| Background:Systemic Lupus Erythematosus(SLE)is a typical autoimmune disease with multiple system involvement.The etiology and pathogenesis of SLE have not been thoroughly studied,which brings great challenges to the diagnosis and treatment of this disease.Therefore,to explore the pathogenesis and further to find new diagnostic markers and therapeutic targets is the key ofSLE treatment.Recent Studies revealed that heredity,hormone,environment,infection and drugs could lead to immune disorder.The immune cells of SLE patients are aberrantly activated,which would generate a large number of pathological antibodies and form immune complexes,and leading to the pathogenesis.Semaphorins,a family of secretory or membrane-bound glycoprotein molecules were widely expressed in various tissues or organs,are divided into 8 types.As a member of Semaphorins family,Sema4A is mainly expressed in immune cells,thus it is termed "immune semaphorins".Sema4A is mainly expressed in dendritic cells(DCs),monocytes and macrophages.Studies proved that Sema4A Sema4A played an important role in T cell-mediated immune response.Recent studies had shown that Sema4A was associated with immune diseases.The expression of Sema4A in serum and in synovial fluid of patients with rheumatoid arthritis was obviously increased,which was closely related to disease activity.In addition,Sema4A can promote the expression of extracellular matrix proteins and α-SMA in fibroblasts by promoting the secretion of IL-17 cytokines.Thus Sema4A could play role in the pathological process of SSc.Recently,it has been reported that SEMA4A gene expression from whole blood cells was up-regulated in patients with SLE.However,the role of Sema4A in the pathogenesis of SLE remains unknown.Therefore,does Sema4A lead to the disorder of T lymphocyte immune function in SLE patients?This study aims to detect the expression of Sema4A in SLE and the correlation with disease-related clinical indicators and preliminarily to explore the effect of Sema4A on proliferation,activation and differentiation of T cells.Therefore,the study could reveal the mechanism ofSema4A in SLE.Objectives:1.To analyze the expression levels of membrane-bound Sema4A in peripheral blood cells and soluble Sema4A in plasma,and their correlation with clinical indicators in patients with SLE.2.To clarify the correlation between Sema4A and the ratio of Th cells,and to clarifythe relationship between Sema4A and Th cell imbalance in SLE patients.3.Toinvestigate whether soluble Sema4A contributes to T lymphocyte activation and proliferation and changes the expression of cytokines,and to determine the effect of Sema4A on T cell immunological effect in SLE patients.Methods:1.37 patients with SLE were recruited as experimental group,28 healthy individuals as control group.The expression of membrane-bound Sema4A on CD4+CD11c+ myeloid dendritic cells(mDCs),T cells,B cells,and Granulocytes were identified by flow cytometry.The concentration of soluble Sema4A in plasma from SLE patients was detected by ELISA.The diagnostic efficiency of Sema4A was analyzed by ROC curve.Clinical and laboratory parameters of SLE patients were collected to analyze the correlation between Sema4A and these parameters.2.The ratio of CD3+CD8-IFN-γ+(Th1),CD3+CD8-IL-4+(Th2),CD3+CD8-IL-17+(Th17)in CD4+T cells(Th cells)were identified by flow cytometry.The correlation between Sema4A and Th cell subtypes was analyzed.3.CD4+T cells from SLE or control groups were sorted by Ficollgradient and magnetic cell separation,and cultured for 5 days with or without recombinant human Sema4A Protein or anti-Sema4A.The expression of CD25 was detected by flow cytometry,and the concentration of cytokines in supernatant was determined by ELISA.CFSE was added into the same culture system above,and cultured for 5 days.CFSE dilution was detected by flow cytometry.Results:1.The expression level of Sema4A in SLE:①Sem4A levels in plasma were significantly higher in samples from patients with SLE than in those from patients(2.26±1.58 vs 13.6±1.33,P<0.05).