Effects And Mechanisms Of Neutrophil Extracellular Traps In Bacillus Calmette-gu(?)rin Immunotherapy Of Bladder Cancer

Part Ⅰ:The formation of neutrophil extracellular traps induced by BCG and and the effects of tumor cells on its formationObjective:To explore BCG induced neutrophil extracellular traps formation an d the influence on the forming process by tumor cells.Methods:Fluorescent staining was for neutrophils after stimulation with BCG,using confocal microscope to observe the formation of NETs and qualitatively analysis the main composition.MicroBCA kit was used to make a quantitative analysis on total protein of NETs.PMNs were cocultured with tumor cells pre-treated with BCG or not The images were taken under the confocal microscope to compare NETs formation rate with the control group using Image J.Supernatants produced by the T24 cells co-culture with BCG,or BCG and T24 cells alone were respectively used to induce neutrophils to form NETs and compare the NETs formation rate.IL-8 in supernatants produced by tumor cells were measured using ELISA kit.Results:1.Neutrophils can form NETs by the stimulation of BCG for 2hrs in vitro,The result of· ·fluorescence staining showed that the main ingredients of NETs were DNA,histones and elastase;2.Compared with the control group,the NETs formation rate induced by tumor cells pre-treated with BCG was higher with statistical significance.(P<0.01)3.The supernatants of T24 cells and BCG co-cultivated for 2hrs can stimulate the neutrophils to form NETs.The formation rate was higher than that of T24 cells or BCG alone.(P<0.01)4.The level of IL-8 in supernatants produced by the T24 cells stumulated with BCG for 2hrs is significantly elevated when compared with the control group.Within the indicated culture time(15 min,30 min,60 min,120 min,240 min,480 min),the expression level of IL-8 peaked after 2h,then gradually reduced When continue to cultured 4h and 8h.Part Ⅱ:The influence of NETs on T24 cell and the cytokine secretion by PBMCObjective:To investigate the effects of NETs on T24 cell in proliferation,apoptosis,expression of apoptosis related protein,and the influence of NETs on the Th1 cytokines secreted by human PBMC.Method:5 X 106 CFU/mL BCG and neutrophils were cocultured for 4hrs to induce NETs formation.Supernatant contains Nets was added to T24 cells for culture.Then CCK-8 kit,LDH kit,FCM and WB were used to detect cell proliferation inhibition effect,direct cytotoxic effect,apoptosis,and the expression of apoptosis related proteins—Bcl-2 and Bax;The levels of IL-2 and TNF-α in supernatants produced by PBMC treated by NETs were measured using ELISA kit.Result:1.The results indicated that NETs can significantly inhibited proliferation of T24 cells in a concentration-and time-dependent manner.2.The results of LDH release experiment showed that the values of T24 cells treated with NETs increased significantly(p<0.05),indicating obvious cytotoxic effects..3.Annexin-V FITC/PI staining revealed the manifestation of apoptosis after NETs-treatment for 24 hrs.(1×NETs 7.88±1.7%,2×NETs 8.1±3.1%VS control group 1.78±0.8%),with significant differences(all p<0.01).4.NETs treatment downregulated Bcl-2 expressions,and enhanced Bax expression when compared to the control group.5.The levels of IL-2 and TNF-α in supernatants produced by PBMC treated with NETs at different times(24h,48h,72h,96h)were significantly higher than thoes of the untreated PBMC(p<0.05).Conclusion:1.BCG can induce neutrophils to form NETs.Additionally,tumor cells contribute to the formation for BCG promote tumor cells secrete IL-8,which has been known as a clear stimulus for NETs formation.2.NETs induced by BCG have the direct cytotoxicity on tumor cells,inhibit its proliferation and induce apoptosis.The effect of induce apoptosis was related with downregulated Bcl-2 and upregulated Bax.3.Nets can stimulate PBMC to secrete Th1 subsets of cytokines,suggestting NETs have an anti-tumor function through strengthen the immunity regulation.4.The results show that PMN can fade by NETosis induced by BCG.NETs may play an important role in immune regulation in the immunotherapy of BCG instillation.Our results makes it necessary to reinterpret the DTH classical theories and provides a scientific basis for BCG of anti-tumor immune mechanism mediated by the neutrophil as the initial effector cells.