Effects And Mechanisms Of NLRP3 Inflammasome In Host Defense Against Neospora Caninum Infection

Neospora caninum is an intracellular protozoan parasite that mainly infects definitive host canids and the intermediate host cattle and caprids,results in abortion in cattle and leads to financial losses worldwide.While at present commercial vaccines or drugs are not available for the prevention and treatment of bovine neosporosis.There are increasing attentions in defining the role of the host immune response in Neospora caninum infection and in exploring the immune functions that influence control of infection and disease development.In intermediate host fast dissemination of replicating tachyzoites is responsible for the acute phase of neosporosis.Innate immune system firstly responses to invading parasite and subsequently initiates appropriate adaptive immune response against this parasite.Upon infection,activation of host pattern recognition receptors expressed by immune cells triggers innate immune response firstly.Intracellular proliferation of Neospora caninum is one of the essential processes during infection.Recently,study showed that NOD2 from cytoplasmic NOD-like receptors?NLRs?family played important roles in eliminating Neospora caninum and inducing pro-inflammatory response.However,how NLRs mediated inflammasomes participate in the recognition of Neospora caninum is still unknown.To explore the molecules in NLRs that could probably recognize Neospora caninum,murine peritoneal macrophages were infected with Neospora caninum tachyzoites at an multiplicity of infection?MOI=3:1,parasite:cell?for different time.Then real-time PCR and Inflammasome RT Profiler PCR Arrays were used to identify the primary genes involved in Neospora caninum infection.Results showed that many NLR molecules participated in the recognition of Neospora caninum,especially the sensor protein NLRP3.These data implied that NLRP3 inflammasome may be activated by Neospora caninum infection,and further studies were needed.To investigate whether NLRP3 inflammasome can be activated,the processing of caspase-1,secretion of IL-1?and expression of the sensor protein NLRP3 were detected by Western Blot;and the production of IL-1?in culture supernatants was determined by ELISA;the distribution of NLRP3 in cytoplasm was detected by immunofluorescence assay.Meanwhile,Nlrp3^-/-macrophages,and inhibitory approaches were used to investigate inflammasome activation and its role in Neospora caninum infection.Results revealed that inflammasome triggered caspase-1 activation and secretion of IL-1?can be induced by Neospora caninum infection in dose-and time-dependent manners.And NLRP3 distribution became punctate in the cell cytoplasm,which facilitated inflammasome activation.Treatment of N.caninum-infected macrophages with caspase-1,pan-caspase and NLRP3inhibitors led to the impaired release of active IL-1?and a failure to restrict parasite replication.And Neospora caninum infected peritoneal macrophages from Nlrp3-deficient mice displayed greatly decreased release of active IL-1?and the failure of caspase-1 cleavage.These data suggest that NLRP3 inflammasome can be activated in Neospora caninum-infected macrophages,and this is the basis to explore the role of NLRP3 inflammasome in Neospora caninum infection.Since cattle are intermediate host of Neospora caninum,understanding the inflammasome activation in bovine immune cells caused by Neospora caninum infection is a prerequisite point in exploring the mechanism of inflammasome in experimental animals.To address this issue,bovine peripheral blood mononuclear cells?PBMCs?and a bovine peritoneal macrophage?BoMac?cell line were used to investigate inflammasome activation and its role in regulating intracellular Neospora caninum replication.Results showed that in Neospora caninum infected PBMCs inflammasome was activated with the maturation of IL-1?.Active caspase-1 can be found in Neospora caninum infected BoMac cells,and caspase-1 dependent cell death was considered to be induced in Neospora caninum infected BoMac cells,because with the pretreatment of zVAD-fmk and VX765 Neospora caninum induced cell death was decreased.Meanwhile,inhibitions of caspase-1 in Neospora caninum infected BoMac cells led to more parasites in vacuole.In contrast,inflammasome activation induced by ATP treatment in Neospora caninum infected BoMac cells contributed to decreased parasite load in vacuole.In addition,pyroptotic