Fig. 4

Foxo1 reciprocally regulates IL-17 and IL-9 in Th17 cells. a, b Naive T cells were differentiated into Th0 and Th17 cells, Il17, Il9, Foxo1 mRNA was determined by qPCR a, total Foxo1 was determined by FACS, data representative of at least two independent experiments b. c Wt B6 mice were immunized with MOG_35–55, 8 day later, cells were restimulated with MOG_35–55 with or without IL-23, mRNA of Il9 (n = 8) and Foxo1 (n = 3) was determined by qPCR. d Naive T cells were differentiated into Th17 cells with or with IL-23, at 72 h mRNA expression of Il9 and Foxo1 was determined. e Wt and Il23r−/− mice were immunized with MOG_35–55 (n = 3), cells were restimulated and then IL-9/IL-17 production was determined by FACS, bars to the right given percentage of IL-9⁺CD4⁺ T cells. f Flow cytometric analysis of pFoxo1 in T cells treated with indicated culture conditions, data represent of two independent experiments. g–j Naive T cells were differentiated into Th0 and Th17 conditions with or without LY294002 (5.0 μM). IL-9 and IL-17 production was determined by flow cytometry g, mRNA expression of Il9, Il17a, Rorc and Il23r was determined by qPCR h–j, IL-17 concentration was determined by ELISA from culture supernatant (i, right panel). Contour plot is representative of two experiments g. k, l Naive T cells were differentiated in Th0 and Th17 condition, transfected with Scr-siRNA or Foxo1-siRNA and treated with or without LY294002 as indicated. At 72 hr, IL-9 and IL-17 concentrations was determined by ELISA. m qPCR analysis of Il9, Il17, Rorc, Il23r from Th17 cells transduced with Scr-shRNA or Foxo1-shRNA. n In vitro differentiated Th17 cells were treated with or without Foxo1 inhibitor (25 nM), and IL-9/IL-17 production was determined by flow cytometry, Il9 and Il23r mRNA was determined o. Bars show + s.d. from combined six or more a, three c–e, h–j, two k, l, three m, o experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t-test and two-way analysis of variance)