Fig. 4
Melatonin inhibited IRBP1-20-specific Th cell responses and promoted Treg cell differentiation in vitro. A, B Cells from DLNs of EAU mice were stimulated with 10 μg/mL IRBP1-20 under T helper 1 (Th1) or T helper 17 (Th17)-polarizing conditions with different concentrations of melatonin (0, 2, 20, or 200 ng/mL) for 72 h, the CD4 + cell population was assessed for interferon (IFN)-γ or interleukin (IL)-17A expression by flow cytometry (n = 4). C, D IFN-γ and IL-17A in supernatants were measured via ELISA. E, F T-bet and RORγt were assayed by flow cytometry (n = 4). G Naïve CD4 + T cells were polarized to Treg cells with or without melatonin for three days. CD25 + Foxp3 + cells expression was analyzed by flow cytometry (n = 4). H, I Cells were isolated from DLNs of EAU mice and stimulated with IRBP1-20 peptide under Th17-polarizing conditions in the absence or presence of melatonin (200 ng/mL). After three days, viable cells were adoptively transferred to C57BL/6J wild-type mice (20 million cells/mouse; n = 4). H Clinical scores and representative pictures on day 14 (green arrow: vasculitis, red arrow: papilledema, blue arrow: linear lesions). I Histology scores and representative pictures of H&E (black arrow: inflammatory cells, red arrow: retinal folding with detachments, scale bars represent 100 μm). The representative data from three independent experiments. Significance was determined by one-way ANOVA (A–G), or Mann–Whitney test (H, I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001