The Modulative Effect Of Histone Demethylase Inhibitor Tranylcypromine On Th1/Th2Lineage Development And Primary Study On The Mechanism

Objective:To explore the effect and mechanism of tranylcypromine (TCP) on Thl/Th2lineage development. To investigate the effect of TCP on T cell proliferation, apoptosis and the influence of LSD1of Th1/Th2lineage development in vitro.Methods:(1)22cases of healthy donors were selected. The peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and purified by magnetic separation anti-CD4MACS microbeads. CD4+T cells were treated by different concentrations (10.30.50、80、100μM) of TCP and the inhibitory effect of TCP on T cell proliferation were observed by CFSE assay. Annexin V-positive apoptotic cells, CD4-IFN-y-positive and CD4-IL-4-positive cells were examined by flow cytometry. The expressions of IFN-y mRNA, IL-4mRNA and T-bet mRNA were detected by RT-PCR and RQ-PCR. ELISA was used to determine the secretion of cytokine IFN-y. Western blot assay the protein levels of T-bet, STAT1and pSTAT1.The purified populations of Thl or Th2cells treat with TCP, CD4-IFN-γ-positive and CD4-IL-4-positive cells were examined by flow cytometry, and the protein levels of T-bet, STAT1and pSTAT1were assayed by western blot.(2) CD4+T cells were treated with different concentrations (10、30、50、80、100μM) of TCP and shLSD1. Light microscope observed the expression of GFP of shLSD1on T lymphocytes. Western blot assayed the protein levels of LSD1, H3K4me2, T-bet, STAT1and pSTAT1. The expressions of IFN-y mRNA and T-bet mRNA were detected by RQ-PCR.Results:(1) TCP treatment induces cellular apoptosis and suppression of proliferation of T lymphocytes in a dose-dependent manner. In presence of anti-CD3and anti-CD28Ab, CD4+T lymphocytes were treated with different concentrations (10、30、50、80、100μM) of TCP. Cells treated with TCP were inhibited in the divisions. There was a dose-dependent suppress of the proliferation in CD4+T cells by TCP, significantly in the dosage over10μM (p <0.01). The assay also showed that TCP was able to dose dependently induced the apoptosis of CD4+T cells significantly (p <0.05). When the dosage of TCP up to100μM, the percentage of apoptotic cells was set to rise dramatically (p<0.001)(2) In response to TCP, the polarization of Th cells is altered. FACS was used to distinguish and quantitatively determine the percentage of IFN-y positive cells. TCP treatment induces significant high percentage of IFN-y positive cells in a dose-dependent manner (p<0.05). Compared with controls,10μM TCP treatment resulted in4folds increases in IFN-y mRNA expression. When the dosage of TCP up to100μM, the expression of IFN-y mRNA was set to rise dramatically (p<0.001),18folds increases than control. By performing ELISA assay, the secretion level of IFN-y of TCP-treated group was significant higher than control (p<0.001)(3) T-bet/STAT1signaling pathway can be activated by TCP. By performing semiquantitative RT-PCR and real-time PCR assay, we dentified that mRNA expression level of T-bet and the protein level of pSTAT1Tyr701were significantly increased. When the dosage of TCP up to100μM, the expression of T-bet mRNA was set to rise dramatically (p<0.001),10folds increases than control. The results demonstrated that TCP induced pSTAT1signaling in purified populations of Th2.(4) Inhibition of LSD1with shRNA can alter expressions of Th1cytokine and transcription factors. Treated with inhibitor TCP could induce an increase of of H3K4dimethylation of T lymphocytes. The expressions of H3K4dimethylation, IFN-y, T-bet and pSTAT1were significantly up-regulated by knockdown of LSD1or treatment with TCP of CD4+T cells.Conclusions:TCP induces polarization of human type1T helper lymphocytes mediated by depression of LSD1activity.