DNA demethylation of CD40L in CD4+ T cells from women with systemic sclerosis: A possible explanation for female susceptibility

Abstract

Objective. Systemic sclerosis (SSc) is an autoimmune disease with a predilection for women. The interaction between CD40 and CD154 (CD40L) is known to be involved in the development of SSc. Although CD40L is overexpressed in patients with SSc, the mechanisms leading to this overexpression are not well understood. We previously demonstrated that DNA demethylation reactivates the silent X chromosome, resulting in CD40L overexpression in healthy women. We hypothesized that CD40L up-regulation by DNA demethylation and subsequent reactivation of the silent X chromosome in female patients with SSc explain the susceptibility of women to SSc. The aim of this study was to investigate the effect of DNA demethylation on CD40L expression in CD4؉ T cells from female patients with SSc.Methods. CD40L expression in CD4؉ T cells from patients with SSc and healthy control subjects was measured by flow cytometry and real-time reverse transcription-polymerase chain reaction. Bisulfite sequencing was performed to determine the methylation status of the CD40L regulatory region.Results. CD40L expression was significantly elevated in female patients with SSc. The methylation levels of the DNA regulatory sequences were reduced in female patients with SSc compared with healthy women, and there was a significant inverse correlation between the average methylation level and CD40L mRNA expression in female patients with SSc. In contrast, no significant difference was observed in the expression of CD40L between male patients with SSc and male control subjects. The DNA regulatory regions in both male patients and male control subjects were largely unmethylated.Conclusion. Demethylation of CD40L regulatory elements on the inactive X chromosome contributes to CD40L overexpression in CD4؉ T cells from female patients with SSc.

Epigenetics and systemic sclerosis

“…Lian and colleagues evaluated the expression levels and DNA methylation within the CD40L gene, which is located on the X chromosome, in men and women with SSc compared to healthy controls. The authors demonstrated increased expression of CD40L in female SSc patients in association with demethylation of the promoter region of CD40L in CD4+ T cells [40]. This study showed that there is no difference in CD40L expression levels between male SSc patients and male controls.…”

Epigenetic suppression of Fli1, a potential predisposing factor in the pathogenesis of systemic sclerosis

“…Thus, DNA methylation results in histone deacetylation leading to the transcriptional repression of target genes through chromatin condensation. In SSc, DNA hypermethylation of the FLI1 and KLF5 genes in dermal fibroblasts (Noda et al, 2014;Wang et al, 2006) and the BMPRII gene in microvascular endothelial cells (Wang and Kahaleh, 2013) and DNA hypomethylation of the ACTA2 gene in lung fibroblasts (Hu et al, 2011) and the CD40L and CD70 genes in CD4 + T cells (Jiang et al, 2012;Lian et al, 2012) have been reported (Broen et al, 2014). Also, hypoacetylation of histone H3 and H4 in the FLI1 and KLF5 promoters is exhibited in SSc dermal fibroblasts (Noda et al, 2014;Wang et al, 2006).…”

Environmental risk factors of systemic sclerosis

“…CD4+ T cell hypomethylation has been shown to be associated with reduced expression of methylation-regulating genes: DNA methyltransferase-1 (DNMT1), methyl-CpG-binding domain (MBD) 3, and MBD4 [6,9,12]. Furthermore, CD40L gene promoter has been shown to be hypomethylated and transcriptionally active in CD4+ T cells [6,9,13]. In female SSc patients, the presence of demethylated CD40L regulatory elements on the inactive X chromosome leads to CD40L overexpression; these latter findings may, in part, explain the predominance of SSc in females [6,9,13]; (2) endothelial cells from SSc patients have been found to show CpG hypermethylation of the bone morphogenetic protein receptor factor type 2 (BMPR2), which may contribute to higher vulnerability of endothelial cells to apoptosis and oxidation damage [14].…”

“…Furthermore, CD40L gene promoter has been shown to be hypomethylated and transcriptionally active in CD4+ T cells [6,9,13]. In female SSc patients, the presence of demethylated CD40L regulatory elements on the inactive X chromosome leads to CD40L overexpression; these latter findings may, in part, explain the predominance of SSc in females [6,9,13]; (2) endothelial cells from SSc patients have been found to show CpG hypermethylation of the bone morphogenetic protein receptor factor type 2 (BMPR2), which may contribute to higher vulnerability of endothelial cells to apoptosis and oxidation damage [14]. Additionally, CD70 is part of the tumor necrosis factor (TNF) α superfamily and is restricted to T and B lymphocytes and mature dendritic cells [6,9,10]; in SSc patients, the promoter of CD70 is hypomethylated, leading to the overexpression of CD70 by CD4+ T cells; and (3) fibroblast global DNA hypermethylation has been found in dermal fibroblasts from SSc patients; histological analyses of skin biopsy specimens of SSc patients showed higher levels of methylation regulatory genes DNMT1, MBD1, and methyl-CpG-binding protein (MECP) 2 than controls [6].…”