Inhibition of interferon-γ production and T-bet expression by menthol treatment of human peripheral blood mononuclear cells

Context: Mentha longifolia (L.) Huds., has shown anti-inflammatory effects. Objective: To evaluate the immunomodulatory effects of menthol, the major constituent of Mentha longifolia on T cells as the main cells affecting the inflammatory responses. Methods: Effect of menthol on: proliferation and viability of the peripheral blood human mononuclear cells (PBMCs) by BrdU and propidium iodide (PI) staining, respectively, interferone (IFN)gamma and interleukin (IL)-4 cytokine production in lymphocytes stimulated with phytohemagglutinin (PHA) and phorbol myristate acetate/calcium ionophore (PMA/CI) by ELISA; intracellular staining of CD4(+) cells for IFN gamma expression by flow cytometry and gene expressions of T heper (Th) cell transcription factors was measured using real time-PCR. Results: Menthol dose-dependently inhibited lymphocytes proliferation from 88.7% at 50 mu g/ml to 3.63% at 800 mu g/ml (p<.05). According to the results of PI staining, this inhibitory effect was not due to cell death. Menthol dose-dependently decreased IFN gamma but not IL-4 production in culture of PHA- and PMA/CI-stimulated lymphocytes to more than 80% at 800 mg/ml. In flow cytometry analysis, menthol reduced the number of IFN-gamma-expressing CD4(+) T cells stimulated either with PHA or PMA/CI. Treatment of PBMCs with 800 mu g/ml of menthol decreased levels of T-bet from 14.5 +/- 2.26 fold in untreated control to 2.76 +/- 1.74 fold (p<.001). Foxp-3 expression decreased to nearly half, but GATA3 did not significantly change. Ratios of T-bet to GATA3 and T-bet to Foxp3 gene expressions were dose-dependently declined. Conclusion: Decreased IFN gamma expression plus T-bet down-regulation suggested the inhibitory effect of menthol on Th1 cells differentiation and hence imply its possible therapeutic usefulness in inflammatory diseases.