Figure 2.

Butyrate enhances FOXP3 expression in CD4+ T cells and impairs the differentiation of Th9 cells. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th9 (TGF-β 2 ng/ml, IL-4 10 ng/ml and anti-IFNγ 10 μg/ml) differentiation condition for 4 days. Butyrate and propionate (250 μM) were added in the culture at day 0. At day 4, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO₂, and analyzed by flow cytometry (A). The frequencies of FOXP3+ and/or IL-9+ CD4+ T cells are represented by bar graphs (B). A correlation analysis of IL-9 and FOXP3 expression was performed (C). The frequencies of IL-9+ and/or IL-10+ CD4+ T cells (D) and FOXP3+ and/or IL-10+ CD4+ T cells (E) were determined and are represented by bar graphs. Data are shown as mean ± S.D. Two-way ANOVA and Tukey's multiple comparison test were used for statistical analysis. NS, not significant.