The Roles Of GBP2 And GBP5 In Resistance To Neospora Caninum Infection In Mouse Macrophages

Neospora caninum is an intracellular protozoan found in recent years,which can infect a variety of animals and cause reproductive disorders in intermediate hosts,such as cattle and sheep,and neurological disorders in terminal hosts such as canines,leading to dyskinesias,meningitis and other diseases in young animals.Neosporosis is distributed worldwide,causing serious economic losses to the world’s breeding industry.In recent years,there are a lot of researches on neosporosis,but specific drugs and vaccines for the prevention and treatment of neosporosis are still limited.Further study of host immunity against N.caninum will provide an important basis for the prevention and treatment for the disease.Guanylate binding proteins（GBPs）are downstream stimulating factors of interferons.As an important part of cell innate immunities,they could target intracellular bacteria,viruses and parasites,or release killer components to combat various pathogen infections.In addition,it was reported that GBPs participate in the pathway of NLRP3 inflammasome activation to resist pathogenic infections.As a key regulator of innate immunity,GBPs played an important role in host defense against pathogen invasion and maintaining body homeostasis,however,the roles and mechanisms of GBPs in N.caninum infection have not been reported yet.This study aimed at exploring the relationship between N.caninum,GBPs and NLRP3inflammasome,and elucidating the role of GBPs in N.caninum infection,this could provide new ideas for the prevention and treatment of N.caninum infection.The main research contents were as follows:1.N.caninum infection promoted the expression of GBP1-GBP11 in peritoneal macrophages.PMs were isolated and N.caninum was purified to establish the N.caninum infection model,the expressions of GBP1-GBP11 m RNA were detected by RT-PCR,and the protein expressions of obvious changed genes were detected by Western blot.The results showed that the expression levels of GBP1-GBP11 m RNA in peritoneal macrophages were increased after N.caninum infection,among which the expression levels of GBP2 and GBP5 protein were increased significantly.2.The functions of GBP2 and GBP5 in regulating the proliferation of N.caninum.The pcDNA3.1 vector,pcDNA3.1-GBP2,pcDNA3.1-GBP5 eukaryotic expression vector,sicontrol,si GBP2 and si GBP5 were transfected into 293T cells and mouse peritoneal macrophages,respectively.Cell viability was detected by CCK8assay,and the protein expression of GBP2 and GBP5 were detected by Western blot.Then these cells were infected with N.caninum,the number of N.caninum was detected by RT-PCR and Giemsa staining counting method,and the localization of GBP2 and GBP5 protein was observed by indirect immunofluorescence.The results showed that GBP2 and GBP5 proteins were successfully overexpressed or interfered in 293T cells and mouse peritoneal macrophages,and the cell viabilities were not affected by these treatments.Compared with the empty vector group,GBP2 or GBP5overexpressions could significantly reduce the number of intracellular N.caninum;on the contrary,the gene interferences of GBP2 or GBP5 could increase the number of N.caninum.In the control group GBP2 or GBP5 proteins were localized in the cytoplasm when observed by confocol assay,while GBP2 or GBP5 were recruited to N.caninum.3.GBP2 and GBP5 mediated the activation of NLRP3 inflammasome to regulate the proliferation of N.caninum.The pcDNA3.1 vector,pcDNA3.1-GBP2 eukaryotic expression vector,pcDNA3.1-GBP5 eukaryotic expression vector,sicontrol,si GBP2 and si GBP5 were transfected into WT type and Nlrp3^-/-type mouse peritoneal macrophages for 24 h,respectively,then the cells in these groups were infected with N.caninum.The maturation of IL-1βand expression of NLRP3 protein were detected by Western blot,the secretion of IL-1βwas detected by ELISA,the release of LDH was used to evaluate cell death and co-localization of GBP2,GBP5 and NLRP3 proteins were observed by immunofluorescence,the number of N.caninum was detected by RT-PCR or immunofluorescence,respectively.Compared with the empty vector group,overexpressions of GBP2 or GBP5significantly increased the expression of NLRP3 and IL-1β,the secretion of IL-1βand the release of LDH in N.caninum-infected WT mouse peritoneal macrophages,but greatly reduced the number of N.caninum.In Nlrp3^-/-mouse peritoneal macrophages GBP2 overexpression were slightly increased the secretion of IL-1βand the release of LDH,while the number of N.caninum and the infection rate were slightly decreased.GBP5 overexpression failed to alter the secretion of IL-1β,the release of LDH,the number of N.caninum and the infection rate in N.caninum-infected Nlrp3^-/-mouse peritoneal macrophages.Compared with the sicontrol group,interference of GBP2 or GBP5 could decrease the expression of NLRP3 and IL-1β,the secretion of IL-1βand the release of LDH in WT mouse peritoneal macrophages,but increase the number of N.caninum and infection rate.The secretion of IL-1βand the release of LDH were slightly decreased in Nlrp3^-/-mouse peritoneal macrophages which were interfered with the expression of GBP2,while the number of N.caninum and infection rate were slightly increased.The secretion of IL-1β,the release of LDH,the number of N.caninum and the infection rate of Nlrp3^-/-mouse peritoneal macrophages interfered with GBP5 had no significant changes.In conclusion,N.caninum could increase the m RNA levels of GBP1-GBP11 in mouse peritoneal macrophages,among which the m RNA and protein expressions of GBP2 and GBP5 were significantly increased.GBP2 and GBP5 could inhibit the proliferation of N.caninum by mediating the NLRP3 inflammasome activation pathway.GBP2-or GBP5-mediated innate immune pathways would be potential targets for the development of drugs against N.caninum.