The Immune Regulation Of Tim-3/Galectin-9 Signaling Pathway In Echinococcus Multilocularis Infection

Objective:To study the variation characteristic of Tim-3/Galectin-9 signal pathway and related molecules in persistent infection by Echinococcus multilocularis,and to investigate their immune regulation.Methods:6-8 week old BALB/c mice were randomly divided into infection group and control group.The infection group was injected protoscoles of Echinococcus multilocularis by intraperitoneally.The control group was received corresponding volume of physiology saline.And then mice were sacrificed at 2days,8 days,30 days,90 days,180 days and 270 days post infection.1)Using HE staining method to observe the pathological changes of liver tissue on each stage.2)ICH was used to observe the expressions of Tim-3 and Galectin-9 in livers of mice.3)mRNA was extracted from lymphocytes of mice by grinding method,and qRT-PCR was used to detect the expressions of Tim-3 and Galectin-9 in lymphocytes of mice,including the expressions of associated IL-12,IFN-? and IL-4.4)FCM was used to detect the expressions of Tim-3 in spleens of mice.CBA was used to detect the serum levels of IL-12,IFN-?and IL-4 in mice on each stage of inflection.5)In vitro activation was used to detect the apoptosis of T cells in spleen of mice,including the changes of the serum levels of IL-12,IFN-?and IL-4.6)ELISA was used to detect the serum levels of sTim-3and Galectin-9 in patients with Alveolar echinococcosis(AE patients)and the healthy control group,meanwhile,CBA was used to detect the serum levels of IL-12,IFN-?and IL-4 of AE patients.Results:1)There were no obvious pathologic changes being seen in hepatic tissue at 2 days and 8 days after mice being infected by Echinococcus multilocularis;the inflammatory cell infiltration inside liver was visible 30 days after infection,and there was enlargement of liver cell;the necrosis of hepatic tissue was visible from 60 days to 90 days after infection,and Echinococcus multilocularis cyst walls were formed;the atrophy and necrosis of liver cell was observed from 180 days to 270 days after infection,and there were Echinococcus.2)ICH results showed that:no difference was found in expressions of Tim-3 and Galectin-9 between the infection group and the control group at 2 days and 8 days after infection;the positive expressions of Tim-3 and Galectin-9 were observed in hepatic sinusoid at 30 days after infection;the increased expressions of Tim-3 and Galectin-9 were observed in the hepatocyte intracytoplasmic,the hepatic portal areas and the areas of cell infiltration from 60 days to 90 days after infection.3)qRT-PCR results showed that:no difference was found in expressions of Tim-3 mRNA and Galectin-9 mRNA between the infection group and the control group at2 days and 8 days after infection;the expression levels of Tim-3 mRNA and Galectin-9mRNA increased gradually with the passage of time from 30 days after infection;the expression levels of Tim-3 and Galectin-9 decreased somewhat from 180 days to 270 days after infection;the expression tendency of IFN-?mRNA and IL-12 mRNA was noticeable rised at 2 days and 8 days after infection;decreased slightly at 30 days to 60 days;significantly decreased at 180 days to 270 days,but still higher than that in control group.The expression of IL-4 mRNA increased slowly with the passage of time after infection.4)The level of CD4~+Tim3~+T/CD4~+T cell in spleen of mice inflected by Echinococcus multilocularis had no significant change at 2 days and 8 days after infection;the level of CD4~+Tim3~+T/CD4~+T cell in spleen of the infection group began to increase from 30 days after infection;the level of CD4~+Tim3~+T/CD4~+T cell in spleen of mice got to the maximum at 90 days after infection;the level of CD4~+Tim3~+T/CD4~+T cell decreased slightly but were still significantly higher than that of control group from 180 days to 270days after infection.The concentrations of IFN-?and IL-12 in serum of mice inflected by Echinococcus multilocularis got to the maximum at 2 days and 8 days after infection,and decreased gradually from 30 days after infection;the concentration of IL-4 increased gradually and slowly from 2 days and 8 days after infection.5)Results for the in vitro experiment showed that add Galectin-9 will increase T cell apoptosis,at the same time IL-12 and IFN-?levels decreased,while the level of IL-4 elevated 6)ELISA results showed that the serum levels of sTim-3 and Galectin-9 of AE patients were significantly higher than those of control group,there was significant statistical difference between the two groups.The serum cytokine of these patients was measured by CBA,and the results showed the levels of IL-12 and IFN-?decreased and the level of IL-4 increased.Conclusions:With prolongated infection time of Echinococcus multilocularis,the expressions of Tim-3,Galectin-9 and CD4~+Tim3~+T cell increased,while the expressions of IL-12 and IFN-?decreased,and the expression of IL-4 increased.The Tim-3/Galectin-9signal pathway may induce Th1/Th2 cell imbalance by regulating secretion of Th1/Th2cytokine.Results for the in vitro experiment showed that increase the ligang Galectin-9will induce apoptosis in T cell and reduce Th1 cell factor,and thus induces immunologic escape of Echinococcus multilocularis infection.