The Role Of ROS-mediated NLRP3 Inflammasome In Neospora Caninum Infection

Neospora caninum is an obligate intracellular protozoan,which causes reproductive disorders in intermediate host,such as cattle and sheep,and neurological disorders in terminal host,such as dogs and canines.N.caninum infection is prevalent all over the world,leading to a significant economic loss on the cattle industry every year.Currently,neither commercial vaccines nor specific drugs have been developed for the prevention and treatment of neosporosis due to the elusive anti-N.caninum immune mechanisms in the hosts.In recent years,the study of innate immune mechanism between host and N.caninum infection have been made some achievements.The innate immune system recognizes the pathogen related molecular pattern?PAMP?of the invading microorganism through the pattern recognition receptor?PRR?expressed by the host cell,and then mediates the innate immune defense response of the host,such as inflammatory response regulates cytokines and chemokines release to identify and eliminate the invading pathogen.Previous studies in our laboratory found that N.caninum-infected significantly upregulated the NLRP3 gene in NOD-like receptors,and activated the NLRP3inflammasome in peritoneal macrophages,and NLRP3 inflammasome activation plays an important role in anti-N.caninum infection.The activation of NLRP3inflammasome secretes IL-1?and IL-18,and mediates their release to outside the cell to exert downstream immune effects.However,the molecular mechanism of N.caninum-induced NLRP3 inflammasome activation was still unclear.N.caninum infection induced ROS-dependent extracellular traps generation in canine polymorphonuclear neutrophils.Therefore,we speculated whether there was the relationship between N.caninum,ROS and NLRP3 inflammasome.In this study,ROS release in N.caninum-infected PMs were investigated,and the effects of ROS on N.caninum-induced NLRP3 inflammasome activation and controlling N.caninum replication in PMs by intervening ROS,to clarify the NLRP3 inflammasome activation and parasites proliferation regulated by N.caninum-induced ROS release,and to target the ROS-mediated NLRP3 inflammasome axis could provide new potential drugs for the treatment of neosporosis.The main research contents are as follows:N.caninum infection elicited ROS generation in PMs.PMs were isolated and the N.caninum was purified to establish the N.caninum infection model,and then the intracellular ROS level was detected.ROS inhibitor NAC pretreatment and different numbers of N.caninum infection,intracellular ROS was labeled with the fluorescent dye DCFH-DA.The results showed that the intracellular ROS was greatly increased in N.caninum-infected PMs,which is a dose-dependent manner with the number of N.caninum infection.ROS regulated NLRP3 inflammasome and N.caninum proliferation in PMs.PMs were pretreated with the ROS inhibitor NAC,and the effects of ROS on NLRP3inflammasome and parasite burden were evaluated by detecting ROS,IL-1?secretion,NLRP3 expression,the LDH release of cell death and intracellular parasites proliferation.Then we detected that the regulation of ROS release and IL-1?secretion by ROS inducers pyrogallol?PG?and piperlongumine?PL?,and found the ROS inducer PG which activated NLRP3 inflammasome.Subsequently,further studies that ROS inducer PG-mediated NLRP3 inflammasome regulated parasite burden in PMs.The results found that ROS inhibitor NAC reduced the production of LDH,IL-1?and NLRP3,but increased parasite burden in PMs,so indicating that N.caninum induces ROS to regulate NLRP3 inflammasome activation and parasite burden in PMs.In N.caninum-infected PMs,ROS inducer PL inhibited intracellular ROS production and IL-1?secretion in dose-dependent manner.However,ROS inducer PG promoted ROS release and IL-1?secretion in dose-dependent manner,and the release of IL-1?,caspase-1,NLRP3 and LDH were most significantly increased and parasite burden was the least in PMs at 30?M PG.Thus,PG induces ROS generation,which mediates NLRP3 inflammasome activation and inhibits N.caninum proliferation in PMs.NLRP3-regulated ROS mediates inflammasome and parasite burden.WT PMs and Nlrp3^-/-PMs were treated with ROS inhibitor NAC,ROS inducer PG,and NADPH Oxidase inhibitor Diphenyleneiodonium chloride?DPI?,respectively,and then the generation of ROS,IL-1?and LDH were detected.And the effects of ROS inhibitor NAC and ROS inducer PG on parasite burden in N.caninum-infected WT PMs and Nlrp3^-/-PMs were examined.The results showed that NAC and DPI treatments decreased the release of ROS,IL-1?and LDH compare to N.caninum infection-only group,but PG treatment promoted the release of ROS,IL-1?and LDH.However,in Nlrp3^-/-PMs,DPI,NAC and PG did not affect the secretion of IL-1?when the trend of regulating ROS release was the same as that of WT PMs,indicating that N.caninum induces NADPH-dependent ROS to activate NLRP3 inflammasome.In WT PMs,NAC increased parasite burden,while PG decreased parasite burden,in Nlrp3^-/-PMs,NAC treatment did not change parasite burden,PG treatment slightly reduced parasite burden,but it was still significantly higher than PG treatment in WT PMs.Therefore,NLRP3 participates in the regulation of ROS-mediated inflammasome and N.caninum proliferation,and PG-induced ROS mediates NLRP3inflammasome activation to inhibit N.caninum proliferation.ROS inducer PG mediates NLRP3 inflammasome to anti-N.caninum infection in vivo.The model of N.caninum-infected WT mice and Nlrp3^-/-mice were established,from day 2 post infection,PG was intraperitoneally injected,IL-18 in serum and parasite burden in peritoneal exudate cells were detected.The effect of PG on anti-N.caninum infection was further studied.PG was applied to WT mice,the weight and survival rate of mice,parasite burden of tissues and histopathological changes were detected.The results showed that PG significantly up-regulated IL-18 in serum and decreased parasite burden of peritoneal exudate cells in WT group,while in Nlrp3^-/-group,PG treatment did not increase IL-18,and parasite burden of peritoneal exudate cells only slightly decreased,but it was still significantly higher than the PG treated with WT group.In further animal studies,found that PG decreased the weight loss caused by N.caninum infection and effectively improved the survival rate of mice compared to the infection-only group,parasite burden and pathological damage of heart,liver,spleen,lung,kidney and brain were reduced.The above results indicate that PG induces NLRP3 inflammasome activation to play an anti-N.caninum role in vivo,and the ROS-mediated NLRP3 pathway could be used to find candidate drugs for the treatment of neosporosis.In conclusion,N.caninum-induced ROS generation plays an important role in NLRP3 inflammasome activation and controlling parasites.The ROS inducer PG mediates NLRP3 inflammasome activation to eliminate N.caninum in vitro and in vivo.ROS-mediated NLRP3 inflammasome axis could be a potential therapeutic strategy for neosporosis.