The Influence Of Blocking Tim-3 Signal Pathway On Immune Pathopoiesis Of H.pylori Infection And Immune Protection Of H.pylori Vaccine And It's Mechanisms

Background and objecyive:The host immune response plays an important role in pathopoiesis of Helicobacter pylori (H.pylori) infection and immune protection of H.pylori vaccine. To clarify the mechanisms of immune pathogenesis of H. pylori infection and immune protection of H.pylori vaccine is great significance for prevention and treatment of H. pylori infection and correlation diseases. 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-3 is an important member of Tim family, is expressed on terminally differentiated Th1 cells, regulate function of different subsets of CD4+ T cell, and influence Toll like receptor (TLR) signaling pathway and the function of regulatory T cells (Treg). Until now, the role of Tim-3 in pathogenesis of H. pylori infection and the immune protection of vaccine remain unclear. In the present study, we focused on influence of blocking Tim-3 signal pathway on the colonization of H. pylori, inflammation, TLR signaling pathways and Foxp3+Treg in gastric mucosa of mice which were vaccinated by H. pylori vaccines with different adjuvants and inoculated by alive H. pylori. This is a new point to discuss the mechanisms of pathopoiesis of H. pylori infection and immune protection of H. pylori vaccine.Methods:1. The best preparation condition and in vitro release characters of Chitosan microspheres-H. pylori antigens:Using Berthold precipitation method to prepare chitosan microspheres, according to different chitosan, different precipitation agents, different acetic acid concentration, different pH, and whether treated with ultrasound to optimize the preparation conditions; Using scanning electron microscope and particle size analyzer to observe the shape and size measurement of chitosan microspheres; Chitosan microspheres with the different quality rate of antigen and chitosan microspheres to load the Chitosan microspheres-H. pylori antigens; Using the BCA Protein Assay Kit to measure the loading efficiency, loading capacity and releasing rate of microspheres.2. The influence of blocking Tim-3 signal pathway on pathopoiesis of H. pylori infection and immune protection of H. pylori vaccine and it's mechanisms .(1)The influence of blocking Tim-3 signal pathway on immune protection of H. pylori vaccine and it's mechanisms: SPF-grade female BALB/c mice(6~8 weeks old)were randomly divided into five groups and immunized by:①Normal control group: PBS solution;②H. pylori antigen + Cholera toxin(CT);③Anti-Tim-3 antibody pretreatment + H. pylori antigen + CT;④Chitosan microspheres-H. pylori antigen;⑤Anti-Tim-3 antibody pretreatment + chitosan microspheres-H. pylori antigen; orally respectively once a week for four weeks. At 4 weeks after the last immunization, the mice from②,③,④and⑤group were challenged by alive H. pylori Sydney Strain 1(1×109CFU/ml, 0.5ml/mice)quartic at two days intervals. At 4 weeks after the last challenge, these mice were all sacrificed and serum, saliva, gastric mucosa were collected.(2)The influence of blocking Tim-3 signal pathway on pathopoiesis of H. pylori infection and it's mechanisms:①Establishing the H. pylori infection model directly;②Establishing H. pylori infection model after tim-3 antibody pretreatment. Model establishment: SPF-grade female BALB/c mice(6~8 weeks old)were inoculated by alive H. pylori Sydney Strain 1(1×109CFU/ml, 0.5ml/mice)at two days intervals for five times. Twelve weeks after the last inoculation, the mice were all sacrificed and serum, saliva, gastric mucosa were collected.(3)Assessment:①H. pylori colonization in gastric mucosa were determined by improved Giemsa staining;②The degree of inflammation in gastric mucosa were determined by HE staining, using Sakagami scoring;③The protein expression of TLR4, MyD88, NF-κBp65, Foxp3 in gastric mucosa were determined by immunohistochemistry;④The mRNA expression of TLR4 and MyD88 in gastric mucosa were determined by RT-PCR;⑤The levels of H. pylori-specific IgG in serum was determined by indirect ELISA.Results:1. The best preparation condition and in vitro release character research of Chitosan microspheres-H. pylori antigens: We choose the best preparation program of chitosan