ReportsMucosal Immunology

Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease

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Science  27 May 2016:
Vol. 352, Issue 6289, pp. 1116-1120
DOI: 10.1126/science.aad9948

Genes and microbes converge in colitis

Both host genetics and intestinal microbes probably contribute to a person's overall susceptibility to inflammatory bowel disease (IBD). The human gut microbe Bacteroides fragilis produces immunomodulatory molecules that it releases via outer membrane vesicles (OMVs). These molecules can protect mice from experimentally induced colitis. Chu et al. now find that OMV-mediated protection from colitis requires Atg16l1 and Nod2 genes whose human orthologs are associated with an increased risk for developing IBD. OMVs trigger an ATG16L1 and NOD2–dependent noncanonical autophagy pathway in dendritic cells (DCs). OMV-primed DCs, in turn, induce regulatory T cells in the intestine that protect against colitis.

Science, this issue p. 1116


Inflammatory bowel disease (IBD) is associated with risk variants in the human genome and dysbiosis of the gut microbiome, though unifying principles for these findings remain largely undescribed. The human commensal Bacteroides fragilis delivers immunomodulatory molecules to immune cells via secretion of outer membrane vesicles (OMVs). We reveal that OMVs require IBD-associated genes, ATG16L1 and NOD2, to activate a noncanonical autophagy pathway during protection from colitis. ATG16L1-deficient dendritic cells do not induce regulatory T cells (Tregs) to suppress mucosal inflammation. Immune cells from human subjects with a major risk variant in ATG16L1 are defective in Treg responses to OMVs. We propose that polymorphisms in susceptibility genes promote disease through defects in “sensing” protective signals from the microbiome, defining a potentially critical gene-environment etiology for IBD.

Intestinal microbiota modulate development and function of the immune system and play a critical role in inflammatory bowel disease (IBD), a family of idiopathic intestinal disorders including Crohn’s disease (CD) and ulcerative colitis (UC) (16). Concordance rates of 40 to 50% between monozygotic twins implicate gene-environment interactions in the pathogenesis of CD (710), albeit in ways that are poorly understood. Advances in DNA-sequencing technologies have empowered unprecedented insights into the human genome and the gut microbiome in IBD, enabling detailed genomic characterization of patients (11) and chronicling alterations in the composition and gene content of the gut microbiome (dysbiosis) (12).

Close to 200 risk loci have been proposed for CD, with several susceptibility genes linked to the regulation of autophagy (e.g., autophagy-related 16–like 1, ATG16L1) (1315) or to microbial sensors that activate autophagy [e.g., nucleotide-binding oligomerization domain–containing protein 2 (NOD2)] (1618). Although previous studies have shown that disruption of ATG16L1 and NOD2 affects CD susceptibility through defects in microbial clearance (1923), recent reports reveal that immune cells impaired in autophagy are hyperinflammatory (2429). This suggests that deficiencies in ATG16L1 or NOD2 may contribute to CD risk through impaired anti-inflammatory responses, a hypothesis not mutually exclusive with microbial clearance functions.

The microbiome of CD patients is altered, with emerging evidence for cause-and-effect relationships to disease. Among other recent examples of host-microbe interactions (3, 5, 6), the human commensal Bacteroides fragilis has evolved beneficial immunomodulatory properties. During colonization of mice, B. fragilis capsular polysaccharide A (PSA) is packaged in outer membrane vesicles (OMVs) and delivered to intestinal dendritic cells (DCs) to induce interleukin-10 (IL-10) production from CD4+Foxp3+ regulatory T cells (Tregs), which protects from experimental colitis (3032). To explore gene-environment interactions during host-microbiota symbiosis, we tested if genetic pathways linked to CD are involved in the immune response to B. fragilis OMVs.

