Methylation Status Of TLR9Gene Promoter In Peripheral Blood Mononuclear Cells Of Patients With Systemic Lupus Erythematosus

[Background]:Systemic lupus erythema tosus is a chronic progressive and multisystem damaged autoimmune disease characterized by the generation of a wide range of autoantibodies,complement activation and immune complex deposition in whole body.Several systems and organs of the body may be damaged. Recent studies on epigenetics, including DNA methylation, could contribute to elucidation of the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). A failure to maintain DNA methylation status in the immune response due to factors including environmental influences, leads to aberrant gene expression, contributing to immune dysfunction and in some cases the development of SLE in genetically predisposed individuals.Toll like receptor9(TLR9) is a member of the TLR family,recognizes the conservative pathogen-associated molecular patterns(PAMPs), participate in specific immune response,therefore,TLR9s bridge innate and adaptive immune responses and have focused attention on the autoimmune disease.TLR9recognizes specific CpG motifs include microbial DNA and synthetic CpG ODN that lead to activation of NF-κB through MyD88-dependent signaling pathways.And then multiple cytokines and immune cells are induced and play a important roles in the pathogenesis of autoimmunity and inflammatory reaction.The process including abnormal B-cell survival,activation,autoantibody production and abnormal plasmacytoid dendritic cell(pDC) activation, releases large amounts of interferon-a.Many studies show TLR9signaling participate in the inflammatory reaction through binding the ligand of CpG motifs. In this experiment,we intend to explore the mechanism of TLR9gene involving in the pathogenesis of SLE from a new point of view to research the correlation between the methylation status of TLR9gene promoters and SLE.[Objective]:Evaluate the correlation between the methylation status of TLR9gene promoters and SLE.[Methods]:1.Blood samples were collected from15newly diagnosed and untreated patients with SLE and32matched controls in Yunnan population.2.DNA and RNA was extracted from the perpheral blood mononuclear cells.3.Quantitative analysis of the difference of methylation status of TLR9gene promoter CpG sites using BSP technique.4.Quantitative analysis of the difference of the expressing of TLR9mRNA were determined by reverse transcription polymerase chain reaction(RT-PCR);5, Statistical analysis using SPSS11.5software.[Results]:1. There were no significant difference in all10CpG sites in the TLR9gene promoter between the two groups (p>0.05).2.There was no significant difference in the combined average methylation status of10CG pairs in TLR9gene promoters between the two groups (p=0.097).3.There were no significant correlations between the combined average methylation status of10CG pairs in TLR9gene promoter and different ages in SLE group (r=0.369. p=0.175).4.There were no significant correlations between the combined average methylation status of10CG pairs in TLR9gene promoter and different ages in health controls (r=0.263,P=0.146).5.There were no significant correlations between the combined average methylation status of10CG pairs in TLR9gene promoter and the expressing level of TLR9mRNA in SLE patients (r=0.097,p=0.730).6. The expressing level of TLR9mRNA in PBMCs of SLE patients increased significantly higher than health controls(p=9.379×10-8<0.05).7.The expressing of TLR9mRNA in PBMCs of active SLE patients increased significantly higher than inactive SLE patients(P=2.664×l0-6<0.05),and the levels in two patient groups were both higher than the health controls(p<0.05).8.The expressing of TLR9mRNA on PBMCs of SLE patients were significant positive related with the SLEDAI scores(r=0.924, P=9.018×l0-7<0.05). [Conclusion]:1.The methylation status of TLR9promoter in PBMCs from newly diagnosed and untreated patients with SLE had no significant difference with health controls,and may do not contribute to the pathogenesis of SLE.2.The expressing of TLR9mRNA on PBMCs may help in assessing disease activity of newly diagnosed and untreated SLE patients in Yunnan population.3.The up-regulated expressing of TLR9mRNA may contribute to the pathogenesis of SLE.