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
  • 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.

  • 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.

  • 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.

  • 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.

Supplementary Materials

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

    Hiutung Chu, Arya Khosravi, Indah P. Kusumawardhani, Alice H. K. Kwon, Anilton C. Vasconcelos, Larissa D. Cunha, Anne E. Mayer, Yue Shen, Wei-Li Wu, Amal Kambal, Stephan R. Targan, Ramnik J. Xavier, Peter B. Ernst, Douglas R. Green, Dermot P. B. McGovern, Herbert W. Virgin, Sarkis K. Mazmanian

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S18
    • Table S1
    • References (42 –47)

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