Bone marrow–derived DCs (BMDCs) differentiated from wild-type (WT) and ATG16L1-deficient (Atg16l1fl/fl Cd11c-Cre; Atg16l1ΔCd11c) mice were pulsed with OMVs harvested from wild-type B. fragilis (WT-OMV), or an isogenic mutant lacking PSA (ΔPSA-OMV), and cocultured with CD4+ T cells. As previously reported (33), WT-OMVs, but not vehicle or ΔPSA-OMVs, promote IL-10 production (Fig. 1, A to C, and fig. S1). Conversely, ATG16L1-deficient DCs do not support IL-10 production in response to WT-OMVs (Fig. 1, A to C). We observe similar results using Atg16l1fl/fl LysM-Cre mice (fig. S3). Purified PSA does not require ATG16L1 for its activity (Fig. 1, A and C, and fig. S2). Next, we tested functional outcomes using in vitro T cell suppression assays. Tregs isolated from cocultures with Atg16l1ΔCd11c BMDCs treated with B. fragilis OMVs exhibit impaired suppressive activity (Fig. 1D and fig. S2A). Neither WT-OMVs nor pure PSA have any effect on IL-10 production among CD4+Foxp3 type 1 regulatory T cells (fig. S4). ATG16L1, ATG5, and ATG7 are components of the autophagy elongation complex; BMDCs deleted in these genes likewise do not induce IL-10 production from Tregs (fig. S5). Further, recent reports reveal a role for autophagy components in Treg homeostasis (34, 35). Our findings indicate that ATG16L1-deficient DCs fail to respond to B. fragilis OMVs, demonstrating that autophagy components in DCs are required for commensal-driven Treg induction and function.

Fig. 1 ATG16L1 signals via a noncanonical autophagy pathway during OMV-mediated Treg induction.

(A) Enzyme-linked immunosorbent assay for IL-10 production during DC–T cell cocultures with WT or Atg16l1ΔCd11c BMDCs treated with phosphate-buffered saline (PBS), B. fragilis WT-OMV, ΔPSA-OMV, or purified PSA. (B and C) Representative flow cytometry plots (B) and frequency (C) of CD4+Foxp3+IL-10+ Tregs from DC–T cell cocultures with WT or Atg16l1ΔCd11c DCs treated with PBS, B. fragilis WT-OMV, ΔPSA-OMV, or purified PSA. (D) T cell suppression assay analyzing in vitro–generated Tregs from WT or Atg16l1ΔCd11c DCs treated with WT-OMVs. (E) Quantification of LC3-GFP accumulation by B. fragilis WT-OMV treatment of Rubicon+/− or Rubicon−/− DCs. Representative flow cytometry histogram plot (inset). PBS, gray; WT-OMV, blue. (F) Frequency of CD4+Foxp3+IL-10+ Tregs from Rubicon+/− or Rubicon−/− DC–T cell cocultures treated with PBS, B. fragilis WT-OMV, ΔPSA-OMV, or purified PSA. Error bars represent SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001. Two-way analysis of variance (ANOVA), followed by Tukey’s post-hoc analysis. Data are representative of at least two independent experiments.

ATG16L1, ATG5, and ATG7 participate in both canonical and noncanonical autophagy pathways (36). Interestingly, the classical autophagy-specific genes Ulk1, Fip200, or Atg14 are not required for CD4+Foxp3+IL-10+ Treg induction upon WT-OMV treatment (fig. S6). We hypothesized that OMVs use the noncanonical autophagy pathway, LC3-associated phagocytosis (LAP), which is specifically activated by microbial ligands delivered as particles rather than as soluble molecules. LAP activation requires RUBICON, which represses canonical autophagy (36). Rubicon+/− but not Rubicon−/− BMDCs display increased accumulation of lipidated, membrane-bound LC3-GFP (green fluorescent protein) (LC3-II) upon B. fragilis WT-OMV treatment (Fig. 1E). As expected, neither ΔPSA-OMVs nor purified PSA are able to activate LAP (fig. S7). Moreover, treatment of Rubicon/– DCs fails to induce Treg responses (Fig. 1F). As RUBICON is upstream of ATG16L1 signaling, OMVs preferentially use the noncanonical autophagy pathway LAP to mediate tolerogenic responses to B. fragilis. Further, these data suggest a reconsideration of previous literature assigning the role of ATG16L1 in IBD to defects exclusively in autophagy.

