Mechanism Of GSDMD-triggered Pyroptosis In Restraining Neospora Caninum Proliferation

Neospora caninum（Neospora）,is an intracellular protozoan parasite,can cause reproductive disorder disease in intermediate hosts such as cattle and sheep,leading to neosporosis.The disease is endemic globally and causes great economic losses to the cattle farming industry.However,there is no specific drug on the market to prevent and treat neosporosis,toltrazuril and its derivative patozuril have good effects on the treatment of neosporosis.Although vaccines are an effective means of preventing pathogenic infections,there is no effective commercial N.caninum vaccine.Understanding of the immune mechanism between host and N.caninum infection is also limited.As a type of programmed cell death,GSDMD-mediated pyroptosis plays an important role in clearing intracellular pathogens.Previous studies in our laboratory have found that the host NLRP3 inflammasome in N.caninum infection can mediate pyroptosis and play an anti-N.caninum infection role;when pyroptosis happened in bovine macrophage,the number of N.caninum in bovine macrophages was reduced.However,the signaling pathway of GSDMD-mediated pyroptosis in N.caninum infection,the mechanism of pyroptosis inhibiting the proliferation of N.caninum and whether pyroptosis can be used as a potential target for screening therapeutics need to be clarified,in order to provide new ideas for the treatment of N.caninum.The main research contents were followed:The signal transduction pathway of GSDMD-mediated pyroptosis in N.caninum infection:The signal pathway of GSDMD-mediated pyroptosis in N.caninum infection was explore by using methods such as Western Blot,the details were as follows:（1）N.caninum induced GSDMD-mediated pyroptosis.Primary peritoneal macrophages and bone marrow-derived macrophages from mice were isolated and infected with N.caninum to establish an in vitro model,the pyroptosis was detected,and the proper macrophage was selected for the following experiments.The results showed that N.caninum infection could induce GSDMD cleavage and caspase-1 activation in both peritoneal macrophages and bone marrow-derived macrophages,suggesting the occurrence of pyroptosis.And the activation of pyroptosis in peritoneal macrophages was more obvious than that of bone marrow macrophages.（2）Signal pathway for N.caninum-induced pyroptosis.Wild-type（WT）,GSDMD-,NLRP3-,ASC-and caspase-1/11-deficient macrophages were infected with N.caninum,changes in pyroptosis pathway-related proteins such as caspase-1,GSDMD,and IL-1βwere detected.The results showed that the expression of GSDMD can be affected by NLRP3,but the results of LDH showed NLRP3,ASC and caspase-1 did not affect N.caninum-induced cell death.Compared with the wild-type group,in the absence of GSDMD,releases of N.caninum-induced IL-1βand active capase-1 were not reduced;the LDH results showed cell death was decreased by about 9%,but the difference was not significant;caspase-8 activation was increased;these indicating that in N.caninum infection GSDMD expression can be affected by NLRP3,but was not responsiable for the releases of IL-1βand caspase-1,and there are other forms of cell death in addition to GSDMD-mediated pyroptosis.（3）N.caninum-induced pyroptosis in mice.Mice were infected with N.caninum to detect whether pyroptosis occurred in mice.The results showed that not only caspase-1 activation and GSDMD cleavage were detected in intraperitoneal lavage cells,but also phosphorylation of necroptosis executor MLKL was detected,indicating that N.caninum infection can cause both pyroptosis and necroptosis.However,the focus of this study was on GSDMD-mediated pyroptosis,providing a basis for the next step of in-depth study of N.caninum inhibition by pyroptosis.The study of N.caninum proliferation inhibition by GSDMD-mediated pyroptosis:The effect of GSDMD-mediated pyroptosis on N.caninum proliferation was detected by q PCR and other methods,the details were as follows:（1）Effects of GSDMD on N.caninum proliferation.WT and GSDMD^-/-mice peritoneal macrophages were infected with N.caninum,the number of intracellular parasite was detected.The results showed that compared with the WT group,in GSDMD^-/-cells the number of intracellular N.caninum were significantly higher,the number of tachyzoites in parasitophorous vacuole was greatly increased,but the infection rates of cells in these two group were not changed.