| Background/Objective:Observations of H. pylori infection and immune protection suggest that there is a distinct interaction between Helicobacter pylori (H. pylori, Hp) and the host immune system. Th immune response play 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. The 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 Th1 and Th2 cell mediated immunity. Tim-1 and Tim-3, both identified in mice and humans, is expressed on terminally differentiated Th2 and Th1 cells, respectively, and participate in the regulation of Th immune response. Until now, the role of Tim in H. pylori infection and immune protection remain unclear. In the present study, we focused on the expression of Tim-1 and Tim-3 in H. pylori infection and immune protection, as well as its relations with Th1 and Th2 immune response in vivo and in vitro. To discuss the mechanism of the effect of H. pylori infection and immune protection 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-1 and Tim-3.Methods:(1) Coculture of H. pylori with splenic lymphocytes of mice: Lymphocytes isolated from spleen of BALB/c mice were cocultured with different concentrations of alive H. pylori (0 CFU/ml, 0.50×10~7CFU/ml, 1.00×10~7CFU/ml, 2.00×10~7CFU/ml and 4.00×10~7CFU/ml), and cells were collected at 3h, 6h, 12h and 24h.(2) H. pylori infection and eradication 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. After identification of H. pylori infection, mice were randomly divided into two groups:①H. pylori infection group: drug excipient 5% gum arabic solution only;②H. pylori eradication group: omeprazole 0.06mg/mice, amoxicillin 2.9mg/mice, clarithromycin 1.5mg/mice, and 5% gum arabic solution; twice a day for 2 weeks. Another group of mice without any drug nor H. pylori was the control group. At 4 weeks after the last administration, mice were sacrificed and samples were taken.(3) H. pylori immune protection: Female BALB/c mice (6~8-week-old) were randomly divided into three groups:①normal control group: without vaccine or H. pylori challenge;②vaccine control group: PBS;③vaccine group: H. pylori antigen, chitosan particles plus CT. Mice in the last two groups were immunized once a week for 4 weeks. At 4 weeks after the last immunization, mice were challenged with alive H. pylori SS1 (1.0×109CFU/ml, 0.5ml/mice) twice at two day intervals. At 4 weeks after the last challenge, mice were sacrificed and samples were collected.(4) Assessment:①H. pylori colonization in gastric mucosa were determined by improved Giemsa staining and H. pylori quantitatively culture;②T he levels of H. pylori-specific antibodies (IgG1 and IgG2α) in serum were determined by indirect ELISA. The levels of cytokines IL-2,IL-12,IFN-γ,IL-4 and IL-10 in gastric mucosa were determined by sandwich ELISA;③The expression of Tim-1 and Tim-3 mRNA in spleen and lymphocytes of mice was determined by RT-PCR.④T he percentage of Tim-3 positive cells were determined by flow cytometry. Results:(1) The effect of H. pylori on Tim-1 and Tim-3 in lymphocytes of mice:①The expression of Tim-1 mRNA in lymphocytes of mice was not significantly different when cocultured with different concentration of alive H. pylori at 3h (P>0.05), nevertheless, it was significantly different at 6h, 12h and 24h (P<0.01,0.05,0.001). At 6h and 12h, the expression of Tim-1 mRNA in 0.50×10~7CFU/ml and 1.00×10~7CFU/ml group was significantly lower than that in control group (P<0.05); but in the higher concentration groups (2.00×10~7CFU/ml and 4.00×10~7CFU/ml), the expression of Tim-1 mRNA rose again, with no significantly different with control group (P>0.05), yet significantly higher than that in the lower concentration groups (P<0.05 , 0.005). At 24h, between 0.50×10~7CFU/ml and 2.00×10~7CFU/ml, the higher the concentration of alive H. pylori, the lower the expression of Tim-1. At 24h, the expression of Tim-1 mRNA in 1.00×10~7CFU/ml and 2.00×10~7CFU/ml group was significantly lower than that in control group (P<0.005); but in the higher concentration group (4.00×10~7CFU/ml), the expression of Tim-1 mRNA rose again, with no significantly different with control group (P>0.05), yet significantly higher than that in the lower concentration groups (P<0.005,0.001, 0,001).②The expression of Tim-3 mRNA in lymphocytes of mice was not significantly different when cocultured with different concentration of alive H. pylori at 3h and 24h (P>0.05), nevertheless, it was significantly different at 6h and 12h (P<0.01,0.001). The higher the concentration of alive H. pylori, the higher the expression of Tim-3. At 6h, the expression of Tim-3 mRNA in 2.00×10~7CFU/ml and 4.00×10~7CFU/ml group was significantly higher than that in control and 0.50×10~7CFU/ml group (P<0.005, 0.05). At 12h, the expression of Tim-3 mRNA in 2.00×10~7CFU/ml group was significantly higher than that in control, 0.50×10~7CFU/ml and 1.00×10~7CFU/ml group (P<0.05); furthermore, the expression of Tim-3 mRNA in 4.00×10~7CFU/ml group was significantly higher than that in control and groups with other concentrations (P<0.001, 0.05).