tract, which is thought to occur as a result ofdysregulated host immune responses to the enteric microbiota withina genetically susceptible host (Abraham and Cho, 2009; Leone et al.,2013). Genome wide association studies have identified a multitudeof genes associated with development of CD, many of which encodefactors involved in the regulation of the autophagy pathway(Baskaran et al., 2014; Cleynen et al., 2013; Hoefkens et al., 2013;Li et al., 2014; Liu et al., 2015; Khor et al., 2011). Among these is theimmunity-related GTPase M (IRGM), which has recently beenshown to play an integral role in the initiation of autophagy duringmicrobial clearance (Chauhan et al., 2015).

We previously reported that mice deficient in Irgm1, a murinehomologue of IRGM, exhibited several phenotypic differenceswithin the gastrointestinal tract, as compared to wild-type (WT) mice(Liu et al., 2013). First, Irgm1−/− mice showed an overall increasedsusceptibility to dextran sodium sulfate (DSS)-induced colonicinjury. Clinically, these knockout (KO) mice demonstrated greaterweight loss, increased fecal blood and worsened stool consistencythan WT mice treated with DSS. Irgm1−/− mice also exhibitedincreased inflammation in the ileum in response to DSS, which is nottypically observed in this injury model (Chassaing et al., 2014).Second, we reported that Irgm1−/−mice expressed an aberrant Panethcell (PC) phenotype. PCs are specialized epithelial cells located in thesmall intestine, which play an important role in innate immunity andregulation of the intestinal microbiota via secretion of antimicrobialpeptides (AMPs). Irgm1−/− mice possessed an increased number ofPCs per crypt, decreased PC granule size and an increased number ofectopically placed PCs. Functionally, the PCs of Irgm1-deficientmice expressed decreased mRNA levels of the AMPs α-defensin 20(Defa20) and lysozyme (Lyz) (Liu et al., 2013).

It is important to note that mice used in the study by Liu andcolleagues were born and raised in conventional (CV) conditions thatdid not exclude, for instance, murine norovirus or speciesof Helicobacter. Because previous work demonstrated thatenvironmental factors, including microbial influences, can affectthe penetrance and severity of intestinal inflammation in murinemodels of inflammatory bowel disease (IBD) (Foltz et al., 1998;Maggio-Price et al., 2006; Ward et al., 1996; Peloquin and Nguyen,Received 23 June 2017; Accepted 14 December 2017

1Center for Gastrointestinal Biology and Disease, University of North Carolina atChapel Hill, Chapel Hill, NC 27599, USA. 2Department of Pediatrics, Division ofGastroenterology, University of North Carolina at Chapel Hill, Chapel Hill, NC27599, USA. 3Geriatric Research, Education, and Clinical Center, VA MedicalCenter, Durham, NC 27705, USA. 4Departments of Medicine; Molecular Geneticsand Microbiology; and Immunology; Division of Geriatrics, and Center for the Studyof Aging and Human Development, Duke University Medical Center, Durham, NC27710, USA. 5Department of Medicine, Division of Infectious Disease, DukeUniversity Medical Center, Durham, NC 27710, USA. 6Department of BioinformaticsandGenomics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA.7Department of Medicine, Division of Gastroenterology and Hepatology, Universityof North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 8Department ofMicrobiology and Immunology, University of North Carolina at Chapel Hill, ChapelHill, NC 27599, USA. 9Department of Pathology and Laboratory Medicine,University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.*These authors contributed equally to this work

‡Author for correspondence (ajay_gulati@med.unc.edu)

A.S.G., 0000-0002-7612-6369

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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2013; Cadwell et al., 2010), we sought to determine whetherenvironmental conditions impact the inflammatory and PCphenotypes in Irgm1-deficient mice. Specifically, we hypothesizedthat Irgm1−/− mice that had been rederived into ‘clean’ housingconditions have an attenuated phenotype in regards to DSSsusceptibility and PC dysmorphology.To test our hypothesis, we established colonies of littermate

Irgm1−/− and WT mice in specific pathogen-free (SPF) and germ-free (GF) facilities. In this study, we demonstrate that environmentalconditions remarkably attenuate the increased susceptibility ofIrgm1−/− mice to DSS-mediated intestinal injury, as well as thedysmorphic PC phenotype observed in these animals. This interplaybetween host genetics and the environment parallels similarinteractions in human CD, thereby enhancing the value of thismurine model in the investigation of gene-environment interactionswithin the context of intestinal inflammatory disorders.

RESULTSIncreased susceptibility to DSS-induced intestinal injury isattenuated in Irgm1-deficientmice housed in SPF conditionsTo determine whether Irgm1−/− mice housed in SPF conditionsexhibit the increased susceptibility to intestinal inflammationpreviously observed in CV KO animals, SPF KO and WTlittermate mice were subject to acute DSS injury as described (Liuet al., 2013). In contrast to our previous findings when applying CVhousing conditions, in which DSS-treated Irgm1KOmice lost moreweight than their WT counterparts, no differences in weight losswere detected between WT and KO groups when mice were housedin an SPF environment (Fig. 1A). Similarly, no differences betweenIrgm1 KO and WT mice were observed when fecal samples werescored for consistency (Fig. 1B). At 7 days post-DSS, theHemoccult index was slightly higher in KO mice compared withtheir WT counterparts, but no differences in stool blood wereobserved at earlier time points (Fig. 1C).To assess colonic inflammation, we first measured colon lengths at

