The Protective Role Of Transcription Factor E4BP4in Systemic Lupus Erythematosus And Its Molecular Mechanisms

Systemic lupus erythematosus (SLE) is an autoimmune disease which involves multiple organs and systems. It is characterized by T and B cells overactivation and the overproduction of autoantibodies against multiple self antigens. Its etiology and pathogenesis is unclear. Recent studies have shown the importance of epigenetic mechanisms contribute to SLE pathogenesis, in addition to various genetic factors.The term "epigenetics" refers to reversible and heritable changes in gene expression without changing the DNA nueleotide sequences. The major mechanisms of epigenetic regulation include DNA methylation, histone modifications, chromatin modifications, microRNA regulation and so on. In recent years, scholars have identified a series of histone acetylation and methylation sites, different modified forms can affect chromatin structure and the ability of conbination with transcription factors, then dynamic regulate gene transcription. Now the epigenetic researches of SLE focuse on DNA methylation. However, studies of histone modifications in the pathogenesis of SLE are very limited.E4BP4, which is also called NFIL3, is a basic leucine zipper transcription represor, and glucocorticoid can induce E4BP4expression dependented on intracellular Ca2+concentration([Ca2+]i). Recently, numerous studies have demonstrated a diverse and important role of E4BP4in the immune system. E4BP4was involved in development of specific immune cell lineages, IgE class switching in B cells, polarisation of T helper responses as well as cytokines production. However, the function of E4BP4in the pathogenesis of autoimmune diseases such as SLE remains to be defined.Our previous studies by high-throughput TFs profiling microarray have shown that E4BP4activity is increased in SLE CD4+T cells compared with normal controls. So we hypothesized that E4BP4played an important role in the pathogenesis of SLE.To prove the hypothesis, we used the following approach:Firstly, to investigate the expression of E4BP4in CD4+T cells from normal controls and SLE patients. Secondly, to study the relationship between abnormal E4BP4expression and autoimmunity:(1) transfected E4BP4expression plasmid pCDNA3.1-E4BP4into normal CD4+T cells activated by anti-CD3/CD28antibodies, to investigate the effect of E4BP4on activation of normal CD4+T cells;(2) knocked down E4BP4expression specifically in SLE CD4+T cells to determine the role of E4BP4in the regulation of self-reactivities of SLE CD4+T cells. Thirdly, to detect whether CD40L is the target gene of E4BP4in CD4+T cells:(1) screened the E4BP4binding sites in the promoter region, and confirm that E4BP4binds to the promoter region of CD40L by ChIP-PCR, measured the CD40L expression in SLE patients CD4+T cells to analyze the corelation between E4BP4and CD40L;(2) analyzed the histone H3acetylation and tri-methylation levels at the CD40L promoter region to observe the effect of E4BP4on CD40L loci. In our study, we found that E4BP4could repress T cell co-stimulator CD40L expression through binding to the promoter region of CD40L and regulating the histone acetylation and methylation status of CD40L promoter. Thus, E4BP4can prevent T cell activation and subsequent autoimmune response by blocking the interaction of T and B cells.Taken together, our findings indicate that overexpression of E4BP4may be a protective mechanisms in SLE CD4+T cells, thereby providing a theoretical basis for more effective therapy of SLE.Part I Expression of E4BP4in CD4+T cells from SLE patientsObjective:To investigate the expression of E4BP4in CD4+T cells from SLE patients.Methods:Peripheral blood mononuclear cells (PBMCs) from15healthy controls and30SLE patients, including15untreated patients and15glucocorticoids (GC) treated patients, CD4+T cells were then isolated by positive selection using magnetic beads. E4BP4mRNA levels were determined by real-time quantitativ PCR, and E4BP4protein levels were examined by western blot, and the correlation between E4BP4and systemic lupus erythematosus disease activity score (SLEDAI) were analyzed.Results:Compared to normal controls, E4BP4mRNA levels were significantly increased both in CD4+T cells from untreated patients and GC-treated patients (untreated:P<0.01; GC-treated:P<0.01), and E4BP4mRNA levels significant increased in CD4+T cells from GC-treated patients compared to untreated patients (P<0.01). In addition, E4BP4protein levels were significantly increased both in CD4+T cells from untreated patients and GC-treated patients (untreated:P<0.05; GC-treated:P<0.01); and E4BP4protein levels significant increased in CD4+T cells from GC-treated patients compared to untreated patients (P<0.01). Furthermore, the E4BP4protein levels was negatively correlated with inereased disease activity in both untreated and GC-treated lupus patients as measured by SLEDAI (untreated:R=-0.771, P=0.01;GC-treated:R=-0.607, P=0.01).Conclusion:E4BP4expression were significantly increased in CD4+T cells from active SLE patients, expression of E4BP4in GC-treated patients were significantly up-regulated. E4BP4expression and disease activity negatively correlated.Part