Fig. 4

Fluvoxamine suppresses the differentiation of Th1 and Th17 cells rather than disturbing their proliferation and apoptosis. Splenic CD4⁺ T cells were isolated and stimulated with fluvoxamine (10 μM) or vehicle. The percentage of proliferated CD4⁺ T cells was defined by A Carboxyfluorescein Succinimidylester (CFSE) assay after 3 days of culture, and B apoptosis of T cells was determined by PI and Annexin V staining. Representative histograms of CFSE in C Th1 and D Th17 cells. Representative FACS plots and frequencies of active caspase-3 in E Th1 and F Th17 cells. Each dot represents the mean of three biological replicates. PLN cells from 12-week-old fluvoxamine- and vehicle-treated mice were harvested and subject to flow cytometry analysis. Representative FACS plots and frequencies of G CD4⁺Ki67⁺ T cells and H apoptotic CD4⁺ T cells determined by PI and Annexin V staining are shown. Percentage of Ki67⁺ proliferative I effector Th1 and J Th17 cells are shown by flow cytometry. K Apoptotic effector Th1 and L Th17 cells were indicated by staining active caspase-3 positive cells (n = 5 per group). Naïve CD4⁺ T cells purified from splenocytes were cultured under Th1 and Th17 conditions in vitro for 3 days in the presence of fluvoxamine or vehicle. M Th1 and N Th17 polarization efficiency was analyzed by flow cytometry. Each dot represents the mean of three biological replicates. Data are expressed as mean ± SEM. Statistical significance was calculated by unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, and ns, not significant