Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10–producing regulatory B cells
Yoshiyuki Mishima, Akihiko Oka, Bo Liu, Jeremy W. Herzog, Chang Soo Eun, Ting-Jia Fan, Emily Bulik-Sullivan, Ian M. Carroll, Jonathan J. Hansen, Liang Chen, Justin E. Wilson, Nancy C. Fisher, Jenny P.Y. Ting, Tomonori Nochi, Angela Wahl, J. Victor Garcia, Christopher L. Karp, R. Balfour Sartor
Yoshiyuki Mishima, Akihiko Oka, Bo Liu, Jeremy W. Herzog, Chang Soo Eun, Ting-Jia Fan, Emily Bulik-Sullivan, Ian M. Carroll, Jonathan J. Hansen, Liang Chen, Justin E. Wilson, Nancy C. Fisher, Jenny P.Y. Ting, Tomonori Nochi, Angela Wahl, J. Victor Garcia, Christopher L. Karp, R. Balfour Sartor
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Research Article Gastroenterology Immunology

Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10–producing regulatory B cells

Abstract

Resident microbiota activates regulatory cells that modulate intestinal inflammation and promote and maintain intestinal homeostasis. IL-10 is a key mediator of immune regulatory function. Our studies describe the functional importance and mechanisms by which gut microbiota and specific microbial components influence the development of intestinal IL-10–producing B cells. Using fecal transplant into germ-free (GF) Il10+/EGFP reporter and Il10–/– mice, we demonstrated that microbiota from specific pathogen–free mice primarily stimulated IL-10–producing colon-specific B cells and T regulatory 1 cells in ex-GF mice. IL-10 in turn downregulated microbiota-activated mucosal inflammatory cytokines. TLR2 and -9 ligands and enteric bacterial lysates preferentially induced IL-10 production and the regulatory capacity of intestinal B cells. Analysis of Il10+/EGFP mice crossed with additional gene-deficient strains and B cell cotransfer studies demonstrated that microbiota-induced IL-10–producing intestinal B cells ameliorated chronic T cell–mediated colitis in a TLR2-, MyD88-, and PI3K-dependent fashion. In vitro studies implicated downstream signaling of PI3Kp110δ and AKT. These studies demonstrated that resident enteric bacteria activated intestinal IL-10–producing B cells through TLR2, MyD88, and PI3K pathways. These B cells reduced colonic T cell activation and maintained mucosal homeostasis in response to intestinal microbiota.

Authors

Yoshiyuki Mishima, Akihiko Oka, Bo Liu, Jeremy W. Herzog, Chang Soo Eun, Ting-Jia Fan, Emily Bulik-Sullivan, Ian M. Carroll, Jonathan J. Hansen, Liang Chen, Justin E. Wilson, Nancy C. Fisher, Jenny P.Y. Ting, Tomonori Nochi, Angela Wahl, J. Victor Garcia, Christopher L. Karp, R. Balfour Sartor

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Figure 8

The PI3K/AKT pathway is involved in TLR2-dependent IL-10 production by CBL-stimulated B cells and their regulatory effect on T cell–mediated colitis.

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The PI3K/AKT pathway is involved in TLR2-dependent IL-10 production by C...
Splenic B cells (1 × 106/well) from 8- to 10-week-old SPF WT, Tlr2−/−, and Tlr4−/− mice were cultured without or with 10 μg/mL CBL. Cells were harvested 0, 15, 30, and 60 minutes after CBL stimulation, and phosphorylation of AKT was analyzed by Western blot. (A) Representative blots and (B) densitometric analysis of phospho-AKT/AKT are shown. n = 5 mice/group, combined from 2 independent experiments. *P < 0.05, **P < 0.01, Kruskal-Wallis test with Dunn’s post hoc test. (C) Splenic B cells (1 × 106/well) from 8- to 10-week-old SPF Il10+/EGFP mice were cultured with 1 μM pan-PI3K inhibitor wortmannin (Wort) or Ly294002 (Ly), 1 μM PI3Kp110δ-specific inhibitor IC-87114 (IC), or DMSO alone, with stimulation with 10 μg/mL CBL for 48 hours. Supernatant levels of IL-10 and IL-12p40 were measured by ELISA. (D) GFP+ population in B cells (live CD45+CD19+B220+) were analyzed by flow cytometry in reference to WT (GFP–) control cells stained with the same antibodies as target samples. Data are presented as median of 6 separate cell cultures, combined from 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test with Dunn’s post hoc test. (E) Splenic B cells (5 × 105/well) from 8- to 10-week-old SPF WT and PI3Kp110δKI mice were cultured with 10 μg/mL CBL for 48 hours. Supernatant levels of IL-10 were measured by ELISA. Data are presented as median. n = 4 mice, combined from 2 independent experiments. *P < 0.05, Mann-Whitney U test. (F) Splenic B cells (1 × 106) from SPF-raised WT or PI3Kp110δKI mice were cotransferred with WT naive CD4+ T cells (5 × 105) isolated by a naive CD4+ T cell isolation kit into SPF Rag2−/− Il10−/− recipients. Six weeks after cell transfer, mice were evaluated for severity of colitis by histology and measurement of spontaneously secreted IL-10 levels in colonic tissue explants by ELISA. n =7–9 mice/ group, combined from 2 independent experiments. Data are presented as median; *P < 0.05, Mann-Whitney U test.