Study On The Mechanism Of Echinococcus Infection Regulating Immune Thrombocytopenia (ITP) Through Polarized Macrophages

Objective:To investigate the immunomodulatory effect of macrophages and related cytokines on immune thrombocytopenia(ITP)in the early stage of Echinococcus granulosus infection.Methods:(1)45 female BALB/c mice were randomly divided into5groups with 9 mice in each group.(2)three groups of mice infected with Echinococcus granulosus were randomly divided into one low-dose group and two high-dose groups for4 weeks.(3)three groups of ITP mice were established by intraperitoneal injection of anti-CD41 platelet antibody(simple ITP group,low dose Echinococcus granulosus infection/ITP group,high dose Echinococcus granulosus infection/ITP group).The peripheral blood,peritoneal lavage fluid,liver and spleen tissue and serum samples of mice were collected.Detection of platelet trend in each group by automatic five-classification hematology analyzer;Observation of liver histopathology in mice infected with Echinococcus granulosus by HE staining;Flow cytometry was used to detect F4/80~+CD11c~+M1 and F4/80~+CD206~+M2 macrophages and lymphocytes in spleen and peripheral blood of mice;The expression and localization of i NOS in liver tissue were detected by immunohistochemistry,and the expression level of i NOS in mouse liver and peritoneal macrophage protein was detected by Western blotting;the levels of IL-1βand IL-6 in serum were detected by ELISA,and the expression levels of i NOS,IL6 and TNF-αin mouse liver were detected by real-time fluorescence quantitative RT-PCR.Results:(1)compared with the control group,the platelet in the injection group showed a downward trend,and the ITP model was established successfully.(2)compared with the control group and the simple ITP group,HE staining showed that the liver of the mice infected with Echinococcus granulosus showed obvious parasitic foci and obvious infiltration of inflammatory cells around the infected foci.Flow cytometry results showed that the proportion of F4/80~+CD11c~+M1 macrophages in spleen and peripheral blood of mice in low-dose and high-dose ITP model group was higher than that in control group and ITP group,while the proportion of F4/80~+CD206~+M2 macrophages was lower than that in control group and ITP group.The difference was statistically significant(P<0.05).The percentages of B,T,CD4~+T,CD8~+T lymphocytes were higher than those of control group and ITP group(4)Immunohistochemical results showed that the expression of i NOS around liver lesions in low-dose and high-dose ITP model group was significantly higher than that in control group and simple ITP group.(5)Western blotting results showed that the expression of i NOS protein in peritoneal and hepatic macrophages of ITP model mice secondary to Echinococcus granulosus infection in low and high dose groups was higher than that in ITP group and control group.(6)RT-PCR results showed that the expression levels of i NOS,IL-6 and TNF-αm RNA in liver of ITP model group secondary to Echinococcus granulosus infection at low and high doses were significantly increased.(7)ELISA results showed that the expression levels of IL-6 and IL-1βin serum of ITP mice secondary to Echinococcus granulosus infection in low and high dose groups were significantly higher than those in ITP group and control group.Conclusion:Echinococcus granulosus infection has immunomodulatory effect on immune thrombocytopenia(ITP)through M1 macrophages and their cytokines,lymphocytes and their subsets.