DNA Methylation Status Of Regulatory Elements Of IL10 And IL13 Gene In CD4~+T Cells Of Patients With Systemic Lupus Erythematosus

Objective:Abnormal DNA methylation contributes to the develpoment of systemic lupus erythematosus (SLE). Interleukin (IL)-10 and IL-13 play important roles in Th2 cell differentiation and production of autoantibodies in patients with SLE. However, the mechanisms leading to IL-10 and IL-13 overexpression in SLE patients are not well understood. In this study, we investigate the DNA methylation status of regulatory elements of IL-10 and IL-13 gene in CD4+ T cells of patients with SLE and the relationship between DNA methylation levels and expression levels of IL-10 and IL-13 genes.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of SLE patients and healthy donors by density gradient centrifugation. CD4+ T cells were isolated using microbeads and protocols provided by the manufacturer. Where indicated PBMCs were stimulated with phytohemagglutinin (PHA), and then cultured with 1μM 5-azaC in RPMI 1640/10% FCS/IL-2 for an additional 72 h. After that, CD4+ T cells were isolated from treated PBMCs using CD4 beads. Real-time RT-PCR was used to detect the expression of IL-10 and IL-13 mRNA. ELISA kit was used to measured IL-10 and IL-13 protein concentrations in the serum from SLE patients and healthy controls. Bisulfite sequencing was used to determine the methylation status of the regulatory sequence of IL-10 and IL-13 gene.Results:Compared to negative controls, the expression levels of IL-10 and IL-13 mRNA were significantly elevated in patient samples compared to healthy controls(IL-10:P=0.001; IL-13:P=0.012). ELISAs showed serum protein levels were significantly increased in SLE patients (IL-10:P=0.005; IL-13:P=0.007). Bisulfite sequencing results showed the DNA methylation level of the regulatory sequence of IL-10 and IL-13 gene was significantly lower in lupus CD4+ T cells than in controls(IL-10: P=0.036; IL-13:P=0.006), it was inversely correlated with the IL-10 and IL-13 mRNA expression(IL-10:p=0.04; IL-13:P=0.006). Moreover, treating healthy CD4+ T cells with the demethylating agent 5-azacytidine (5-azaC) increased IL-10 and IL-13 mRNA transcription (IL-10:p= 0.017; IL-13:p=0.025).Conclusion:DNA Hypomethylation contributes to overexpression of IL-10 and IL-13 in CD4+ T cells of SLE patients. Aberrant DNA methylation modification plays an important role in the pathogenesis of SLE.