Study On Aberrant Epigenetic Modification In CD4~+T Cells From Patients With Systemic Sclerosis

Systemic sclerosis (SSc) is complex autoimmune connective tissue disease and the etiology and pathogenesis of SSc remain incompletely understood. The pathogenic processes of SSc including severe and extensive fibrosis of skin and other visceral organs, pronounced pathological changes in the microvasculature, and cell-mediated immunity and humoral immune dysfunction. Especially T lymphocytes play a crucial role in the development process in SSc.Although the precise aetiology and pathogenesis had not been elucidated, a number of studies have demonstrated that activation of the immune system is a key stimulus to vascular abnormalities and fibrosis. Lymphocyte activation, release of various cytokines, and autoantibody production are all important components of abnormal active immune responses that lead to pathological disorder of SSc. Increasing evidence suggests that interactions between genes and environment might play a critical role in the pathogenesis of complex polygenic disease such as SSc. According to the related epigenetic theory that epigenetic marks can be affected by environmental triggers, providing a plausible link between environmental factors and the onset and development of various autoimmune diseases including SSc.Recent researches have focused on the DNA methyl ation, the most prevalent and best-described epigenetic modification. In general, DNA methylation in the promoter region is associated with condensed heterochromatin and transcriptional repression; while DNA hypomethylation is responsible for gene activation. Studies have shown that T cell DNA methylation changes are closely related with autoimmune diseases. Global hypomethylation was observed to be significantly down-regulated in SSc T cells by our previous study. Further more,we demonstrated that we demonstrated that DNA demethylation of T cell results in overexpression of CD40L and CD70genes playing an critical pathogenic role in SSc. However, the molecular basis of DNA demethylation in SSc T cells are still unclear. These studies provide new insights into the mechanisms causing DNA demethylation in SSc T cells, and suggest novel approaches for the treatment of SSc and may be a target for more effective SSc treatment.Histone modifications, another important aspect of epigenetic mechanisms. Histone acetylation can neutralize alkaline amino acid’s positive charge of core histones and therefore lessens their interaction with DNA backbone with the negatively charge resulting in chromatin structure more open and accessible for transcriptional factors. In contrast, histone hypoacetylation that restores the positive charge of histones leading to a closed chromatin configuration and subsequent inhibition of gene expression. It is possible that changes in DNA methylation have a crosstalk with histone modifications because DNA does not exist naked in the nucleus but is associated with histone proteins in the form of chromatin. Our group has studied the global pattern of histone modifications in SSc and declared a global histone H3hypoacetylation. However, global histone modification patterns might be different from that of gene-specific ones. And DNA methyltransferase inhibitor or histone deacetylase inhibitor affects the status of histone H3acetylation modifications at the promoter region of the TNFSF7gene, and increases CD70mRNA expression in CD4+T cells. The purpose of this study is to explore the status of histone H3acetylation occupancy of TNFSF7gene promoter in SSc CD4+T cells.MicroRNAs are endogenous short noncoding molecules able to regulate gene expression at the post-transcriptional level. MicroRNAs are genome-encoded21-23nt RNAs targeting the3’untranslated region (3’-UTR) of specific messenger RNAs (mRNA) for degradation or translational repression. microRNAs exist in the majority of multi-cell organisms and account for2%among the genome. It is reported that more than30%the gene encoding the protein regulated by microRNAs. It has been proved that microRNAs play important roles in growth, development, cell proliferation, apoptosis, inflammation, tumor formation, immune response as well as many other physiological and pathological processes. Chromatin-based mechanisms responsible for epigenetic regulation of gene expression also include RNA-based mechanisms commonly referred as microRNAs. Microarray is the best choice to detect the expression of microRNA. However, It’s unclear that the miRNA expression profiling in CD4+T cells from SSc patients. So in this part,we aim to explore the profiling of microRNAs expression and to confirm the altered microRNAs and predict target genes of altered microRNAs in CD4+T cells from patients with systemic sclerosis.The aim of this study is to investigate the alterations of epigenetic modifications including DNA methylation, histone modifications, and microRNAs in SSc CD4+T cells. Our study will contribute to uncover whether altered epigenetic modifications are involved in the occurence and development of SSc and establish foundations for comprehensive and deep study on the pathogenesis of SSc thereby providing the novel clues for the diagnosis and treatment for SSc. Part I DNA Demethylase Regulating DNA hypomethylation in CD4+T cells and its role in the pathogenesis of Systemic SclerosisSection I Expression of Tets in CD4+T cells from SSc patientsObjective Recent studies have shown that the CD4+T cell DNA demethylation play an important role in the pathogenesis of SSc. The mechanisms of demethylation in SSc CD4+T cells remain unknown. Recent study showed that Tets can remove methylation marks, thereby reduce epigenetic silence of genes. The aim of these study was to investigate the expression of Tets, global hydroxymethylation level in SSc CD4+T cells and their correlations with disease phenotype.Methods Peripheral blood mononuclear cells (PBMCs) from18SSc patients and20healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation, CD4+subsets were isolated by positive selection using Miltenyi beads. Activity (Valentini) and severity(Medsger) scores