NKT Cell–Deficient Mice Harbor an Altered Microbiota That Fuels

NKT Cell−Deficient Mice Harbor an Altered Microbiota That Fuels Intestinal Inflammation during Chemically Induced Colitis This information is current as of September 25, 2021. Thirumahal Selvanantham, Qiaochu Lin, Cynthia Xinyi Guo, Anuradha Surendra, Stephanie Fieve, Nichole K. Escalante, David S. Guttman, Catherine J. Streutker, Susan J. Robertson, Dana J. Philpott and Thierry Mallevaey

J Immunol published online 31 October 2016 Downloaded from http://www.jimmunol.org/content/early/2016/11/04/jimmun ol.1601410 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2016/10/31/jimmunol.160141 Material 0.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 4, 2016, doi:10.4049/jimmunol.1601410 The Journal of Immunology

NKT Cell–Deﬁcient Mice Harbor an Altered Microbiota That Fuels Intestinal Inﬂammation during Chemically Induced Colitis

Thirumahal Selvanantham,*,1 Qiaochu Lin,*,1 Cynthia Xinyi Guo,* Anuradha Surendra,†,‡ Stephanie Fieve,* Nichole K. Escalante,* David S. Guttman,†,‡ Catherine J. Streutker,x Susan J. Robertson,* Dana J. Philpott,* and Thierry Mallevaey*

NKT cells are unconventional T cells that respond to self and microbe-derived lipid and glycolipid Ags presented by the CD1d molecule. Invariant NKT (iNKT) cells influence immune responses in numerous diseases. Although only a few studies have examined their role during intestinal inflammation, it appears that iNKT cells protect from Th1-mediated inflammation but exacerbate Th2- mediated inflammation. Studies using iNKT cell–deficient mice and chemically induced dextran sodium sulfate (DSS) colitis have Downloaded from led to inconsistent results. In this study, we show that CD1d-deficient mice, which lack all NKT cells, harbor an altered intestinal microbiota that is associated with exacerbated intestinal inflammation at steady-state and following DSS treatment. This altered microbiota, characterized by increased abundance of the bacterial phyla Proteobacteria, Deferribacteres, and TM7, among which the mucin-eating Mucispirillum, as well as members of the genus Prevotella and segmented filamentous bacteria, was transmissible upon fecal transplant, along with the procolitogenic phenotype. Our results also demonstrate that this proinflammatory microbiota influences iNKT cell function upon activation during DSS colitis. Collectively, alterations of the microbiota have a major http://www.jimmunol.org/ influence on colitis outcome and therefore have to be accounted for in such experimental settings and in studies focusing on iNKT cells. The Journal of Immunology, 2016, 197: 000–000.

