NETs Induce Alveolar Macrophage Pyroptosis Via Regulating NLRP3 Deubiquitination Modification For Aggravating The Development Of Septic Lung Injury

Objective:Uncontrolled inflammation is a typical feature of sepsis-related lung injury.The major key role in the progression of lung injury is Caspase-1-dependent alveolar macrophages(AMs)pyroptosis.Similarly,neutrophils are stimulated to extrude extracellular traps(NETs)to participate in the innate immune response.Moreover,both NETs and macrophages are related to the development of inflammatory disease.However,the specific mechanisms by which NETs activate macrophages and maintain lung inflammation are still unclear.This study aims to illustrate the specific mechanisms by which NETs activate alveolar macrophages at the posttranslational level and maintain lung inflammation.Methods:We established a septic lung injury model by cecal ligation and puncture(CLP).Confocal microscopy and enzyme-linked immunosorbent assay were used to detect the contents of NETs and IL-1β in lung tissue of sham-operated group,CLP group,and DNAse Ⅰ + CLP group;Western blotting and immunofluorescence analysis were utilized to determine the effect of NETs on AMs cell pyroptosis;Immunohistochemical staining and Western blotting to determine the protective effect of degrading NETs or targeting NLRP3 inflammasome on alveolar macrophage pyroptosis and lung injury;Immunofluorescence analysis verified the effect of NETs on ROS generation in macrophages;The effects of NETs on the ubiquitination of NLRP3 and the effect of down-regulation of ROS levels on the binding of NLRP3 to ubiquitin molecules were detected by coimmunoprecipitation.Results:1.The increased NETs production and interleukin-1b(IL-1β)release in septic mice are correlated with aggravation of lung injury.Inhibition of NETs can significantly reduce the level of IL-1β in plasma and BALF.2.Immunohistochemistry and immunofluorescence staining showed that the infiltration of neutrophils and macrophages in the lung tissue of septic mice increased,and both were adjacent to each other in space.3.NETs were identified by immunofluorescence staining in vitro.ELISA assay revealed that NETs induce LPS-pretreated macrophages to secrete a large amount of IL-1β and IL-18.DNAse I inhibited the release of IL-1β and IL-18 induced by NETs combined with LPS.4.NETs upregulated the level of NLRP3,followed by ASC foci production,NLRP3 inflammasome assembly,caspase-1 activation,and leading to AMs pyroptosis executed by the activated fragment of full-length Gsdermin D(FH-GSDMD).However,the opposite effect of the above results was observed in the inhibition of MCC950 or DSF.5.Immunohistochemistry and Western Blot techniques showed that intraperitoneal injection of DNAse I could reduce the expression of NLRP3 and N-GSDMD in the lung tissue of septic mice.Meanwhile,H&E staining showed that DNAse I could attenuate sepsis-induced lung injury in mice.6.Co-immunoprecipitation suggested that NETs could induce deubiquitination of NLRP3 in LPS-primed alveolar macrophages.Furthermore,the deubiquitinase inhibitor G5 facilitated NETs-induced the binding of NLRP3 and ubiquitin.7.Western Blot technique showed that G5 could inhibit the expression of NLRP3 and N-GSDMD in LPS-primed macrophages induced by NETs.Similarly,confocal microscopy revealed that NETs-induced ASC foci generation was inhibited by G5.Flow cytometry showed that the number of dead macrophages in the LPS +NETs + G5 group was significantly lower than that in the LPS + NETs group.8.Immunofluorescence staining found that NETs can markedly elicit an increase in reactive oxygen species(ROS)generation,The level of ROS in the cytoplasm was significantly reduced in the inhibition of NETs with DNAse I.9.Co-immunoprecipitation assay showed that the ROS scavenger NAC could inhibit NETs-induced deubiquitination of NLRP3 in LPS-primed macrophages.Removal of ROS can promote the binding of NLRP3 and ubiquitin,and inhibit NLRP3 binding to ASC.10.Western Blot assay showed that NAC could inhibit the expression of NLRP3 and N-GSDMD in LPS-primed macrophages induced by NETs.Likewise,confocal microscopy revealed that NETs-stimulated ASC foci production was inhibited by NAC.Flow cytometry showed that the number of dead macrophages in the LPS + NETs + NAC group was significantly lower than that in the LPS + NETs group.11.Immunohistochemical assay showed that intraperitoneal injection of NAC could reduce the NLRP3 positive area in the lung tissue of septic mice.Meanwhile,H&E staining found that NAC had a protective effect on CLP-induced lung injury in mice.Conclusion:In summary,these findings indicate that NETs prime ROS generation,which promotes NLRP3 inflammasome activation at the posttranslational level to mediate AMs pyroptosis and sustain lung injury in septic mice.