As a CD-risk gene, we investigated the in vivo requirement for ATG16L1 in CD11c+ DCs during OMV-mediated protection from experimental colitis. Indeed, WT mice treated by oral gavage with WT-OMVs are protected from 2,4-dinitrobenzenesulfonic acid (DNBS) colitis (33), whereas Atg16l1ΔCd11c mice exhibit acute weight loss and increased mortality similar to that of untreated mice (Fig. 2A and fig. S8A). WT, but not Atg16l1ΔCd11c, mice orally administered OMVs are protected from shortening of the colon, a hallmark of colitis models (Fig. 2B), with colitis scoring and cytokine profiles verifying protection from disease (Fig. 2C and fig. S8B). Prevention of colitis is not due to an overall defect in Treg development in Atg16l1ΔCd11c mice (fig. S9). Further, although proportions of CD4+Foxp3+ cells are comparable in all groups of mice during colitis (fig. S10), Atg16l1ΔCd11c mice produce significantly less IL-10 from gut Foxp3+ Tregs compared to WT mice after WT-OMV treatment (Fig. 2D and fig. S8C). Thus, WT-OMVs require ATG16L1 within DCs to induce IL-10 expression from Foxp3+ Tregs and to suppress intestinal inflammation in a colitis model.

Fig. 2 B. fragilis OMVs require ATG16L1 in CD11c+ DCs for protection from colitis.

(A and B) Weight loss (A), colon length, and gross pathology (B) of WT and Atg16l1ΔCd11c mice orally treated with PBS or B. fragilis WT-OMV during DNBS colitis. Sham groups were treated with ethanol. (C) Colitis scores by a blinded pathologist using a standard scoring system, and representative hematoxylin and eosin (H&E) images. Scale bar, 100 μm. (D and E) MLN lymphocytes isolated post-DNBS analyzed for IL-10 (D) and IL-17A (E) production among CD4+Foxp3+ Tregs, as assessed by flow cytometry. Error bars represent SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001. Two-way ANOVA, followed by Tukey’s post-hoc analysis. Data are representative of at least three independent experiments, with three to nine mice per group.

In addition to impaired IL-10 production in response to OMV treatment, Atg16l1ΔCd11c mice display an increase in IL-17A expression (Fig. 2E), but not IFN-γ (fig. S11), among mucosal CD4+Foxp3+ T cells during colitis. Further, in vitro cocultures of OMV-pulsed Atg16l1ΔCd11c BMDCs result in impaired IL-10 expression among Tregs (Fig. 1C) and increased IL-17A production in CD4+Foxp3+ T cells (fig. S12). Interestingly, whereas OMVs from other enteric bacteria each elicited a unique ATG16L1-dependent immune profile, only B. fragilis OMVs exclusively induce an anti-inflammatory response (fig. S13). Together, these data suggest that ATG16L1 deficiency in DCs alters the quality of the T cell response to OMVs.

As DCs coordinate adaptive immunity, we sought to determine how Atg16l1ΔCd11c DCs are impaired in promoting tolerogenic responses. After OMV stimulation, we observe no differences by WT or Atg16l1ΔCd11c DCs in internalizing OMVs or in surface expression of major histocompatibility complex class II (MHC II), CD80, and CD86 (fig. S14) (27). However, stimulation with OMVs results in an increase transcription of multiple proinflammatory cytokines in Atg16l1ΔCd11c DCs compared to WT cells (fig. S15). These data are consistent with previous reports of a hyperinflammatory response in ATG16L1-deficient macrophages and DCs stimulated with other microbial ligands (24, 26). Abrogation of Treg responses by ATG16L1-deficient DCs is likely due to increased proinflammatory cytokine production, which may impair DC–T cell interactions. Atg16l1ΔCd11c mice do not display more severe colitis than WT mice in the absence of OMV treatment (Fig. 2), suggesting that lack of protection is not due to more fulminant inflammation, but rather to an inability to induce Tregs in mice deficient in ATG16L1 among CD11c+ DCs.

