Cd200 And Microrna Role In The Pathogenesis Of Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease that affects many tissues and organs. It has been suggested that failure of phagocytes to remove apoptotic cells allows excessive release of autoantigens and leads to the induction of autoimmunity, although the underlying mechanisms remain unclear. CD4+CD25hihg FoxP3+regulatory T cells (Tregs), that are pivotal in the maintenance of T cell homeostasis and are critical regulators of immune tolerance, exhibit quantitative and/or qualitative deficiencies in SLE thatmay contribute to the development of lupus pathogenesis. CD200is a type Ⅰ transmembrane glycoprotein that can regulate the activation threshold of inflammatory immune responses, polarize cytokine production and maintain immune homeostasis. While available evidence highlighted an important role of CD200/CD200R1in experimental autoimmune diseases, the role of CD200/CD200R1in human autoimmune diseases, such as SLE remains unknown. We, therefore, explored the expression and function of CD200/CD200R1in subjects with SLE.Method161patients diagnosed of SLE were recuited,95sex and age matched health volunteers were accepted as health controls (HC). Anti-coagulated peripheral blood was collected and peripheral blood mononuclear cells (PBMC) were isolated.The expression of CD200and CD200R1by cell subsets was examined by flow cytometry.Serum CD200level was detected by ELISA.Western Blot was used to examine the expression of DOK2and the level of p-DOK2in CD4+T cells.The effect of CD200on the differentiation of Th17and Treg was evaluated by cell coculture in vitro.PBMC were stimulated and stained with CFSE for evaluation of the effect of CD200on cell proliferation.PBMC were stained with Annexin V/propidium iodide for evaluation of the effect of CD200on cell apoptosis.CD200+/CD200-live cells, CD200+/CD200-early apoptotic were sorted by flow cytometerand then cocultured with dendritic cells in vitro. Binding and phagocytosis of apoptotic cells were compared between groups using fluorescence microscope and flow cytometer.The effect of CD200on DC migration was determined by transwell migration assay.Results1. The proportion of CD200+cells in PBMC of SLE patients was significantly higher than that in HCs(p=0.026), especially in the CD4+T cell population (p=0.012), the CDllc-CD123high plasmacytoid DC (pDC)(p=0.004), and the CDllc+CD123-myeloid DC (mDC)(p=0.016), but not in CD8+T cells, CD19+B cells, CD38bright plasma cells or CD14+monocytes.2. The percentage of CD3+CD200+cells was negatively correlated with serum C3(r=0.486, p<0.05).3. The circulating levels of CD200in SLE patients were also significantly higher than that in HCs(p<0.0001), Furthermore, the serum CD2001evel negatively correlated with serum C3level (r=-0.45, p=0.014) but not SLEDAI score or the levels of serum BAFF, IL-6, IFN-a, or anti-dsDNA(p>0.05)4. SLE patients had a decreased proportion of CD200R1+cells in PBMC compared with HCs (p=0.001). This reduction was evident in CD4+T cells (p=0.004), pDC (p<0.001), and mDC (p<0.001). CD4+CD45RA+naive T cells had less CD200R1expression than CD4+CD45R0+memory T cells in both HCs and SLE patients (p<0.05), and there was no significant difference between SLE patients and HCs.5. CD200Fc induced phosphorylation of DOK2in CD4+T cell.6. Anti-CD200R1antibody promotes TCR-driven proliferation of CD4+T cells in SLE patients7. CD200reduces CD4+T cell differentiation into Thl7cells8. CD200signaling rescues the defective generation of CD4+CD25high FoxP3+T cells in SLE patients9. Increased lymphocyte apoptosis with upregulation of CD200expression in SLE patients. 10. CD200expression on early apoptotic cells is associated with decreased binding and phagocytosis by DCs11. CD200Fc inhibits DC migration.ConclusionWe demonstrated that in SLE patients, the number of CD200+cells as well as the serum level of CD200were significantly higher than in HCs, whereas CD200R1expression was significantly lower than in HCs, especially in CD4+T cells and DCs. In addition, in SLE patients, exogenous CD200Fc reduced the proportion of Thl7cells and rescued the defective generation of CD4+CD25hifih FoxP3+T cells, whereas anti-CD200R1antibody promoted anti-CD3/CD28-induced CD4+T cell proliferation. Moreover, we found that in SLE patients, CD200on early apoptotic cells was increased in SLE patientsand may serve to limit their binding and phagocytosis by DCs. These data collectively indicate that CD200and CD200R1expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in this