DNA hypermethylation of the forkhead box protein 3 (FOXP3) promoter in CD4+ T cells of patients with systemic sclerosis

BACKGROUND: Systemic sclerosis (SSc) is a complex autoimmune disease that involves dysregulation of immune homeostasis. The failure of impaired regulatory T cells (Tregs) to maintain immune homeostasis plays a major role in the development of SSc. Transcriptional silencing of the forkhead box protein 3 gene (FOXP3) via hypermethylation of regulatory regions has been identified as a hallmark of committed Tregs and several autoimmune disorders. OBJECTIVES: To investigate whether aberrant expression and methylation of FOXP3 occurs in CD4+ T cells of patients with SSc and their roles in the pathogenesis of SSc. METHODS: FOXP3 expression in CD4+ T cells was measured by real-time quantitative reverse-itranscriptase polymerase chain reaction and western blot. Bisulfite sequencing was performed to determine the methylation status of the FOXP3 proximal promoter sequence. The percentage of Treg cells was estimated by flow cytometry. RESULTS: Decreased FOXP3 expression was observed in CD4+ T cells from patients with SSc. The methylation levels of the FOXP3 regulatory sequences were elevated and inversely correlated with FOXP3 mRNA expression in patients with SSc. The number of Tregs was significantly reduced in patients with SSc. Treatment of SSc CD4+ T cells with a DNA methylation inhibitor, 5-azacytidine, reduced the mean methylation levels, and enhanced FOXP3 expression and Treg generation. The promoter methylation status and expression level of FOXP3 are significantly associated with disease activity. CONCLUSIONS: The contribution of the hypermethylation of the FOXP3 promoter to decreased FOXP3 expression and the subsequent quantitative defects of Tregs may mediate the immune dysfunction in SSc.