nflammatory bowel disease (IBD) is a spectrum of chronic and chemotaxis, such as NOD2, ATG16L1, and IL23R (2). How- disorders of the gastrointestinal tract characterized by re- ever, the functional relevance of most of these genetic variants I curring inflammatory bouts (1). Crohn disease (CD) and remains to be established. It is generally admitted that deregulated ulcerative colitis (UC) are the two major forms of IBD and T cell responses toward the intestinal microbiota are at the core of broadly differ in the spread and depth of inflammatory lesions, as IBD (3). Reciprocally, perturbations of host–microbiota homeo- by guest on September 25, 2021 well as their immune profile, broadly Th1 and Th2, respectively. stasis induced by the host genetics and/or environmental factors IBD has genetic and environmental components. More than 160 (e.g., diet, antibiotics) can alter the makeup of the microbiota and IBD susceptibility loci have been identified and comprise genes fuel inflammation at mucosal surfaces (1, 3). involved in pathways of innate and adaptive immunity, autophagy, NKT cells are unconventional T cells that respond to lipid and glycolipid Ags presented by the MHC class Ib molecule CD1d, and *Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, they generally express some activating and/or inhibitory receptors Canada; †Department of Cell and Systems Biology, University of Toronto, Toronto, found on bona fide NK cells. NKT cells can be divided into type 1 or Ontario M5S 1A8, Canada; ‡Centre for the Analysis of Genome Evolution and x invariant NKT (iNKT) cells (4, 5) as well as type 2 or diverse NKT Function, University of Toronto, Toronto, Ontario M5S 3B2, Canada; and Depart- ment of Laboratory Medicine and Pathology, University of Toronto and St. Michael’s (dNKT) cells (6), based on TCR diversity and lipid specific- Hospital, Toronto, Ontario M5B 1W8, Canada ity. iNKT cells respond to the prototypical glycosphingolipid 1T.S. and Q.L. contributed equally to this work. a-galactosylceramide (aGC) or related compounds and can be ORCIDs: 0000-0002-9771-1954 (T.S.); 0000-0003-2106-1147 (C.X.G.); 0000-0001- accurately identified using aGC-loaded CD1d tetramers (7, 8). 8479-3869 (D.S.G.). iNKT cells are innate lymphocytes capable of producing copious Received for publication August 12, 2016. Accepted for publication October 3, 2016. amounts of a large array of cytokines and chemokines only minutes This work was supported by a Crohn and Colitis Canada grant-in-aid of research, following stimulation, and they also exert cytotoxic functions (5). Canada Foundation for Innovation Physical Infrastructure Grant 29186 (to T.M.), and Based on their functional properties, iNKT cells modulate immune by a Canadian Institutes of Health Research grant (to D.J.P.). T.S. was supported by a Canadian Institutes of Health Research Banting and Best Doctoral Research Award. responses and have been reported to play protective or deleterious T.M. is supported by a Canada Research Chair in NKT Cell Immunobiology. functions during infection, autoimmunity, cancer, and inflamma- Address correspondence and reprint requests to Dr. Thierry Mallevaey, University of tion, and they therefore constitute an appealing target for immu- Toronto, Department of Immunology, Medical Sciences Building, Room 7308, 1 notherapy (4, 5). The biology of dNKT cells remains more elusive King’s College Circle, Toronto, ON M5S 1A8, Canada. E-mail address: thierry. [email protected] due to the paucity of reagents to track these cells. Some of these The online version of this article contains supplemental material. cells respond to self and/or microbial sulfated b-galactosylceramides Abbreviations used in this article: B6, C57BL/6; B6B6, C57BL/6 mice grafted with (9–11) or phospholipids (12, 13). Whether dNKT cells are innate or C57BL/6 cecal contents; B6CD1d, C57BL/6 mice grafted with CD1d KO cecal contents; more reminiscent of conventional T cells remains an open question. CD, Crohn disease; dNKT, diverse NKT; DSS, dextran sodium sulfate; aGC, a-galacto- Only a few studies have explored the role of NKT cells during sylceramide; IBD, inflammatory bowel disease; IEL, intraepithelial lymphocyte; iNKT, invariant NKT; KO, knockout; OTU, operational taxonomic unit; SFB, segmented intestinal inflammation, and most have focused on iNKT cells. It filamentous bacteria; UC, ulcerative colitis. appears that these cells protect from Th1-mediated inflammation Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 (14–17) (CD) but fuel Th2 type responses such as those typically

