B Cells Contribute to Mucosal Homeostasis and Prevention of Colitis Through IL-10 Production

Abstract

The role of IL-10-producing B cells in regulating intestinal homeostasis and inflammatory bowel diseases (IBD) is poorly understood. Several studies showed that B cell depletion by rituximab (anti-CD20 antibody) might contribute to developing colitis (IBD 2007, Gut 2008). Hypothesis: Intestinal B cells contribute to mucosal homeostasis and protection against IBD through IL-10 secretion. Wild-type (WT) or IL-10−/− splenic CD4+ T cells were co-transferred with purified splenic B cells from WT or IL-10−/− mice into Rag2−/−IL-10−/− (DKO) mice. 6 weeks after co-transfer, these mice were evaluated for colitis severity by histology (0: non-inflamed, 12: severe inflammation), colonic tissue explant cytokine secretion (gut cult.), mesenteric lymph nodes (MLN) cytokine production (MLN cult.), and Foxp3 expression in MLN CD4+ T cells. To investigate suppressive mechanisms of B cells on the bacteria-activated differentiation of naïve T cells in vitro, B cells from WT or IL-10−/− mice were co-cultured with a) CD25−CD4+ T cells from IL-10EGFP reporter mice and IL-10−/− antigen-presenting cells (APC) or b) CD25−CD4+ T cells and IL-10−/− APC co-cultured with either WT or IL-10−/− CD25+CD4+ and/or B cells from WT or IL-10−/− mice stimulated by cecal bacterial lysates (CBL). IL-10, IFNγ and IL-17a supernatant levels were measured by ELISA and IL-10, IFNγ, IL-17a, Foxp3 and GFP expression were assessed by flow cytometry (FACS). In vivo, histology showed WT CD4+ T cell recipient DKO mice that received co-transferred WT B cells developed less severe colitis than those receiving either IL-10−/− B cells or no B cells (4.3 ± 1.0, 7.2 ± 1.1 and 7.6 ± 0.7, p<0.02). Gut cult. and MLN cult. demonstrated that either spontaneous or bacteria-induced IFNγ and IL-17a secretion was significantly lower and IL-10 levels were higher in DKO mice that received WT B cells than those receiving IL-10−/− B cells or no B cells. Foxp3 expression in MLN CD4+ T cells was induced by cotransferring either WT B cells (10.9±1.0%, p<0.05) or IL-10−/− B cells (11.6±0.8%, p<0.05), compared to animals without B cells (7.4±1.2%). However, all DKO mice with transferred IL-10−/−CD4+ T cells developed severe colitis with no evidence of suppression by WT or IL-10−/− B cells. In vitro, CBL-stimulated WT but not IL-10−/− B cells produced abundant IL-10 and suppressed IFNγ and IL-17a production by CD25−CD4+ WT T cells and IL-10−/−CD25+ T cells equivalent to co-cultured WT or IL-10−/−CD25+ regulatory T cells. FACS data demonstrated that% of either CBLstimulated IL-17a+ or IFNγ+ unfractionated CD4+ T cells were significantly lower when co-cultured with WT but not IL-10−/− B cells. Interestingly, although both WT and IL-10−/− B cells induced Foxp3+CD4+ T cells, only WT B cells could induce GFP+IL-10-producing regulatory T cells (Tr1 cells) (3.7±0.3% with WT B cells, 2.2±0.2% with IL-10−/− B cells, and 2.0±0.3% without B cells, p<0.01). WT but not IL-10−/− B cells ameliorate T cell-mediated colitis despite B cell induction of Foxp3+CD4+ T cells being IL-10 independent. IL-10-producing B cells may contribute to intestinal homeostasis by suppressing effector T cells directly (by IL-10 secretion) and indirectly (by induction of IL-10-producing Tr1 cells).