microspheres from 32 species preparation programs, the best preparation program is the material of sea shell chitosan, 1% concentration of acetic acid, sodium sulfate for precipitating agent and 5.0 pH value, not used ultrasonic treatment. The scanning electron microscope of microspheres show smooth round and dense, diameters ranged from 1.0μm to 5.0μm; When the quality rate of antigen and microspheres is 1:5, loading time is 3h, the loading efficiency is maximum; antigen loading efficiency is 79.92%, loading capacity is 16.47%; Release experiment in vitro shows that the total antigen releasing rate is 20.39%, showing a slow-release status.2. The influence of blocking Tim-3 signal pathway on pathopoiesis of H. pylori infection and immune protection of H. pylori vaccine and it's mechanisms:(1)The colonization density of H. pylori in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants were significantly lower in groups with anti-Tim-3 antibody pretreatment than those in group without anti-Tim-3 antibody pretreatment(P<0.05); The colonization density of H. pylori in gastric mucosa were not significant different among of mice vaccinated by H. pylori vaccines with different adjuvant(P>0.05).(2)The colonization density of H. pylori in gastric mucosa of mice inoculated with alive H. pylori was significantly higher than those in control group (P<0.001), anti-Tim-3 antibody pretreatment were not affect the colonization density of H.pylori (P>0.05) .(3)The inflammatory degree in gastric mucosa of mice vaccinated by H.pylori, two H. pylori vaccines with different adjuvants were higher than those in control group (P<0.05, 0.001), and in groups with anti-Tim-3 antibody pretreatment were higher than groups without anti-Tim-3 antibody pretreatment (P<0.05, 0.01); The inflammatory degree in gastric mucosa were not significant different among of groups vaccinated by H. pylori vaccines with different adjuvant (P>0.05).(4)The inflammatory degree in gastric mucosa of mice inoculated with alive H. pylori were significantly higher than those in control group (P<0.001), and in group with anti-Tim-3 antibody pretreatment were significantly higher than group without anti-Tim-3 antibody pretreatment (P<0.05).(5)The expression of TLR4 mRNA and the TLR4-positive cells score in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants were significantly higher than those in control group (P<0.05, 0.001), and the TLR4-positive cells score in groups with anti-Tim-3 antibody pretreatment were significantly higher than those in groups without anti-Tim-3 antibody pretreatment (P<0.05, 0.01, 0.001). In mice vaccinated by H. pylori vaccine with chitosan microspheres as adjuvant, the expression of TLR4 mRNA were significantly higher in group with anti-Tim-3 antibody pretreatment than those in groups without anti-Tim-3 antibody pretreatment(P<0.001); But in mice vaccinated by H. pylori vaccine with CT as adjuvant, there were no significant different between two groups( P>0.05). The TLR4-positive cells score in gastric mucosa of mice vaccinated by H. pylori vaccines with different adjuvants were not significant different (P>0.05). In mice without anti-Tim-3 antibody pretreatment, the expression of TLR4 mRNA in group vaccinated by H. pylori vaccines with CT as adjuvant were significantly higher than those in group vaccinated by H. pylori vaccines with chitosan microspheres as adjuvant (P<0.05), but in mice with anti-Tim-3 antibody pretreatment, there were no significant different between two groups (P>0.05).(6)The expression of TLR4 mRNA and TLR4-positive cells score in gastric mucosa of mice inoculated with alive H. pylori were significantly higher than those in control group (P<0.001); and in group with anti-Tim-3 antibody pretreatment were significantly higher than group without anti-Tim-3 antibody pretreatment(P<0.05, 0.001).(7)The expression of MyD88 mRNA and the MyD88-positive cells score in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants were significantly higher than those in control group(P<0.05, 0.001); And in groups with Anti-Tim-3 antibody pretreatment were higher than groups without anti-Tim-3 antibody pretreatment (P<0.05, 0.001); The MyD88-positive cells score in gastric mucosa of mice vaccinated by H. pylori vaccines with different adjuvants were not significant different (P>0.05). In mice without anti-Tim-3 antibody pretreatment, the expression of MyD88 mRNA in group vaccinated by H. pylori vaccines with CT as adjuvant were significantly higher than those in group vaccinated by H. pylori vaccines with chitosan microspheres as adjuvant (P<0.05), but in mice with anti-Tim-3 antibody pretreatment, there were no significant different between two groups (P>0.05).