Fig 1: Melatonin suppresses Th17 and enhances Treg differentiation via SIRT1 activation. Sorted naïve CD4+ T cells (CD3+ CD4+ CD45RA+ CD45RO-) from umbilical cord blood of healthy newborns were transduced with lentivirus-containing control-shRNA (CTRL shRNA) and SIRT1-shRNA (SIRT1 shRNA) (A-I), or control lentivirus (CTRL LV), and SIRT1-expressing lentivirus (SIRT1 LV) (J-R). At 24 h after transduction, CD4+ T cells were differentiated under Th17 or Treg conditions for 4 days in the presence of melatonin (20 ng/mL). (A) Representative flow cytometric plots of IL-17 and Foxp3 expression in gated CD4+ T cells under Th17 (top) and Treg (bottom) conditions following transduction with CTRL or SIRT1 shRNA. (B, C) Quantification of the frequency of Th17 (B) and Treg (C) cells in (A). (D-I) Real-time qRT-PCR analysis of relative mRNA expression of ROR?t (D), IL-17 (E), IL-22 (F), Foxp3 (G), TGF-ß (H), and IL-10 (I) in Th17 (D-F) and Treg (G-I) conditions. (J) Representative flow cytometric plots of IL-17 and Foxp3 expression in gated CD4+ T cells under Th17 (top) and Treg (bottom) conditions following transduction with CTRL LV or SIRT1 LV. (K, L) Quantification the frequency of Th17 (K) and Treg (L) cells in (J). (M-R) Real-time qRT-PCR analysis relative mRNA expression of ROR?t (M), IL-17 (N), IL-22 (O), Foxp3 (P), TGF-ß (Q), and IL-10 (R) in Th17 (M-O) and Treg (P-R) conditions (as described in the Methods). Each symbol (B-I, K-R) represents an individual experiment (n = 5); column graphs represent the mean with error bars indicating standard deviation (SD), *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. P values were derived from one-way ANOVA followed by the Bonferroni multiple comparison test (B-I, K-R).