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CD4 + T cell subsets including regulatory T cells (Tregs), Th1 and Th17 are critical for control and development of inflammation and autoimmunity. We investigated the in vitro and in vivo effects of silymarin, a well-known herbal medicine on differentiation and function of Tregs and Th1 and Th17 responses. For in vitro study, mice splenocytes treated with 20-30 μg/ml silymarin were evaluated for gene expressions of specific transcription factors and cytokines of CD4 + T cell subsets using real-time PCR. Induction of Treg cell development in the presence of silymarin was performed on isolated naïve CD4 + T cells. Effect of silymarin-induced Tregs on T cell suppression was determined by CFSE labeling method. Results of this part showed that silymarin significantly decreased IFNγ, RORγt and IL-17 gene expressions and upregulated Foxp3, TGF-β and IL-10 mRNA. More silymarin-enhanced naïve CD4 + T cells differentiated to Tregs (67%) than the control (47%). Silymarin-induced Tregs reduced proliferation of naïve activated T cells (&lt;50%). For in vivo study, mice were immunized with ovalbumin (Ova) on days 1 and 14. Silymarin (100 mg/Kg) was intraperitoneally administered two days before the first Ova challenge followed by on every day for two weeks. Splenocytes were then isolated for assessment of CD4 + T cell subsets and ex vivo analysis using flow cytometry. Treatment of Ova-immunized mice with silymarin increased Tregs (11.24 ± 1.2%, p &lt; 0.01(but decreased Th1 (1.72 ± 0.4%, p &lt; 0.001) and Th17 (1.07 ± 0.04%, p &lt; 0.001) cells. Ex vivo Ova challenge of splenocytes from Ova-immunized mice treated with silymarin decreased proliferation of splenocytes, IFNγ (2.76% of control) and IL-17 (&lt;8%) along with increased TGF-β (59.7%) expressions in CD4 + T-bet + , CD4 + RORγt + and CD4 + Foxp3 + T cells, respectively. In conclusion, silymarin promoted Treg differentiation and function and decreased Th1 and Th17 cells. Silymarin may differentially regulate CD4 + T cell responses which can provide potential benefits for its use as treatment of immune-related diseases. Graphical abstract ᅟ.

Differential regulation of CD4+ T cell subsets by Silymarin in vitro and in ovalbumin immunized mice