The Study Of Correlation Between Macrophage Polarization And Toxoplasma Gondii Infection

Toxoplasma gondii is an intracellular parasite that almost able to infect all warm-blooded animals. The toxoplasmosis is one of the most popular zoonotic parasitic diseases worldwidely. The complex life cycle of T.gondii with widespread hosts leads to difficultly diagnosis and do much threat to the development of human and animal husbandry. As the first barrier against pathogens, macrophages are one of the most vulnerable to be infected in the process of defense. At the same time, macrophages are the main carrier of pathogen to spread disease in acute infection, which also provide a favorable place for T.gondii proliferation. The study concerning the interactions between macrophages and T.gondii maybe helpful to clarify the mechanism about infection, dissemination and pathogenesis, and can also be helpful for disease prevention, control and treatment. Isolated mouse bone marrow cells were differentiated into macrophages by M-CSF. The purity of macrophages identified by flow cytometry was 90 % or more. The resting state macrophages stimulated by LPS/IFN-γ or IL-4 respectively. The expression level of polarization-related markers were detected by semi-quantitative PCR, western blot and flow cytometry. The activity of iNOS and Arginase-1 was also detected by Griess assay and arginase activity assay. The results showed that the bone marrow-derived macrophages were successfully induced into two polaried subtypes, M1 and M2. Based on the above results, we also wonder whether the infection with T.gondii induced bone marrow-derived macrophages into M1 or M2 polarization. Bone marrow-derived macrophages were infected with T.gondii, and cells and supernatant were collected at 6, 12, 24, 36 and 48 hours after inoculation. The activity of iNOS and Arginase-1 was detected by Griess assay and arginase activity assay. The expression level of M1 and M2 polarizationrelated markers related polarization were also detected by real-time PCR, western blot and flow cytometry at different time points post T.gondii infection. The results showed that arginase activity of macrophages post T.gondii infection was up-regulated. However, the activity of iNOS was not outstanding changed. Besides, in concert with activity assay, the expression of markers related M2 macrophage were remarked elevated. Althought there were increases in mRNA level of some genes related M1 macrophages post infection, the expression of protein levels were not significantly different with control at all time points. Furthermore, the activated state of JAK-STAT and Akt-mTOR signaling pathway involved in polarization was investigated and demonstrated by western blot analysis. The results showed that JAK-STAT and Akt-mTOR signaling pathway were activated by T.gondii. On the other hand, to evaluate the effects of macrophage polarization on the infection and proliferation of T.gondii, we performed western blot analysis and fluorescence assay. The results indicated that different subtype macrophages had no effect on the infection of T.gondii. But proliferation of T.gondii in M1 macrophages was significantly attenuated when compared with in M0 and M2 macrophages.