autoimmune disease. BackgroundSystemic lupus Erythematous(SLE) is a chronic, multisystem-involved typical autoimmune disease in which abnormally activated and proliferated B cells act as one of the important characteristics of SLE. MicroRNAs (miRNAs) are small, singal-stranded noncoding RNAs, many of which have been highly conserved throughout evolution. Mature miRNAs have been identified as regulators that modulate target gene expression and are involved in many diseases. In the previous study of our lab, mir-7expresion was abnormal in SLE patients. However, studies of mir-7focused on tumor, while the knowledge of mir-7in autoimmune disease especially in SLE patients was less collected. In this research we try to investigate the expression and functional of mir-7in SLE patients.Method20patients diagnosed of SLE were recuited.20sex and age matched health volunteers were accepted as health controls (HC). Anti-coagulated peripheral blood was collected and mononuclear cells (PBMC) were isolated.Taqman real time quantitative PCR was used for detecting the levels of mir-7in PBMC,B cell,T cell and monocyte in lupus patients and controls.Bioinformatics prediction and dual luciferase report gene assay system were performed to identify mir-7targets.B cell,T cell and monocyte were separated from PBMC by flow cytometer or micro beads.SYBR Green RT-PCR was used to evaluate the mRNA expression levels of phosphatase and tensin homologdeleted onchromosome ten (PTEN) in B cell and T cell in SLE patients and HC.Peripheral B cell were divided into3group, electroporated with pre-mir-7,anti-mir-7or control-microrna using a Nucleofector device respectively, SYBR Green RT-PCR was used to detect the mRNA expression levels of PTEN. The transfected B cells were also stained with CFSE and stimulated for5days; the status of proliferation was evaluated by flow cytometer.PBMC was transfected with Ad-mir-7or Ad-GFP control, the expression of CD200R1was detected by flow cytometer48hours later.Results1. The expression of miR-7in PBMC of SLE patients was significantly higher than that in HCs(0.86±0.03vs0.10±0.08, p=0.0001). especially in the B cell population(0.59±0.08vs0.31±0.06, p=0.027), but not in T cells (0.91±0.18vs0.99±0.18, p=0.78) or monocytes (1.30±0.36vs0.98±0.02, p=0.70).2. The target genes of mir-7were predicted by bioinformatic method first,8genes (PTEN,FOXO1,CD200R1,XIAP,RELA,CD72,BCL2L11,IL17RB) which related with SLE were picked out and validation by dual luciferase report gene assay system. We found that miR-7significantly inhibited the luciferase activity of the8genes3'UTR reporter (P<0.05) 3. Detectable PTEN mRNA on peripheral B cells was significantly down-regulated in SLE patients compared to HC (0.92±0.24vs2.20±0.49, p=0.041), but not on peripheral T cells (1.23±0.24vs1.55±0.53, p=0.579)4. Electroporated peripheral B cell with pre-mir-7or anti-mir-7result in overexpression or inhibition of mir-7effectively.5. Overexpression of miR-7could reduce the PTEN mRNA level and promote the proliferation of B cells. Correspondingly, inhibion of mir-7increased PTEN mRNA level and blocked the proliferation of B cells. Thus, the results suggested that miR-7negatively regulates the production of PTEN and induce efficient proliferation of human B cells.6. PBMC were transtected with Ad-mir-7or Ad-GFP Ctrl. Results showed that the positive rate of CD200R1was25.13%and Mean Fluorescent Intensity (MFI) was406.55in the cells transfected with Ad-GFP Ctrl. While the positive rate of CD200R1was7.51%and MFI was262.86in the cells transfected with Ad-mir-7, indicated that mir-7could down-regulate CD200R1directly.ConclusionThe expression of miR-7in PBMC of SLE patients was significantly higher than that in HCs, especially in the B cell population. PTEN mRNA on peripheral B cells was down-regulated in SLE patients compared to HC. PTEN was identified as the target of mir-7, which was proved by bioinformatics, dual luciferase report gene assay and transfection. Over-expression of miR-7could reduce the PTEN mRNA level and promote the proliferation of B cells. Correspondingly, inhibion of mir-7increased PTEN mRNA level and blocked the proliferation of B cells. Conclusively, mir-7is supposed to affect the proliferation of B cells via regulating the expression of PTEN. On the other hand, CD200R1was identified as the target of mir-7as well. Since the involvement of CD200/CD200R1signaling in the immunopathogenesis of SLE has been stated well in the previous section, mir-7contributes to the progression of SLE by targeting CD200R1as well.