③The ratio of Tim-1/Tim-3 mRNA in lymphocytes of mice was not significantly different when cocultured with different concentration of alive H. pylori at 3h (P>0.05), nevertheless, it was significantly different at 6h, 12h and 24h (P<0.001,0.05,0.001). At 6h, the ratio of Tim-1/Tim-3 mRNA in 0.50×10~7CFU/ml and 1.00×10~7CFU/ml group was significantly lower than that in control group (P<0.05, 0.01); at 12h, the ratio of Tim-1/Tim-3 mRNA in 1.00×10~7CFU/ml group was significantly lower than that in control group (P<0.005); at 24h, the ratio of Tim-1/Tim-3 mRNA in 0.50×10~7CFU/ml, 1.00×10~7CFU/ml and 2.00×10~7CFU/ml group was significantly lower than that in control group (P<0.005, 0.001, 0.001). In the higher concentration group (4.00×10~7CFU/ml) at 6h, 12h and 24h, the ratio of Tim-1/Tim-3 mRNA rose again, with no significantly different with control group (P>0.05), yet significantly higher than that in the lower concentration groups (P<0.001). ④Flow cytometric analysis showed that, the percentage of Tim-3 positive cells in lymphocytes of mice was not significantly different when cocultured with different concentration of alive H. pylori at 24h (P>0.05), nevertheless, it was significantly different at 6h and 12h (P<0.01,0.001). The higher the concentration of alive H. pylori, the higher percentage of Tim-3 positive cells. At 6h, the percentage of Tim-3 positive cells in 4.00×10~7CFU/ml group was significantly higher than that in control and 0.50×10~7CFU/ml group (P<0.01, 0.001), and the percentage of Tim-3 positive cells in 2.00×10~7CFU/ml group was significantly higher than that in 0.50×10~7CFU/ml group (P<0.05). At 12h, the percentage of Tim-3 positive cells in 2.00×10~7CFU/ml group was significantly higher than that in control, 0.50×10~7CFU/ml and 1.00×10~7CFU/ml group (P<0.05, 0.005, 0.05).(2) The expression of Tim-1 and Tim-3 mRNA in H. pylori infection and eradication in mice:The expression of Tim-1 mRNA in spleen of mice in H. pylori infection group was significantly lower than that in normal control group (P<0.05). The expression of Tim-1 mRNA in spleen of mice in H. pylori eradication group was significantly increased, higher than that in H. pylori infection group (P<0.05), yet with no significant difference with normal control group (P>0.05). The expression of Tim-3 mRNA in spleen of mice in H. pylori infection group was significantly higher than that in normal control group (P<0.05). The expression of Tim-3 mRNA in spleen of mice in H. pylori eradication group was significantly decreased, lower than that in H. pylori infection group (P<0.05), yet with no significant difference with normal control group (P>0.05). The ratio of Tim-1/Tim-3 mRNA in spleen of mice in H. pylori infection group was significantly lower than that in normal control group (P<0.05). The ratio of Tim-1/Tim-3 mRNA in spleen of mice in H. pylori eradication group was significantly increased, higher than that in H. pylori infection group (P<0.05), yet with no significant difference with normal control group (P>0.05).(3) The expression of Tim-1 and Tim-3 mRNA in H. pylori immune protection in mice:The expression of Tim-1 and Tim-3 mRNA in spleen of mice in vaccine group was significantly higher than that in normal control and vaccine control group (P<0.001~0.05). The expression of Tim-3 mRNA in spleen of mice in vaccine control group was significantly higher than that in normal control group (P<0.05). The ratio of Tim-1/Tim-3 mRNA in spleen of mice in vaccine control group was significantly lower than that in normal control group (P<0.05). Whereas, the ratio of Tim-1/Tim-3 mRNA in spleen of mice in vaccine group was significantly increased, higher than that in vaccine control group (P<0.001), yet with no significant difference with normal control group (P>0.05).(4) Tim-1 and Tim-3 as well as Th immune response in H. pylori infection and eradication, and H. pylori immune protection in mice:1) Tim-1 and Tim-3 as well as Th immune response in H. pylori infection and eradication in mice:①The level of anti-H. pylori IgG1 and IgG2a in serum in H. pylori infection and eradication group was significantly higher than that in normal control group (P<0.001, 0.01, 0.05), and there was no significant difference between H. pylori infection group and H. pylori eradication group (P>0.05). In each group, the expression of Tim-1 mRNA in spleen was significantly positively correlated with the level of anti-H. pylori IgG1 in serum (reflect Th2 immune response) (P<0.001, 0.05), whereas had no correlation with the level of anti-H. pylori IgG2a in serum (reflect Th1 immune response) (P>0.05); the expression of Tim-3 mRNA in spleen was significantly positively correlated with the level of anti-H. pylori IgG2a in serum (P<0.01, 0.05), whereas had no correlation with the level of anti-H. pylori IgG1 in serum (P>0.05).