The Role And Mechanism Of CD40L Escaping From X Chromosome Inactivation In Systemic Sclerosis

Objective:Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by autoantibody production, progressive skin and muti-organ fibrosis and microangiopathy. The morbidity rate has significant gender difference and more than80%of SSc patients are female. The pathogenesis of SSc is unclear. Elevated CD40ligand (CD40L) expression is found in SSc patients and tight-skin mice. However, little is known about the mechanisms underlying the overexpression of CD40L in SSc. Our early research demonstrated that DNA methyltransferase inhibitors reactivates original inactivated X chromosomes and double CD40L expression in healthy women. So our study mainly compared the CD40L expression and DNA methylation status of CD40L gene regulatory sequences in SSc patients, aim to explore the reasons for CD40L overexpression in SSc and provide new ideas for pathogenesis of SSc.Methods:PBMCs (peripheral blood mononuclear cells) were isolated from the peripheral venous blood of26SSc patients(16females,10males) and20healthy donors(15females, lOmales) by density gradient centrifugation, and the flowcytometry was used to detect the expression levels of CD40L in CD4+T cells. CD4+T cells were isolated from the PBMC using magnetic cell separation technique, then the mRNA levels of CD40L in CD4+T cells were measured by real-time quantitative reverse transcriptase-polymerase chain reaction (real-time RT-PCR). Bisulfite sequencing was used to determine the methylation status of CD40L regulatory sequence in CD4+T cells.Results:CD40L mRNA and protein levels are significantly increased in CD4+T cells of female SSc patients compared to female controls(P<0.05, P<0.05; respectively). In heathy female, half of the cloned fragments of CD40L regulatory sequences are unmethylated, while the other half are methylated, but the same sequences are mostly unmethylated in female SSc. The mean methylation levels of the CD40L promoter and enhancer from female SSc patients were much lower than from healthy female controls(P<0.001,P<0.001; respectively). Moreover, there was a significant inverse correlation between the average methylation level and CD40L mRNA expression in female SSc patients (r=-0.629, P<0.01; r=-0.727, p<0.01, respectively). We observed no significant increase of CD40L expression in male SSc patients compared with male controls(P>0.05, P>0.05; respectively). The CD40L regulatory regions were largely unmethylated in both male patients and male controls, and there was no significant difference in the methylation level of CD40L promoter and enhancer between them (p>0.05,p>0.05, respectively).Conclusion:CD40L was overexpressed in CD4+T cells of female SSc patients but not males. Demethylation of CD40L regulatory elements contributes to X chromosome reactivation and CD40L overexpression in CD4+T cells from female SSc patients, that maybe a important reason for female susceptibility to SSc.