Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10–producing regulatory B cells
Yoshiyuki Mishima, … , Christopher L. Karp, R. Balfour Sartor
Yoshiyuki Mishima, … , Christopher L. Karp, R. Balfour Sartor
Published June 18, 2019
Citation Information: J Clin Invest. 2019;129(9):3702-3716. https://doi.org/10.1172/JCI93820.
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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 1

Resident intestinal microbiota increases the frequency of intestinal IL-10–producing immune cells and enhance IL-10 production.

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Resident intestinal microbiota increases the frequency of intestinal IL-...
(A) Left: Spontaneous IL-10 secretion by colonic tissue explants; middle: number of total IL-10–producing (GFP+) colon LP cells; right: Il10 mRNA expression in distal colon tissue normalized by expression in GF mice in GF, SPF-raised, or GF-conventionalized Il10+/EGFP reporter mice 3 days and 7 days (D3 and D7) after fecal transplantation (TP) with SPF feces. n = 6–9 mice/group, combined from 2 independent experiments. (B and C) Phosphorylation levels of STAT3 in the distal colon were evaluated by Western blot analysis and quantified by densitometry. n = 4 mice/group. (D) IL-10–producing (GFP+) colon LP cells were characterized by flow cytometry identifying cell subsets with antibodies to the indicated cell surface proteins. CD25–CD4+ T cells (CD25–CD4+CD3+), B cells (B220+CD19+), and Tregs, including GFP+Foxp3+RORγt+CD4+ T cells within the total GFP+Foxp3+CD4+ T cell population. n = 5–7 mice/group, combined from 2 independent experiments. (E) Representative dot plots for colon LP GFP+CD25–CD4+ T cells (CD4+ T cell–gated) and GFP+ B cells (B cell–gated) in GF and conventionalized day 7 Il10+/EGFP mice. For flow cytometry, the GFP+ population in live CD45+ colon LP cells was assessed using WT (GFP–) cells stained with the same antibodies as target samples as a control (see Flow cytometry in Methods). All data are presented as median values; *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test with Dunn’s post hoc test.