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601410 2 CD1d KO MICROBIOTA FUELS COLITIS seen in UC (18) and asthma (19), which is consistent with their colons fixed in 4% formalin, embedded in paraffin, and stained with H&E. overall yin-yang functions in disease. Using the T cell transfer colitis Colon histology was assessed for infiltration by immune cells and tissue model, Hornung et al. (14) showed that CD4+DX5+ T cells (a phe- damage, by a trained pathologist, as described by Smith et al. (36) notype reminiscent of NKT cells) could protect from colitis. In the Salmonella enterica serovar Typhimurium infection and dextran sodium sulfate (DSS) model, one study found that NKT cell– histological examination deficient (CD1d knockout [KO]) mice developed a similar colitis to Mice were treated with 20 mg of streptomycin following a 3-h fast. Twenty- that in control mice (15). However, another study reported that CD1d four hours later, mice were fasted for 3 h and then infected with 5 3 107 KO mice were more sensitive to colitis (17). The basis for this dis- CFU S. enterica serovar Typhimurium SL1344. Bacterial load was mea- crepancy is unclear, but it may relate to the controls used. What is sured in spleens or livers homogenized in 1% Triton X-100 and plated on more established is that pharmacological activation of iNKT cells 50 mg/ml streptomycin/MacConkey agar plates. Cecum histology was assessed on cecum fixed in 4% formalin, embedded in paraffin, and stained through injection of aGC or the Th2-biasing lipid OCH9 confers with H&E. H&E-stained, formalin-fixed cecum tissues were assessed for consistent protection in the DSS model (15, 16), revealing iNKT cells edema, epithelial erosion, neutrophil recruitment, and goblet cell deple- as a potential target for immunotherapies to treat IBD. Moreover, tion, as described by Geddes et al. (37, 38). iNKT cell–derived IL-13 mediates the development of intestinal in- Quantitative RT-PCR to analyze relative abundance of flammation during oxazolone-induced colitis, a Th2-driven model of bacterial groups intestinal pathology resembling human UC (18). Of note, a recent study demonstrated that deregulation of dNKT cells led to the de- The abundance of bacterial groups was analyzed as described by Robertson et al. (39). Briefly, total DNA was extracted from the colon fecal matter velopment of spontaneous colitis in mice (20). using the MO BIO Laboratories PowerSoil kit following the manufac- Vertebrates are colonized by complex communities of mutualistic turer’s specifications. Bacterial DNA was analyzed by quantitative RT-PCR Downloaded from organisms that reside on mucosal surfaces (lungs, intestines, and using 16S rDNA primers (Integrated DNA Technologies) for specific genitals) and on the skin and outnumber host cells and genes by bacterial groups (39). The relative abundance of each bacterial group was 2DDCt factors of 10 and 100, respectively. Although the term microbiota normalized to Eubacteria using the 2 method. classically refers to bacterial species, it also includes fungi, viruses, Manipulation of the microbiota by prenatal gavage bacteriophages, and other more elusive microorganisms (21, 22). We used a method adapted from Markle et al. (25). Timed pregnancies were Perturbations of the microbiota composition, a phenomenon called set using 7- to 8-wk-old B6 mice. Pregnant females were transferred into http://www.jimmunol.org/ dysbiosis, have been associated with several human diseases, in- clean cages and gavaged with cecal content from adult female B6 or CD1d cluding metabolic diseases (type I diabetes mellitus and obesity) KO mice. Briefly, the cecum of donor mice was dissected, opened along its (23) and IBD (3). Whether dysbiosis is a cause or a consequence of length, and the contents were transferred to a sterile tube and diluted 1:50 (v/v) in sterile water. Two hundred microliters of this suspension was given disease remains unclear. However, there is mounting evidence that to each recipient by oral gavage using 22- to 24-gauge rounded edge the transfer of altered microbiota into healthy mice through needles. Recipients were rested for 24 h, and this procedure was repeated cohousing, cross-fostering, or fecal transplant can transfer disease once. We think that the longitudinal transfer upon natural delivery allows in some cases (24–26). Importantly, fecal transplantation from for a greater stability of the altered microbiota in the newborn mice. These healthy donors has recently been shown to induce remission in UC mice were used at 7–8 wk of age. patients in a clinical trial (27). One mechanism by which the Ex vivo tissue culture by guest on September 25, 2021 microbiota can influence disease outcome is through the shaping of Mice were administered with 2% DSS in drinking water followed by regular the immune system, and individual bacteria or groups of bacteria water for 24 h. Proximal colon biopsy punches (2 mm) were cultures in have been shown to modulate the development and/or function of complete RPMI 1640 medium supplemented with 10% FCS for 48 h with immune cells, including Th17 cells (28), regulatory T cells (29), or without recombinant mouse IL-23 (10 ng/ml). IL-22 was analyzed using neutrophils (30), and more recently iNKT cells (31–34). a Ready-Set-Go! ELISA set purchased from eBioscience/Affymetrix. In this study, we describe that CD1d-deficient mice, which lack Microbiota 16S rRNA amplification and sequencing iNKT and dNKT cells, harbor an altered intestinal microbiota char- 16S rRNA sequencing was performed by the Centre for the Analysis of Genome acterized by the increased abundance of several proinflammatory Evolution and Function at the University of Toronto. The V4 region was bacterial groups. This altered microbiota is associated with a amplified using primers V4-515 forward (59-AATGATACGGCGACCACC- colitogenic phenotype at steady-state, which is exacerbated upon DSS GAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA-39)and treatment. We also provide evidence that the intestinal microbiota V4-806 reverse (59-CAAGCAGAAGACGGCATACGAGATXXXXXXXXX- modulates iNKT cell function upon activation during intestinal XXXAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT-39)(40).PCRre- actions were generated in triplicate for each sample using KAPA2G Robust inflammation. HotStart ReadyMix (Kapa Biosystems, Wilmington, MA) according to the manufacturer’s instructions. A template-minus control was also included for Materials and Methods each sample. Replicate PCRs for each sample were pooled. Equal quantities of Mice each pool were determined using the Quant-iT PicoGreen dsDNA assay kit (Thermo Fisher Scientific, Waltham, MA) and combined. The final pool was C57BL/6 (B6) mice were purchased from The Jackson Laboratory (Bar purified using Agencourt AMPure XP beads (Beckman Coulter, Mississaugua, Harbor, ME). CD1d2/2 mice were generated and provided by Dr. Chyung- ON, Canada) before paired-end sequencing. 16S rRNA sequencing was per- Ru Wang (Northwestern University Feinberg School of Medicine) (35). All formed on an Illumina MiSeq2 using V2 chemistry. animal procedures were approved by the Faculty of Medicine and Phar- macy Animal Care Committee at the University of Toronto (animal use Bioinformatic analysis of the microbiota composition protocols 20009889 and 20010032 to D.J.P., and protocols 20009158, The PANDAseq (41) assembler was used for the quality trimming and 20009741, and 20010379 to T.M.). Mice were housed in specific pathogen- assembly of the paired-end reads, with a minimum overlap of 25 nucle- free conditions at the Division of Comparative Medicine in the Centre for otides between the forward and reverse reads. The USEARCH (42) ref- Cellular and Biomolecular Research (University of Toronto). Unless erence method was used, along with the Greengenes database version 13.8 specified otherwise, 12- to 13-wk-old mice were used in all experiments. (43), for chimera detection and removal. The removed chimeric sequences Littermate and nonlittermate mice were used, as specified. were clustered at 97% identity using USEARCH in Quantitative Insights DSS-induced colitis and histological examination Into Microbial Ecology (44) version 1.8.0 against the Greengenes database version 13.8 (43). A representative sequence from each operational taxo- Mice were administered 2% DSS (MP Biomedical, Santa Ana, CA) in nomic unit (OTU) was selected and aligned by PyNAST (45) to the drinking water (w/v) for 7 d followed by regular water, unless specified Greengenes database version 13.8 (43). The Ribosomal Database Project otherwise. Mice were weighed daily. Histological scoring was performed on Bayesian algorithm, executed within Quantitative Insights Into Microbial The Journal of Immunology 3

