Molecular Mechanisms Of IFN-? Production And Effect In Neospora Caninum Infection

Neospora caninum(N.caninum)is a newly discovered intracellular parasitic protozoan,with dogs as its definitive hosts as well as bovines and sheep as its intermediate hosts,causing neosporosis.N.caninum results in serious damage to dairy cows,mainly causing abortion,stillbirth,and motor nerve diseases in newborn calves,which causes huge economic losses to the livestock industry worldwide.However,there are no effective drugs or vaccines against N.caninum.In the parasite-host relationship,the host resists parasites by inducing immune responses,and parasites can evade the immune attack of the host.Therefore,an in-depth study of the host immune response to N.caninum infection is molecular fundamental to controlling parasitic diseases by immunological means.IFN-?is an important cytokine in the type I interferon family and plays an important role in resistance to viral and bacterial infections.IFN-?could induce interferon-inducible proteins such as immune-related guanosine triphosphatase M proteins(IRGMs)and guanine nucleotide-binding proteins(GBPs)to inhibit pathogen proliferation by binding to type I interferon receptors.Mitochondria are multifunctional intracellular organelles,and mitochondrial homeostasis is closely related to IFN-?production.Mitochondrial damage has been reported to activate innate immune signaling,thereby inducing IFN-?expression in response to pathogenic infection or cellular injury.Infectious gastroenteritis viruses could hijack host mitophagy to remove damaged mitochondria,thereby attenuating the activation of immune signals for immune evasion.Recent studies have shown that IFN-?played different roles in parasitic infections such as Toxoplasma gondii and Plasmodium.However,whether IFN-?production can be induced by N.caninum and the role and mechanism of IFN-?on N.caninum infection are not clear.Therefore,in this study,to address the unclear role of IFN-?infection and the molecular mechanism of IFN-?production in N.caninum infection,IFN-?was as an entry point to analyze N.caninum-induced IFN-?expression,host cell mitochondrial damage,and mitophagy assays.We explained the roles of IFN-?,mitochondrial damage,and mitophagy in N.caninum infection and also investigated that the mechanism of N.caninum-regulated IFN-?expression was through the c GAS signaling pathway.The study investigated the host immune response to N.caninum infection to provide a new theoretical basis for controlling N.caninum infection.The main contents and results are as follows:1.The role of IFN-?against N.caninum(1)Detection of N.caninum-induced IFN-?expression.In N.caninum-infected mice,mouse peritoneal macrophages,and bovine macrophages,IFN-?expression levels were detected using fluorescence quantitative PCR.The results showed that N.caninum was able to induce IFN-?expression.(2)The role of IFN-?in N.caninum infection.IFN-?^-/-mice,WT mice,mouse peritoneal macrophages,and bovine macrophages were infected with N.caninum,as well as the IFN-?recombinant protein treatment group was set up.The mouse survival rate,weight gain rate,change of charge volume,and the integrity of the parasitophorous vacuole membrane were detected.The results showed that IFN-?deficiency accelerated the death of mice,increased the level of body weight loss,and led to an increase in parasite loads;Adding IFN-?recombinant protein significantly increased the survival rate of mice,alleviated the weight loss,and reduced the number of parasites;IFN-?disrupted the integrity of parasitophorous vacuole membrane and inhibited the proliferation of N.caninum;Parasite number also decreased in bovine macrophages after treating with IFN-?recombinant protein.(3)Screening effector molecules of IFN-?against N.caninum infection.N.caninum-infected mouse peritoneal macrophages were treated with IFN-?recombinant protein,and the high expression of IGRMs and GBPs proteins were screened by fluorescence quantitative PCR;N.caninum-infected IFN-?^-/-and WT mouse peritoneal macrophages were further validated for IRGM3 and GBP9 expression;IRGM3 and GBP9overexpression vectors and si RNA were constructed and transfected into mouse peritoneal macrophages or 293T cells.The results showed that IFN-?could promote IRGM3 and GBP9 expression in N.caninum-infected macrophages,while IFN-?deficiency led to decreased IRGM3 and GBP9 expression;Overexpression of IRGM3and GBP9 led to the disruption of parasitophorous vacuole membrane integrity and significantly inhibited parasite proliferation,while the knockdown of IRGM3 and GBP9 up-regulated parasite numbers.2.N.caninum-induced IFN-?production through the host cell mitochondrial damage-c GAS signaling pathway(1)Detection of N.caninum-induced macrophage mitochondrial damage.Macrophage mitochondrial structure was observed by transmission electron microscopy,mitochondrial membrane potential was detected by flow cytometry,and cytoplasmic mt DNA content was detected by fluorescence quantitative PCR in mouse macrophages infected with N.caninum.The results revealed that the mitochondria in N.caninum-infected macrophages showed obvious wrinkling and mitochondrial cristae were damaged and vacuolated;The mitochondrial membrane potential was significantly reduced and the cytoplasmic mt DNA content increased in N.caninum-infected macrophages.