(8)The expression of MyD88 mRNA and MyD88-positive cells score in gastric mucosa of mice inoculated with alive H. pylori were significantly higher than those in control group (P<0.001); And in group with anti-Tim-3 antibody pretreatment were significantly higher than group without Anti-Tim-3 antibody pretreatment(P<0.05, 0.001).(9)The percentage Foxp3-positive cells in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants were significantly higher than those in control group (P<0.05, 0.001); And in groups with anti-Tim-3 antibody pretreatment were lower than groups without anti-Tim-3 antibody pretreatment (P<0.05, 0.001); The percentage Foxp3-positive cells in gastric mucosa of mice vaccinated by H. pylori vaccines with different adjuvants were not significant different (P>0.05).(10)The percentage Foxp3-positive cells in gastric mucosa of mice inoculated with alive H. pylori were significantly higher than those in control group (P<0.001); And in group with anti-Tim-3 antibody pretreatment were significantly lower than group without anti-Tim-3 antibody pretreatment (P<0.001).(11)The NF-κBp65-positive cells score in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants were significantly higher than those in control group (P<0.001); And in groups with anti-Tim-3 antibody pretreatment were higher than groups without anti-Tim-3 antibody pretreatment (P<0.05, 0.001); The NF-κBp65-positive cells score in gastric mucosa of mice vaccinated by H. pylori vaccines with different adjuvants were not significant different (P>0.05).(12)The NF-κBp65-positive cells score in gastric mucosa of mice inoculated with alive H. pylori were significantly higher than those in control group (P<0.001, 0.01); And in group with anti-Tim-3 antibody pretreatment were significantly higher than group without anti-Tim-3 antibody pretreatment (P<0.001).(13) The level of anti-H. pylori IgG in serum of mice vaccinated by two H. pylori vaccines with different adjuvants were significantly higher than those in control group (P<0.001), anti-Tim-3 antibody pretreatment were not affect the level of anti-H. pylori IgG in serum (P>0.05). The level of anti-H. pylori IgG in serum were not significant different among of groups vaccinated by H. pylori vaccines with different adjuvant (P>0.05). (14) The level of anti-H. pylori IgG in serum of mice inoculated with alive H. pylori were significantly higher than those in control group (P<0.001), anti-Tim-3 antibody pretreatment were not affect the level of anti-H. pylori IgG in serum (P>0.05). The level of anti-H. pylori IgG in serum of mice vaccinated by H. pylori vaccines were significantly higher than H. pylori infection (P<0.05).Conclusion:1. This study examined the preparation conditions of chitosan microspheres, the chitosan microspheres, which was manufactured by this experiment, were loaded the H. pylori whole cell protein with high encapsulation efficiency and better release effect.2. Blocking Tim-3 signal pathway can improve the H. pylori vaccine protection rate, but does not reduce the H. pylori colonization density of gastric mucosa of mice naturally infected with H. pylori.3. Blocking Tim-3 signal pathway can exacerbate the inflammatory degree in gastric mucosa of mice vaccinated by H. pylori vaccine and mice infected with H. pylori.4. Blocking Tim-3 signal pathway can upregulate TLR4, MyD88 expression and promote the NF-κB activation, reduced the numbers of CD4+CD25+Foxp3+Treg, this could be the mechanism that it enhanced H. pylori vaccine immune protection, but not impact the colonization density of H. pylori in gastric mucosa of mice naturally infected with H. pylori .5. H. pylori vaccine with Chitosan microspheres as adjuvant, the same as classical adjuvant CT, had immunoprotection effect to H. pylori infection. These suggested that chitosan microspheres could be used as mucosal adjuvant of H. pylori vaccine.