the time of necropsy (after 7 days of DSS treatment). There were nobaseline differences between SPF WT and Irgm1 KO mice thatreceived water control; however, both groups demonstrated shortenedcolons relative to controls when treated with DSS (Fig. 2A). Thiscolon shortening was more pronounced in KO mice receiving DSS(P<0.01), though the absolute difference between DSS-treated WTand KO mice was relatively small. Histologically, the vehicle-treatedcontrols of both the KO and WT mice lacked any discerniblehistologic inflammation or ulceration of the colon. Within the DSS-treated groups (Fig. 2B,C), mild inflammation occurred in theproximal colon of both KO and WT mice; however, there were nodifferences in severity between these two groups. Similarly, the cecaof both KO and WT DSS-treated groups showed moderateinflammation, but the genotype had no discernible effect. In themiddle colon, both KO andWT groups treated with DSS exhibited anequally severe inflammatory response. The only differences inhistologic inflammatory scores that was noticed in SPF mice were inthe distal colon, where KO animals did display increasedinflammation relative to their WT counterparts. Notably, this starklycontrasts with our previous finding in CV-raised mice, in which thegenotype contributed to differences in inflammation throughout themiddle colon, distal colon and cecum (Liu et al., 2013).In the ileum, we found no significant differences in inflammation

between DSS-treated WT and KO mice raised in SPF conditions(Fig. 2D,E). Ileal tissues were also assessed for expression of TNFαmRNA; however, neither Irgm1 KO nor WT mice housed in SPFconditions yielded detectable levels of TNFαmRNA as compared to

positive controls (data not shown). Again, this contrasts our previousfindings in CV KOmice, which showed increased ileal inflammationand TNFα mRNA expression in response to DSS, as compared totheir WT littermates (Liu et al., 2013). In summary, the increasedsusceptibility to ileal and colonic inflammation in response to DSSthat is displayed by CV Irgm1-deficient mice is markedly attenuatedin SPF housing conditions.

Environmental conditions influence the PC phenotype inIrgm1-deficient miceIn addition to enteric inflammatory responses to DSS, SPF Irgm1−/−

mice were also evaluated for the PC anomalies previously observedin CV-raised KO animals. Similar to CV Irgm1−/− mice, SPF KO

Fig. 1. Clinical response of Irgm1-deficient mice receiving dextransodium sulfate. Male SPF wild-type (WT) and Irgm1−/− (KO) mice wereadministered 3% dextran sodium sulfate (DSS) in drinking water or drinkingwater alone (control) for 7 days. (A) Weight loss shown as a percentage ofinitial weight prior to treatment. (B) Average stool consistency score.(C) Hemoccult score measuring occult fecal blood. Values are means±s.e.m.;data are combined from four separate experiments. Combined cohort sizes areas follows: n=22 WT DSS, 21 KO DSS, 11 WT control, 10 KO control. *P<0.05(KO DSS versus WT DSS).

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mice exhibited an increased number of PCs per histologic crypt-villus unit as compared to their WT controls (Fig. 3A,B). We alsoobserved a trend towards increased ectopic PCs in SPF KO micerelative to their WT counterparts (Fig. 3C), though this did notreach statistical significance as we previously reported in CV KOmice (Liu et al., 2013). Notably, Irgm1−/− mice housed in SPFconditions failed to show differences in PC granule sizecompared to WT animals (Fig. 3D), a difference that wasapparent in CVmice. Finally, we also measured the expression ofgenes related to PC function (including the antimicrobialpeptides Lyz and Defa20) that have previously shown to bedecreased in CV Irgm1−/− mice. Again, SPF Irgm1−/− miceshowed no differences in expression of either of these genescompared to WT littermates (Fig. 3E,F). In summary, the PCabnormalities originally observed in CV Irgm1−/− mice areattenuated in SPF conditions.

Irgm1-deficient and wild-type littermate mice display nodifferences in goblet cell numbers or epithelial proliferationin SPF conditionsIn addition to its role in PC biology, autophagy has also beenimplicated in the regulation of goblet cell function (Patel et al., 2013)and cellular proliferation (Sbrana et al., 2016). Therefore, we assessedgoblet cell numbers and epithelial cell proliferation by using periodicacid–Schiff (PAS) and Ki67 staining, respectively, in the ileum ofIrgm1−/− and WT littermates. As shown in Fig. 4A,C, goblet cellnumbers were similar in WT and Irgm1−/− mice; however, we didobserve an increased intensity of PAS staining in the goblet cells of KOanimals. This was consistent across multiple sections from numerousmice. In regards to epithelial proliferation, we found no differencesin the Ki67-positive zones of WT and Irgm1−/− mice (Fig. 4B,D).Importantly, there were also no ectopic foci of proliferation that wouldsuggest aberrantly located crypt formation in the KO animals.

Fig. 2. Intestinal inflammation inspecific pathogen-free Irgm1-deficientmice receiving dextran sodium sulfate.Male specific pathogen-free (SPF) wild-type (WT) and Irgm1−/− (KO) mice wereadministered 3% dextran sodium sulfate(DSS) in drinking water or drinking wateralone (control) for 7 days. (A) Gross colonlength. (B) Representative histologicaltissue sections from the colon of WT andKO mice treated with DSS (H&E stain, 4×magnification). (C) Average histologicinflammation scores from indicatedsegments of colon. (D) Representativehistological tissue sections from the ileumof WT and KO mice treated with DSS(H&E stain, 20× magnification).(E) Average histologic inflammationscores in ileum. Results from watercontrols were omitted from B-D, as therewas no inflammation present in thesesamples. Values are means±s.e.m.; dataare combined from two separateexperiments. Combined cohort sizeswere as follows: n=14 WT DSS, 9 KODSS, 8 WT control, 9 KO control.*P<0.01.

