RESUMO
Objective: To investigate the role and mechanism of DNA methylation in Mycobacterium tuberculosis (MTB lysate) -induced downregulation of interleukin-6 receptor(IL-6R) expression in CD4+T cells. Methods: A prospective study was conducted. Bisulfite sequencing (BSP) was applied to determine the methylation levels of CpG island in IL-6R promoter region and 3'untranslated region (3'UTR) region in CD4+T cells from peripheral blood mononuclear cells (PBMC) of control group (healthy person, n=10) and TB group (tuberculosis patients, n=10) in Shenzhen Third People's Hospital between 2019 and 2020. Quantitative reverse transcription-PCR (RT-qPCR) and Western blotting were used to detect the expression of IL-6R, DNMT1, DNMT3A and DNMT3B in MTB lysate-stimulated CD4+T cells and Jurkat E6-1 cells. Furthermore, PBMC in control group and Jurkat E6-1 cells activated by anti-CD3/CD28 antibody were stimulated by MTB lysates to detect the methylation levels of CpG island and IL-6R and DNMT expression. Transcriptional activity of differently methylation regions of IL-6R 3'UTR was detected by using luciferase reporter gene system. Results: IL-6R expression in TB group was lower than that in control group, but DNMT1 and DNMT3B expressions were higher than those in control group in CD4+T cells isolated from PBMC. There was no significant difference in the methylation rate of IL-6R promoter CpG island of CD4+T cells between control and TB group. However, the methylation rates of CpG island in 3'UTR region were significantly higher (P<0.001) in TB (69.5%±3.4%), compared with control (54.3%±4.7%). Besides, IL-6R expression was lower than unstimulated, while DNMT1 and DNMT3B expression was higher than unstimulated after MTB lysate-stimulation of activated control PBMC in vitro. The methylation rate of CpG island in IL-6R 3'UTR region of CD4+T cells increased from 58.8%±11.6% to 79.4%±10.9% (P<0.001) after MTB lysate-stimulated PBMC of the control. The same results were observed in the MTB lysate-stimulated CD4+T cells isolated from PBMC in control and Jurkat E6-1 cell line. Furthermore, IL-6R expression after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysate was higher than that stimulated by MTB lysate alone. In addition, the methylation levels of CpG islands in the 3' UTR region of IL-6R were lower than those stimulated by MTB lysates alone after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysates. The transcriptional activity of the fully unmethylated IL-6R 3'UTR CpG island reporter gene was higher than that of the fully methylated IL-6R 3'UTR CpG island. Conclusions: MTB lysates stimulation inhibited IL-6R expression transcriptionalely as well as on the protein level by inducing hypermethylation of CpG island in IL-6R 3'UTR region of CD4+T cells. The hypermethylation of CpG island in IL-6R 3'UTR region of CD4+T cells induced by MTB may be related to the increased expression of DNMT1 and DNMT3B.