NOD2 encodes an intracellular sensor of bacterial peptidoglycan, and polymorphisms in this gene contribute to the largest fraction of genetic risk for CD (13). NOD2 has been shown to physically recruit ATG16L1 (20, 21), a process that is impaired in human cells homozygous for a NOD2 frameshift mutation (20). Accordingly, Nod2−/− BMDCs pulsed with WT-OMVs are unable to support IL-10 production from Foxp3+ Tregs during in vitro cocultures (Fig. 3, A and B), revealing a crucial role for NOD2 signaling in microbiome-mediated immune tolerance. This notion is supported by in vivo studies showing that Nod2−/− mice are not protected from colitis by WT-OMV treatment (Fig. 3, C and D). Similar to Atg16l1ΔCd11c animals, Nod2−/− mice produce significantly less IL-10 from Foxp3+ Tregs of the mesenteric lymph node (MLN) after WT-OMV treatment (fig. S16A), and proportions of Tregs remain unchanged during DNBS colitis (fig. S16B). Previous studies have shown that Toll-like receptor 2 (TLR2) is required for the PSA response (33, 37). Although the role of NOD2 in inducing LAP is currently unknown, signaling through TLR2 potently activates LAP (36, 38). B. fragilis OMVs induce reactive oxygen species from WT DCs, a known product of LAP activation (36), but at significantly reduced levels in Nod2−/− or Tlr2−/− DCs (fig. S17). Though further studies are needed to define the mechanism of LAP activation by OMVs, these data reveal that NOD2 and ATG16L1 may cooperate as part of a common pathway to promote anti-inflammatory immune responses to the microbiome.

Fig. 3 NOD2 is required for OMV-mediated Tregs induction and protection from colitis.

(A and B) Representative flow cytometry plots (A) from WT-OMV (left)– and ΔPSA-OMV (right)–treated BMDCs cocultured with CD4+ T cells, and frequency (B) of CD4+Foxp3+IL-10+ Tregs from DC–T cell cocultures. (C and D) Weight loss (C), colon length, and gross pathology (D) of WT or Nod2−/− mice treated with PBS or B. fragilis WT-OMV during DNBS colitis. Error bars represent SEM. *P < 0.05, ****P < 0.0001. Two-way ANOVA, followed by Tukey’s post-hoc analysis. Data are representative of at least three independent experiments, with three to five mice per group.

To extend and validate gene deletion approaches, we tested responses to OMVs by immune cells carrying the CD-associated variant of ATG16L1 (13, 14, 39). The ATG16L1 T300A variant leads to protein instability and altered cellular responses (23). BMDCs from transgenic mice expressing the T300A allele are also unable to promote IL-10 expression from Foxp3+ Tregs in response to WT-OMVs (fig. S18A). Further, ATG16L1 T300A transgenic mice are not protected from DNBS colitis and do not mount a potent Treg response when administered WT-OMV compared to WT mice (fig. S18, B to G). These findings prompted us to investigate if human immune cells from CD patients with the ATG16L1 T300A risk variant (table S1) are also defective in promoting Foxp3+ Treg development by B. fragilis OMVs. Monocyte-derived dendritic cells (MoDCs) from CD patients and healthy controls harboring either the protective allele (A300) or the risk allele (A300) were pulsed with OMVs or PSA and cocultured with syngeneic CD4+ T cells. Consistent with our mouse data, human cells homozygous for the risk allele are unable to support induction of IL-10 from Foxp3+ Tregs by WT-OMVs compared to MoDCs carrying the protective allele (Fig. 4). Notably, all samples tested display the predicted outcome based on genotype and not disease status. However, cells from most subjects, regardless of genotype, respond to purified PSA (Fig. 4). Collectively, we conclude that mouse and human DCs require functional ATG16L1 for induction of CD4+Foxp3+IL-10+ Tregs in response to B. fragilis OMVs.

