The Preliminary Study On The Polarization Of Peritoneal Macrophages Of BALB/c Mice Infected With Neospora Caninum

Neospora caninum(N.caninum)is an obligate intracellular Apicomplexa parasite.It has been reported that domestic animals such as cattle,sheep,horses,pigs,dogs,cats,etc.;wild animals such as deer,foxes,bears,primates,etc.;some marine animals such as walruses,sea lions and seals can be naturally infected with N.caninum.Among them,the cattle and sheep were the most common intermediate host,and caused miscarriage or stillbirth pregnant female after infection,and even lead to fetal nervous system diseases,which has brought huge economic losses to the aquaculture industry.The innate immune system is the first line of defense and antagonism during the invasion of the sheep and cattle organism by N.caninum.The macrophage is one of the main immune cells in the innate immune system,and differentiate into the classical activated macrophages(M1)and the alternative activated macrophages(M2)according to the different ways of activation.The M1 macrophages are characterized by expression of high levels of pro-inflammatory cytokines,such as IL-12?IL-17?IL-23?TNF-? and i NOS,which enhance inflammatory response and the ability of antigen presentation,and kill parasites.In contrast,M2 macrophages are thought to promote the survival of parasites by producing IL-10?Arg-1?CD206?Ym1 and Fizz1.In the process of pathogen infection,macrophages can be polarized into M1 or M2 depending on the pathogen.This polarization process is regulated by a variety of signaling molecules and their pathways.Among them,PPAR? is a ubiquitous nuclear transcription factor in the monocyte macrophage system and has antagonistic effects with NF-?B,which plays a key role in regulating the polarization of macrophages in M2.It has been reported that Leishmania and Trypanosoma could induced the polarization of macrophages in M2 through activating PPAR?.However,the effect of N.caninum on macrophage polarization has not been reported.Therefore,this study analyzed the changes of peritoneal macrophage subtypes(M1/M2)by establishing a mouse model of N.caninum infection.To verify the changes of macrophage subsets,we construct N.caninum infection model in vitro by J774 A.1 cells.And to further explore the mechanism of PPAR?/NF-?B signaling pathway in the regulation of macrophage polarization in the process,in order to find the key nodes macrophage polarization,for further research on how to improve the host to provide ideas and experimental according to the new resistance of N.caninum infection.Firstly,we investigated the dynamics of macrophage polarization in mice during different stages of N.caninum infection.BALB/c mice were randomly divided into four groups as follows: a blank control group,two weeks of N.caninum infection group,four weeks of N.caninum infection group and eight weeks of N.caninum infection group.The N.caninum infection groups were administered with 1�107 tachyzoites.The blank control group was treated with an equal volume of PBS.The pathological tissue sections were used to determine the different periods of the mice infected with the new spore;Flow cytometry was used to identify the subtypes of mouse peritoneal macrophages.The results showed that the pathological changes were gradually increased in brain tissue with the passage of time.And the peritoneal macrophages were polarized to M1 type in the early stage of N.caninum infection in mice,and then the peritoneal macrophages were gradually changed from M1 to M2 type.Secondly,we investigated the role of PPAR?/NF-?B signaling pathway in the regulation of macrophage polarization in J774 A.1 cells.The experiment was divided into 4 groups as follows: a blank control group,GW9662+ N.caninum group,RGZ+ N.caninum group and N.caninum group.The concentrations of TNF-?,IL-12,IL-10,i NOS and Arg-1 were measured by ELISA,and the subtypes of J774 A.1 cells were detected by FCM,and PPAR?/NF-?B signaling pathway were analyzed by Western blot.The results showed that the expressions of CD206,IL-10 and Arg-1(P < 0.01)of the M2 marks were increased by activating PPAR-? in RGZ and N.caninum groups.Meanwhile,the activity of NF-?B was inhibited in RGZ and N.caninum groups.In conlusion,we determined that macrophages differentiated into M2 at the late stage of N.caninum infection through mediating PPAR?/NF-?B signaling pathway,or further research on how to improve the host to provide ideas and experimental according to the new resistance of N.caninum infection.