for SSc were calculated for all patients. Expression of Tets mRNA in CD4+T cells was detected by real-time RT-PCR,and western blot was used for protein expression in the same samples. The global hydroxymethylation level was measured by DNA global hydroxymethylation kit. The correlation between Tetl mRNA, global hydroxymethylation level and activity and severity indices of the disease were performed.Results The expression of mRNA and protein in CD4+T cells from SSc patients were significantly Tetl elevated, Compared with controls (P=0.021,P=0.035,respectively). There was no significant difference between SSc patients and controls in the mRNA levels of Tet2and Tet3mRNA in CD4+T cells(P<0.05). The global hydroxymethylation level in SSc patients CD4+T cells in increased(P=0.026). There was a significant positive correlation between Tet1mRNA expression and global hydroxymethylation level and (R=0.594,P=0.009). Furthermore, the Tet1mRNA level was positively correlated with and increased disease activity in SSc patients as measured by SDAI (R=0.479,P=0.042).Conclusion Tetl expression were significantly increased in SSc CD4+T cells, which is associated with global hydroxymethylation level and increased disease activity playing an important role in the pathogenesis of SSc. Section Ⅱ Inhibiting T cells autoreactivity by downregulating Tetl expressionObjective To investigate the effect of downregulating Tetl expression by transfecting Tetl-siRNA on expression and methylation status of autoimmunity related genes CD40L、CD70, and autoreactivity in SSc CD4+T cells.Methods Peripheral blood mononuclear cells (PBMCs) from5SSc patients and5healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation, CD4+subsets and B cells were isolated by positive selection using Miltenyi beads. Tet1-siRNA was transfected into SSc CD4+T cells by transient electroporation. Real-time RT-PCR was used to detect the expression of Tetl and methylation sensitive genes CD40L and CD70mRNA. CD40L and CD70protein levels were measured by flow cytometry. Cell proliferation assay and production of IgG antibody were performed following the protocol of kits. The global hydroxymethylation level and methylation status of CD40L and CD70 promoter regulatory elements were detected by the bisulfite sequencing and sodium bisulfite sequencing, respectively.Results The mRNA expression of Tetl, CD40L and CD70mRNA were significantly downregulated in SSc CD4+T cells transfected with Tet1-siRNA (P<0.05). The protein level of CD40L and CD70were decreased too (P<0.05). Reduced cell proliferation activity and IgG production, and accompanied with global hydroxymethylation were also found in SSc CD4+T cells transfected with Tet1-siRNA (P<0.05). The average methylation of CpG pairs at TNFSF5promoter sequence (-402to+150) and TNFSF7promoter element (-581to-288) were significantly increased (P<0.05) in SSc CD4+T cells transfected with Tetl-siRNA.Conclusion Downregulation of Tetl expression can inhibit expression of methylation sensitive genes, increase the methylation levels of these genes and reduce autoreactivity in SSc CD4+T cells. Part II Aberrant Histone H3acetylation modifications at TNFSF7promoter in Systemic Sclerosis CD4+T cellsObjective Histone acetylation often work together with DNA methylation playing key roles in epigenetic gene regulation, resulting in inappropriate expression or silencing of genes without corresponding changes in their DNA sequence in mammalian cells. CD70is encoded by TNFSF7gene, and TNFSF7promoter regulatory sequence is hypomethylated in SSc CD4+T cells. The purpose of this study is to explore the status of histone histone H3acetylation modifications occupancy of TNFSF7gene promoter in SSc CD4+T cells.Methods PBMCs from10SSc patients and10healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+subsets were isolated by positive selection using Miltenyi beads and protocols provided by the manufacturer. Real-time PCR were used to detect the mRNA level of CD70. Chromatin immunoprecipitation (ChIP) analysis and Real-time PCR were used to detect the status of histone H3acetylation at the TNFSF7promoter region.Results Histone H3acetylation level is significantly elevated in SSc patients CD4+subsets compared with normal controls (P=0.008). And Histone H3acetylation level positively correlated with both CD70mRNA expression and increased disease activity measured by SDAI (R=0.691, P=0.027; R=0.719, P=0.019, respectively).Conclusion Aberrant histone modifications such as H3hyperacetylation targeted to TNFSF7promoter region may contribute to the development of SSc by increasing CD70expression in CD4+T cells. Part Ⅲ Abnormal microRNAs expression in CD4+T cells from patients with Systemic SclerosisObjective To investigate the profiling of microRNAs expression CD4+T cells from patients with systemic sclerosis.Methods PBMCs from5patients with systemic sclerosis and5normal controls were extracted by Ficoll-Hypaque density gradient centrifugation. CD4+subsets were isolated by positive selection using Miltenyi beads and protocols provided by the manufacturer. Total RNA including microRNAs was extracted using TRIzol reagent. MicroRNAs with differential expression were detected by μParaflo microRNAs microarray by a service provider (LC Sciences) and the Altered microRNAs were confirmed in18patients with systemic sclerosis and20healthy controls by real-time PCR. And then to predict target genes of altered microRNAs.Results According to μParaflo microRNAs microarray,16microRNAs including miR-451a, miR-126, miR-21and miR-223were significantly up-regulated in CD4+T cells from SSc patients;8microRNAs including miR-5100, miR-146a, miR-150and miR-142-5p were significantly down-regulated in CD4+T cells from SSc patients compared to normal controls (p<0.01), all of which were confirmed in18patients and20healthy controls by real-time PCR. In addition, These differentially expressed microRNAs have a relationship with DNA methylation, histone modifications and related immune and inflammatory signaling pathways.Conclusion Aberrant microRNAs expression in CD4+T cells may play a role in the pathogenesis of systemic sclerosis.