The Mechanism Of NLRP3 Inflammasome Activation And Occurrence Of Pyroptosis Induced By Pasteurella Multocida Infection

Pasteurella multocida(P.multocida)is a zoonotic pathogen that can cause respiratory tract infections in different animals,P.multocida type A is an important pathogen causing bovine respiratory disease complex,it has brought huge economic losses to the beef cattle breeding industry.Inflammasomes are complexes assembled from multiple proteins in the cytoplasm and play an important role in host defense against microbial infection.The NLRP3 inflammasome mediates innate immunity,it is also implicated in the pathogenesis of multiple autoinflammatory and metabolic diseases.Subsequent triggering of caspase-1 activation is critical for host defense against pathogens.Previous studies have shown that macrophages infected by P.multocida setotype A high virulence strains(Pm CQ2)can activate the NLRP3 inflammasome and induce the secretion of cytokine IL-1β,but the mechanism of it is still unclear.Potassium ion(K~+)efflux and NIMA-related kinase 7(Nek7)are hotly discussed conditions for the activation of the NLRP3 inflammasome.Cells can induce K~+efflux under certain infection or stimulation conditions and activate Nek7 to interact with NLRP3,thereby activating inflammasome,and the downstream reactions also include the cleavage of pyroptosis-executing molecule gasdermin D(GSDMD).Therefore,this experiment took K~+efflux,Nek7 and GSDMD as the starting point to investigate the underlying mechanism of P.multocida-induced NLRP3 inflammasome activation and pyroptosis.1.The role of K~+efflux in Pm CQ2-induced NLRP3 inflammasome activationTo explore the role of potassium efflux in Pm CQ2 infection-induced NLRP3inflammasome activation.In this study,different concentrations of KCl and potassium channel inhibitors Qunine and Glibenclamide were used to inhibit K~+efflux.ELISA was used to detect the secretion of related inflammatory cytokines,and Western-bolt was used to detect the activation of NLRP3 inflammasome-related components the activation of caspase-1,the oligomerization of ASC,and the mature secretion of IL-1βand GSDMD.At the same time,the formation of ASC specks was detected by immunofluorescence,and the transcription of NLRP3 was detected by q PCR.The results showed that these K~+channel inhibitors significantly reduced Pm CQ2-induced IL-1βsecretion in macrophages in a concentration-dependent manner,suggesting that K~+efflux is essential for IL-1βsecretion in Pm CQ2-infected macrophages.In addition,under the action of the inhibitor,the oligomerization/specks formation of ASC was significantly reduced,and the active form of caspase-1 and GSDMD-N were also significantly inhibited,resulting in reduced secretion of the mature form of IL-1βprotein.Interestingly,inhibition of K~+efflux did not affect the transcription of NLRP3,indicating that K~+efflux only affected the activation of the NLRP3 inflammasome,not its expression.In conclusion,K~+efflux adequately mediates Pm CQ2-induced NLRP3 inflammasome activation and promotes its activation by affecting the oligomerization of ASC in the NLRP3 inflammasome fraction,which subsequently triggers caspase-1 activation and mature secretion of IL-1β,and cause pyroptosis.2.The role of Nek7 in Pm CQ2-induced NLRP3 inflammasome activationSeveral studies have shown that the combination of Nek7 and NLRP3 can promote the activation of NLRP3 inflammasome after pathogenic microorganism infection,but the role of Nek7 in the mechanism of NLRP3 inflammasome activation by P.multocida infection is still unclear.In order to explore the role of Nek7 in the activation of NLRP3inflammasome by Pm CQ2,interfering RNA(si-RNA)was used to silence the nek7 gene to restrict its expression at the transcriptional and protein levels.The results showed that the secretion level of IL-1βin the si-Nek7 group was significantly lower than control group,while the secretion of other cytokines TNF-α,IL-6 and IL-1αwas not affected.Similarly,caspase-1,a kinase upstream of IL-1β,was secreted at reduced levels after Nek7 silencing and markedly reduced ASC specks formation.In addition,Pm CQ2-induced combination of Nek7 and NLRP3 was abrogated in KCl-treated macrophages while Nek7 protein expression was not affected.Furthermore,deletion of relevant components of the NLRP3 inflammasome did not affect Nek7-NLRP3 binding,suggesting that Nek7 specifically binds NLRP3 to mediate inflammasome activation.In conclusion,our study shows that Nek7 just binds to NLRP3 protein to regulate inflammasome in response to Pm CQ2 infection,and this process is mediated by K~+efflux.This finding provides a new insight on P.multocida-induced host immune responses.3.Pm CQ2 infection induces GSDMD activation and pyroptosis through theclassical or non-canonical inflammasome pathwayThe activation of NLRP3 inflammasome includes classical and non-canonical pathways,the main difference between the two ways is the involvement of caspase-11,and both pathways may eventually lead to pyroptosis.Previous studies have shown that Pm CQ2 infection activates the NLRP3 inflammasome through the classical pathway.To further explore whether Pm CQ2 infection of macrophages induces IL-1βsecretion to induce pyroptosis and whether non-classical pathway is involved,related proteins and cytokines were analyzed.The results showed that the secretion of inflammatory cytokine IL-1βdecreased in a dose-dependent manner after using GSDMD inhibitor Necrosulfonamide(NSA),while the secretion of TNF-α,IL-6 and IL-1αwas not affected,suggesting that GSDMD is involved Pm CQ2 infection induces IL-1βsecretion and pyroptosis.The production of the active form of GSDMD,GSDMD-N,was still detected after knockdown of nlrp3 and asc genes,and the activated form of caspase-11 was detected under this condition,indicating that showed that Pm CQ2 infection of macrophages also activates the non-canonical pathway of the NLRP3 inflammasome.The above results suggest that Pm CQ2-infected macrophages trigger pyroptosis through GSDMD,and non-canonical pathways are involved in the mechanism of NLRP3inflammasome activation.