Fig. 4 The T300A risk variant of ATG16L1 in human cells is unable to support OMV responses.

(A and B) MoDCs with either the protective (A) or risk (B) allele were treated with PBS, B. fragilis WT-OMV, ΔPSA-OMV, or purified PSA; washed; and cocultured with syngeneic CD4+ T cells. IL-10 expression was analyzed by flow cytometry among CD4+Foxp3+ Tregs. Human samples were processed and analyzed in a blinded fashion. CTL, control subjects; CD, Crohn’s disease subjects. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. One-way ANOVA, followed by Tukey’s post-hoc analysis.

IBD affects more than 1.5 million people in the United States, with rates of diagnosis increasing and treatment options remaining limited (40, 41). The etiology of IBD is complex and incompletely resolved (1). Here, we describe how interactions between genetic (ATG16L1/NOD2) and environmental (microbiome) factors cooperate to promote beneficial immune responses. B. fragilis OMVs use LAP, an ATG16L1-dependent cellular trafficking and signaling pathway, to induce mucosal tolerance. The hyperinflammatory responses that occur with mutations in ATG16L1 likely alter antigen-processing pathways and impair signaling by DCs to T cells and may explain why CD-associated polymorphisms abrogate Treg induction by OMVs. Collectively, discovery of genetic circuits co-opted by the microbiome to engender health provides unprecedented functional insights into gene-environment interactions relevant to the pathogenesis of IBD. We propose an additional role for genes previously implicated in killing bacteria—namely, mutations in genetic pathways linked to IBD result in an inability to sense and/or respond to beneficial microbes. This hypothesis may represent a new perspective for the etiology of microbiome-related diseases.

Supplementary Materials

Materials and Methods

Figs. S1 to S18

Table S1

References (4247)

References and Notes

Acknowledgments: We thank L. Hwang, E. Park, and M. Salas for clinical research coordination (Cedars-Sinai); A. Maskell, L. Sandoval, and C. Rumaldo for animal husbandry (Caltech); and members of the Mazmanian laboratory for discussions and critical reading of the manuscript. The data presented in this manuscript are tabulated in the main paper and in the supplementary materials. This work was supported by the National Institutes of Health (NIH) under a Ruth L. Kirschtein National Research Service Award (DK100109) to H.C.; NIH DK097485 to R.J.X.; NIH PO1DK046763, the Cedars-Sinai F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute Research Funds, and The Feintech Family Chair in IBD to S.R.T.; Wayne and Gladys Valley Foundation and NIH AI079145 to P.B.E.; The Lupus Research Institute and NIH AI40646 to D.R.G; NIH U19 AI109725 to H.W.V.; The Lisa Z. Greer Endowed Chair in IBD Genetics, NIH DK062413, NIH DE023789-01, grant 305479 from the European Union, The Crohn's and Colitis Foundation of America, and The Leona M. and Harry B. Helmsley Charitable Trust to D.P.B.M.; NIH AI109725 to H.W.V.; and NIH DK078938, NIH GM099535, The Crohn's and Colitis Foundation of America, and the Heritage Medical Research Institute to S.K.M. Rubicon and ULK1 knockout mice were obtained from D. R. Green and M. Kundu, respectively, under a materials transfer agreement with St. Jude Children’s Research Hospital. A provisional patent application entitled “Beneficial Activation of Autophagy Components by the Microbiome” has been filed by H.C., H.W.V., and S.K.M.
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