Mesenchymal Stem Cells Alleviate SLE Through Upregulating Treg Cells By SHLA-G

Background:Systemic lupus erythematosus(SLE)is an autoimmune disease,mainly characterized by hyperactivation of lymphocytes,immune complex deposition and secreting of inflammatory cytokines.Most studies have reported a reduced number and impaired function of regulatory T(Treg)cells in active SLE patients.Human leukocyte antigen-G(HLA-G)is a non-classical HLA class-I molecule and exhibits strong immunosuppressive properties,playing an important role in autoimmune diseases.Studies have found that mesenchymal stem cells(MSCs)can secrete soluble HLA-G(sHLA-G),which is involved in MSCs-mediated immunoregulation.MSCs transplantation(MSCT)alleviates disease progression in SLE patients and animal models.However,the underlying mechanisms are unknown.Objectives:To explore the expression of sHLA-G in SLE and analyze the correlation between sHLA-G and clinical features.To explore the efficacy and mechanism of MSCT in the treatment of lupus mice and SLE patients.To explore whether sHLA-G is involved in upregulating effects of MSCs on Treg cells,which contributes to therapeutic effects of MSCT in SLE.Methods:1)The expressions of serum sHLA-G in SLE patients and healthy controls were detected by ELISA.The correlations between the levels of sHLA-G and clinical features were analyzed,respectively.Peripheral blood mononuclear cells(PBMC)were isolated from 15 SLE patients and 15 healthy controls.The percentages of CD4+ILT2+,CD8+ILT2+,CD19+ILT2+ and Treg cells were examined by flow cytometry.2)The expression of HLA-G of MSCs were interfered by small interfering RNA(siRNA).Female B6.MRL-Faslpr mice were divided into three groups:B6.MRL-Faslpr mice infused with phosphate buffer saline(PBS),B6.MRL-Faslpr mice infused with MSCs and B6.MRL-Faslpr mice infused with HLA-G siRNA MSCs.C57BL/6 mice were served as normal control.1×106 cells or equivoluminal PBS were injected into the tail vein of mice,respectively.Six weeks later the mice were sacrificed.The therapeutic effects of MSCs transplantation were evaluated by the analyses of spleen index,24-hour proteinuria,the renal pathology and lymphocyte subpopulation.Ten SLE patients were infused with umbilical cord-derived MSCs(UC-MSCs),106 cells/kg body weight.The serum levels of sHLA-G and TGF-?1 concentrations were measured 24 hours after infusion.The percentages of CD4+ILT2+,CD8+ILT2+,CD19+ILT2+ cells in peripheral blood,Treg cells and the levels of PD-1 and CTLA-4 on Treg cells were detected by flow cytometry.3)Bone marrow-derived MSCs(BM-MSCs)from healthy donors and SLE patients were isolated and the expression of sHLA-G was measured by ELISA and PCR.PBMC were isolated from SLE patients and co-cultured with UC-MSCs for 3 days at different ratios with or without HLA-G antibody,and the frequencies of CD4+CD25+Foxp3+ T cells and proliferation of T cells were then determined by flow cytometry.CD4+ T cells and CD4+CD25-T cells were purified from PBMC of SLE patients,and were co-cultured with UC-MSCs for 3 days with or without HLA-G antibody.The frequencies of CD4+CD25+Foxp3+ T cells were then determined by flow cytometry.MSCs were pre-treated with IFNy for 48 h and the HLA-G mRNA expression was detected.The macrophages of SLE patients were induced from monocytes and co-cultured with MSCs for 48 h.The expression of HLA-G mRNA was detected by PCR.Results:The sHLA-G levels were significantly lower in SLE patients with renal involvement(19.6±10.9ng/ml)than those without renal involvement(25.3±8.8ng/ml).There was a negative correlation between sHLA-G levels and SLE disease activity index(SLEDAI)scores in active SLE patients(SLEDAI>4).We found that serum sHLA-G levels were negatively correlated with the levels of blood urea nitrogen,serum creatinine and 24-hour proteinuria in SLE patients.The expression of ILT2 on CD4 +T cells from SLE patients decreased significantly compared to that of healthy controls.A positive correlation between the frequencies of Treg and CD4+ILT2+ T cells was found in SLE patients.The spleen index,24-hour proteinuria and renal pathology were significantly improved in MSCs-treated B6.MRL-Faslpr mice,but there were comparable results in B6.MRL-Faslpr mice infused with HLA-G siRNA MSCs,which indicated that MSCs was a potential therapeutic strategy for lupus mice,but may not rely on sHLA-G.Compared with B6.MRL-Faslpr mice infused PBS,the percentages of splenic Th17 and Tfh cells were decreased,whereas the percentages of splenic Treg cells,Breg cells and macrophages were increased in B6.MRL-Faslpr mice infused with MSCs.HLA-G siRNA MSCs showed an impaired ability in downregulating plasma cells.The level of serum sHLA-G was significantly increased in SLE patients after MSCs transplantation.The percentages of Treg cells and the functional molecules,PD-1 and CTLA-4 were significantly increased.The percentages of CD4+ILT2+ T cells were also significantly improved.MSCs could not upregulate the HLA-G mRNA expression of macrophages in vitro.Conclusions:The expressions of sHLA-G and its receptor ILT2 were altered in SLE,which might participate in the pathogenesis of the disease.MSCs may alleviate SLE through upregulating Treg cells,which was partly dependent on sHLA-G.However,the beneficial effects of MSCs on lupus mice may be independent on sHLA-G.