Nicotine Promotes Thymocyte Apoptosis By TET2-regulated Fas Demethylation

Background:Nicotine,the major addiction and toxic component in cigarettes,plays a key and leading role in smoking-related immune system damage and related diseases.Our previous studies have shown that nicotine exposure during pregnancy can promote fetal thymocyte and CD4 single positive(SP)cell apoptosis,causing CD4+Naive T cells output decreased and leading to offspring immune dysfunction,among which Fas pathway played an important role in promoting cell apoptosis.In addition,studies have shown that nicotine can regulate the DNA methylation level of fetal tissue genes,and Fas expression can be regulated by DNA methylation.Therefore,we explore the molecular mechanism of nicotine to promote thymocyte apoptosis from the perspective of DNA methylation in vitro,laying the foundation for the early diagnosis or treatment of immune diseases.Objective:This study was designed to observe the effect of nicotine on thymocyte apoptosis,and to explore the underlying molecular mechanism of Fas mediated thymocyte apoptosis by nicotine from the perspective of DNA methylation.Methods:(1)Thymocyte apoptosis and Fas pathway-related genes expression:3 weeks weaning ICR mice were sacrificed to prepare for primary thymocytes which were divided into a control group and five nicotine groups at different doses(0,25,50,100,200 and 400 ?M),with nicotine treating for 48 h.The activity of thymocytes was detected by Cell Counting Kit-8(CCK-8).Then the appropriate doses of nicotine(0,25,50 and 100 ?M)were selected to treat thymocytes for 48 h.The total thymocytes and CD4SP cell apoptosis were analyzed by Fluorescence Activating Cell Sorter(FACS).According to the dose-effect result,50 ?M nicotine was selected to treat thymocytes at 0,24,48,72 hours,respectively.The total thymocytes and CD4SP cell apoptosis were analyzed by FACS.In addition,the mRNA expression of a7 nAChR and Fas apoptotic pathway related genes(Fas,caspase 8,caspase 3)were detected by Real-time quantitative PCR(qPCR),Fas protein expression was analyzed by FACS and the activity of caspase 3 and 8 by corresponding Caspase Activity Detecting Kit.(2)Fas methylation and methylation-related enzymes involved in the apoptosis of thymocytes induced by nicotine:First,50?M nicotine was selected to treat thymocytes for 48 h,Sequenom MassARRAY was used to detect DNA methylation of Fas promoter,qPCR was used to detect the mRNA expression of methylation-related enzymes(TET1,TET2,TET3,Dnmt 1,Dnmt 3a,Dnmt 3b).The expression significantly altered gene(TET2)was selected for siRNA interference,the expression of TET2 protein was analyzed by Western blot,the expression of Fas protein in the total thymocytes and CD4SP cells were detected by FACS and Fas methylation by Sequenom MassARRAY.(3)To verify the role of ?7 nAChR in nicotine-induced thymocyte apoptosis:According to the dose-effect and time-effect of nicotine on thymocytes apoptosis,50 ?M was selected as the dose of nicotine and 48 h as the time of administration of nicotine,and one hour after administration of nicotine receptor-specific inhibitor a-bungarotoxin(?-Btx,1 ?/mL).FACS was used to analyze the thymocyte apoptosis and the expression of Fas protein,Caspase Activity Detecting Kit was performed to measure the activity of caspase 3 and caspase 8,Western blot was used to detect the TET2 expression,and Sequenom MassARRAY was utilized to analyze the methylation of Fas promoter.Results:(1)Nicotine promoted thymocyte apoptosis and activated the expression of ?7 nAChR and Fas pathway-related genes:?The results of cell viability:25 ?M nicotine had no significant effect on thymocyte viability compared to the control group,whereas 50 to 200?M nicotine reduced the viability of thymocytes in a dose-dependent manner(P<0.05,P<0.01).? Dose-effect and time-effect of nicotine on thymocyte apoptosis:After 48 h of treatment with nicotine,25 ?M nicotine had no effect on the apoptosis of total thymocytes and CD4SP cells in mice compared with the control group,whereas 50 and 100 ?M nicotine significantly increased the apoptosis rate of total thymocytes and CD4SP.The result of time-effect of nicotine on thymocyte apoptosis showed that nicotine treatment for 24 h had few effect on apoptosis of thymocytes,while with prolonged duration of action of nicotine,the apoptosis rate was significantly increased(P<0.01)in a time-dependent manner at 48?72 h.?The mRNA expression of ?7 nAChR and Fas apoptotic pathway-related genes:50 and 100 ?M nicotine increased the mRNA and protein expression of ?7 nAChR,Fas,caspase 8,caspase 3 and TET2 in a dose-dependent manner.(2)Fas methylation and methylation-related enzymes mediated the apoptosis of thymocytes induced by nicotine:? The results of Fas methylation and the expression of methylation-related enzymes:The methylation level of nuleotide(nt)-2394,nt-2441 and nt +295 at Fas gene promoter was significantly decreased(P<0.05),and the methylation level of nt +456 showed a declining trend(P = 0.06).Besides,50 and 100 ?M nicotine significantly increased the expression of TET2,however,not the expression of TET1,TET3,Dnmt 1,Dnmt 3a and Dnmt 3b.?The results of siRNA transient transfection:Nicotine-induced total thymocyte and CD4SP cell apoptosis,Fas demethylation and Fas expression were inhibited after TET2 knockdown.(3)?7 nAChR mediated Fas demethylation and thymocyte apoptosis:After addition of ?-Btx,nicotine-induced total thymocyte and CD4SP cell apoptosis,caspase 8 and caspase 3 active protein and TET2 protein expression,Fas demethylation and protein expression were inhibited.Conclusion:Nicotine can activate ?7 nAChR on the surface of mouse thymocyte membrane,and then decreases the methylation level of Fas promoter by enhancing the expression of desmethylated protein TET2,which activating the apoptotic pathway and finally leading to increase of thymocyte apoptosis.