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 7

TLR2/MyD88 signaling increases the frequency of IL-10–producing B cells upon bacterial product stimulation ex vivo and mediates the suppression of experimental colitis by IL-10–producing B cells in vivo.

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TLR2/MyD88 signaling increases the frequency of IL-10–producing B cells ...
(A) Colonic LP B cells from 8- to 10-week-old WT Il10+/EGFP, Tlr2−/− Il10+/EGFP, Tlr4−/− Il10+/EGFP, and Myd88−/− Il10+/EGFP mice were cultured without (–) or with 10 μg/mL CBL, 50 ng/mL Pam3, or 200 ng/mL LPS for 24 hours, after which IL-10 levels in culture supernatants were measured by ELISA (top panel). GFP+ populations in B cells (live CD45+CD19+B220+) were analyzed with flow cytometry in reference to WT (GFP–) control cells stained with the same antibodies as target samples. Data are presented as median of 7–8 separate cell cultures, with cells in each culture pooled from 2–3 mice, combined from 3 independent experiments (bottom panel). *P < 0.05, **P < 0.01. (B) Eight-week-old GF Il10+/EGFP reporter mice were conventionalized with SPF fecal bacterial transplantation. Mice were given anti-TLR2 antibody (αTLR2) or isotype control i.v. on days 0 and 4 after fecal bacterial transplantation and harvested on day 7 for IL-10 analysis. Colonic LP cells were isolated, and live CD45+GFP+ B cells (CD19+B220+) were analyzed by flow cytometry (left) as described above; Il10 mRNA levels were normalized with Actb (right). n = 4–5 mice. Data are presented as median, Mann-Whitney U test. (C and D) Splenic B cells (1 × 106) from SPF-raised WT, Tlr2−/−, Tlr4−/−, Myd88−/−, or Il10−/− mice were cotransferred with 5 × 105 WT naive CD4+ T cells isolated by a naive CD4+ T cell isolation kit (see Methods, Cell purification) into SPF Rag2−/− Il10−/− recipients. Six weeks after cell transfer, mice were evaluated for (C) severity of colitis by histology and (D) measurement of spontaneously secreted IL-10 and IFN-γ in colonic tissue explants by ELISA. n = 7–9 mice/group, combined from 2 independent experiments. Data are presented as median; *P < 0.05 and **P < 0.01 (vs. WT mice), Kruskal-Wallis test with Dunn’s post hoc test.