Ecology, produced taxonomy assignment for each OTU. The aligned se- CD1d and/or iNKT cell deficiency, were dominantly influencing quence data were used to generate a phylogentic tree. The above phylogenetic intestinal inflammation in this model. tree was constructed with the FastTree (46) algorithm. An OTU table was To substantiate this finding, we studied B6 and CD1d KO non- created using taxonomy assignment and aligned representative sequences. +/2 2/2 Sequences that failed to align to the reference database were removed from littermate mice as well as CD1d and CD1d littermate mice the OTU table. Low-abundance OTUs with a fraction count ,0.005% were during intestinal inflammation driven by infection with S. enterica removed and the retained OTUs were used for downstream analysis (47). serovar Typhimurium. Pretreatment of mice with streptomycin al- Then, a diversity plots were generated and data were rarefied to a depth of lows Salmonella to trigger an acute colitis, and this model has been 40,000 based on the plots. Weighted UniFrac distances were calculated through the R phyloseq library and used for principal coordinates analysis. widely used to tease out bacterial and host factors that regulate inflammation. Using this model, we found that CD1d KO mice Statistical analysis developed a marginally yet significantly milder cecal inflammation Student t tests were used when two groups were compared. For four-group mainly characterized by reduced goblet cell depletion (Fig. 2C, 2 2 2 comparisons, we used one-way ANOVA tests with a Tukey multiple com- 2D). However, CD1d+/ and CD1d / littermate mice showed no parison tests to compare individual pairs. Statistical analyses were per- difference in intestinal inflammation that was also comparable to p , formed using GraphPad Prism. A value 0.05 was considered significant. that of the original CD1d KO mice (Fig. 2C, 2D). Additionally, bacterial translocation to the spleen, measured by CFUs, was sim- Results ilar between the four groups of mice (data not shown), suggesting CD1d-deficient mice are more susceptible to DSS-mediated that neither CD1d deficiency nor environmental factors in our ex- colitis perimental settings impact Salmonella infection and Salmonella- iNKT cells are present in many lymphoid (e.g., bone marrow, driven inflammation. Taken together, these observations suggested Downloaded from thymus, spleen, lymph nodes) as well as nonlymphoid (e.g., liver, that a dominant nongenetic factor present in CD1d KO mice lungs, skin) tissues. Consistent with previous studies (32, 33, 48), we influenced colitis susceptibility in a context-dependent fashion. detected iNKT cells in the small intestine, cecum, and colon lamina CD1d KO mice have an altered, dominant, and transmissible propria of B6 naive mice (Supplemental Fig. 1). However, we could microbiota not detect iNKT cells within the intraepithelial lymphocyte (IEL) compartment (data not shown). Nevertheless, we were able to detect The composition of the indigenous intestinal microorganisms is a http://www.jimmunol.org/ iNKT cells within the IEL population in Va14 transgenic mice (not major factor that can vary between wild-type and gene-targeted mice shown), which have increased numbers of iNKT cells, raising the and influence disease outcome. Variations of microbiota composition possibility that iNKT cells may be present at very low numbers can be due to a direct impact of genetic landscape variation or to within the small intestine IELs in B6 mice. stochastic longitudinal transmission that can result in ecological The role iNKT cells play during DSS-mediated colitis has been drifts across generations in adjacently caged animals (49, 50). previously studied but yielded inconsistent results (15, 17). In an Additionally, differences in microbiota composition have been as- attempt to clarify this controversy, we subjected mice deficient for sociated with many disease models, including colitis (3). To analyze the two CD1d genes (hereafter named CD1d KO) to a 2% DSS and compare the microbiota between our littermate and non- regimen. After 8 d, CD1d KO mice showed macroscopic signs of littermate mice, we quantified the relative bacterial abundance of by guest on September 25, 2021 sickness, such as lethargy, reluctance to move, and hunched pos- several bacterial groups in the colon of naive mice by quantitative ture, compared with B6 mice (Supplemental Video 1). Addition- real-time PCR using primers targeting 16S rDNA (39). Compared ally, CD1d KO mice had greater and faster weight loss (Fig. 1A) to B6 nonlittermate mice, CD1d KO mice had higher abundance of and shorter colons (Fig. 1B) compared with B6 mice. Finally, Bacteroides, Bifidobacterium,andLactobacillus but similar levels histological examination of H&E-stained colon sections revealed of mouse intestinal Bacteroides (Fig. 3). We also analyzed the mice more severe inflammation in CD1d KO mice (Fig. 1C, 1D). To- for the presence of segmented filamentous bacteria (SFB), which gether with previous work using both CD1d KO and Ja18 KO have been associated with increased Th17 (28) responses. Whereas mice (17), our data suggested that CD1d and CD1d-restricted SFB were virtually undetectable in B6 mice, they were consistently +/+ +/2 2/2 T cells, including iNKT cells, exerted protective functions dur- found in CD1d KO mice (Fig. 3). CD1d ,CD1d , and CD1d ing chemically driven intestinal inflammation. littermate mice were found to be similar to CD1d KO mice for the Bacteroides, Lactobacillus, and SFB groups, but not the Bifido- CD1d-deficient and -sufficient littermate mice have similar bacterium group (Fig. 3). Taken together, these findings revealed intestinal inflammation sensitivity that adjacently caged B6 and CD1d KO nonlittermate mice had The controversial role of iNKT cells in colitis could reflect different profound microbiota differences, and they suggested that the CD1d environmental conditions (e.g., housing conditions, diet). There- KO colitogenic microbiota was both transmissible and dominant. fore, we hypothesized that these discrepancies could be attributed to the use of nonlittermate control mice. Indeed, although the use of The CD1d KO microbiota is proinflammatory littermate mice with different genotypes is the best way to control To directly assess whether the differences in microbiota composition for environmental differences, nonlittermate control mice are often in CD1d KO mice were responsible for the increased colitis sen- used for practical as well as budgetary reasons. To test this hy- sitivity, we performed fecal transplant experiments. B6 pregnant pothesis, we crossed CD1d KO mice with B6 mice to generate dams were gavaged with diluted cecal contents from either B6 or CD1d+/2 mice that were subsequently crossed to generate CD1d+/+, CD1d KO female mice. Markle et al. (25) recently demonstrated that CD1d+/2,andCD1d2/2 littermate mice. These animals were then the gavage of weanlings resulted in microbiota alterations that were subjected to 2% DSS to trigger intestinal inflammation, as described durable until at least 11 wk of age. We reasoned that natural ma- above. Surprisingly, mice displayed identical weight loss regardless ternal transmission would result in durable engraftment of the al- of genotype (Fig. 2A). Additionally, histological examination of tered microbiota in newborn mice. Adult offspring from these H&E-stained colon sections revealed identical inflammation in all gavaged dams were subjected to 2% DSS in the drinking water ad mice studied (Fig. 2B). Interestingly, all mice showed increased libitum for 8 d, at which point they were switched to regular water. sensitivity compared with our original B6 mice (compare Figs. 1C, In the absence of DSS, B6 mice grafted with either B6 or CD1d KO 1D, 2A, 2B). This suggested that environmental factors, and not cecal contents (B6B6 and B6CD1d mice, respectively) had similar 4 CD1d KO MICROBIOTA FUELS COLITIS

