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

Abstract

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 (T(regs)) to suppress mucosal inflammation. Immune cells from human subjects with a major risk variant in ATG16L1 are defective in T(reg) 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.

Fig. 1. ATG16L1 signals via a non-canonical autophagy pathway during OMV-mediated T_reg induction

(A) ELISA for IL-10 production during DC–T cell co-cultures with WT or Atg16L1^ΔCD11c BMDCs treated with 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⁺ T_regs from DC–T cell co-cultures with WT or Atg16L1^ΔCD11c DCs treated with PBS, B. fragilis WT-OMV, ΔPSA-OMV or purified PSA. (D) T cell suppression assay analyzing in vitro generated T_regs from WT or Atg16L1^ΔCD11c 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, grey; WT-OMV, blue. (F) Frequency of CD4⁺Foxp3⁺IL-10⁺ T_regs from Rubicon^+/− or Rubicon^−/− DC–T cell co-cultures treated with PBS, B. fragilis WT-OMV, ΔPSA-OMV or purified PSA. Error bars represent S.E.M. * 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 2 independent experiments.

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 Atg16L1^ΔCD11c 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 H & E images. Scale bar represents 100 μm. (D and E) Mesenteric lymph node (MLN) lymphocytes isolated post-DNBS analyzed for IL-10 (D) and IL-17A (E) production among CD4⁺Foxp3⁺ T_regs, as assessed by flow cytometry. Error bars represent S.E.M. * 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 3 independent experiments, with 3-9 mice/group.

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

(A and B) Representative flow cytometry plots (A) from WT-OMV (left) and ΔPSA-OMV (right) treated BMDCs co-cultured with CD4⁺ T cells, and frequency (B) of CD4⁺Foxp3⁺IL-10⁺ T_regs from DC–T cell co-cultures. (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 S.E.M. * p < 0.05, ****p < 0.0001. Two-way ANOVA, followed by Tukey’s post-hoc analysis. Data are representative of at least 3 independent experiments, with 3-5 mice/group.

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 co-cultured with syngeneic CD4⁺ T cells. IL-10 expression was analyzed by flow cytometry among CD4⁺Foxp3⁺ T_regs. Human samples were processed and analyzed in a blinded fashion. CTL, control subjects; CD, Crohn’s Disease subjects. Error bars represent S.E.M. * 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.