| Objective:Primary membranous nephropathy(PMN)is an organ-specific autoimmune disease caused by Th2 immune responses with glomerular lesions as the main pathological changes.It is a common etiology of nephrotic syndrome in adults.As to the etiology of PMN,some studies have shown that the majority of cases(about 85%)of PMN can be mediated by the phospholipase A2 receptor(PLA2R)antibody,and the others are related to the thrombospondin type-1 domain-containing 7A(THSD7A)or unknown mechanism.Th17 cells are key players in renal autoimmunity by mediating fundamental inflammatory cascades and promote formation of autoantibodies which are pivotal to most renal autoimmune diseases,and enhance tissue inflammation and have direct effects on renal parenchymal cells.However,it is still unclear whether Th17 cells are involved in the occurrence and development of PMN.Myeloid-derived suppressor cells(MDSC)are a group of immunosuppressive cells and promoted Th17 differentiation and disease progression in lupus nephritis patients.However,it have not been studied so far the change of MDSC and its interaction with Th2 and Th17 cells in PMN.This study aims to reveal the relationship between MDSC and Th2 or Th17 immune responses in disease progression of PMN,which could be explored to develop monitoring and therapeutic strategies.Methods:(1)The renal tissues of PMN patients were stained with paraffin and frozen sections,and the pathologic features of PMN lesions were observed by light microscope.The deposition of immune complexes was detected by IF in the glomerular basement membrane(GBM).The location and intension of IL-4,CD3 and CD11b antibodies were determined by multiplex immunohistochemistry(IHC)in PMN pathological tissues.Enzyme-linked immunosorbent assay(ELISA)was used to assay the content of anti-PLA2R in plasma from HC and PMN patients,and its sensitivity and specificity for disease diagnosis were analyzed respectively.(2)The percentage of CD4+IL-4+(Th2)and CD4+IFN-?+(Th1)cells,and IL-4,IL-6,IL-10,IL-13 and IFN-y level in plasma from HC and PMN patients were detected by FCM and ELISA,and the correlation between the percentage of Th2 cell and disease activity and Th1/Th2 ratio were analyzed respectively.The percentage of regulatory T cells(Treg)in HC and PMN patients were also analyzed.(3)The percentages and numbers of MDSC and their subsets from peripheral blood mononuclear cell(PBMC)of healthy control(HC)and PMN patients were measured using flow cytometry(FCM).The correlation was analyzed between both the percentage and number of MDSC and disease activity or Th2 percentage from PMN patients.(4)Naive CD4+T cells from HC and PMN patients were isolated by magnetic beads and labeled with CFSE.At the same time,the respective MDSC were isolated from PBMCs of HC volunteer and PMN patients by cell sorting using a cell sorter.Naive CD4+T cells were co-cultured in the absence or presence of autologous MDSCs at a 1:1 ratios from HCs or PMN patients with anti-CD3 mAb and soluble anti-CD28 mAb in 96-well flat-bottom plate for three days,and T cell proliferation was determined by measuring CFSE.(5)Naive CD4+T cells from HC and PMN patients were cultured for five days under the Th2 differentiation condition in vitro.The percentage of CD4+IL-4+(Th2)cells was detected with MDSC and/or Arg-1 and iNOS inhibitor or with IL-6 and IL-10 by FCM and ELISA.(6)Co-localization of IL-17+cells and CD11b+cells in kidney tissue of PMN patients was detected by immunofluorescence staining(IF).The percentage of Th17 cells and IL-17A content level in HC and PMN patients were detected by FCM and ELISA,and the correlation between both and disease activity was analyzed respectively.(7)Naive CD4+T cells from HC and PMN patients were cultured for six or seven days under Th17 differentiation conditions in vitro.The percentage of CD4+IL-17A+(Th17)cells and IL-17A in cultured supernatant were detected with MDSC and/or Arg-1 inhibitors by FCM and ELISA.(8)The plasma Arg-lcontent in HC and PMN patients was detected by ELISA,and the correlation was analyzed between Arg-1 content and disease activity or number of MDSC in PMN patients.MDSC from PMN patients were sorted by flow sorting and co-culture with IL-6 or IL-10 factor,Arg-1 mRNA content in MDSC was detected by real-time quantitative PCR(qRT-PCR).(9)PMN mouse model were established with non-collagenous(NC)domains of the ?3 chain of recombinant human type ? collagen(rh-?3(?)NC1)protein by subcutaneous injection in DBA/1 mice.MDSC were removed with gemcitabine(GEM)by intraperitoneal injection.the percentage of MDSC,Th2,Th17,Th1 and Treg cells in the peripheral,spleen and lymph nodes from mice were detected by FCM.The levels of urine albumin,urine creatinine and serum urea nitrogen in mice were detected by ELISA.The expressions of Th2 and Th17 related cytokines IL-13 and IL-17A and related transcription factors GAT A3,RORyt and ROR? mRNA in the spleen,lymph node and kidney were detected by qRT-PCR.The deposition of IgG was detected by IF staining in renal tissue.Pathological features in kidney were observed by light microscope and the ultrastructural features of renal tissues were observed by electron microscope.Results:(1)Compared with HC,the percentage of IL-4+CD4+(Th2)cells in peripheral blood of PMN patients and IL-6,IL-10 and IL-13 levels in plasma were significantly increased,and the percentage of Th2 cells was positively correlated with disease activity.(2)Compared with HC,the percentages and numbers of MDSC and their subsets from PBMC of PMN patients were significantly increased and which was positively correlated with disease activity.the inhibitory function of MDSC was enhanced.(3)MDSC from both HC and PMN patients inhibited naive CD4+T differentiation into Th2 cells.IL-6 and IL-10 both enhanced the ability of MDSC to inhibit Th2 differentiation in vitro.(4)Compared with HC,Th17 cells and plasma IL-17A levels from PBMC of PMN patients were significantly increased,which both were positively correlated with disease activity.(5)The levels of MDSC,Arg-1 and IL-17 from PBMC of PMN patients were correlated.(6)MDSC from PMN patients were significantly more potent than those from HC in promoting Th17 cell differentiation,and MDSC-mediated Th17 differentiation was completely abrogated in the presence of nor-NOHA,a selective Arg-1 inhibitor in vitro.(7)Plasma Arg-1 level in PMN patients was higher than that of HC,and which was positively correlated with disease activity.IL-6 and IL-10 could promote the secretion of Arg-1 in MDSC.(8)The mouse PMN model was successfully established with rh-?3(?)NC1 protein.The percentage of MDSC,Th17 and Th2 cells in the peripheral blood of PMN mice increased,but the proportion of Thl and Treg cells decreased with the prolongation of immune time.(9)The renal injury were alleviated by removal of MDSC.(10)The proportion of Th17 and Th2 cells and related transcription factors of Th17 and Th2 cells reduced,but the proportion of Thl and Treg cells increased with removal of MDSC.Conclusions:The percentages and numbers of MDSC in PMN increased significantly,and MDSC may contribute to the disease progression of PMN mainly by enhancing the Th17 response.Th17 differentiation is Arg-1-dependent with MDSC secretion.The renal injury were alleviated by reducing Th17 and Th2 immune responses with removal of MDSC in mouse PMN model. |
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