Study On The Role Of Transcriptional Factor RFX1 In The Pathogenesis Of Systemic Lupus Erythematosus

Background Systemic lupus erythematosus (SLE) is a chronic and potentially fatal autoimmune disorder characterized by T lymphocyte autoreactivity and the production of autoantibodies that cause widespread tissue damage.Its etiology and pathogenesis is unclear. Recent studies have shown the importance of altered DNA methylation, especially hypomethylation in CD4+ T cells, in the pathogenesis of SLE.Epigentic marks can represent an addition to the genetic templates without changing the DNA nucleotide sequences.Epigenetics processes include DNA methylation, acethylation and phosphorylation. DNA methylation is an epigenetic regulator in several biological processes, including embryonic development, gene transcription, X chromosome inactivation, genomic imprinting and chromatin modification. Our previous studies showed that DNA methylation defect correlate with upregulated expression of some autoimmune-related genes such as CDlla (ITGAL), perforin (PRF1), CD70(TNFSF7) and CD40 ligand(TNFSF5). Recently it was reported the expression of CD70 and CD11a were up-regualted in CD4+ T cells from SLE patients, which cause B-cell over-stimulation. Overstimulated B cells can induce to produce the autoreactive T cell and autoantibodies, which result in tissue damage and SLE pathogensis.The mechanism of overexpression of these genes in SLE is not clear. Transcription factors play important role in regualting gene expression. In this study, we identified some transcription factors (TFs) such as RFX1 and SRF with different activities in SLE CD4+ T cells, among which the expression and acitivity of RFX1 were confirmed downregulated in SLE CD4+ T cells. Moreover, the prediction from bio-software showed that RFX1 may bind the promoter of target genes CD1 la and CD70, suggesting RFX1 may be involved in regulating CD11a and CD70 expression. In this study, we demonstrated the role of RFX1 in the pathogenesis of SLE through transgene and RNA interference, which provide the clues for understanding the molecular mechanisms of autoimmune dieases and the novel target for treatment of SLE.Part I Profiling of transcription factors (TFs) activities in SLE CD4+ T cells and healthy controlsObjective To compare the TFs activities in CD4+ T cells between SLE and healthy controls.Methods A total of 60 ml of venous peripheral blood was withdrawn from SLE patients and healthy controls and preserved with heparin. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+ T cells were isolated by positive selection using CD4 beads according to protocols provided by the manufacturer (Purity was generally higher than 95%).Nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents, Protein concentrations were determined by using BCA Protein Assay Kit. Total RNA was isolated using the RNeasy mini Kit. The transcription factor profiling microarray system consisting of 225 TF-binding sequences was purchased from CapitalBio Corporation. The DNA-binding activity of a TF in SLE samples was deemed significantly different from control if the median ratio was>1.5 or<0.67.The activity of one TF was detected by ELISA. mRNA level was detected by real-time quantitative RT-PCR and protein level was detected using western blot.Results The results from TFs chip showed that the binding activities of RFX1,SRF and MIF1 were decreased by factors of 2.55±0.18,2.85±0.28 and 3.03±0.4 respectively; whereas E4BP4 binding was increased by a factor of 1.58±0.25.The results from ELISA confirmed that RFX1 and SRF activities are significantly decreased in SLE CD4+ T cells relative to healthy controls, which are identical with the results from chip assay. Moreover, both the mRNA and protein levels of RFX1 were significantly downregulated in SLE CD4+ T cells compared to controls (P<0.01).The protein level of SRF was decreased in SLE CD4+ T cells compared to controls (P<0.01),but the change of SRF mRNA level is no significant.Conclusion There are some TFs with different activities such as RFX1,SRF, MIF1 and E4BP4 in CD4+ T cells between SLE and healthy controls.The expression and activities of RFX1 and SRF are down-regulated in SLE CD4+ T cells. The results from TFs chip experiment are validated.Part II The role of RFX1 in the pathogenesis of SLESection I Reducing RFX1 levels leads to CD4+ T Cell autoreactivityObjective To investigate the role of RFX1 in regulating CD11a and CD70 expression and autoreactivity of CD4+ T cells.Methods The RFX1 RNAi plasmid (pSUPER.RFX 1)and negative control plasmid (pSUPER) were provided by Dr. Yosef Shaul (Weizmann Institute of Science, Rehovot, Israel), which were transfected into CD4+ T cells from three healthy controls respectively using nuclefector program V-024 in the Amaxa nucleofector. The cells transfected were cultured in human T cell culture medium and harvested at 48 hours.Two days later the cells were harvested and the efficiency of gene silencing was quantitated by western blot. Real-time RT-PCR was used to detect the expression of CD11a and CD70 mRNA. CD11a and CD70 protein levels were measured by flow cytometry. The detection of IgG antibody production was performed using the ELISA kit.Results Compared to negative controls, RFX1 RNAi reduced RFX1 protein levels by approximately 60% (P<0.05),which caused a significant upregulation of CD11a and CD70 mRNA levels (P<0.01)and CD11a and CD70 protein levels (P<0.01).The auto-antibody production from RFX1-deficient CD4+ T cells and B cells is higher than that from negative control CD4+ T cells and B cells (p<0.05).Conclusion Reducing RFX1 in CD4+ T cells is sufficient to cause CD11a and CD70 overexpression and lupus-like T and B cell hyperactivity.Section II RFX1 overexpression rescues SLE CD4+ T Cell autoreactivityObjective To determine whether overexpression RFX1 can downregulate CD11a and CD70 expression and inhibit the autoreactivity in SLE CD4+ T cells.Methods The plasmid encoding the human RFX1 cDNA (pSG5-RFX1) and the blank control plasmid (pSG5)were provided by Dr. Yosef Shaul (Weizmann Institute of Science, Rehovot, Israel), which were transfected into CD4+ T cells from three SLE patients respectively using nuclefector program V-024 in the Amaxa nucleofector. The cells transfected were cultured in human T cell culture medium and harvested at 48 hours.The expression of RFXl protein was quantitated by western blot,Real-time RT-PCR was used to detect the levels of CD11a and CD70 mRNA. CD11a and CD70 protein levels were measured by flow cytometry.The detection of IgG antibody production was performed using ELISA kits.Results Compared to negative controls, RFX1 protein were significantly increased in cells transfected with RFX1 expression vector pSG5-RFX1 (P<0.05).The increase of RFX1 lead to a concurrent decrease in CD11a and CD70 mRNA and protein levels (P<0.01),which caused a reduction of autoantibody IgG secretion (P<0.05).Conclusion Overexpressing RFX1 suppresses the expression of CD11a and CD70 and rescues SLE CD4+ T cell autoreactivity.