Aim: The promoter of human interleukin-10 (IL10), a cytokine crucial for suppressing inflammation and regulating immune responses, contains an interspecies-conserved sequence with CpG motifs. The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA).
Methods: Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human, macaque and mouse IL10 genes. Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected. The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 μmol/L). The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA, respectively. The methylation of CpGs in the IL10 promoter was determined by pyrosequencing. Chromatin immunoprecipitation (ChIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions.
Results: One interspecies-conserved sequence was found within the IL10 promoter. The upstream CpGs at -408, -387, -385, and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls. In contrast, the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016), which was correlated with higher IL10 mRNA and serum levels. In the 5-azacytidine-treated PBMCs, the CpG motifs were demethylated, and the expression levels of IL10 mRNA and protein was significantly increased. CHIP assays revealed increased phospho-CREB binding to the IL10 promoter.
Conclusion: The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.