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Environment but not genotype drives microbial compositionand diversity in Irgm1-deficient mice and wild-typelittermatesGiven the differences in the inflammatory and PC phenotypesbetween populations of mice raised in different housing facilities,we next sought to determine whether the intestinal microbiotavaried between groups. Deep sequencing of the V6 region of the16S rRNA gene was performed on stool pellets and ileal tissuecollected from Irgm1 KO and WT mice reared in both housingconditions. Interestingly, principal coordinates analysis (PCoA)using Bray–Curtis dissimilarity showed no clustering of microbialcommunities based on Irgm1 status; however, distinct differencesbetween microbial communities from SPF and CV mice wereobserved in both stool and ileal tissue compartments (Fig. 5A,D).This is depicted statistically in Table 1. As indicated, the Irgm1genotype did not significantly impact microbial composition instool or ileal samples for the first three PCoA axes, based on theirP-values corrected for false discovery rate (FDR) (Table 1A). Incontrast, housing conditions did significantly influence microbialcomposition for the first two PCoA axes in both stool and ilealcompartments (Table 1B). Notably, similar results were foundthrough a parallel analysis using the QIIME platform (http://qiime.org/) (Fig. S1A).In addition to its effects on PCoA clustering (β-diversity), the

housing environment also significantly impacted the richness of theileal microbiota, as measured by observed operational taxonomic units(OTU) numbers (Fig. 5B,E) and the Chao1 index (Fig. 5C,F).Specifically, CV mice had a decreased richness of their ilealmicrobiota (FDR=0.024) when compared to SPF mice. Therichness of the fecal microbiota between these two groups wassimilar (FDR>0.66). Again, Irgm1 gene status did not appear to

influence the richness of the ileal (FDR=0.817) or fecal (FDR=0.375)microbiota. Similar results were obtained using the QIIME platform(Fig. S1B,C). In total, these findings suggest that environmentalfactors, as opposed to Irgm1 expression, are the primary drivers of gutmicrobial composition and diversity in these animals.

Microbial influences modulate the PC morphology in Irgm1-deficient miceGiven our finding that SPF and CV housing facilities supportmarkedly distinct enteric bacterial communities in both KO andWT mice, we speculated that a microbial driver is responsible for thedifferences in PC phenotype we observed in these two environmentalconditions. To test this hypothesis, we rederived our Irgm1 colonyinto GF conditions and assessed their PCs for both morphology andAMP expression. Similar to the SPF colony (but in contrast to CV-raised mice), there were no significant differences in PC number,location and granule size between WT and KO mice (Fig. 6A-D).Intriguingly, however, we found that GF KO mice showed increasedmRNAexpression ofLyz andDefa20 compared toWTmice (Fig. 6E,F). These findings suggest that the gut microbiota influences PCmorphology in Irgm1-deficient mice, though there appears be aneffect of Irgm1 itself on gene expression of AMPs.

H. hepaticus is not sufficient to reconstitute theinflammatory phenotype of conventional Irgm1-deficientmiceThe observed differences in gut-bacterial communities in CV versusSPF mice also allowed us to generate hypotheses regarding theimpact of specific bacterial groups on DSS susceptibility in Irgm1-deficient animals. Specifically, we were able to determine whichbacterial groups were increased in relative abundance in CV versus

Fig. 3. Histologic and functional Paneth cell measurements of specific pathogen-free Irgm1−/− and wild-type mice. (A) Representative ileal tissuesections from male specific pathogen-free (SPF) wild-type (WT) and Irgm1−/− (KO) mice (Lyz IHC, 20× magnification). (B) Number of Lyz+ cells per histologiccrypt/villus unit. (C) Number of ectopic Lyz+ cells existing outside the base of the crypt per crypt/villus unit. (D) Average individual PC granule size. (E,F) Results ofquantitative RT-PCR measurement of ileal tissue transcript levels of the antimicrobial peptides Lyz and Defa20 normalized to β-actin. n=6-12 mice/group,**P<0.008; error bars indicate s.e.m.; N.S., not significant.

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SPF housing conditions. A complete list of these organisms can befound in Table S1. One bacterial group of particular interest that wasincreased in the CV versus SPF samples was theHelicobacter genus(Fig. 7A). Because various Helicobacter spp. have been shown toenhance intestinal inflammation in numerous murine models ofcolitis (Chichlowski and Hale, 2009), we sought to determinewhether this organism could be a driver of the inflammatoryphenotype observed in our CV Irgm1 KO mice.Quantitative PCR testing for specific Helicobacter groups

identified H. hepaticus as the most abundant species in the stoolsamples of CV mice. Therefore, we next assessed whetherH. hepaticus colonization of SPF Irgm1 KO mice would enhancetheir inflammatory response to treatment with DSS. To accomplishthis, we colonized SPF Irgm1 KO and WT mice with H. hepaticus,administered DSS, and then assessed for intestinal inflammation. Inthese studies, we found no differences in weight loss betweenDSS-treated Irgm1 KO and WT mice that had been pre-infectedwith H. hepaticus (Fig. 7B). Moreover, all experimental groupsdeveloped similar histologic inflammation in both the ileum and thecolon (Fig. 7C,D). Intriguingly, contrary to our original hypothesis,less inflammation was seen in the cecum of KO mice receiving

H. hepaticus+DSS, as compared to similarly treated WT animals.Regardless, the introduction of H. hepaticus was not sufficient torecrudesce the inflammatory phenotype previously shown in CVKO mice.

DISCUSSIONTo date, genome-wide association studies (GWAS) have linked 206genetic loci to the development of IBD (Ellinghaus et al., 2016).However, the cumulative disease risk described by these studies is∼25% (Denson et al., 2013), a relatively small fraction of overallIBD heritability. Moreover, the concordance rates for IBD inhomozygous twins is ∼50% for CD and ∼20% for ulcerative colitis(Halfvarson et al., 2003). These findings suggest that environmentalinfluences play a key role in dictating the penetrance of IBDphenotypes. In this present study, we demonstrate that housingconditions modulate the susceptibility of Irgm1-deficient mice tointestinal injury. Housing environment also interacts with Irgm1 toregulate PC morphology and function. These findings are essentialknowledge for investigators using the Irgm1−/− mouse model andset the stage for further understanding the cross talk between theenvironment and Irgm1 to regulate mucosal homeostasis.

