The Study Of Autophagy Induced By Neospora Caninum In Macrophage And Its Effect On Neospora Caninum Invasion And Proliferation

Neospora cannium is an obligate intracellular apicomplexan protozoan parasite,which leads to abortions in pregnancy and impair motor nervous system in newborn animals,is responsible for significant financial losses to the dairy industry,particularly in the beef industry.To date,there is no available effective commercial vaccine for control.The etiology and pathogenesis of neosporosis are complicated,especially to avoid immune recognition and escape from the strong immune response,tachyzoites can convert to bradyzoites and form cyst.The relationship between the N.caninum and host immune system has not been clearly elucidated.Autophagy is a highly conserved mechanism that is responsible for maintaining the stability of the intracellular environment,and plays an important role in a variety of physiological processes in living organism.However,the interaction between N.caninum and autophagy remains unclear.In this study,it is mainly studied in the following aspects:1.Detection of autophagy induced by N.caninum in mouse macrophages.To investigate whether N.caninum infection modifies the autophagy of mouse macrophages,several assays were used.The formation of autophagic vesicles was mainly detected by transmission electron microscopy,the aggregation of GFP-LC3fluorescent spots was observed by laser confocal microscopy,the expression of autophagy-related protein genes was detected by fluorescence quantitative PCR,and the conversion rate of LC3-II and the expression of autophagy-related proteins were analyzed by Western blot.It was found that N.caninum infection activated the initial phase of autophagy,as a large number of autophagic vesicles were detected in macrophages during the early stages of infection.Then,the autophagic flux was further examined,the degradation of p62/SQSTM l was analyzed by Western blot,and the formation of autolysosome was observed by laser confocal microscopy in combination with m Cherry-GFP-LC3 and lysosomal marker protein LAMP1.The results indicated that fusion of autophagosome and lysosome was blocked in mouse macrophages during N.caninum infection,indicated that autophagic flux was impaired.In addition,the AKT/m TOR signaling pathway,a negative regulatory pathway of cellular autophagy was detected.The results indicated that AKT/m TOR,an autophagy-negative regulatory pathway,was activated during infection,suggesting that N.caninum activated autophagy through an m TOR-independent pathway.2.The role of TLR2 in N.caninum-induced autophagy in mouse macrophagesTo further understand the mechanism of N.caninum-induced host cell autophagy,in this section we further explored the host TLRs and the function of autophagy in infection.Fluorescence quantitative PCR and PCR arrays were performed to explore the activation of TLRs and related molecules in macrophages infected by N.caninum.Results showed that several TLRs were upregulated in N.caninum-infected macrophages,among which TLR2 was the most significantly upregulated and was identified as a candidate for our further study.To verify the activation of TLR2/NF-κB signaling pathway,the nuclear translocation of NF-κB p65 protein was detected by laser confocal microscopy,the expression of TLR2 and NF-κB signaling pathway-related proteins was detected by Western blot.Results showed that during N.caninum infection phosphorylation of NF-κB p65 was significantly increased,nuclear translocation of NF-κB p65 was observed,production levels of IFN-γ,TNF-α,IL-12and IL-6 were greatly elevated,when compared to that in N.caninum-infected TLR2^-/-macrophages,indicating that the TLR2/NF-κB signaling pathway was activated and played a central role in the regulating inflammatory cytokines in N.caninum-infected macrophages.To determine whether TLR2 was participated in manipulating N.caninum-induced incomplete autophagy,the expression and accumulation of LC3 and p62 were analyzed by Western blot and immunofluorescence.The results showed that TLR2 deletion did not disrupt autophagosome formation,but p62 accumulation in the cytoplasm was rescued,suggesting that TLR2 may be involved as an upstream signal in the N.caninum-triggered inhibition of autophagosome binding to lysosomes.In addition,the phosphorylation of AKT/m TOR was reduced in TLR2-deficient macrophages infected by N.caninum,suggesting that TLR2 participated in regulating the activation of AKT/m TOR.Survival assays and pathological analysis were performed to further analyze the role of TLR2 during the infection.The results showed that TLR2-deficient mice suffered more serious damage in the major organs and had a reduced survival rate,indicating that TLR2 was involved in the host defense against N.caninum infection.3.The effect of N.caninum on autophagy and TLR2 expression in bovine macrophagesBovine is the natural host of N.caninum.In order to determine the changes and roles of host autophagy and TLR2 in N.caninum-infected bovine macrophages,bovine peripheral blood mononuclear cells（PBMCs）and a bovine peritoneal macrophage cell line were employed as N.caninum infection models.TLR2/NF-κB signaling pathway was found to be activated by N.caninum infection via fluorescent quantitative PCR,Western blot,and immunofluorescence.The increased secretions of inflammatory cytokines were detected in N.caninum-infected bovine macrophages with ELISA.Notably,the activation of autophagy was delayed by N.caninum infection in bovine macrophages compared to that in mouse macrophages,but similar with the results in mouse macrophages that incomplete autophagy with an impaired autophagic flux was also found in N.caninum-infected bovine macrophages.Moreover,the activation of autophagy was found to be independent of the parasite viability.4.The role of autophagy in the N.caninum infectionTo assess the role of autophagy in N.caninum infection,small interfering RNAs and compounds which inhibit or induce autophagy were employed.It was shown that N.caninum invasion and proliferation were promoted after the treatment of autophagy inducer rapamycin,whereas decreased by the autophagy inhibitor 3-MA when the formation of autophagosome was reduced.In addition,impaired autophagosome formation in macrophages transfected with si ATG5 resulted in a significant reduction in both N.caninum invasion and proliferation.Secretions of cytokines induced by N.caninum infection were significantly reduced after host autophagy was inhibited.Further in vivo experiments,in which mice were injected with autophagy regulators,were carried out to explain the contribution of cellular autophagy in N.caninum infection.According to the results,at the initial location of infection,the infection rate and the proliferation of N.caninum were significantly higher in the Rapa-infected group compared with the normal-infected group,and the survival rate of mice was significantly lower.Interestingly,the reduced infection rate and N.caninum proliferation were found in the 3-MA infected group,although the survival rate was similar to that of the normal-infected group.In summary,this study revealed that N.caninum can induce the formation of autophagosome in host cells and lead to incomplete autophagy via a m TOR-independent pathway.TLR2 signaling pathway was involved in the inflammatory response and regulating the autophagic flux inhibition triggered by N.caninum.In addition,activation of classical autophagy was favorable for N.caninum infection,whereas inhibition of autophagosome formation was benefit to control N.caninum infection.The present study provided further insight into the mechanisms of host-N.caninum interaction and suggested that modulating the host autophagy could be a new strategy to control N.caninum infection.