| Background:Systemic lupus erythematosus(SLE)is the prototypic systemic autoimmune disease characterized by the production of autoantibodies and inflammatory cytokines.At present,SLE is prone to relapse,because the therapeutic efficacy of drugs remains limited.Therefore,to study its pathogenesis is particularly necessary.The pathogenesis of SLE includes genetic,hormonal,environmental and other factors,and the key pathological mechanism is the exposure of autoantigens and the lack of immune tolerance,which leads to the disorder of immune regulation including the abnormal activation and proliferation of T and B lymphocytes.Neutrophils are as the frontline cells of innate immunity,and their abnormal apoptosis are involved in the development of SLE.Recently,neutrophil extracellular traps(NETs)was discovered as a new kind of cell death.During the process of NETs formation,the abundant self-antigens release and promote the development of SLE.However,the pathogenic mechanism is still unclear.Can NETs promote the initiation of SLE by altering the functions of T and B cells?Therefore,the effects of NETs on T and B cells in SLE patients were studied.In addition,the signaling pathways of NETs formation in SLE patients were explored.As outlined above,the study aims to elucidate preliminarily the immunological role of NETs in SLE,and find molecular targets for reducing the formation of NETs in SLE patients,so as to provide new ideas for the treatment of SLE.Objectives:1.To clarify the immunological role of NETs in SLE,the level of T and B cells activation in peripheral blood of patients with SLE and the correlation between the activation level of cells and the level of NETs were detected,and whether NETs can induce the activation,proliferation and differentiation of T and B cells was further analyzed in vitro.2.To identify the role of PI3K/Akt during NETs formation in SLE patients,whether PI3K/Akt signaling pathway is activated in the process of NETs was explored.Methods:1.The fasting venous blood of patients with SLE and healthy donors was collected and anticoagulated with EDTA.In the anticoagulative samples,the positive expression rates of early activated T cell CD4+CD69+T,late activated T cell CD4+HLA-DR+T and activated B cell CD19+CD69+B[(CD69+(%CD4+)T,HLA-DR+(%CD4+)T and CD69+(%CD19+)B],and the average expression of activation markers on the corresponding single cell(Mean Fluorescence Intensities,MFI)were detected by flow cytometry.Neutrophils were separated from anticoagulative blood and the level of NETs were detected by fluorescence photometry.Then the correlation between NETs and the lymphocytes activation level was analyzed.2.Polymorphonuclear neutrophils(PMNs)and peripheral blood mononuclear cells(PBMCs)from 10 healthy donors were isolated.PMNs were induced by 25nM Phorbol 12-myristate 13-acetate(PMA)for 4 h to form NETs.After PBMCs co-cultured with NETs for 24h,the expression of activation indexes on T and B cells was detected as above.Similarly,after co-cultured for 72h,the ratio of CD4+/CD8+T and CD38+(%CD19+)B cell subgroups were detected.Meanwhile,PBMCs stained by the cell proliferation dye CFSE were co-cultured with NETs for 72 h,the level of PBMCs proliferation were detected.3.After PMNs were isolated from peripheral blood of 6 healthy donors,the level of NETs of PMNs treated by PMA or sera from SLE patients with or without LY294002 or MK2206,PI3K/Akt specific inhibitor,was detected by fluorescence photometry.Results:1.The activation level of T and B cells and their correlation with NETs in SLE patients:the ratio of early activated T cell CD4+CD69+T in SLE group(58 cases)and HC group(36 cases)was respectively[2.10%(1.10%4.08%)VS 1.20%(0.80%,2.03%),P=0.001],and the MFI of CD69 on CD4+T was individually[(105.72±47.32)VS(72.75,13.65),P=0.000].There was a significant difference between the two groups,suggesting that the early activation level of T cells in SLE group was significantly higher than that in the control group.Compared with HC group(36 cases),the proportion of late activated T cell CD4+HLA-DR+T in SLE group(65 cases)was significantly higher[7.60%(5%,13.5%)VS 4.75%(3.43%,5.78%),P=0.000],and the MFI of HLA-DR on individual cell increased[(163.82±72.72)VS(99.97±26.21),P=0.000].The ratio of activated B cell CD19+CD69+B in SLE group(58 cases)and HC group(36 cases)was respectively[3.85%(1.70%,6.18%)VS 1.30%(1%,1.88%),P=0.000],and the MFI of CD69 on single cell