Regulation Of Pyroptosis Mediated By GSDMD And GSDME In Inflammatory Diseases

Pyroptosis belongs to programmed cell death,which is crucial for regulating innate immune response against infections and maintaining homeostasis of the host.As previously reported,the activation of GSDMD and GSDME serves as the determinate step of pyroptosis pathways.Silicosis is a progressive pulmonary fibrotic disorder induced by inhaled crystalline silica.Previous studies indicated that NLRP3/Caspase-1 pathway-mediated macrophage pyroptosis is associated with the development and progression of silicosis.However,the mechanism of Gasdermin-dependent pyroptosis in silicosis is still unclear.We analyzed the clinical samples of silicosis patients and found the cleavage of GSDMD and GSDME,as well as the activation of multiple caspases(Caspase-1,-3,-6 and-8).Then we compared the results from human patients with that of a mouse silicosis model.To confirm whether these genes are crucial for developing silicosis,we analyzed the susceptibility of Nlrp3-/-,Gsdmd-/-,Caspase-1-/-,Gsdme-/-and Gsdmd-/-Gsdme-/-mice to experimental acute silicosis.It has been demonstrated the Nlrp3-deficient mice strongly resisted to silica induced silicosis.And our data showed Gsdmd-/-Gsdme-/-mice also exhibited low susceptibility to silicosis.Gsdmd-/-,Caspase-1-/-and Gsdme-/-mice displayed pulmonary inflammation and silicosis pathology like wild-type mice.In vitro experiments were also carried out to systematically dissect the signaling pathway in silica-stimulated macrophage.Our work verified that silica mediated cleavage of GSDMD by Caspase-1 depending on the activation of NLRP3 inflammasome,along with cleavage of GSDME by apoptosis-related Caspase-3 /-6 /-8 to induce macrophage pyroptosis.Caspase-1 and Caspase-8 were involved in the maturation of IL-1β mediated by NLRP3 inflammasome through a similar mechanism.Inhibition of GSDMD and GSDME-mediated pyroptosis reduced the secretion of inflammatory cytokines,alleviated lung inflammation and the silicosis pathology in mice.Furthermore,we also found the collapse of MMP and mitochondrial ROS generation during the GSDMD-mediated macrophage pyroptosis.To verify the interaction between GSDMD and ROS,using mass spectrometry analysis,we identified that 4 cysteine residues in human GSDMD(Cys38,Cys56,Cys268 and Cys467)and mouse GSDMD(Cys39,Cys57,Cys265 and Cys487)were oxidized by mt ROS.Oxidative GSDMD potentiated its cleavage efficacy by Caspase-1,subsequently promoted pyroptosis.However,in vitro experiments limit results to the cellular level,and we look forward to future verification of this finding in animals.In conclusion,our study found both cleavage of GSDMD and GSDME are required for silica-induced cell death and pulmonary inflammation.In addition,oxidative modification of GSDMD enhanced its cleavage efficacy by Caspase-1 in canonical inflammation signaling pathway.Our work advances the further understanding of GSDMD and GSDME in mediating cell death and inflammatory diseases.