The Role Of Tims And Tim-3-Galectin-9 Pathway In Regulation Of Th1/Th2 Immune Polarization In Schistosoma Japonicum Infection Of Mice

ObjectiveSchistosomiasis is one of the most prevalent and severe parasite diseases, particularly in developing countries and impoverished regions. The most serious outcome of schistosomiasis is the immune responses to schistosome eggs, resulting in hepatic granuloma and consequential hepatic fibrosis. The mechanism of host Th2-biased immune response to schistosome infection remains unclear. In this paper, the expressions of Tim molecules and the role of Tim-3-Galectin-9 pathway in Th1/Th2 immune polarization during S. japonicum infection in mice are investigated.MethodsEach mouse of experimental groups was percutaneously infected with 35 cercariae to build models of schistosomiasis. Liver sections were cut from the formalin-fixed paraffin-embedded block in each case and stained with HE/antibodies prior to histopathology examination and immunohistochemical analysis. After infection, the single cell suspension of splenocytes was prepared by grinding the spleen within Hanks' solutions and lymphocytes were separated by density gradient centrifugation.To detect the expressions of Tims and cytokines at different stages of S. japonicum infection, 1×10⁷ lymphocytes from each mouse were incubated in 1 ml complete RPMI 1640 medium that contains 10% fetal calf serum. After 24 hours, lymphocytes were collected by centrifugation and lysed by TRIZOL reagent for cellular RNA abstraction. In addition, lymphocyte supernatant were collected by centrifugation and conserved in -80?C for ELISA.To detect the role of Tim-3-Galectin-9 pathway in Th1/Th2 polarization of infected mice, 1×10⁷ lymphocytes from each mouse were seeded in 2 ml nutrient solution. The lymphocytes were incubated in the presence of Tim-3-Fc fusion protein or PBS for 48 hours. Subsequently, these lymphocytes were prepared for FACS analysis.To induce in vitro Th1 cells apoptosis from normal mice, 3×10⁷ lymphocytes from each mice were cultured in nutrient solution. The lymphocytes were pre-stimulated overnight at 37℃in 5% CO₂ in the presence of anti-mouse CD3 and anti-mouse CD28 and subsequently incubated within SWA/SEA/PBS respectively. After 36 hours of incubation, lymphocytes incubated with Galectin-9 or PBS, preparing for further stages of FACS analysis and fluorescence microscope observation.ResultsTim-2 and IL-4 expressions in lymphocytes of mice at different infection stages were observed, in which a negative correlation between Tim-2 and IL-4 expressions was found, whereas there was no strong correlation between the expressions of Tim-3 and IFN-γ. In addition, the expressions of Tim molecules increased during the appearance of hepatic fibrosis after week 9, accompanying an increasing severity of tissue damage. At last, immunohistochemistry staining showed the expressions of Tims around the egg nodes from infected mice.Our observation showed that the IFN-γexpression in lymphocytes stimulated by Tim-3-Fc was dramatically higher than PBS controls. An obvious increase of IFN-γexpression was found as the concentration of Tim-3-Fc was enhanced, while the similar but mild trend can be observed for the IL-4 expression. In addition, there were more increase in Th1 than Th2 cells when the co-incubated substrate was Tim-3-Fc from the statistical analysis of FACS.Our data from FACS indicated that the percentage of early apoptosis incubated with Galectin-9 was higher than the one in PBS control group. This variation tendency of apoptosis rate was same in different pre-stimulated antigen substrates. Besides, the pictures from fluorescence microscope also illustrated that more early apoptotic cells were found in Galectin-9 group and some late apoptotic cells showed in terms of co-incubation time. In addition, to figure out the Th type response that was inhibited by Tim-3-Galectin-9, the expressions of IFN-γand IL-4 in lymphocytes stimulated by PBS/Galectin-9 were measured using ELISA. It can be found that the reduction of IFN-γexpression between PBS column and Galectin-9 column was more prevailing, no matter what the pre-stimulated substrate was, i.e. PBS or SWA or SEA.ConclusionsThe Th2 immune polarization is a predominant phenomenon of down-regulated immune response of host to the later stage of S. japonicum infection. Tim-3-Galectin-9 pathway involves in Th1 cells apoptosis and, as a consequence, constitute a major reason for Th2-biased immune response. This finding provides a new contribution to the mechanism of down-regulation of immune response in schistosome infection and a potential significance of new therapeutic target to schistosomiasis and its related immunopathy.