The Effect Of Inhibition Of The NLRP3 Inflammasome-mediated Pyroptosis On The Systemic Lupus Erythematosus And Toluene Diisocyanate-induced Asthma

Objective:Pyroptosis is recognized as gasdermin-mediated inflammatory programmed necrosis.NLRP3 inflammasome activation can induce pyroptosis.The aberrant activation of NLRP3 inflammasome plays a vital role in the pathogenesis of immune and inflammatory diseases including systemic lupus erythematosus(SLE)and toluene diisocyanate(TDI)-induced asthma.Therefore,in this study,we aim to investigate the effect of MCC950(a selective inhibitor of NLRP3 inflammasome activation)on pyroptosis and further determined whether MCC950 was effective on lupus mouse model and TDI-induced asthma mouse model.Methods:1.In vitro,human peripheral blood monocytes were pretreated with MCC950 for 1h,and then stimulated with LPS and ATP or LPS and Nigericin.The percentage of caspase-1+PI+cells was tested by flow cytometry,the release of LDH measured by SpectraMax M3 fluorescent plate reader at 450 nm,and the levels of IL-1?and IL-18 in the supernatant examined by ELISA.2.In vivo,MRL/lpr mice were treated with MCC950 or saline,and then 24h proteinuria,plasma antibodies,IL-1? and IL-18,the ratios of T cell subsets,kidneys histopathology and IgG deposition were detected.3.Similarly,human bronchial epithelial cell line(16HBE)were administrated with MCC950,and then the percentage of caspase-1+PI+cells,the release of LDH,and the levels of IL-1? and IL-18 were measured.4.TDI-induced asthma model mice were also treated with MCC950 or saline,and then the serum levels of IgE,IL-1?,IL-18,the levels of IL-4,IL-5 and IL-13 in bronchoalveolar lavage fluid(BALF),the airway reactivity,and the lung histopathology were tested.Results:1.Compared with LPS+ATP or Nigericin group,the percentage of caspase-1+PI+cells,the release of LDH,and the levels of IL-1? and IL-18 were significantly decreased in MCC950 group.In addition,MCC950 treatment partly stabilized the mitochondria membrane potential and reduced the ROS production.2.In bright field,swelling,blurred membrane edges and bubbles were seen in LPS+ATP or Nigericin stimulated cells.However,MCC950-treated cells retained complete cell membrane.LPS+ATP or Nigericin stimulated cells showed pale blue fluorescence in cytoplasm while MCC950-treated cells with strong and concentrated blue fluorescence by Hoechst staining analysis.In addition,MCC950 administration decreased the percentage of PI+cells in LPS+ATP or Nigericin stimulated monocytes,but increased the percentage of Annexin V+PIcells.3.To evaluate the effects of MCC950 treatment on SLE development,we treated MRL/lpr mice with PD for 8 weeks.Importantly,MCC950 treatment resulted in a distinct reduction in 24h proteinuria levels.Compared with saline-treated MRL/lpr mice,MCC950 treatment markedly reduced the levels of circulating anti-dsDNA and anti-Sm antibodies and histopathological scores of kidney.Less deposition of IgG was observed in the kidneys of MCC950-treated MRL/lpr mice.4.In addition,MCC950 treatment reduced the protein expression levels of cl-caspase-1 and GSDMD-N,the serum levels of IL-1?,IL-18,TNF-?,IFN-?and the ratio of Th1 and Th2,but increased the ratio of Treg and Th 17.5.TDI-HSA induced 16HBE pyroptosis time and concentration dependently.In vitro,MCC950 treatment also significantly decreased the protein expression levels of cl-caspase-1,the LDH release and the levels of IL-1?,IL-18 and HMGB-1.6.Consistently,the serum levels of IgE,the total number of inflammatory cells,the percentage of eosinophils and neutrophils in BALF,the airway reactivity were reduced in MCC950-treated asthma mice induced by TDI.And MCC950 improved the lung pathology.7.MCC950 also decreased the caspase-1 protein expression levels of bronchial epithelial cells,the serum levels of IL-1? and IL-18 and the levels of IL-4,IL-5 and IL-13 in bronchoalveolar lavage fluid(BALF)in TDI-induced asthma mice.Conclusion:1.Inhibition of NLRP3 inflammasome ameliorated lupus-like manifestations in MRL/lpr mice by inhibiting pyroptosis of monocytes,reducing the subsequent release of inflammatory cytokines and modulating the ratio of T cell subset.2.Inhibition of NLRP3 inflammasome also alleviated the airway reactivity and reduced inflammatory responses in TDI-induced asthma mice by suppressing pyroptosis of bronchial epithelial cells.3.These results indicated the important role of pyroptosis in the pathogenesis of SLE and TDI-induced asthma.It provided novel and potential therapeutic targets for these pyroptosis-related diseases.