Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40(hi)CD5(+) regulatory B cells in vitro and in vivo

Abstract

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40(hi)CD5(+) B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40(hi) is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10(-/-)CD5(+)CD19(+) B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40(hi)CD5(+) Breg cells in mice. However, the population of CD40(hi)CD5(+) B cells was minimal in IL-10(-/-) mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40(hi)CD5(+) B cells and the autocrine effect of IL-10 is critical to the formation of CD40(hi)CD5(+) Breg cells.

Fig. 1.. LPS-induced CD40 high expression is dependent on IL-10-producing B cell differentiation. (A) Representative images of LPS-induced IL-10⁺ B cell activation in a time-dependent manner. (B) Frequencies of IL-10 in mouse splenic B cells stimulated with LPS. (C) Quantitative analysis of secreted IL-10 from LPS-stimulated B cells. Cells were stimulated with LPS for the indicated times and then single cells or supernatant were harvested. Data are the means ± SEM from three independent experiments. *P ＜ 0.05; **P ＜ 0.01. n.s., not significant. (D) Representative images and (E) mean fluorescence intensity (MFI) analysis of CD40 expression on LPS-stimulated B cells. (F) Levels of LPS-stimulated B-cell secretion of IL-10 and surface expression of CD40. Representative images are shown from three independent experiments. (G) Comparison of CD40 expression on IL-10⁺ or IL-10⁻ B cells and IL-10 expression in CD40^hi or CD40^lo B cells. (H) Phenotypic analysis of CD40^hi or CD40^lo B cells by LPS stimulation for 5 or 48 h. (I) Representative image and MFI of IL-10 expression in CD40^hi or CD40^lo B cells from indicated tissue-derived cells. MFI values are the means ± SEM from three independent experiments: *P ＜ 0.05; **P ＜ 0.01. n.s., not significant.

Fig. 2.. The predominant IL-10-producing B cells are CD40^hiCD5⁺ B cells in long-term LPS stimulation. (A) Representative images and (B) frequencies of IL-10 expression in CD5⁺ or CD5⁻ B cells induced by LPS treatment for 5 or 48 h. (C) Representative histogram and (D) mean fluorescence intensity (MFI) of surface CD40 expression on CD5⁺ or CD5⁻ B cells treated with LPS for the indicated times. MFI values are the means ± SEM from three independent experiments: *P ＜ 0.05; **P ＜ 0.01. n.s., not significant. (E) Flow cytometric analysis based phenotypic separation of CD40^hiCD5⁺, CD40^loCD5⁺, and CD5⁻ LPS-induced B cells. (F) MFI, (G) representative gene expression images, and (H) numeric values for IL-10 protein band density in lysates of CD40^hiCD5⁺, CD40^loCD5⁺, and CD5⁻ B cells.

Fig. 3.. The formation of CD40^hiCD5⁺ B cells is controlled by autocrine IL-10. (A) Frequencies of IL-10 production and (B) CD40^hiCD5⁺ expression in splenic B cells induced by LPS (10 μg/ml), IL-10 (10 ng/ml), CD40L (1 μg/ml), and BAFF (100 ng/ml) treatment for 5 or 48 h. Representative images for CD40^hiCD5⁺ expression on B cells induced by LPS, IL-10, CD40L, and BAFF treatment for the indicated times. (C) Representative images and frequency of CD40^hiCD5⁺ expression in B cells stimulated with LPS or IL-10 for 5 or 48 h. (D) Representative images and frequency of CD40^hiCD5⁺ B cells after treatment with LPS for 5 or 48 h. (E) Representative images and frequency of CD40^hiCD5⁺ B cells from WT (IL-10^+/+) or IL-10^−/− mice. Data are the means ± SEM from three independent experiments. **P ＜ 0.01. n.s., not significant. (F) Representative images of LPS-induced IL-10 production and (G) CD40^hiCD5⁺ expression in B cells treated with AG490 for 5 or 48 h.

Fig. 4.. The formation of LPS-induced CD40^hiCD5⁺ B cells is dependent on endogenous IL-10 production in mice. (A) Representative images, (B) frequency and number of IL-10-producing splenic B cells in mice treated with LPS (1 mg/kg, i.v.) for 0 to 3 days. For intracellular IL-10 detection, the cells were cultured with LPS, PMA, ionomycin, and brefeldin A for 5 h. (C) Representative images and frequency of CD40^hiCD5⁺ splenic B cells from WT and IL-10^−/− mice treated with LPS for 3 days. Data are the means ± SEM from three independent experiments. *P ＜ 0.05; **P ＜ 0.01. n.s., not significant. (D) Schematic of the proposed mechanism for the differentiation of IL-10-producing B cells into CD40hiCD5+ B cells by autocrine IL-10. The activation of Breg and Breg progenitor cells can be initiated by LPS and autocrine IL-10 produced by Breg cells. Costimulation by LPS and autocrine IL-10 leads to the differentiation of IL-10-producing B cells to the CD40^hiCD5⁺phenotype via the JAK/STAT signaling pathway.