Methylation Of Forkhead Box Protein 3 Gene In CD4+T Cells In Patients With Chronic Hepatitis B

Background:Hepatitis B virus(HBV)infection has been a serious public health problem.HBV has chronically infected approximately 2.57 million people worldwide and persistent HBV infection leads to 786,000 deaths annually.The clinical outcome of HBV infection is largely dependent on the complex interplay between virus replication and host immune response.Adaptive immunity,especially HBV-specific cytotoxic T lymphocytes and CD4+ T lymphocytes,has been proved to be essential for the control of HBV infection.However,chronic carriers of HBV are unable to generate sufficient adaptive immunity against the virus.It has been indicated that CD4+CD25+ regulatory T cells(Tregs)play a vital role in the maintenance of immunologic tolerance to viral antigens by inhibiting aggressive T cell response.Emerging data suggest that pathogenesis of persistent virus infections may be closely associated with the levels of Tregs.Furthermore,there is a large amount of evidence that increased frequency of Tregs may be an underlying cause of chronic HBV infection.Forkhead box transcription factor 3(Foxp3)is essential for the development and function of regulatory T cells(Tregs).It has been shown that a stable Foxp3 expression is a prerequisite for the development and maintenance of the suppressive capacity of Tregs.Elevated Foxp3 expression has been observed in patients with chronic HBV infection and plays a role in the pathogenesis of chronic HBV infection.At present,the expression regulation mechanism of Foxp3 gene in CD4+ T cells in HBV infected patients remains unclear.Epigenetic regulation plays an important role in cell development and differentiation.Several studies have clearly demonstrated that DNA methylation plays a major role in the regulation of stable Foxp3 expression in naturally occurring Tregs.Foxp3 hypermethylation suppresses Foxp3 expression while hypomethylation allows stable Foxp3 expression.Therefore,there is a possibility that demethylation of Foxp3 gene may exist in CD4+ T cells of CHB,which down-regulate gene expression and contribute to the maintenance of chronic HBV infection.Aim:This study aimed to investigate the methylation status and expression of Foxp3 in CD4+ T cells of patients with chronic hepatitis B(CHB).Methods:Fifty nine patients with CHB,and 22 healthy controls(HC)were randomly enrolled.The methylation status of Foxp3 was determined by methylation-specific polymerase chain reaction.The percentage of CD4+CD25+Foxp3+Tregs in CD4+ T cells was estimated by flow cytometry.Foxp3 expression was measured by quantitative real time-polymerase chain reaction(RT-qPCR)and western blot.Results:1.The percentage of Tregs in CD4+ T cells was significantly higher in patients with CHB(4.6%[3.2%,6.4%])than HC(3.6%[2.75%,4.3%])(P<0.05).The percentage of CD4+CD25+Foxp3+Tregs in CHB patients with HBeAg-positive(5.30%[3.73%,7.98%])was significantly higher than that in CHB patients with HBeAg-negative(3.90%[3.10%,4.90%])and HC(3.6%[2.75%.4.3%])(P<0.05),however,there was no significant difference between CHB patients with HBeAg-negative and HC(P>0.05).2,The Foxp3 mRNA and protein expression levels in CD4+ T cells were significantly higher in patients with CHB(0.0032[0.0019,0.0069];0.59[0.4,0.93])than HCs(0.0017[0.0015,0.0030];0.45[0.37,0.55])(P<0.05).The expression of Foxp3 mRNA(0.0046[0.0040,0.0061])and protein(0.73[0.66,0.93])in CD4+ T cells of CHB patients with HBeAg-positive was significantly higher than in patients with HBeAg-negative(0.0024[0.0022,0.0041];0.49[0.41,0.66])and HC(0.0017[0.0015,0.0030];0.45[0.37,0.55])(P both<0.05),The Foxp3 mRNA levels in CHB patients with HBeAg-negative were also significantly higher than in the HC group(P<0.05),however,there was no significant difference in expression of Foxp3 protein between CHB patients with HBeAg-negative and HC(P>0.05).In addition,the expression of Foxp3 gene in CD4+ T cells was positively correlated with the ratio of CD4+CD25+Foxp3+ Tregs cells in CHB patients by Spearman rank correlation test.3.The methylation frequency of Foxp3 was significantly lower in CHB patients than HCs(P<0.05).The methylation frequencies of Foxp3enc and Foxp3cpg in CHB patients with HBeAg-positive(35.3%;32.4%)were significantly lower than those in HC group(72.7%;63.6%)(P<0.05).However,The methylation frequencies of Foxp3enc and Foxp3cpg in CHB patients with HBeAg-negative(52.0%;44.0%)was no significant difference between CHB patients with HBeAg-positive and HC(P>0.05).In addition,Foxp3 mRNA levels and the percentage of Tregs in CD4+ T cells were significantly lower(P<0.05)in CHB patients with gene methylation than those without.(P<0.05).4.Patients with high serum HBV DNA(HBV DNA load>1000 IU/ml)had a significantly higher percentage of Tregs(5.00%[3.80%,7.60%])and Foxp3 mRNA level(0.0037[0.0024,0.0080])in CD4+ T cells compared with patients with low serum HBV DNA(HBV DNA load<1000 IU/ml)(3.70%[2.95%,5.08%];0.0024[0.0017,0.0038])(P<0.05).However,the methylation frequency of Foxp3 was not significantly lower in patients with high serum HBV DNA than that in patients with low serum HBV DNA(Foxp3enc 34.3%vs 56.5%,P = 0.129;Foxp3cpg vs 45.8%,P>0.05).Conclusions:Aberrant demethylation of Foxp3 gene existed in CD4+ T cells of CHB,which contributed to an elevation in Foxp3 expression and percentage of CD4+CD25+Foxp3+Tregs.It might provide a new target for prevention and treatment of CHB.