FIGURE 1. CD1d-deﬁcient mice are more susceptible to DSS-mediated colitis. B6 and CD1d KO (KO) mice were treated with 2% DSS and were monitored for signs of colitis. The data show normalized daily weight measurements (A) as well as colon length (B) and colon histopathology (C and D) at day 8. The data depict mean values 6 SEM of two to four independent experiments. Representative images (original magniﬁcation 310) of H&E-stained colonsectionsofB6andCD1dKOmiceare shown (D). Mouse numbers are indicated. *p ,

0.05, **p , 0.01. Downloaded from http://www.jimmunol.org/

weight gain (Fig. 4A). However, although they also showed similar colons from DSS-treated B6CD1d mice failed to produce IL-22 initial weight loss upon DSS treatment, B6CD1d mice appeared to (Fig. 4D), a cytokine that is involved in the resolution of intesti- have delayed recovery upon DSS removal, compared with B6B6 nal inflammation and wound repair (51, 52). However, this defect mice (Fig. 4A). Additionally, B6CD1d mice had elevated colon could be corrected by the addition of IL-23, which is known to weight-to-length ratios (Fig. 4B) and worse pathological scores induce IL-22 production. Taken together, these results suggested (Fig. 4C) upon histological examination compared with B6B6 mice. that the microbiota found in CD1d KO mice was proinflammatory This indicates that colonization of mice by the CD1 KO microbiota and affected the resolution of inflammation. by guest on September 25, 2021 resulted in increased intestinal inflammation. B6CD1d mice also showed a trend toward elevated colon weight-to-length ratios and Fecal transplant creates a third state of microbiota pathological scores in the absence of DSS exposure, although the Next, we compared microbial communities between B6 and CD1d latter did not reach statistical significance. Finally, we found that KO mice pre- and post-transplant by sequencing bacterial 16S