Fig. 4. Goblet cells and intestinal epithelialproliferation in specific pathogen-free Irgm1−/−

and wild-type mice. Male specific pathogen-free(SPF) wild-type (WT) and Irgm1−/− (KO) mice wereadministered 3% DSS in drinking water or drinkingwater alone (control) for 7 days. (A) Representativeileal tissue sections from DSS-treated SPF WT andKOmice (PAS, 20×magnification). (B) Representativeileal tissue sections from DSS-treated SPF WT andKOmice (Ki67 IHC, 20× magnification). (C) Number ofgoblet cells per crypt/villus unit, based on PASstaining. (D) Number of proliferating epithelial cells percrypt/villus unit, based on Ki67 staining. n=4-5 mice/group; error bars indicate s.e.m.

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Although numerous IBD susceptibility genes have beenidentified, the precise mechanisms by which these moleculesenhance disease risk remain unclear. In vitro studies have shownthat human IRGM regulates key molecular processes, includingmitochondrial fission (Singh et al., 2010), autophagy (Chauhanet al., 2015) and the clearance of intracellular pathogens (Kim et al.,

2012). Limited studies of murine Irgm1 demonstrate similarfunctions of this mouse homologue. Specifically, mouse Irgm1localizes to mitochondria and regulates fission (Henry et al., 2014).Irgm1 is also a mediator of IFNγ-induced autophagy and promotesthe clearance of intracellular bacteria in cultured cells. Despite thisin vitro knowledge, however, our understanding of how IRGM (andIrgm1) regulate intestinal inflammation in vivo is unclear.

We previously reported that Irgm1-deficient mice raised in CVhousing conditions develop extensive intestinal injury after DSSexposure, as compared to WT mice (Liu et al., 2013). Specifically,DSS-treated CVKOmice develop more-severe disease in the ileum,middle colon, distal colon and cecum relative to WT animals. Incontrast, the present study demonstrates that ‘cleaner’, DSS-treatedSPF KO mice only incur more significant inflammation in the distalcolon. Importantly, there is no increased susceptibility to ileitis inSPF KO mice treated with DSS. These data suggest that anenvironmental component modulates DSS responses in Irgm1-deficient animals.

A potential mechanism by which the environment modulatesDSS susceptibility is via the enteric microbiota. It is well establishedthat environmental influences can affect the composition andfunction of the gut microbiota (Ivanov et al., 2008; Thoene-Reinekeet al., 2014). Moreover, variations in the enteric microbiota caninfluence the severity of DSS colitis (Brinkman et al., 2013;Nagalingam et al., 2011). Therefore, we characterized gut bacterial

Fig. 5. Gut microbial composition and diversity differ based on housing conditions, but are not significantly influenced by genotype. (A,D) Principalcoordinates analysis of stool and ileal microbial composition of wild-type (WT) and Irgm1−/− (KO) mice in conventional (CV) and specific pathogen-free(SPF) conditions based on Bray–Curtis dissimilarity statistic. Richness measured using observed OTUs (B,E), and Chao1 index (C,F). n=11 CVWT, 11 CV KO, 7SPF WT, 10 SPF KO. *FDR=0.024.

Table 1. Effects of Irgm1 genotype and housing conditions on gutbacterial composition

Stool Ileum

Axis Standard FDR-corrected Standard FDR-corrected

A. Genotype effects1 0.6022 0.8173 0.8112 0.82322 0.3373 0.8173 0.8054 0.82323 0.6539 0.8173 0.1181 0.2952B. Housing effects1 0.0144 0.0361 0.0007 0.00182 1.62×10−10 8.09×10−10 0.0003 0.00133 0.7433 0.7433 0.5010 0.8349

Standard and false discovery rate (FDR)-corrected P-values are shown for thefirst three axes of principal coordinates analysis of stool and ileal microbialcomposition of (A) wild-type versus Irgm1-deficient mice, and (B) conventionalversus specific pathogen freemice, based onBray–Curtis dissimilarity statistic.n=11 CV WT, 11 CV KO, 7 SPF WT, 10 SPF KO.

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communities in WT and KO mice in both CV and SPF housingconditions. Remarkably, we found no impact of Irgm1 itself onglobal enteric microbial composition in either CV or SPFenvironments. This is intriguing in light of previous worksuggesting IRGM can promote the clearance of intracellularbacteria, such as Mycobacterium tuberculosis (Singh et al., 2006).That said, it is unclear how defects in intracellular bacterial killingaffect the composition of the luminal/mucosal commensalmicrobiota, which is the focus of the present study. Such defectsmay be more relevant to pathogenic organisms, such as Listeriamonocytogenes (Collazo et al., 2001) and Salmonella typhimurium(Henry et al., 2007), which do result in increased disease andbacterial burdens in Irgm1−/− mice. Additionally, Irgm1 may beprimarily involved in mediating inflammatory responses to entericbacteria, rather than shaping the composition of those bacteria.Despite the lack of a genotype effect, our microbiota analyses diddemonstrate the gut microbiota of mice reared in CV conditionsdiffered profoundly from those in SPF housing. This supports thepossibility that the enteric microbiota may influence the variableinflammatory phenotypes exhibited between CV and SPF KO micein response to DSS.A complete list of gut bacterial taxa exhibiting differential

abundances in CV versus SPFmice is shown in Table S1. Of interestis the Helicobacter genus, which is increased in CV mice comparedto SPF counterparts. Numerous Helicobacter species have beenshown to induce intestinal inflammation in susceptible mice, themost common of which include H. hepaticus and H. bilis(Chichlowski and Hale, 2009). By using species-specific, targetedqPCR, we identified H. hepaticus as the primary Helicobacterspecies present in our CV housing facility. This organism is a

known trigger of intestinal inflammation in murine IBD models,such as the IL10-deficient mouse (Kuhn et al., 1993; Kullberg et al.,1998). Based on these findings, we postulated that H. hepaticusinfection is able to recapitulate an inflammation-susceptiblephenotype in our SPF Irgm1 KO animals.

