Abnormal DNA Methylation In T Cells From Patients With Subacute Cutaneous Lupus Erythematosus (SCLE)

Background Subacute cutaneous lupus erythematosus(SCLE) is a subtype of lupus erythematosus(LE).Most patients with SCLE have a chronic or relapsing but benign condition,with few of the serious manifestations associated with systemic lupus erythematosus(SLE),such as renal disease or central nervous system symptoms.However,in some patients SCLE develops into SLE,causing progressive systemic damage over a period of years.Although the genetic and epigenetic factors that trigger SLE or the transition from SCLE to SLE remain unknown,studies indicate the importance of altered DNA methylation,especially hypomethylation in CD4+ T cells,in the pathogehesis of SLE.DNA methylation is an epigenetic regulator of several biological processes,including embryonic development,gene transcription,X chromosome inactivation,genomic imprinting and chromatin modification.Methylation involves the transference of a methyl group from the methyl donor S-adenosyl-L-methionine to the 5th carbon position in the cytosine ring of deoxycytosine(dC) bases in CG pairs. Methylation of CpG islands within promoter or enhancer regions suppresses transcription of target genes.Patterns of DNA methylation are established and maintained by DNA methyl transferases(DNMTs).In humans,the three enzymes DNMT1,DNMT3a,and DNMT3b are known to have DNA methyltransferase activity.DNMT3a and DMMT3b are responsible for de novo methylation and modify unmethylated DNA whereas DNMT1 is thought to be required for maintaining methylation patterns and acts on hemimethylated DNA.Methylated DNA is recognized by a conserved family of methyl-DNA binding domain(MBD) proteins,of which there are five known members in mammalian genomes:MBD1,MBD2,MBD3, MBD4,and MECP2.All MBDs share a common methyl CpG binding domain and associate with proteins that play active roles in regulating DNA methylation,heterochromatin formation and gene transcription.In CD4+T cells of patients with SLE,MBD2 and MBD4 mRNA expression are elevated,and it has been proposed that this plays an active role in compromising T cell methylation.Evidence linking aberrant DNA methylation with SLE has been accumulating over the past two decades.Drugs that inhibit methylation trigger lupus-like autoimmune responses in mice and human lymphocytes, and T cells from patients with active lupus have genome-wide decreases in deoxymethylcytosin and gene-specific hypomethylation.These methylation deficits correlate with upregulated expression of autoimmune-related genes such as CD11a,CD70,perforin and CD40 Ligand.Overexpression this genes inducing excessive B cell stimulation, autoreactive monocyte and macrophage killing and contribute to the disease pathogenesis.SCLE is characterized by clinical features distinct from those of SLE and the genetic and epigenetic factors causing the disorder are unknown. In this work,we set out to investigate whether T cell hypomethylation is a determinant of the pathogenesis of other,less severe forms of erythematosus lupus conditions such as SCLE.Firstly we investigated the global DNA methylation levels as well as the expression of DNMTs and MBDs in CD4+ and CD8+ T cells of patients with SCLE,secondly we compared the expression level of some autoimmune-related genes such as CD11a,CD70,perforin and CD40 Ligand in CD4+ and CD8+ T cells of patients with SCLE and normal controls,and lastly we investigated whether gene-specific hypomethylation induces autoimmune-related genes overexpression in SCLE CD4+ T Cells.Part one Abnormal DNA methylation in T cells from patients with subacute cutaneous lupus erythematosusObjective To investigate the global DNA methylation and the expression of genes that regulate methylation in T cells of patients with SCLE.Methods PBMC(peripheral blood mononuclear cells) cells were isolated from the peripheral venous blood of 12 SCLE patients and 9 healthy donors by density gradient centrifugation.CD4+ and CD8+T cells were isolated from the PBMC using magnetic cell separation technique.We quantified global methylcytosine levels in CD4+ and CD8+T cells from patients with SCLE and healthy controls using the Methylamp^TM Global DNA Methylation Quantification Kit.mRNA levels of DNA methyltransferases(DNMTs) and methylated CpG binding proteins(MBDs) were measured by real-time quantitative polymerase chain reaction(RT-PCR).Results Global DNA methylation was significantly reduced in CD4+ T cells from SCLE patients relative to controls(P=0.002).No statistically significant differences were found between patients and controls when the methylation status of CD8+ T cells was assessed(p=0.231).DNMT1 and DNMT3a mRNA levels were significantly lower in CD4+T cells from SCLE patients than that in controls(P=0.027;P=0.004, respectively).MBD1,MBD3 and MBD4 mRNA levels were significantly higher in CD4+ T cells from SCLE patients compared to controls (p＜0.001;p＜0.001 and p=0.001,respectively).Additionally,MECP2 and MBD4 mRNA expression was also significantly increased in CD8+ T cells from SCLE patients than that in controls(p=0.001;p=0.001, respectively).We also found DNMT1 mRNA expression and CD4+T cell DNA methylation to be positive correlated within our SCLE patient cohort(r=0.590,P=0.044).Conclusion These data suggest that globe DNA hypomethylation in CD4+T cells contributes to the development of SCLE. Part two Abnormal expression and methylation pattern of regulatory sequences of some autoimmune-related genes in T cells from patients with subacute cutaneous lupus erythematosusObjective To investigate the expression levels and methylation patterns of some autoimmune-related genes in CD4+ T cells from SCLE patients.Methods PBMC(peripheral blood mononuclear cells) cells were isolated from the peripheral venous blood of 12 SCLE patients and 9 healthy donors by density gradient centrifugation.CD4+ and CD8+T cells were isolated from the PBMC using magnetic cell separation technique.Using the flowcytometry to detect the expression levels of CD11a,CD70,CD40L and Perforin in CD4+ and CD8+T cells,mRNA levels of CD11a,CD70,CD40L and Perforin were measured by real-time quantitative polymerase chain reaction(RT-PCR).Perforin protein was analysed by western-blot.Bisulfite sequencing was used to determine the methylation status of the regulatory sequence in which gene the expression are significantly difference.Results CD70 and Perforin is overexpressed on SCLE CD4+T cells. Demethylation of the CD70 and Perforin promoter regulatory sequence were seen in CD4+T cells from patients with SCLE.Conclusion Demethylation of regulatory sequence contributes to CD70 and Perforin overexpression in SCLE CD4+T cells.