FIGURE 2. CD1d-deficient and -sufficient littermate mice develop similar intestinal inflammation during chemical and S. typhimurium– induced colitis. (A and B)CD1d+/+,CD1d+/2, and CD1d2/2 littermate mice were treated with 2% DSS and were monitored for signs of colitis. The data show daily normalized weight measurements (A) as well as colon pathological scores at day 8 (B). The data show the mean 6 SEM of three independent experiments. (C and D) B6 mice, CD1d KO nonlittermate mice, as well as CD1d+/2 and CD1d2/2 littermate mice were infected orally with S. typhimurium SL1344. The degree of cecal histopathology was determined 3 d postinfection from an H&E- stained cecum section. The data show the mean values 6 SEM of the total pathological score (C) and the breakdown of epithelial damage, goblet cell depletion, neutrophil infiltration, and edema (D)oftwoindependentexperiments.*p , 0.05. The Journal of Immunology 5

FIGURE 3. CD1d KO mice have an altered, dominant, and transmissible microbiota. Rela- tive abundance of the indicated bacterial groups in the proximal colon of B6 and CD1d KO (KO) nonlittermate mice as well as CD1d+/+,CD1d+/2, and CD1d2/2 littermate mice. The data show individual values as well as the mean values 6 SEM for each group of mice. *p , 0.05, **p , 0.01, ***p , 0.001. MIB, mouse intestinal Bacteroides. Downloaded from

rRNA libraries prepared from proximal colon contents from B6 and controls. At the phylum level, we found that members of the CD1d KO nonlittermate mice as well as transplanted B6B6 and Proteobacteria, Deferribacteres, and the candidate group TM7 B6CD1d mice. Principal coordinates analysis revealed differences were elevated in CD1d KO and B6CD1d mice compared with their http://www.jimmunol.org/ in gut microbiota composition. Although B6 and transplanted respective controls (Fig. 5B, 5C). Among Proteobacteria, we B6B6 mice were essentially indistinguishable from each other, our found that only Betaproteobacteria and Deltaproteobacteria were analysis revealed that CD1d KO mice and transplanted B6CD1d elevated in CD1d KO and B6CD1d mice, whereas Alphaproteo- mice were different from their B6 and B6B6 counterparts, as well bacteria and Gammaproteobacteria remained unaffected (Fig. 5D as from each other (Fig. 5A). This suggested that transfer of the and not shown). Of note, Epsilonproteobacteria, including the CD1d KO microbiota into B6 mice only partly recapitulated the genus Helicobacter, were more abundant in CD1d KO mice original microbiota, and created a “third state” of microbiota, as compared with B6 mice, but these bacteria were not transmissible

previously reported (25). The results showed that the fecal trans- via fecal transplant (not shown). Deferribacteres contains the by guest on September 25, 2021 plant procedure in itself induced a shift in microbiota composition mucin-degrading Mucispirillum species, which were significantly characterized by an overall decrease in Bacteroidetes and increase elevated in CD1d KO and B6CD1d mice (Fig. 5E). Interestingly, in Firmicutes (Fig. 5B), although this shift did not parallel in- the other known group of mucin-eating bacteria Akkermansia creased DSS sensitivity. Next, we reasoned that proinflammatory were found abundantly only in B6B6 mice (Fig. 5F). Finally, al- bacterial groups may be similarly increased in CD1d KO and though overall levels of Bacteroidetes were not affected between B6CD1d mice and/or that anti-inflammatory bacterial groups may B6 and CD1d KO mice, members of the genus Prevotella were be decreased in these mice, compared with their respective significantly more abundant in CD1d KO mice and were transmissible