In contrast to our hypothesis, however, we found that H. hepaticusinoculation was not sufficient to increase the susceptibility of SPFIrgm1 KO mice to DSS-induced intestinal injury over their WTcounterparts. There are numerous putative explanations for thisfinding. First, it is possible that H. hepaticus is not the sole organismresponsible for the increased susceptibility of CV Irgm1 KO miceto DSS injury. Additional microbes, perhaps in concert withH. hepaticus, might be required to drive the inflammatoryphenotype of CV KO animals. Indeed, previous work has shownthat the composition of the commensal microbiota can regulate hostinflammatory pathways that drive intestinal disease in H. hepaticusmodels of colitis (Nagalingam et al., 2013). A second possibleexplanation for these findings is that the strain ofH. hepaticus used inour study might not be identical to that found in the CV animals.Ample evidence exists supporting the concept that different bacterialstrains within a given species differ in their ability to induce colitis inanimal models of IBD (Moran et al., 2009). However, it should benoted that the specific H. hepaticus strain used in this study (MU-94)has been shown to induce intestinal inflammation in susceptiblemouse strains (Cook et al., 2014; Livingston et al., 2004). Finally, italso possible that the increased susceptibility to intestinalinflammation observed in CV Irgm1 KO mice is not due to atransmissible microbial factor. Numerous environmental factors havebeen implicated in IBD pathogenesis (i.e. smoking, nonsteroidal anti-inflammatory drugs, appendectomy, diet, stress), and it is not clear

Fig. 6. Histologic and functional Paneth cell measurements of germ-free Irgm1−/− and wild-type mice. (A) Representative ileal tissue sections from germ-free (GF) wild-type (WT) and Irgm1−/− (KO) mice (Lyz IHC, 20× magnification). (B) Number of Lyz+ cells per histologic crypt/villus unit. (C) Number ofectopic Lyz+ cells existing outside the base of the crypt per crypt/villus unit. (D) Average individual PC granule size. (E,F) Results of quantitative RT-PCRmeasurement of ileal tissue transcript levels of the antimicrobial peptides Lyz andDefa20 normalized to β-actin. n=6-12mice/group, **P<0.005; error bars indicates.e.m.; N.S., not significant.

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whether these exert an impact on intestinal inflammation via the gutmicrobiota (Frolkis et al., 2013). Similarly, our housing facilitiesvaried in regards to food, water, bedding and caging (Table S2).While it is likely that these differences may contribute to microbialvariations between the colonies, it is also possible that they mayimpact host inflammatory responses through amicrobial-independentmechanism.In addition to its impact on inflammation susceptibility, housing

environment also appears to interact with Irgm1 to influence PCmorphology and AMP transcription. We have shown that, in CVhousing, Irgm1-deficient mice display multiple PC abnormalitiesincluding increased PC numbers, ectopic PCs, decreased PCgranule size and decreased transcript levels of specific AMPs (Lyzand Defa20) (Liu et al., 2013). In the present study, Irgm1 KO micehoused in SPF conditions continued to demonstrate increased PCnumbers per crypt and a trend towards increased ectopic PCs.However, their granule morphology was identical to that of WTmice. Moreover, differences in Lyz and Defa20 mRNA expressionbetweenWT and KOmice were abrogated in SPF conditions. Giventhe differences in bacterial communities between our CV and SPFfacilities, these data suggest that the gut microbiota modulate PCfunction in Irgm1-deficient mice.In order to dissect the relative contributions of genotype (Irgm1

status) and the microbiota to PC regulation in this model, we nextrederived Irgm1-deficient mice and WT littermates in GF housingconditions. Elimination of the microbiota resulted in complete lossof the morphological PC differences between WT and KO mice.Specifically, GF WT and KO mice displayed similar PC numbers,location and granule morphology. Intriguingly, transcriptexpression of Lyz and Defa20 were increased in GF KO mice

compared to their WT counterparts. Historically, these AMPs werenot thought to be transcriptionally induced (Bevins and Salzman,2011). For the α-defensins, however, elegant work by Wehkampand colleagues has demonstrated transcriptional regulation of thesemolecules through Wnt signaling [via Tcf-1 (Beisner et al., 2014)and Tcf-4 (Wehkamp et al., 2007)]. Indeed, polymorphisms of theTCF-4 promoter have been associated with ileal CD (Koslowskiet al., 2009). It is possible that the Wnt/β-catenin/Tcf pathway isupregulated in Irgm1-deficient mice, and future experiments areplanned to explore this possibility. Presently, however, we cansurmise that these findings contrast with the decreased Defa20 andLyzmRNA expression observed in CV KOmice, and the equivalentAMP expression shown between SPF KO mice and their WTcounterparts. This suggests that the gut microbiota influences themanner in which Irgm1 regulates AMP expression.

