The Influence Of H. Pylori On Tim-3 In Lymphocytes And Gastric Muconsa Of Mice And Th Immune Response And The Correlation Between Them

Background/ObjectiveThe host immune response plays an important role in pathopoiesis of Helicobacter pylori (H. pylori) infection. To clarify the mechanisms of immune pathogenesis of H. pylori infection is great significance for prevention and treatment of H. pylori infection and correlation diseases.Th immune response plays an important role in H. pylori infection and immune protection, nevertheless,the mechanisms of the effect of H. pylori on Th immune response is still not understood. Recently identified T-cell immunoglobulin and mucin-domain-containing molecule (Tim) gene family encodes type I membrane glycoproteins expressed on T cells that are involved in the differentiation of CD4+ T cells, and the regulation of Thl and Th2 cell mediated immunity. Tim-3 is an important member of Tim family, is expressed on terminally differentiated Thl cells, regulates function of different subsets of CD4+ T cell, and influences the function of regulatory T cells (Treg) and TLR signaling pathway.Until now, the role of Tim-3 in pathogenesis of H. pylori infection remains unclear.In the present study, we focused on the expression of Tim-3 in H. pylori infection, as well as its relations with Th1 immune response in vivo and in vitro. To discuss the mechanism of the influence of H. pylori infection on Th immune response, thus provide theoretical and experimental evidences for the prevention and treatment of H.pylori infection and correlation diseases through regulation of Tim-3.Methods:(1) Coculture of H.pylori with splenic lymphocytes of mice:The ratio of SS1 H. pylori bacteria and Lymphocytes isolated from spleen of BALB/c mice respectively is 0:1CFU/(control group),100:1CFU/(A group),400:1 CFU/(B group),700:1 CFU/(C grooup), and cells were collected at 12h.(2) H. pylori infection in mice:Female BALB/c mice (6-8-week-old) were infected with alive H. pylori SS1 (1×109CFU/ml,0.5ml/mice) at two day intervals for five times. Twelve weeks after the last inoculation, the mice were all sacrificed and gastric mucosa were collected.Another group of mice without H. pylori was the control group.(3) Assessment:①H. pylori colonization in gastric mucosa were determined by improved Giemsa staining and H. pylori culture;②The levels of cytokines IL-2, IL-12, IFN-y, IL-4 and IL-10 in gastric mucosa and cultural supernatant were determined by sandwich ELISA;③The expression of Tim-3, MyD88 and Foxp3 protein in gastric mucosa and lymphocytes of mice was determined by Western blot.Results:(1) The influence of H. pylori on Tim-3 in lymphocytes of mice and Th immune response.①H. pylori could up-regulate the expression of Tim-3 and MyD88 protein in lymphocytes of mice in vitro at 12h significantly (P<0.005). The expression of Tim-3 protein in each experimental group was significantly higher than that in control group (P<0.005); The expression of MyD88 protein in experimental groups (B, C) was significantly higher than that in control group (P<0.05); The expression of MyD88 protein in experimental group (A)was no significantly different with control group (P>0.05). The expression of Foxp3 protein in all experimental groups was no significantly different with control group (P>0.05).②In the level of IL-4, IL-10, IL-12 and IFN-y in cultural supernatant, there was significant difference among all groups. The level of IL-4 and IL-10 in cultural supernatant in all experimental groups was significantly lower than that in control group, the more the number of alive H. pylori, the lower the level of IL-4 and IL-10.(P<0.001,0.005,0.05); The level of IL-12 and IFN-y in cultural supernatant in all experimental groups was significantly higer than that in control group(P<0.005, 0.05), and that in experimental groups (B, C) was significantly higer than that in control group (P<0.001); The level of IL-12 in cultural supernatant in all experimental groups was no significantly different with control group (P>0.05).(2) The influence of H. pylori on Tim-3 in H. pylori infection in mice and Th immune response:①The expression of Tim-3, MyD88 and Foxp3 protein in gastric mucosa of H. pylori infection group was significantly higher than that of normal control group (P<0.01,P<0.05).②The level of IL-4, IL-10, IL-12 and IFN-γin gastric mucosa was significantly different between normal control and H. pylori infection group (P<0.01,0.05), the level of IL-2 and IFN-γof Thl cytokine was significantly higher in H. pylori infection group than that in normal control(P<0.005,0.05), the level of IL-4 and IL-10 of Th2 cytokine was significantly lower in H. pylori infection group than that in normal control(P<0.001,0.05).③In H. pylori infection group, the expression of Tim-3 protein in gastric mucosa was significantly positively correlated with the level of IFN-γand IL-12 of Thl cytokine in gastric mucosa (Rs=0.902,0.933, P<0.005), and was significantly negatively correlated with the level of IL-4 and IL-10 of Th2 cytokine in gastric mucosa (Rs=-0.922,-0.954, P<0.005), whereas in gastric mucosa among control group, the expression of Tim-3 protein had no significant correlation with the level of IL-4, IL-10, IFN-γand IL-12(Rs=-0.727,-0.531,0.746,0.630, P>0.05).④In H. pylori infection group, the expression of Tim-3 protein in gastric mucosa was significantly positively correlated with that of MyD88 protein in gastric mucosa (Rs=0.905, P<0.05), and had no significant correlation with that of Foxp3 protein (Rs=0.723, P>0.05); There was no significant correlation among the expression of Tim-3 protein, MyD88 protein and Foxp3 protein in control group(Rs=0.819 and 0.508, P>0.05).Conclusion:1. H. pylori could up-regulate the expression of Tim-3 in vitro, suggesting that H. pylori could inhibit the differentiation of T lymphocytes towards Th2 cells, promote the Thl cell differentiation,thus induce Thl-biased immune response.2. The expression of Tim-3 is positively correlated with the level of Thl cytokine in gastric mucosa of H. pylori infection group, and that is nagetively correlated with the level of Th2 cytokine, which suggests that the expression of Tim-3 could reflect Thl immune response in H. pylori infection and is positively correlated with MyD88 protein, which is the important molecule of Toll like receptor (TLR) signaling pathway,in gastric mucosa, which indicates that there is interaction in Tim-3 and TLR signaling pathway.