FIGURE 4. The CD1d KO microbiota is proinflammatory. B6 mice were colonized with B6 (B6B6)orCD1dKO(B6CD1d) microbiota and treated with or without 2% DSS for 8 d. The data show daily normalized weight measurements of (A) colon weight-to-length ratios (B) and colon histopathology at day 12 (C). (D) Colon biopsy punches were cultured with or without IL-23 (10 ng/ml) from 48 h, and IL-22 production was assessed by ELISA. The data show the mean 6 SEM of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. 6 CD1d KO MICROBIOTA FUELS COLITIS to B6 mice upon fecal transplant (Fig. 5G). Collectively, our Discussion analysis revealed profound differences in the microbiota found in Our study provides a possible explanation for the published dis- CD1d KO mice that could be partially transmitted into B6 re- crepancies regarding the function that CD1d-restricted T cells, in- cipient mice upon fecal transplant. cluding iNKT cells, play during DSS-mediated colitis (15, 17), and it emphasizes the need to use littermate control mice in these ex- Microbiota influences iNKT cell function during colitis perimental settings. We found that the microbiota has a major in- It was recently shown that the intestinal microbiota affects the fluence on experimental colitis outcome during DSS treatment, and phenotype and function of iNKT cells (31–34), including during to a lesser extent following S. typhimurium infection. Alternatively, Th2-driven airway and intestinal inflammation (33). Additionally, the mere absence of CD1d-restricted T cells appeared to have little pharmacological activation of iNKT cells through the injection of effect on inflammation in these models. Finally, our data showed aGC or OCH9 was shown to decrease the severity of DSS- that CD1d-deficient mice harbor a proinflammatory and transmis- mediated colitis (15, 16). We next assessed whether the protec- sible microbiota that also modulated iNKT cell function upon tive outcome from lipid-mediated iNKT cell activation would pharmacological activation during DSS-mediated colitis. differ based on microbiota differences. For this, B6B6 and B6CD1d Perturbations in the gastrointestinal microbiota have been mice were placed on 2% DSS for 8 d and at the onset of symptoms reported in CD and UC patients, generally characterized by a (day 7) received a single i.p. injection of either OCH9 or vehicle. reduction in ecological diversity, expansion of Gammaproteo- As described above, B6CD1d mice treated with vehicle experienced bacteria, and a contraction of Bacteroidetes and certain Firmicutes greater weight loss and delayed recovery compared with B6B6 (3). Additionally, studies comparing conventional and gnotobiotic mice (Fig. 6A), confirming the proinflammatory nature of the genetically susceptible mouse strains, as well as fecal transplant Downloaded from CD1d KO microbiota. Contrary to previously published data, we and colonization experiments, demonstrated that certain com- found that OCH9 treatment slightly increased weight loss in B6B6 mensal bacterial species can modulate the immune system as well mice (Fig. 6B). Finally, we found that OCH9 increased weight as intestinal inflammation (24, 28, 29, 53–55). In agreement with loss and impaired recovery in B6CD1d mice compared with both this, we found that transfer of the CD1d KO microbiota triggered vehicle-treated B6CD1d mice (Fig. 6C) and OCH9-treated B6B6 basal inflammation at steady-state, which was greatly exacerbated mice (Fig. 6D). Of note, we found no difference in colon weight- upon DSS exposure, demonstrating that CD1d KO mice are col- http://www.jimmunol.org/ to-length ratios and pathological scores in these settings (Fig. 6E, onized with commensal bacteria with proinflammatory potential. 6F). Taken together, these results suggested that the intestinal Our analyses revealed that CD1d KO mice harbor several bacterial microbiota influenced iNKT cell function upon pharmacological groups that have been described as proinflammatory. First, Elinav activation during DSS-mediated colitis. et al. (24) reported that NLRP6 inflammasome deficiency leads to by guest on September 25, 2021

FIGURE 5. Fecal transplant creates a third state of microbiota. (A) 16S bacterial rRNA sequencing was used to deﬁne the microbiota proﬁles of B6, CD1d KO, B6B6, and B6CD1d mice. These groups were separated by principal coordinates PC1–PC3, collectively explaining 81% of the total similarity between samples, based on weighted UniFrac distances. (B) The graphs depict global bacterial composition at the phylum level. Additional selected graphs show differences at the phylum (C), class (D), or genus levels (E–G). Statistical differences were calculated using a one-way ANOVA test. Eight mice per group were analyzed. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from