②The ratio of membrane-bound Sema4A on CD4+CD11c+mDCs in SLE patients were significantly higher than that in healthy individuals(77.67±12.38vs 55.35±8.32,P<0.01).2.Correlation between Sema4A and Clinical Indexes:①Membranebound Sema4A expression levels on CD4+CD11c+mDCs positively correlated with SLEDAI scores(r=0.677,P<0.01).②The concentration of soluble Sema4A negatively correlated with C3(P<0.01,r=-0.511)and C4(P<0.01,r=-0.541)levels.③The levels of soluble Sema4A and membrane-bound Sema4A on CD4+CD11 c+mDCs negatively correlated with Hb concentration(P<0.05,r=-0.331;P<0.05,r=-0.335).④The ratio of membrane-bound Sema4A on CD4+CD11 c+mDCs was significantly higher in the proteinuria positive group than that in the proteinuria negative group(81.79 ± 12.34vs73.41±11.89,P<0.05).3.Proportion of Th1,Th2,Th17 cells in CD4+ T cells in peripheral blood of SLE patients:①SLE patients had significantly higher percentage of Th2 cells(4.64±2.08 vs 2.64±0.50,P<0.01)and Th17 cells(3.58±1.42 vs 2.52±0.81,P<0.01),but only slight statistically insignificant increment of Th1 cells,compared to the healthy subjects.The proportion of Th1/Th2 in SLE patients was significantly lower than that in the healthy subjects(8.28±5.30 vs 11.52±4.77,P<0.05).②The concentration of soluble Sema4A positively correlated with the ratio of Th2 cells(P<0.05,r=0.345)and Th17 cells(P<0.01,r=0.455).A negative correlation was observed between soluble Sema4A and proportion of Th1/Th2(P<0.05,r=-0.373)4.Analysis the ability of soluble and membrane-bound Sema4A to recognize SLE:The concentration of soluble Sema4A had diagnostic value for SLE,and its diagnostic efficiency was moderate.An area under the curve(AUC)was 0.70 to 0.87 Membrane-bound Sema4A expression levels on CD4+CD11c+ mDCs hada high diagnostic accuracy.The AUC was 0.91.5.Effects of soluble Sema4A on T cell activation and proliferation:①Sema4A could promote the expression of CD25 in SLE(62.73±3.98 vs 90.33±1.39,P<0.001)and healthy individuals(55.97±5.98 vs 79.95±4.84,P<0.01),which were markedly suppressed by adding soluble anti-Sema4A antibody(P<0.001).②Sema4A could promote the proliferation of CD4+T cells in SLE(58.95±3.60vs 91.72±1.80,P<0.001)and healthy individuals(47.46±2.17 vs 78.19±4.57,P<0.001),which were markedly suppressed by adding soluble anti-Sema4A antibody(P<0.001).6.Effects of soluble Sema4A on the expression of cytokines:The expression of IL-4(90.51±15.22 vs 168.36±8.13,P<0.01)and IL-17(1262.03±58.16 vs 2530.09±168.05,P<0.01)of SLE patients were significantly increased.The expression of IL-4(63.87±6.31 vs 140.11±14.69,P<0.01)and IL-17(1022.13±130.00 vs 1888.72±136.56,P<0.01)of healthy individuals were significantly increased,which were markedly suppressed by adding soluble anti-Sema4A antibody(P<0.01).The expression of IFN-γ of SLE patients(1628.77±460.24 vs 1318.56±109.51,P<0.05)and of healthy individuals(1606.64±96.76 vs 1309.18±386.68,P<0.05)wereinhibited,but the inhibitory effect could not be neutralized by anti-Sema4A antibody.Conclusions:1.Elevated soluble and membrane-bound Sema4Awas closely related to SLEDAI,complement and hemoglobin,which suggesting that Sema4A played an important role in the pathogenesis of SLE.Furthermore,Soluble Sema4A and membrane-bound Sema4A have a strong ability to recognize SLE.Therefore,Sema4A was expected to become a new diagnostic marker.2.Soluble Sema4A was associated with Th cell subsets in SLE patients,which revealed that Sema4A promoted immune inflammatory response by regulating the distribution of Th cellssubsets.Thus Sema4A was involved in the pathogenesis of SLE.3.Sema4A could induce the activation of CD4+Tcells and promote cell proliferation,which indicated that abnormally elevated Sema4A contributed to the activation and proliferation of T cells.Thus,Sema4A play an important role in initiating and activating inflammatory diseases.4.Sema4A could promote the differentiation of Th2 and Th17 cells and inhibit the differentiation of Th1 cells,which revealed that the increase expression of Sema4A in SLE patients participates in immune regulation by affecting the imbalance of Th cells and cytokines,thus participating in the pathogenesis of SLE. |
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