Ⅱ The relationship between abnormal E4BP4expression and autoimmunitySection Ⅰ Overepression of E4BP4inhibits activation of CD4+T cellsObjective:To investigate the effect of E4BP4on activation of normal CD4+T cells.Methods:PBMCs from3healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells and CD19+B cells were then isolated by positive selection using magnetic beads. E4BP4expressed plasmid (pcDNA3.1-E4BP4) and control plasmid (pcDNA3.1) were transfected into anti-CD3/CD28antibody activated CD4+T cells by transient electroporation. Protein levels were examined by western blot, the expression level of CD69was detected by flow cytometry. Furthermore, we measured the mRNA and protein levels of CD11a, CD70and CD40L by realtime PCR and flow cytometry. Then detected the levels of autoantibody IgG by T and B cell costimulation assays and ELISA.Results:The E4BP4protein levels were significantly up-regulated in anti-CD3/CD28antibody activated CD4+T cells transfected with E4BP4expression plasmid (P<0.01), CD69protein expression was inhibited in CD4+T cells transfected by pcDNA3.1-E4BP4compared with negative controls (P<0.01), CD40L mRNA (P<0.01) and protein(P<0.01) expression was down-regulated significantly in activated CD4+T cells with E4BP4overexpression compared with negative controls. No significant change was observed in the expression levels of CD70and CD11a. Production of IgG was decreased significantly when E4BP4was overexpressed in activated CD4+T cells (P<0.01).Conclusion:Overepression of E4BP4inhibits activation of normal CD4+T cells induced by anti-CD3/CD28antibodies in vitro.Section II Knock-down of E4BP4aggravates the self-reactivity of SLE CD4+T cellsObjective:To determine the role of E4BP4in the regulation of self-reactivities of SLE CD4+T cells.Methods:PBMCs from3SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells and CD19+B cells were then isolated by positive selection using magnetic beads. E4BP4-siRNA was transfected into SLE CD4+T cells by transient electroporation, E4BP4protein levels detected by western blot, detected the mRNA and protein expression levels of CD11a, CD70and CD40L by flow cytometry. Then detected the levels of autoantibody IgG by T and B cell costimulation assays and ELISA.Results:E4BP4protein expression was inhibited in SLE CD4+T cells transfected with E4BP4-siRNA-3compared with negative controls (P<0.01), and the mRNA (P<0.01) and protein (P<0.05) levels of CD40L decreased significantly, the mRNA and protein expression levels of CD11a, CD70were no significant change. SLE CD4+T cells with E4BP4knockdown can stimulate autologous B cells to produce much more IgG antibody than negative controls (P<0.01).Conclusion:Inhibition of E4BP4expression aggravates the self-reactivity of SLE patients CD4+T cells.Part III E4BP4regulates CD40L expression and its mechanisms in SLE CD4+T cellsSection I CD40L is the target gene of E4BP4in CD4+T cellsObjective:To detect whether CD40L is the target gene of E4BP4in CD4+T cells.Methods:Screened the E4BP4binding sites in the promoter region (up-stream2kb of transcription starting site) of CD40L by Mat-Inspector software. Peripheral blood mononuclear cells (PBMCs) from3healthy people, CD4+T cells were then isolated by positive selection using magnetic beads. pcDNA3.1-E4BP4and control pcDNA3.1were transfected into CD4+T cells by transient electroporation. ChIP-PCR analysis to confirm that E4BP4binds to the promoter region of CD40L in CD4+T cells. Real-time PCR to detect the CD40L mRNA expression in SLE patients, and analyzed the correlation between E4BP4and CD40L expression.Results:MAT-Inspector software predicted several E4BP4binding sites in CD40L promoter region. ChIP-PCR confirmed E4BP4combined with the promoter region of CD40L. CD40L expression levels were higher in untreated and treated SLE patients compared with normal controls(untreated:P<0.01; GC-treated:P<0.05), meanwhile, we also found that the mRNA levels of CD40L were decreased in treated SLE patients compared with untreated patients(P<0.05), and the mRNA levels of CD40L were negatively correlated with protein levels of E4BP4in CD4+T cells from SLE patients (R=-0.758, P=0.01).Conclusion:CD40L is the target gene of E4BP4. E4BP4may inhibit expression of CD40L directly to attenuate the self-reactivities of SLE CD4+T cells, thus it might be a protective molecular in the SLE desease.Section II E4BPE regulates the histone modification status of CD40L promoter Objective:To observe the effect of E4BP4on histone modifications at the CD40L loci in normal CD4+T cellsMethods:Three pairs of primers were designed for the the CD40L promoter region which may be protential binding sites of E4BP4. Histone H3acetylation and tri-methylation levels at the CD40L promoter were evaluated by ChIP and real-time PCR.Results:CD4+T cells overexpressing E4BP4showed decreased histone H3acetylation at Lys9and Lys14and H3methylation at Lys4within the region including potential E4BP4binding sites in the promoter region of CD40L gene. In addition, we also showed increased histone H3methylation at Lys9in the same region.Conclusion:E4BP4regulates CD40L gene expression by altering the histone acetylation and methylation status of its promoter region.