DNA methylation status of the forkhead box protein 3 gene in CD4+ T cells of patients with Henoch-Schönlein purpura and its correlation with regulatory T cells

Abstract

Objective To determine the proportion of CD4+CD25+ regulatory T (Treg) cells, mRNA expression of the forkhead box protein 3 (Foxp3) gene, and DNA methylation status of the Foxp3 promoter in peripheral CD4+ T cells from patients with Henoch-Schonlein purpura. Methods Totally, 20 inpatients with Henoch-Schonlein purpura and 20 healthy controls were enrolled from Department of Dermatology, the Second Xiangya Hospital of Central South University between 2015 and 2016, and there were no significant differences in the gender and age between the two groups (both P > 0.05) . CD4+ T cells were isolated from the peripheral blood samples of these subjects. Real-time fluorescence-based quantitative PCR was performed to detect the mRNA expression of the Foxp3 gene, flow cytometry to determine the proportion of CD4+CD25+ Treg cells, and sodium bisulfite sequencing PCR (BSP) to determine the DNA methylation status of the Foxp3 promoter. Statistical analysis was carried out with SPSS16.0 software by using two-sample t test for the comparison between the two groups, and linear correlation analysis for evaluating the correlations of the DNA methylation status of the Foxp3 promoter with clinical severity scores and the proportion of CD4+CD25+ Treg cells. Results Compared with the healthy control group, the Henoch-Schonlein purpura group showed significantly decreased mRNA expression of the Foxp3 gene in CD4+ T cells (0.380 ± 0.226 vs. 1, t = 9.503, P < 0.01) , proportion of CD4+CD25+ Treg cells (1.668% ± 0.959% vs. 2.741% ± 1.131%, t = 2.552, P < 0.05) , but significantly increased DNA methylation status of the Foxp3 promoter (0.712 ± 0.164 vs. 0.453 ± 0.147, t = 3.610, P < 0.01) . In the Henoch-Schonlein purpura group, the DNA methylation status of the Foxp3 promoter was negatively correlated with the percentage of CD4+CD25+ Treg cells (r = -0.490, P < 0.05) , but positively correlated with the clinical severity scores (r = 0.486, P < 0.05) . The DNA methylation level of the Foxp3 promoter was significantly higher in the patients with renal impairment than in those without renal impairment (P < 0.05) . Conclusion The patients with Henoch-Schonlein purpura showed increased DNA methylation status of the Foxp3 promoter in CD4+ T cells, decreased mRNA expression of the Foxp3 gene and proportion of CD4+CD25+ Treg cells, which may be related to the occurrence of Henoch-Schonlein purpura, and affect disease development and prognosis. Key words: Purpura, Schoenlein-Henoch; T-lymphocytes, regulatory; DNA methylation; Genes, Foxp3