To date, the precise microbial constituents that regulate PCphenotype in the Irgm1-deficient mouse model remain unclear.Previous work, studying the autophagy-related CD risk alleleATG16L1, has demonstrated that Atg16l1 hypomorph mice have asimilar PC phenotype to our CV Irgm1 KO animals. Specifically,Atg16l1HM mice possess dysmorphic PCs with reduced numbers oflysozyme granules (Cadwell et al., 2008, 2009). Remarkably, thisabnormal PC phenotype is dependent on a distinct murine norovirusstrain (MNV-CR6) (Cadwell et al., 2010). Notably, murinenorovirus is excluded from our SPF mouse colony and, hence,SPF Irgm1 KO mice are not exposed to this microbe (Table S3).Future studies will attempt to recapitulate a dysmorphic PCphenotype in Irgm1 KO mice by colonizing with the MNV-CR6strain. Intriguingly, we did observe subtle differences in the ilealgoblet cells of SPF Irgm1−/− mice. Although goblet cell numbers

Fig. 7. Clinical and pathologic response of H. hepaticus-infected Irgm1−/− mice to treatment with DSS. (A) Relative abundance of Helicobacter species instool and ileal samples of untreated conventional (CV) and specific pathogen-free (SPF) mice. *FDR<0.009 (ileum); FDR=0.125 (stool). n=11 CV wild-type (WT),11 CVKO, 7 SPFWT, 10 SPF KO. For B-D, male SPFWTand KOmicewere infected withH. hepaticus (HH) for 6 weeks, followed by administration of 3%DSS indrinking water for 7 days. (B) Weight loss shown as a percentage of initial weight prior to DSS treatment, P>0.1 (two-way ANOVA). (C-D) Average histologicinflammation scores in the ileum and large intestine. For B-D, values are means±s.e., combined from two separate studies. Cohort sizes were in the range of 7-14mice/group, *P<0.05; error bars indicate s.e.m.; N.S., not significant.

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were similar between WT and KO mice, the increased intensity ofPAS staining in KO animals could indicate differences in the mucincomposition of these cells. This will also be explored further infuture studies.The findings presented in this study demonstrate that environmental

conditions interact with Irgm1, putatively through the entericmicrobiota, to direct PC function and host-susceptibility to intestinalinjury. This might be a critical consequence of the role Irgm1 plays inregulating autophagy. An established function of autophagy is to assistin the clearance of certain intracellular organisms (Levine et al., 2011).This antimicrobial process, known as xenophagy, can be triggered bypathogen-associatedmolecular patterns (Deretic et al., 2015). As such,amechanism is required to transmit microbial inputs to the initiation ofxenophagic processes. Recently, human IRGM has been shown todirectly interact with the pattern-recognition receptor NOD2, forminga core complex that can regulate autophagy in response to microbialproducts (Chauhan et al., 2015). The interaction between theenvironment and Irgm1 demonstrated in the present study maysupport a similar role for this murine homologue. It is possible thatIrgm1 serves to integrate environmental inputs, such as themicrobiota,into signals that can regulate autophagy within host cells. While acomprehensive evaluation of the impact of environment on autophagyin Irgm1-deficient mice is beyond the scope of the present study,future work will be directed at dissecting the mechanistic aspects ofthis putative Irgm1 function.Overall, the findings described in this study highlight the

importance of the Irgm1-deficient mouse model for the study ofhuman CD. The pathogenesis of CD is multifactorial, involvingboth environmental and host factors that interact to drive disease in asusceptible individual. Irgm1 KO mice appear to display a similarphenotype, in which environmental influences are able to modulatethe impact of Irgm1 deficiency on PC morphology andinflammation susceptibility. The findings presented also parallelwork in human CD, which has demonstrated that abnormal PCmorphology in ileal biopsies may predict a more-rapid time torecurrence in patients with CD (VanDussen et al., 2014). Althoughdirect causality between dysmorphic PCs and susceptibility to DSShas not been shown in the Irgm1 KO model, a similar phenomenonis observed in these mice: CVKOmice with abnormal PCs are moresusceptible to DSS-mediated injury, while SPF KO mice haverelatively normal PCs and less-severe responses to DSS.In summary, our present study characterizes novel aspects of the

Irgm1−/− mouse model that are important to the scientificcommunity and are highly relevant to the understanding of humanCD. In particular, environmental conditions strongly impact theinfluence of Irgm1 on PC and inflammatory phenotypes in themouse small intestine. In contrast, Irgm1 itself does not appear toregulate gut microbial composition. Future work using this modelmay now focus on how, mechanistically, abnormal PCs drive thisincreased susceptibility to intestinal injury. Importantly, the role ofenvironmental factors in this process may also be elucidated.Characterizing these gene-environment interactions will enhanceour understanding of the role of IRGM in human CD and,ultimately, promote the development of novel preventative andtherapeutic approaches for these patients.

MATERIALS AND METHODSMiceAll animals used in this study were Mus musculus, C57BL/6, sex-matched,adult (2-4 months old) mice. SPF and GF Irgm1 heterozygous mice wererederived from mice housed in CV conditions (Liu et al., 2013; Collazoet al., 2001), by implantation of two-cell stage embryos into WT SPF or GF

pseudopregnant females. Heterozygous mice were then mated to produceIrgm1 KO and WT littermate animals for experimental use. Husbandrydetails for each facility are provided in Table S2. Importantly, our SPFfacility excludesHelicobacter spp. and murine norovirus. A complete list oforganisms excluded in our CV and SPF facilities is shown in Table S3. Allmice were housed under AAALAC-accredited facilities and IACUC-approved protocols at the Durham VA Medical Center, Duke UniversityMedical Center, and the University of North Carolina Medical Center,respectively.

