Abnormal DNA Methylation In CD4~+T Cells From Patients With Autoimmune Diabetes

Part one Abnormal genomic DNA methylation in CD4+T cells from patients with autoimmune diabetesObjective To investigate global DNA methylation in CD4+T cells from patients with autoimmune diabetes.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of20patients with classical type1diabetes (T1ADM),15patients with latent autoimmune diabetes in adults (LADA) and19healthy controls by density gradient centrifugation. CD4+T cells were isolated from the PBMCs using magnetic cell separation technique. The global methylcytosine levels in CD4+T cells from patients with autoimmune diabetes and healthy controls were quantified using the MethylampTM global DNA methylation quantification kit.Results Global DNA methylation was significantly increased in CD4+T cells from patients with T1ADM relative to healthy controls (P=0.047). In CD4+T cells from patients with LADA, global DNA methylation was as well significantly increased relative to healthy controls(P=0.046).Conclusion These datas suggest that the global DNA methylation status is aberrant in CD4+T cells from patients with autoimmune diabetes. Part two Expression levels of genes regulating DNA methylation in CD4+T cells from patients with autoimmune diabetesObjective To investigate the mRNA expression levels of genes regulating DNA methylation in CD4+T cells from patients with autoimmune diabetes.Methods PBMCs were isolated from the peripheral venous blood of20patients with T1ADM,15patients with LAD A patients and19healthy controls by density gradient centrifugation. CD4+T cells were isolated from the PBMCs using magnetic cell separation technique. mRNA levels of DNA methyltransferases (DNMTs) and methyl-CpG-binding domains (MBDs) were measured by real-time quantitative polymerase chain reaction (real time-PCR).Results DNMT3b mRNA levels were significantly higher in CD4+T cells from patients with T1ADM and LAD A than that in healthy controls (P=0.038, P=0.040, respectively). MBD1mRNA level was significantly lower in CD4+T cells from patients with T1ADM than in healthy controls (P=0.042). No significant differences were found between patients with LADA and healthy controls when mRNA levels of MBDs were assessed. Moreover, DNMT3b mRNA expression positively correlated with CD4+T cell DNA methylation within our T1ADM patient and LADA patient cohorts (r=0.567,P=0.009; r=0.422, P=0.032; respectively).Conclusion These datas suggest that abnormal genomic DNA methylation in CD4+T cells from patients with autoimmune diabetes was associated with aberrant expression of genes regulating DNA metylation. Part three Abnormal expression and methylation pattern of regulatory sequences of some autoimmune-related genes in CD4+T cells from patients with autoimmune diabetesObjective To investigate the expression levels and methylation patterns of some autoimmune-related genes in CD4T+cells from patients with autoimmune diabetes and healthy controls.Methods PBMCs were isolated from the peripheral venous blood of20patients with T1ADM,15patients with LAD A and19healthy controls by density gradient centrifugation. CD4+T cells were isolated from the PBMCs using magnetic cell separation technique. mRNA levels of Foxp3, ICOS, CTLA-4, IFNG, IL10, IL2, IL17and IL4were measured by real time-PCR. The expression levels of CD11a and CD40L in CD4+T cells were detected by using the flowcytometry. Foxp3protein was analyzed by western-blot. Bisulfite sequencing was used to determine the methylation status of the regulatory sequence of Foxp3.Results The expression levels of Foxp3were significantly lower in CD4+T cells from patients with autoimmune diabetes than healthy controls. The mean methylation levels of eight CG pairs within the promoter of Foxp3gene in CD4+T cells from patients with autoimmune diabetes were higher relative to healthy controls.Conclusion Hypermethylation of regulatory sequence contributes to Foxp3low expression in CD4+T cells from patients with autoimmune diabetes.