The Therapeutic Effects Of Mesenchymal Stem Cells On Macrophages In Systemic Lupus Erythematosus

Background:Systemic lupus erythematosus(SLE)is an inflammatory autoimmune disease,involving in many organs dysfunctions.It is manifested by overactive T/B cells,accumulation of autoantibodies and overexpression of pro-inflammatory cytokines.Umbilical cord(UC)-derived mesenchymal stem cells(MSC)have been confirmed to exert therapeutic effects on systemic lupus erythematosus(SLE).Deficiency in SLE macrophages exhibits excessive activation and inefficient clearance of self nuclear antigens.However,whether the benefit effects of UCMSC on SLE were mediated by regulatory effects on macrophages remains to be elucidated.Objective:We aim to explore whether UCMSC could educate SLE macrophages to become a kind of alternatively activated macrophages,increase their immunomodulatory function and enhance the phagocytic activity.Methods:CD14+ monocytes were isolated from peripheral blood of HC and SLE patients.We cultured human monocytes for 7 days with macrophage colony-stimulating factor(M-CSF)to generate macrophages,and then cocultured with or without UCMSC(5:1)for 2 more days in transwell systems.The level of CD206,the marker of alternatively activated macrophages,was detected by FACS.The expressions of IL-10,TGFβ1 TNFα and iNOS,stimulated by LPS for 24-hours or not,were determined by Real-time PCR and ELISA.CD4+ T cells were isolatedfrom PBMC of one healthy donor,and then CD4+ T cells were cocultured with prepared HC,SLE or UCMSC-educated SLE macrophages in 5:1 ratio with anti-CD3/CD28 stimulation for 4 days.The proliferation of T cells was detected by FACS.To determine whether UCMSC had effects on the phagocytic activity of SLE macrophages,flurospheres and apoptotic cells were added into the cultures.The uptake of flurospheres and apoptotic cells were determined by FACS.Meanwhile,we also detected the effects of SLE macrophages on the generation of immunomodulation factors in UCMSC.Female B6.MRL-Faslpr mice were divided into control group,UCMSC transplantation group,cyclophosphamide(CTX)treated group,and fibroblast like synoviocytes(FLS)transplantation group.Female C57BL/6 mice were served as control.The mice in CTX treated group received intraperitoneal injections of CTX at a dose of 100mg/kg/day for 2 days at 24 weeks.Meanwhile,1×106 UCMSC or FLS were injected into the UCMSC and FLS groups via tail vein respectively.All mice were sacrificed at the age of 28 weeks.The therapeutic effects of UCMSC transplantation were evaluated by the renal pathology,spleen index,T lymphocyte subpopulation and plasma cells.Murine peritoneal and renal macrophages were isolated to detect the proportion of CD206+ macrophages.The levels of IL-10,TGFβ1,TNFa and iNOS,stimulated by LPS for 24hours or not,were determined by ELISA.CD4+ T cells were isolated from 8 weeks female C57BL/6 mice,and then CD4+ T cells were cocultured with prepared murine peritoneal and renal macrophages in 5:1 ratio with anti-CD3/CD28 stimulation for 4 days.The proliferation of T cells was detected by FACS using CFSE-labelled assay.To determine the phagocytic activity of murine peritoneal/renal macrophages,flurospheres were added into the cultures.The uptake of flurospheres was determined by FACS.Results:SLE macrophages exhibited lower level of CD206,the marker of alternatively activated macrophages.The anti-inflammatory cytokines IL-10 and TGFβ1 of SLE macrophage were decreased in both mRNA and protein level.However,the level of inflammatory factors TNFa and iNOS did not change.SLE macrophages could not suppress the proliferation of CD4+ T cells effectively.SLE macrophages were deficient in the engulfment of flurospheres and apoptotic cells.UCMSC-educated SLE macrophages consistently increased the expression of CD206.UCMSC-educated SLE macrophages suppressed the proliferation of CD4+ T cells effectively.The phagocytic activity of UCMSC-educated SLE macrophages was improved in engulfment of flurospheres and apoptotic cells.However,the cytokine profiles assay of SLE macrophagesshow little change,except the level of TNFa was decreased in UCMSC-educated SLE macrophages in the presence of LPS.SLE macrophages promoted the generation of some immunomodulatory factors(HGF/TGFβ1/HO-1)in UCMSC.UCMSC rescued the low-level of CD206+macrophage in B6.MRL-Faslpr mice.The murine peritoneal and renal macrophages from UCMSC-treated group suppressed the proliferation of CD4+ T cells effectively.The phagocytic activity of murine peritoneal and renal macrophages from UCMSC-treated group was improved in the engulfment of flurospheres.Consistant with the results in vitro,UCMSC transplantation had few effects on the the cytokine profiles of B6.MRL-Faslpr mice macrophage.Conclusions:In our experiment,we found that SLE macrophages were deficient in both phenotype and function and UCMSC could alleviate the deficiency of SLE macrophagesboth in vitro and vivo.The results indicate the important role of deficient macrophages in the pathogenesis of SLE,and UCMSC could be effective in treating SLE through targeting the deficient macrophages.