Fig. 9.

SCFAs enhance mTOR activity. (a) Activation of the mTOR pathway based on rS6 phosphorylation in CD4⁺ T cells. T cells were activated with anti-CD3/CD28 and IL-2 in the presence or absence of C2 or C3 and examined by flow cytometry. (b) Acetylation of S6K in activated T cells in the presence of C2, C3 or TSA. The antibodies used for immunoprecipitation (IP) and western blotting (WB) were anti-acetylated-lysine and anti-S6K respectively. (c) The effect of rapamycin on SCFA-dependent generation of Th cell subsets was assessed. Naïve CD4⁺ T cells were cultured in a Tnp, Th1, or Th17 polarization condition for 5-6 days in the presence of C2 (10 mM), C3 (1 mM), and/or rapamycin (25 nM), and the cytokine phenotype of cultured T cells was examined by flow cytometry and ELISA (for IL-10). (d) AMPK activation suppressed the SCFA effect on T cell differentiation. Naïve CD4⁺ T cells were cultured with SCFAs and/or metformin in indicated polarization conditions for 5-6 days, and frequencies of indicated T cell subsets were examined. The dot plots were from the data obtained in a Th1 condition. (e) Activation of STAT3 by C2 or C3 was examined. CD4⁺ T cells were activated for 3 days with anti-CD3/28 and then examined for phosphorylation of STAT3. Representative and pooled data obtained from at least three experiments are shown. *Significant differences from blank or control groups (P≤0.05).