FIGURE 6. The microbiota influences iNKT cell function during DSS-mediated colitis. B6 mice were colonized with B6 (B6B6) or CD1d KO (B6CD1d) http://www.jimmunol.org/ microbiota and treated with or without 2% DSS for 8 d. On day 7, mice received a single i.p. injection of OCH9 or vehicle (Ctrl). The data show daily normalized weight measurements (A–D), colon weight-to-length ratios (E), and colon histopathology at day 12 (F). The data represent the mean 6 SEM of four independent experiments. *p , 0.05, **p , 0.01. increased basal colonic inflammation that is exacerbated upon DSS deficiency fosters an ecological drift over time that leads to the treatment, a phenotype very similar to that of CD1d KO mice. accumulation of proinflammatory microorganisms. These authors ascribed this elevated risk for colitis to an increased iNKT cells are activated by S. typhimurium both in vitro and abundance of bacteria from the phylum TM7 and the family Pre- in vivo (64–67). Oral infection of mice with S. typhimurium was by guest on September 25, 2021 votellaceae (24), which were similarly increased in CD1d KO mice. previously shown to activate iNKT cells and induce IFN-g pro- Second, we found that SFB were present in CD1d KO mice, in duction in peripheral lymphoid tissues and the cecum lamina agreement with a previous study (56), and transmissible to their propria (67, 68). In this study, we found that CD1d-restricted progeny. SFB were previously reported to increase inflammation T cells, which include iNKT cells, do not seem to play a role in (57, 58), perhaps by increasing the development of Th17 cells (28). S. typhimurium–driven acute inflammation following infection of Third, we found that the mucin degraders of the genus Mucispir- streptomycin-pretreated mice, which is in agreement with a pre- illum but not Akkermansia were significantly increased in the CD1d viously published study focusing on oral infection in the absence KO microbiota. Although both genera are increased in DSS-treated of antibiotic treatment (68). mice (59, 60), Akkermansia has been shown to protect from the CD1d-sufficient or -deficient mice displayed no differential development of colitis (61), and its absence in CD1d KO mice susceptibility to DSS treatment, which is in agreement with a study could explain their increased sensitivity. Finally, Betaproteobacteria from the Blumberg and colleagues (15) but in stark contrast with a and Deltaproteobacteria, which are involved in nitrogen fixation more recent study (17). Our work clearly shows that the micro- and sulfate/sulfur reduction, respectively, were more abundant in biota is a confounding factor that can lead to misleading conclu- the CD1d KO microbiota, although their contribution to inflam- sions. Despite funding and time constraints in a challenging and mation remains elusive (62). aggressive research environment, using littermate control mice Another interesting question arising from this work is whether (69) and/or standardization of the microbiota (70, 71) is of para- the proinflammatory microbiota found in CD1d KO mice results mount importance, especially during infection and inflammation from serendipitous familial transmission (49) or is fostered by the studies. Although cohousing of weanlings or adult experimental lack of CD1d and/or iNKT cells in these mice. Our analyses show mice can be an attractive cost-effective alternative, it is well that second generation littermate mice have an identical micro- known that the adult microbiota is more stable and resilient to biota regardless of their genotype, which is in stark contrast with perturbations (72). Additionally, the timing and method of colo- another study (56). The basis for these discrepancies is unclear. To nization can affect colonization efficiency and alter immune pa- the best of our knowledge, Blumberg and colleagues (56) are rameters as well (39, 73). using CD1d KO mice generated by Sonoda et al. (63), and we are The prevalence of iNKT cells is highly variable in humans and using CD1d KO mice generated by Chen et al. (35). Although between different strains of mice. Additionally, the existence of both strains lack the two copies of the CD1d gene, it is possible functionally discrete iNKT cell populations (i.e., Th1, Th2, Th17, that genetic differences between the mice used play a role. Ad- Th10) have recently been revealed in mice (74, 75), with strain- ditionally, housing and husbandry conditions that differ between dependent relative prevalence (75). It is not known whether similar animal facilities may explain the difference between the two functional iNKT cell subsets are present in humans. It is likely that studies. Nonetheless, it is possible that CD1d and/or NKT cell differences in iNKT cell prevalence and functional differentiation 8 CD1d KO MICROBIOTA FUELS COLITIS are the consequence of both genetic and environmental factors, in the presence of va14 natural killer T cells in mice. Inflamm. Bowel Dis. 11: 35–41. among which the microbiota could be a contributing factor. In 17. Kim, H. S., and D. H. Chung. 2013. IL-9-producing invariant NKT cells protect stark contrast with previous studies, we found that iNKT cell against DSS-induced colitis in an IL-4-dependent manner. Mucosal Immunol. 6: activation via OCH9 treatment exacerbated colitis, especially in 347–357. 18. Heller, F., I. J. Fuss, E. E. Nieuwenhuis, R. S. Blumberg, and W. Strober. 2002. mice colonized with the CD1d KO microbiota. This suggests that Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated the gut microbiota affects iNKT cell function during intestinal by IL-13-producing NK-T cells. Immunity 17: 629–638. inflammation and is in direct agreement with recent studies (31– 19. Akbari, O., P. Stock, E. Meyer, M. Kronenberg, S. Sidobre, T. Nakayama, M. Taniguchi, M. J. Grusby, R. H. DeKruyff, and D. T. Umetsu. 2003. Essential 34). A better characterization of the impact of the CD1d KO role of NKT cells producing IL-4 and IL-13 in the development of allergen- microbiota on iNKT cell homeostasis and function will be the induced airway hyperreactivity. Nat. Med. 9: 582–588. focus of further studies. 20. Liao, C. M., M. I. Zimmer, S. Shanmuganad, H. T. Yu, S. L. Cardell, and C. R. Wang. 2012. Dysregulation of CD1d-restricted type ii natural killer T cells In conclusion, we have identified an altered microbiota within leads to spontaneous development of colitis in mice. Gastroenterology 142: 326– NKT cell–deficient CD1d KO mice that exacerbates intestinal in- 34.e1–2. 21. Underhill, D. M., and I. D. Iliev. 2014. 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