Background/Purpose: Systemic sclerosis (SSc) is a systemic autoimmune disease of which the etiology and pathogenesis remains complex and poorly understood.  Attention has recently been directed towards regulatory T cells (Tregs) because they are important in maintenance of immunologic self-tolerance as well as negatively regulating immune responses against non-specific stimuli.  The normal development and suppressive phenotype of Tregs depends on stable expression of FOXP3.  The methylation status of the CpG residues in the proximal promoter region has an essential role in FOXP3 expression. Transcriptional silencing of FOXP3 through hypermethylation in regulatory elements regions has been identified as a hallmark of committed Treg cells and several human diseases.  The aim of this study was to investigate on how DNA methylation status of CpG islands in the proximal promoter region within the FOXP3 locus affect FOXP3 expression and defects in Tregs from patients with SSc.

Methods: 18 SSc patients and 16 healthy control subjects(HC) were recruited from the outpatient clinics,inpatient services and medical staff at the Second Xiangya Hospital of Central South University in China. Patients and controls were age- and sex-matched in all experiments.Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density-gradient centrifugation,and CD4+T cells were isolated by positive selection using immunomagnetic beads,according to protocols provided by the manufacturer.  The percentage of CD4+CD25+FOXP3+ Treg cells in peripheral blood mononuclear cells was measured by flow cytometry from SSc patients and HC.   FOXP3 expression in CD4+ T cells from patients with SSc and HC was measured by RT-PCR and western blot.  Bisulfite sequencing was performed to determine the methylation status of the FOXP3 proximal promoter in CD4+ T cells from SSc patients, HC and in CD4+ T cells with DNA methylation inhibitors.

Results: The percentage of CD4+CD25+FOXP3+ Treg cell was decreased in SSc patients.  FOXP3 expression was significantly reduced in patients with SSc. The methylation levels of the DNA regulatory sequences were elevated in patients with SSc compared with healthy controls, and there was a significant inverse correlation between the average methylation level and FOXP3 mRNA expression in patients with SSc.  Further more, there was a significant inverse correlation between the the average methylation level and the percentage of Tregs in a cohort of SSc patients. Treatment with a DNA methylation inhibitor led to FOXP3 hypomethylation in the FOXP3 promoter resulting in FOXP3 overexpression and expansion of Tregs. The percentage of Tregs was positively correlated with disease activity.

Conclusion: Hypermethylation of the FOXP3 promoter contributes to the decreased FOXP3 expression and quantitative defects of Tregs in SSc patients correlating with disease activity.  These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of SSc. We propose that this reduction per se could sustain autoimmunity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dna-hypermethylation-of-forkhead-box-protein-3-foxp3-locus-leads-to-quantitative-defects-of-regulatory-t-cells-in/