(2)N.caninum activated the c GAS signaling pathway to regulate IFN-?expression.The activation of c GAS-related pathways was analyzed by fluorescence quantitative PCR and Western Blot in mouse peritoneal macrophages infected by N.caninum after treatment with mt DNA removal agents and the si RNA and inhibitor of c GAS.The results showed that N.caninum infection activated c GAS signaling and promoted the phosphorylation of TBK1 and IRF3;The si RNA and inhibitor of c GAS and mt DNA removal agents inhibited N.caninum-induced c GAS signaling,decreased the phosphorylation of TBK1 and IRF3,and inhibited IFN-?expression.(3)Role of c GAS signaling in N.caninum infection.N.caninum-infected mouse macrophages were treated with c GAS si RNA and inhibitors,and the number of parasites was detected by fluorescence quantitative PCR;N.caninum-infected mice were treated with the inhibitor of c GAS,and the survival rate and weight gain rate were detected,as well as parasite loads and the expression of IFN-?was detected by fluorescence quantitative PCR.The results showed that inhibiting c GAS signaling significantly increased parasite numbers in mouse macrophages.And inhibiting c GAS signaling in mice accelerated the death of mice,increased the level of weight loss,led to increased parasite loads,and decreased expression of IFN-?during N.caninum infection.(4)Identifying the role of c GAS in bovine macrophages infected with N.caninum.The expression level of c GAS in N.caninum-infected bovine macrophages was detected by fluorescence quantitative PCR;After adding the inhibitor and agonist of c GAS,fluorescence quantitative PCR was performed to measure the number of parasites and IFN-?expression.The results showed that N.caninum promoted the expression of c GAS in bovine macrophages;Inhibiting c GAS signaling decreased IFN-?expression and increased the number of parasites while adding the c GAS agonist led to increased IFN-?expression as well as decreased parasite numbers.3.The mechanism by which N.caninum-induced host cell mitophagy inhibited IFN-?expression was through regulating the c GAS signaling pathway.(1)The detection of N.caninum-induced host cell mitophagy.Mitochondrial states were observed by transmission electron microscopy,the co-localization of LC3 and mitochondria was detected by immunofluorescence,the mt DNA/n DNA ratios were detected by fluorescence quantitative PCR,and the change of mitochondrial marker proteins was detected by Western Blot in mouse macrophages during N.caninum infection.Mitochondrial marker proteins and mt DNA/n DNA ratios were detected in peritoneal lavage cells and brain tissues from N.caninum-infected mice.The results showed that a double-layer membrane structure appeared around the damaged mitochondria,LC3 co-localized with mitochondria,mt DNA/n DNA ratios were significantly reduced,and the expression of mitochondrial marker proteins(Hsp60 and Tim23)significantly decreased during N.caninum infection.(2)Effect of host mitophagy on N.caninum-induced c GAS signaling pathway and IFN-?expression.The inducer(CCCP)and inhibitor(Mdivi-1)of mitophagy were used to analyze the role of N.caninum-induced mitophagy in the activation of c GAS signaling,the expression of IFN-?,and parasite proliferation.CCCP treatment significantly reduced the expression of IFN-?and led to increased parasite loads,while the opposite results were observed in mouse macrophages after Mdivi-1 treatment.(3)The role of N.caninum-induced host cell mitophagy in mice.After treatment with CCCP and Mdivi-1 in N.caninum-infected mice,the survival rate,body weight change,parasite loads,and IFN-?expression were examined respectively.The results showed that CCCP treatment significantly accelerated the death of mice,reduced body weight,increased parasite loads,and inhibited the expression of IFN-?,while the results of Mdivi-1 treatment were opposite compared to CCCP treatment.(4)Bovine macrophages were used to analyze the effect of mitophagy on N.caninum infection.Mt DNA/n DNA ratios,IFN-?expression,and the number of N.caninum were detected in N.caninum-infected bovine macrophages treated with CCCP and Mdivi-1.The results showed that N.caninum infection decreased the mt DNA/n DNA ratios in bovine macrophages and transmission electron microscopy data showed host mitophagy occurred in bovine macrophages;The expression of IFN-?was significantly decreased and the number of N.caninum was increased in bovine macrophages after CCCP treatment,while the opposite results were obtained in bovine macrophages treated with Mdivi-1.In summary,the study established that N.caninum infection could induce host IFN-?expression and that IFN-?exerted anti-N.caninum effects through regulating the expression of IRGM3 and GBP9,which disrupted the parasitophorous vacuole membrane of N.caninum.Meanwhile,N.caninum caused mitochondrial damage in host cells,and mt DNA was released into the cytoplasm to activate the c GAS-STING-TBK1-IRF3 signaling pathway to promote the expression of IFN-?.Besides,N.caninum induced mitophagy of host cells,which inhibited the c GAS-STING-TBK1-IRF3 signaling pathway to reduce IFN-?expression.The above studies clarified the role of IFN-?on N.caninum infection and revealed that the mechanism of IFN-?expression was through mitochondrial damage,and the immune evasion mechanism of N.caninum was through inducing host mitophagy to inhibit IFN-?expression.And the study provided new targets for preventing and controlling N.caninum infection through IFN-?.