Abnormal DNA Methylation In CD4+T Cells From Patients With Primary Sj(?)gren's Syndrome(pSS)

Objective:Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by lymphocytic infiltration and the production of autoantibodies, leading to the destruction of lacrimal and salivary glands. However, very little is known about the pathogenesis of this disorder. In recent years, evidence linking aberrant DNA methylation with regulation of genes expression in T cells and autoimmune diseases has been accumulating. For instance, genome-wide decreases in deoxy-methylcytosine and hypomethylation of specific genes, such as CD11a(ITGAL), CD70(TNFSF7), CD40 Ligand(TNFSF5) and perforin (PRF1), have been confirmed in systemic lupus erythematosus(SLE). Overexpression of these genes can induce autoreactive T cells, excessive B cell stimulation and macrophage killing, and contributes to the pathogenesis in SLE. In this study, we investigate the expression levels and methylation patterns of some autoimmune-related methylation sensitive genes in CD4+T cells from pSS patients and healthy controls, aiming to discover the aberrant epigenetic regulations in pSS and open up new avenues for revealing the pathogenesis of pSS.Methods:PBMCs (peripheral blood mononuclear cells) were isolated from the peripheral venous blood of 17 pSS patients and 14 healthy donors by density gradient centrifugation, and the flowcytometry was used to detect the expression levels of CD 11 a, CD70 and CD40L in CD4+T cells. CD4+T cells were isolated from the PBMC using magnetic cell separation technique, then the mRNA levels of CD70 and perforin in CD4+T cells were measured by real-time quantitative reverse transcriptase-polymerase chain reaction (real-time RT-PCR). Perforin protein was analysed by Western blot. Bisulfite sequencing was used to determine the methylation status of the regulatory sequence in which gene the expression are significantly difference.Results:CD70 mRNA and protein levels are significantly increased(P=0.001, P=0.003; respectively) in CD4+T cells of pSS patients compared to controls. Perforin mRNA level in CD4+T cells is significantly increased (P=0.018) compared to the controls, but no significantly difference was found in protein level. And the expression of CD11a and CD40L have no significantly difference between two groups. The TNFSF7 promoter is demethylated in CD4+ T cells of pSS patients (P=0.007) compared to controls. At the same time, we found there was a significantly negative correlation between the expression of CD70 and TNFSF7 promoter methylation level in pSS CD4+ T cells(r=-0.698, P= 0.008).Conclusion:Demethylation of the CD70 promoter regulatory elements contributes to CD70 overexpression in pSS CD4+ T cells, indicating that abnormal DNA methylation of CD70 promoter may play a crucial role in the pathogenesis of pSS.