（2）Effects of pyroptosis-induced supernatant on N.caninum.WT and GSDMD^-/-mouse peritoneal macrophages were treated with LPS plus ATP to obtain supernatants,N.caninum incubated with these supernatant liquids was used to re-stimulate fresh WT peritoneal macrophages,then intracellular tachyzoites in parasitophorous vacuole were observed.The results showed that compared with WT pyroptosis supernatant-incubated N.caninum,the infection rate of GSDMD^-/-pyroptosis supernatant-incubated N.caninum in fresh macrophage was significantly increased,and the number of tachyzoites in parasitophorous vacuole was increased and mostly 8 or more,indicating that N.caninum proliferation can be inhibited by GSDMD-mediated pyroptosis,and the ability of N.caninum to re-infect cells can be reduced by the supernatant released after GSDMD-mediated pyroptosis.The effect of the pyroptosis agonists on the proliferation of N.caninum:The effect of pyroptosis agonists on N.caninum proliferation was explored by q PCR,electron microscopy and other methods,the details were as follows:（1）Preliminary screening of pyroptosis agonists:N.caninum-infected mice with were treated with three pyroptosis agonists niclosamide,ivermectin and berberine,repsectively,toltrazuril was selected as a positive control.The survival rates and weight changes of mice were detected in order to select the appropriate pyroptosis agonist with anti-N.caninum potential.The results showed that niclosamide and ivermectin could improve the survival rate of mice and had a certain therapeutic effect.（2）Evaluation of the effect of pyroptosis agonists on the treatments of N.caninum infection.The N.caninum-infected mice were treated by different concentrations of niclosamide and ivermectin,the therapeutic effect of these two agonist was further evaluated by survival rates,etc.The results showed that 0.5mg/kg ivermectin was the optimal concentration selected,and survival rate of the mice was about 50%,which was better than that of niclosamide.（3）Effect of ivermectin on N.caninum-induced pyroptosis.Macrophages were infected with N.caninum,and then treated with the pyroptosis agonist ivermectin,and the release of IL-1βwas detected to explore whether ivermectin promoted N.caninum-induced pyroptosis.The results showed that N.caninum-induced IL-1βwas greatly increased by ivermectin,indicating that the pyroptosis was promoted to a certain extent.（4）Evaluation of effect of ivermectin on intracellular N.caninum proliferation in vitro.N.caninum-infected VERO cells and N.caninum were treated with ivermectin,respectively,to evaluate the inhibition effects.The results showed that the intracellular parasite number,infection rate of cells,and number of tachyzoites in parasitophorous vacuole were significantly reduced by ivermectin,and the effect was better than toltrazuril（positive control）.Transmission electron microscopy observations showed that morphological changes,such as structural edge deformation and membrane rupture,were observed in ivermectin-treated N.caninum,indicating that the normal morphological structure can be directly damaged by ivermectin.These showed that the screened pyroptosis agonist ivermectin could inhibit the proliferatin of N.caninum,and had a certain therapeutic effect on N.caninum infection.In summary,this study found that GSDMD-mediated pyroptosis can be induced by N.caninum both in vitro and in vivo,affected by NLRP3.GSDMD-mediated pyroptosis and its released contents can inhibit the intracellular N.caninum proliferatin and reduce the ability of N.caninum to re-infect cells.As a pyroptosis agonist,ivermectin could promote N.caninum-induced pyroptosis,significantly inhibit intracellular N.caninum proliferation,and had a direct damage effect on the parasite,and also had a certain therapeutic effect on N.caninum infection.This study suggests that pyroptosis pathway can be used as a potential target for screening drugs for the treatment of neosporosis.