DSS-induced intestinal injuryAcute intestinal injury was induced as previously described (Liu et al.,2013). Briefly, male mice received 3% (w/v) dextran sodium sulfate (DSS)(MP Biomedicals, #160110) in their drinking water for 7 days. Control micereceived drinking water without DSS. Mice were weighed and fecescollected on days 0, 3, 5, 6 and 7. Fecal blood and stool consistency wereassessed at each time point as previously described (Liu et al., 2013). Micewere killed on day 7, and their intestinal tissues collected for analysis.Colons were removed and length measured prior to being openedlongitudinally and content removed. Colons were then rolled in a Swiss-roll fashion. Ceca were also opened longitudinally, their content removedand rolled for histology. Ileal tissue was opened longitudinally, grosscontents were removed and the remaining tissue fixed flat prior to paraffinembedding. Tissues bound for histologic analysis were fixed in 10%formalin solution for 48 h prior to paraffin embedding. Sections were cut(5 µm) and stained with hematoxylin and eosin (H&E). Histologicinflammation was scored in a blinded fashion using validated systems forileum (Moran et al., 2009) and DSS-induced colitis (Erben et al., 2014).

ImmunohistochemistryImmunohistochemistry (IHC) was performed for Ki67 and Lyz using 5 µm,formalin-fixed, paraffin-embedded ileal tissue sections as described above.De-paraffinization was accomplished by using fresh xylene, followed by re-hydration via a series of ethanol dilutions. Heat-induced epitope retrieval wassubsequently utilized for antigen retrieval (ThermoFisher, #TA-135-HBL).This was followed by treatment with 3% hydrogen peroxide to quenchendogenous peroxidase activity. Blocking was performed with 10% normalgoat serum. After sections were prepared in this fashion, they were incubatedovernight at 4°C with either anti-Ki67 (1:250, Vector Laboratories, #VP-RM04) or anti-Lyz (1:100, Diagnostic Biosystems, #RP028) rabbit primaryantibodies. Sections were next incubated for 1 h at room temperature with abiotinylated goat anti-rabbit IgG secondary antibody (1:500, JacksonImmunoResearch, #111-065-144). Signal amplification was accomplishedusing the Vectastain Elite ABC-HRP Kit per manufacturer’s instructions(1:50, Vector Laboratories, #PK-6100). Finally, staining was visualized withDAB chromoreagent (ThermoFisher, #TA-125-QHDX).

Epithelial cell analysesEpithelial cell subtypes were enumerated in a blinded fashion and reportedas number of cells per crypt-villus unit. To be included in the analysis, acrypt-villus unit was required to contain a properly oriented crypt (full cryptwith lumen visible in cross section) and visibly adjacent villi. At least 10crypt-villus units were evaluated for each mouse. Paneth cells (PCs) wereenumerated by counting the number of Lyz IHC positive cells within ahistologic crypt-villus unit. Ectopic PCs were recorded as the number of PCsidentified outside their normal position within the crypt base. PC granulesizes were measured on H&E stained sections, using the ellipse tool inImageJ version 1.48 software (NIH). At least 70 granules from varyingregions were measured for each mouse. Goblet cells were enumerated percrypt-villus unit using periodic-acid Schiff (PAS) staining (ThermoFisher,#SS32-500), which readily identifies deep purple cells with classic gobletcell morphology. Finally, proliferating epithelial cells were assessed bycounting the number of Ki67 IHC positive cells per crypt-villus unit.

Quantitative RT-PCRIleal tissues were collected into RNAlater solution (Qiagen, #76104) forsubsequent RNA isolation. Total RNA was extracted from flushed ileal

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tissue using an RNeasy Mini Kit per manufacturer’s instructions (Qiagen,#74104). Complementary DNAwas generated using SuperScript II ReverseTranscriptase (Invitrogen, #18064014). Quantitative reverse-transcriptasepolymerase chain reaction (RT-PCR) was conducted for Lyz (AppliedBiosystems, Mm00657323_m1) and Defa20 (Applied Biosystems,Mm00842045_g1). These specific AMPs were chosen because they havepreviously been shown to differ between Irgm1KO andWTmice (Liu et al.,2013). Each sample was run in triplicate via quantitative RT-PCR usingTaqMan Gene Expression Master Mix and the appropriate primer/probe setfor the gene of interest (ThermoFisher Scientific, #4369016). Results wereanalyzed by using the comparative threshold cycle (ΔΔCt) method,normalized against β-actin (Applied Biosystems, Mm02619580_g1) andcompared with the baseline WT group.

Deep sequencing of gut-bacterial communitiesBacterial DNA was extracted from 100 mg of frozen tissue or feces aspreviously described (Shanahan et al., 2014). Briefly, samples wereincubated in a sterile lysis buffer [200 mM NaCl, 100 mM Tris pH 8.0,20 mM EDTA, 20 mg/ml lysozyme (Sigma-Aldrich, #12671-19-1)] for30 min at 37°C. Lysis was completed by addition of 40 µl of proteinase K(20 mg/ml) and 85 µl of 10% SDS to the mixture, and incubation for anadditional 30 min at 65°C. Samples were next homogenized by bead beatingfor 2 min using 300 mg/sample of 0.1 mm zirconium beads (BioSpecProducts, #11079101Z). DNAwas extracted from the resultant supernatantsusing phenol/chloroform/isoamyl alcohol (25:24:1), and precipitated withabsolute ethanol for 1 h at −20°C. Precipitated DNAwas purified by usinga DNeasy Blood and Tissue extraction kit (Qiagen, #69504) permanufacturer’s instructions.