cell supernatant collected from ATP stimulated BoMac cells also impaired the ability of this parasite to infect new cells.These data suggest that Neospora caninum could trigger inflammasome activation in both bovine PBMCs and BoMac cells.And this inflammasome activity promoted the elimination of intracellular Neospora caninum.These results were in accordance with that in murine macrophages,and it was beneficial to explore role of NLRP3 inflammasome in Neospora caninum infection in mice.To explore the role of NLRP3 inflammasome during the acute period of Neospora caninum infection,NLRP3,ASC,caspase-1/11 deficient mice were used.In vitro results showed that Neospora caninum infection of murine bone marrow-derived macrophages activated the NLRP3 inflammasome,accompanied by the release of IL-1?and IL-18,cleavage of caspase-1,and induction of cell death.Similar with positive control ATP,K⁺efflux also participated in the signals that activate NLRP3 inflammasome in Neospora caninum infected BMDMs.And this inflammasome activation was dependent on NLRP3,ASC,caspase-1/11.Infection of mice deficient in NLRP3 and ASC resulted in high mortality rates;mice deficient in ASC and caspase-1/11 resulted in increased parasite loads in tissues.Interestingly,production of IL-1?cannot be detected,but high level of IL-18 was examined in serum of WT mice.However,infection of mice deficient NLRP3,ASC,caspase-1/11led to decreased production of IL-18 and reduction of IFN-?in serum,and elevated amounts of monocytes/macrophages and neutrophils were found in the initial infection site,but failed to limit Neospora caninum replication.Thus,these findings suggest that NLRP3 inflammasome can be involved in the host response to Neospora caninum infection at the acute stage,and NLRP3,ASC,caspase-1/11 play different roles in limiting parasite dissemination and promoting host survival.Meanwhile,NLRP3 inflammasome may enhance Th1 response by inducing production of IFN-?and encourage immune cells to eliminate parasites.And these findings also suggest that inflammsome mediated pyroptosis could participate in the clearance of Neospora caninum.To further investigate whether pyroptosis promotes the clearance of Neospora caninum,GSDMD deficient mice and WT mice were infected with Neospora caninum,GSDMD is the direct executor of pyroptosis.Results showed that the mortality rate of Gsdmd^-/-mice was extremely higher,and parasite burdens in tissues were also significantly higher,however,productions of IL-18 and IFN-?were significantly reduced when compared to that in WT mice.Meanwhile,elevated amounts of monocytes/macrophages and neutrophils were found in the initial infection site in Gsdmd^-/-mice,but failed to limit Neospora caninum replication.To further explore whether GSDMD contributes to the production of IFN-?in serum and clearance of N.caninum by mediating IL-18 production.Gsdmd^-/-mice were intraperitoneally injected with recombinant IL-18.During N.caninum infection high level of IL-18 and IFN-?in serum were observed in WT mice,but production of both IL-18 and IFN-?were significantly decreased in the absence of GSDMD,as expect,production of IL-18 and IFN-?were greatly increased in Gsdmd^-/-mice with the treatment of rIL-18.High level of IL-18 was detected in peritoneal exudate fluids,but IL-18 production was significantly reduced in the absence of GSDMD,with rIL-18 treatment production of IL-18 was greatly increased.Notably,compared with WT mice parasite load in peritoneal exudate cells of Gsdmd^-/-mice was significantly increased,interestingly,administration of rIL-18 could greatly reduce the number of N.caninum in Gsdmd^-/-mice.These data suggest that GSDMD mediated pyroptosis plays an important role in promoting murine survival,clearing Neospora caninum within immune cells,controlling the dissemination of parasite,inducing IFN-?production in serum and promote the clearance of Neospora caninum by mediating IL-18,and triggering Th1 immune response during Neospora caninum infection.In summary,NLRP3 inflammasome can be activated,and mediate caspase-1activation and IL-1?/IL-18 secretion,as well as induce pyroptosis during Neospora caninum infection.NLRP3 inflammasome participates in host response against Neospora caninum infection by promoting Th1 immune response and eliminating parasite.This study suggests that NLRP3 inflammasome may be a potential target for future development of drugs or vaccines against N.caninum infection in cattle.