| Objective:The present study was performed to explore the role of XCT/GPX4/GSH in the expression of inflammatory cytokines induced by pneumococcal protein which will enrich our knowledge on the pathogenesis of Streptococcus pneumoniae(S.pneumoniae)and provide new targets for the treatment of pneumococcal infection.Methods:1.Macrophages were infected with pneumococcal protein.Transcriptome analysis was performed to analyze differentially expressed genes(DEGs)in macrophages before and after infection with pneumococcal protein.RT-q PCR was employed to demonstrate the transcription levels of M1-type polarization markers NOS2 and CD86,and M2-type polarization markers CD206 and IL-10.Western-blot and ELISA were used to analyze the levels of NLRP3 and inflammatory cytokines,respectively.2.Transmission electron microscopy(TEM)was used to reveal changes of organelles in macrophages.The expression of XCT/GPX4/GSH and levels of reactive oxygen species(ROS)in macrophages infected with pneumococcal protein was examined,which was verified by addition of the inhibitor,Ferrostatin-1(Fer-1).3.In animal experiments,mouse lung infection models were established by nasal inoculation with 2×10~7 CFU of S.pneumoniae D39 strain.The inflammatory response of lung tissues was analyzed by HE staining of lung tissue sections and measuring the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF).The expression levels of XCT and GPX4 in lung tissues were detected by Western-blot.4.Macrophages were treated with different pneumococcal protein,or pneumococcal lysates with anti-PLY serum,and levels of GPX4 were analyzed by Western-blot.5.Macrophages were treated with concentrations of wild-type PLY and non- hemolytic PLY mutant,and levels of XCT/GPX4/GSH and reactive oxygen species ROS were analyzed.Results:1.A total of 1615 DEGs were obtained by transcriptomic analysis,including 926 up-regulated genes and 689 down-regulated genes.Of them,inflammation related genes,such as TNF,CD86,NOS2 and IL-6,were significantly up-regulated,while antioxidants GPX4 and NRF2 were significantly down-regulated.The expression of M1-type polarization marker NOS2 was significantly increased in macrophages infected with S.pneumoniae(P<0.05),accompanied by the activation of NLRP3 and secretion of inflammatory cytokines.2.TEM results showed obvious changes in mitochondria,including mitochondrial swelling,crest reduction and chromatin looseness.Pneumococcal infection also reduced the expression of XCT,GPX4 and GSH in macrophages,but increased intracellular ROS level,and XCT and GPX4 could be partially restored following the addition of Ferrostatin-1.The addition of Fer-1 also attenuated the activation of NLRP3 signaling and reduced the levels of inflammatory cytokines.3.In the mouse model,lung tissue of mice intranasally infected with S.pneumoniae showed obvious hyperemia and a large number of inflammatory cell infiltrations,a large number of inflammatory factors could be detected in BALF,and levels of XCT and GPX4 were significantly reduced.4.Compared with pneumococci grown in N-acetylglucosamine,bacteria grown in glucose produced higher amount of PLY.S.pneumoniae significantly reduced the expression of GPX4 which could be reversed by the addition of PLY antiserum in macrophages.5.The recombinant PLY protein also significantly inhibited the expression of XCT, GPX4 or GSH and increased the levels of ROS in macrophages,which was dependent of its concentrations and hemolytic activity.Conclusion:S.pneumoniae induces a PLY-dependent activation of NLRP3inflammasome and secretion of inflammatory cytokines by inhibiting the XCT/GPX4/GSH axis in macrophages. |
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