The purified DNA samples were used to construct an Illumina V6 16SrRNA library for deep sequencing, as previously described (Arthur et al.,2012). Briefly, an initial PCR was performed using barcoded universal V6primers (Table S3). This allowed for amplification of the V6 region, withsimultaneous addition of unique barcodes for multiplex analyses. 15 μl ofthis PCR product was then used in a second PCR reaction using PCRF1/PCRR1 primers (Table S4), to add Illumina flow cell adaptor sequences tothe final product. The final PCR amplicons were purified using a QiagenPCR Purification Kit and visualized on a 1.5% agarose gel to confirm sizeand purity. Finally, equal amounts of DNA from each sample were pooled(final concentration 21 ng/μl) and subject to 100 bp paired-end readsequencing, using the Illumina HiSeq2000 platform.

Analysis of 16S rRNA sequencesA total of 88,015,334 paired-end reads were generated and processed asdescribed previously (Arthur et al., 2014). Forward and reverse reads weremerged using a minimum of 70 continuous matching bases between them,which resulted in 48,999,102 merged sequences for the current study,representing 78 samples. We used AbundantOTU+ v.0.93b (http://omics.informatics.indiana.edu/AbundantOTU/out+.php) with the ‘-abundantonly’option to cluster these sequences into 1468 Operational Taxonomic Units(OTUs), incorporating 99.86% of the input sequences. UCHIME (http://www.drive5.com/uchime/) (Edgar et al., 2011) and the Gold referencedatabase were used to screen for the presence of chimeras in our OTUsequences, and a total of eight OTUs were removed. The remaining 1460OTUs were used for downstream analysis. Taxonomic assignments weredone using uclust through QIIME assign_taxonomy.py (Caporaso et al.,2010). A parallel analysis using QIIME v.1.9.1 was also conducted,utilizing the close-reference OTU picking approach (at 97% similarity levelusing the Greengenes 97% reference dataset, release 13_8). We excludedOTUs that had ≤0.005% of the total number of sequences according toBokulich and colleagues (Bokulich et al., 2013).

PCoA plots were generated from both Bray–Curtis dissimilarity statisticon the normalized and log10 transformed reads (Arthur et al., 2014), andUniFrac after rarefying the counts to the minimum number of reads found inall samples (32,698 for Ileum and 78,957 for stool samples). Alpha diversity(observed OTU estimate and Chao1 diversity index) was calculated afterrarefying the raw counts to a depth of the minimum count in all samples(32,698 for Ileum and 78,957 for stool samples). We used a linear mixed-effects model utilizing the package nlme_v. 3.1-128 in R (v. 3.3.1) (http://

www.R-project.org/) to analyze the data and account for possiblecontributions that may arise from co-housing the mice in the same cage.In our model, cage was modeled as a random effect and both Irgm1 statusand housing condition as fixed effects (McCafferty et al., 2013). P-valuesare reported from the linear mixed-effects model using F-test. We controlledfor false discovery rate (FDR) by correcting the P-values using theBenjamini and Hochberg (BH) approach (Benjamini et al., 2001).

Helicobacter studiesTesting of fecal matter for Helicobacter colonization was conducted viacommercial PCR assays utilizing Helicobacter genus-level primers,followed by speciation using species-specific primers (IDEXXBioResearch, Columbia, MO). For H. hepaticus colonization experiments,the MU-94 strain was obtained as a kind gift from Dr Robert Livingstone(IDEXX BioResearch). SPF mice received 1×108 colony forming units(CFU) of H. hepaticus suspended in 200 μl of Brucella broth that wasgavaged on three separate occasions spaced 3-4 days apart (Livingston et al.,2004). After each gavage dosing, an additional 1 ml of H. hepaticus-infected broth was added to the floor of each cage. Helicobactercolonization using this protocol was confirmed by PCR. Mice were heldfor 6 weeks before being exposed to a 7-day DSS treatment and then killedas described above.

Statistical analysisAll statistical analyses, other than sequencing analysis described above,were performed using Graphpad Prism version 6 (Graphpad software Inc.,San Diego, CA). Results are expressed as standard error of the mean (s.e.m.).Clinical data were analyzed using two-way analysis of variance (ANOVA)with other comparisons analyzed using the Kruskal–Wallis and Mann–Whitney tests. A P<0.05 was considered significant.

AcknowledgementsWe are grateful to Dr Robert Livingstone for providing the H. hepaticus used in ourcolonization experiments. We also appreciate the assistance of the UNC Center forGastrointestinal Biology and Disease gnotobiotic (Maureen Bower) and histology(Carolyn Suitt) cores. Immunohistochemistry services were provided by theHistology Research Core Facility in the Department of Cell Biology and Physiology atthe University of North Carolina, Chapel Hill NC (Ashley Ezzell).

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: A.R.R., A.A.F., R.B.S., G.A.T., A.S.G.; Methodology: A.R.R.,A.A.S., M.J.J., S.R., G.A.T., A.S.G.; Formal analysis: A.R.R., A.A.S., B.E.F., V.A.C.,M.J.J., R.Z.G., S.R., A.A.F., G.A.T., A.S.G.; Investigation: A.R.R., A.A.S., B.E.F.,V.A.C., G.A.T., A.S.G.; Writing - original draft: A.R.R., G.A.T., A.S.G.; Writing - review& editing: R.Z.G., S.R., A.A.F., R.B.S., G.A.T., A.S.G.; Supervision: G.A.T., A.S.G.;Project administration: G.A.T., A.S.G.; Funding acquisition: G.A.T., A.S.G.

FundingThis work was supported by the National Institutes of Health [P30 DK034987 andT32 DK007737 to R.B.S., R01 AI57831 to G.A.T., K08 DK095917 and R03DK104005 to A.S.G.]; and the U.S. Department of Veterans Affairs [BX002369 toG.A.T.].

Data availability16S rRNA sequences have been uploaded to the National Center for BiotechnologyInformation (NCBI) under BioProject ID PRJNA431281.

Supplementary informationSupplementary information available online athttp://dmm.biologists.org/lookup/doi/10.1242/dmm.031070.supplemental

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