was[98(81.50,121)VS 66(58.50,73.25),P=0.000].There was a significant difference between the two groups,showing that the activation level of B cells in SLE group was significantly increased.In addition,the MFI of CD69 on CD4+T cell in patients with SLE was positively correlated with NETs[(r=0.42,P=0.026)],and the same to CD19+B[(r=0.45,P=0.024)].2.The effect of NETs on the activation of lymphocytes in vitro:PMNs and PBMCs from peripheral blood of 10 healthy donors were isolated.And PMNs were induced by PMA in vitro to form NETs.Then PBMCs were co-cultured with NETs for 24 hours,compared with PMNs group,CD69+(%CD4+)T and the MFI of CD69 on single CD4+T cell in NETs group increased[(81.41±15.48)%VS(1.99±1.30)%,P<0.001]、[(4663.50±2192.65)VS(93.70±46),P<0.001],suggesting that NETs can induce early activation of CD4+T cells.But there was no difference between two groups on HLA-DR+(%CD4+)T[(11.37±6.04)%VS(11.23±3.20)%,P=0.95].The MFI of HLA-DR on single CD4+T cell increased in NETs group[(3294.60±2789.37)VS(245.80±87.57),P<0.001],indicating that NETs can promote the expression of HLA-DR on CD4+T cells.The proportion of CD 19+CD69+B of CD 19+B cells in NETs group was higher than PMNs group[(57.58±11.24)%VS(22.13±16.13)%,P<0.001].And the MFI of CD69 on single CD 19+T cells increased as well[(2268.90±571.79)VS(247.6±96.70),P<0.001],suggesting that NETs can induce the activation of CD 19+B cells.3.The effect of NETs on the proliferation of PBMCs in vitro:PBMCs and PMNs from the peripheral blood of 10 healthy donors were isolated.After PBMCs incubated with the cell proliferation fluorescent dye CFSE,PBMCs was stained with green fluorescence observed by fluorescence microscope and flow cytometry.After PMA-induced NETs co-cultured with CFSE-stained PBMCs for 72 hours,there was no significant difference on the proliferation of PBMCs between NETs and PMN group[(184.40±76.47)VS(198.40±68.90),P=0.93],showing that NETs could not induce the proliferation of PBMC in vitro.4.The effect of NETs on the subgroups of T and B cells in vitro:after NETs co-cultured with PBMCs for 72 hours,there was no significant difference in CD4+/CD8+T ratio between NETs and PMNs group[(1.76±0.38)VS(2.09±0.51),P=0.64],and there was also no difference in the proportion of CD38+(%CD19+)B cells in the two groups[(74.11±4.86)%VS(73.31±7.19)%,P=0.96],suggesting that NETs can not cause the imbalance of CD4+/CD8+T ratio and CD 19+CD38+B subgroups in vitro.5.The activation of PI3K/Akt in the formation of NETs:PMNs from peripheral blood of 6 healthy donors were isolated.During the process of PMNs to form NETs stimulated by PMA and sera from SLE patients,after adding PI3K inhibitor LY294002,the level of NETs decreased significantly[(310±182.77)VS(840.20±334.54),P=0.005]、[(419.33±196.83)VS(703±266.71),P=0.004].Similarly,after the addition of Akt inhibitor MK2206,the production of NETs decreased[(593±224.93)VS(842.60±307.04),P=0.007]、(500.67±271.53)VS(784.50±348.28),P=0.009],indicating that LY294002 and MK2206 could reduce the formation of NETs stimulated by PMA and sera from SLE patients by inhibiting the activation of PI3K/Akt.In the process of spontaneous NETs formation of PMNs,compared with the cultured PMNs,NETs formation was inhibited by Akt inhibitor MK2206[(138.671±52.11)VS(210.33±80.66),P=0.03),but the PI3K inhibitor LY294002 did not[(168±78.08)VS(220.60±87.03),P=0.15],indicating that Akt activation is involved in the spontaneous NETs formation,but its upstream protein may not be PI3K.Conclusions:1.The positive correlation between the level of T and B cells activation and NETs in peripheral blood of patients with SLE,which indicates that NETs may involved in the pathogenesis of SLE by causing abnormal activation of T and B cells,suggests that innate immunity mediated by neutrophils and adaptive immunity mediated by lymphocytes may play a synergistic role in the occurrence of SLE.2.NETs can promote the activation of T and B cells except the proliferation of PBMCs in vitro.It indicates that NETs may participate in the development and progression of SLE by priming cellular and humoral immunity.3.The production of NETs stimulated by PMA and sera from SLE patients could be initiated by PI3K/Akt signaling pathway.It suggests that to inhibit the activation of PI3K/Akt signaling molecules may regulate the production of NETs and thus further control the pathological process of SLE. |
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