Figure 4

Downregulation of Th1 and Th17 and induction of regulatory T cells in CIA by ergotope peptide treatment. (A) Splenic mononuclear cells were isolated on d 14 post first immunization from untreated (CIA) or C-P, P5-6 or P14-16 treated mice with CIA and stimulated in vitro with CII (20 µg/mL) for 48 h and analyzed for CD4 and intracellular cytokine expression by flow cytometry. Dot plots (left) show representative double staining for IL-17A and IFN-γ in gated CD4 + T cells. Percentages of CD4 + IFN-γ + (Th1) and CD4 + IL-17A + (Th17) are shown on the right. Values are the mean ± SD of eight mice per group and pooled from three independent experiments. *: P < 0.05 versus C-P group; #: P < 0.05 versus P5-6 group. (B) Cytokine production was assessed by ELISA in sera and in supernatants by splenic mononuclear cells with CII stimulation (20 µg/mL) for 48 h. Values are the mean ± SD of eight mice per group. (C) Regulatory T cells were analyzed in the splenocytes from indicated groups by flow cytometry. Percentages of CD4 + CD25 + T cells are shown in the representative dot plots (upper panel). Histograms show the expression of FOXP3 + cells (dark gray lines) in gated CD4 + CD25 + cells from splenocytes, while light gray lines indicate antibody isotype controls. Data are representative of eight mice per group. Statistical analysis is displayed on the right. Values are the mean ± SD. *: P < 0.05 versus C-P group. (D) Treg-secreted cytokines TGF-β and IL-10 were measured by ELISA in sera and in supernatants by splenic mononuclear cells with CII stimulation (20 µg/mL) for 48 h. Values are the mean ± SD of eight mice per group. (E) The expressions of Th cell related mRNA, IFN-γ, T-bet, IL-17, STAT3, RORγt, FOXP3, IL-10 and TGF-β in splenic mononuclear cells were detected by real-time qPCR at CII (20 μg/mL) stimulation for 24 h. *: P < 0.05; **: P < 0.01; ***: P < 0.001 versus C-P group.