②The level of IL-4, IL-10 and IL-12 in gastric mucosa was not significantly different among normal control, H. pylori infection and H. pylori eradication group (P>0.05), but the level of IL-2 and IFN-γwas significantly different (P<0.01, 0.05). Furthermore, the level of IL-2 and IFN-γwas significantly higher in H. pylori infection group than that in normal control and H. pylori eradication group (P<0.005, 0.01, 0.05), yet with no significant difference between H. pylori eradication group and normal control group (P>0.05). In each group, the expression of Tim-1 mRNA in spleen was significantly positively correlated with the level of IL-4 and IL-10 in gastric mucosa (Th2 cytokine) (P<0.005, 0.01, 0.05), whereas had no correlation with the level of IL-2, IFN-γand IL-12 in gastric mucosa (Th1 cytokine) (P>0.05); the expression of Tim-3 mRNA in spleen was significantly positively correlated with the level of IL-2, IFN-γand IL-12 in gastric mucosa (P<0.005, 0.05), whereas had no correlation with the level of IL-4 and IL-10 in gastric mucosa (P>0.05).2) Tim-1 and Tim-3 as well as Th immune response in H. pylori immune protection in mice:①The level of anti-H. pylori IgG1 and IgG2a in serum in vaccine group was significantly higher than that in vaccine control and normal control group (P<0.001); furthermore, the level of anti-H. pylori IgG1 in serum in vaccine control group was significantly higher than that in normal control group (P<0.01). In each group, the expression of Tim-1 mRNA in spleen was significantly positively correlated with the level of anti-H. pylori IgG1 in serum (P<0.05), whereas had no correlation with the level of anti-H. pylori IgG2a in serum (P>0.05); the expression of Tim-3 mRNA in spleen was significantly positively correlated with the level of anti-H. pylori IgG2a in serum (P<0.001, 0.05), whereas had no correlation with the level of anti-H. pylori IgG1 in serum (P>0.05).②The level of IL-4 in gastric mucosa was not significantly different among H. pylori vaccine groups (P>0.05), but the level of IL-10, IL-2, IFN-γand IL-12 was significantly higher in vaccine group than that in normal control and vaccine control group (P<0.001, 0.005, 0.05). Furthermore, the level of IFN-γand IL-12 was significantly higher in vaccine control group than that in normal control group (P<0.01, 0.05), and the level of IL-10 was significantly lower in vaccine control group than that in normal control group (P<0.05). In each group, the expression of Tim-1 mRNA in spleen was significantly positively correlated with the level of IL-4 and IL-10 in gastric mucosa (P<0.001, 0.005, 0.01, 0.05), whereas had no correlation with the level of IL-2, IFN-γand IL-12 in gastric mucosa (P>0.05); the expression of Tim-3 mRNA in spleen was significantly positively correlated with the level of IL-2, IFN-γand IL-12 in gastric mucosa (P<0.005, 0.01, 0.05), whereas had no correlation with the level of IL-4 and IL-10 in gastric mucosa (P>0.05).Conclusion:1. H. pylori down-regulate the expression of Tim-1, up-regulate the expression of Tim-3, and reduce the ratio of Tim-1/Tim-3 in lymphocytes of mice in vitro, indicating that H. pylori could inhibit the differentiation of T lymphocytes towards Th2 cells, promote the Th1 cell differentiation, thus induce Th1-biased immune response.2. H. pylori infection down-regulate the expression of Tim-1, up-regulate the expression of Tim-3, and decrease the ratio of Tim-1/Tim-3 in spleen of mice in vivo, whereas after H. pylori eradication, the expression of Tim-1 rise, the expression of Tim-3 descend, and the ratio of Tim-1/Tim-3 rise, indicating that H. pylori infection could induce Th1-biased immune response, yet H. pylori eradication could reverse the Th1-biased immune response induced by H. pylori infection.3. H. pylori vaccine up-regulate the expression of Tim-1 and Tim-3 mRNA and increase the ratio of Tim-1/Tim-3 in spleen of mice, indicating that H. pylori vaccine could induce both Th1 and Th2 immune response, reverse the inhibition of Th2 immune response induced by H. pylori infection and recover the Th1/Th2 balance.4. In each group of H. pylori infection and vaccination, the expression of Tim-1 is positively correlated with the level of anti-H. pylori IgG1 reflecting Th2 immune response and Th2 cytokine, but with no correlation with the level of anti-H. pylori IgG2a reflecting Th1 immune response and Th1 cytokine, yet the expression of Tim-3 is on the contrary, which indicate that the expression of Tim-1 and Tim-3 could reflect Th2 and Th1 immune response respectively in H. pylori infection and vaccination, thus provide evidences for the prevention and treatment of H. pylori infection and correlation diseases through regulation of Tim-1 and Tim-3. |
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