The Influence Of Blocking TLRs Signal Pathway And Treg 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. Toll like receptors are recently found to play an essential role in the first line of host defense by recognition of microbial components. These receptors rcognize conserved molecular patterns that are expressed by infectious agents. By this way TLRs mediate the production of proinflammatory cytokines and chenokines resulting in inflammation. Regulatory T cells(Tregs) are thought to be important in suppressing deleterious immune and inflammatory responses. There is the interaction between TLRs single pathway and Treg. By the TLRs single pathway,the immune suppression was resisted. Thus, Treg can degrade the level of cytocine induced by TLRs. But the relation of TLRs single pathway and Treg is not clear during H. pylori infection and immunited by H. pylori vaccine. Then, we researched the relation between TLRs single pathway and Treg in gastric mucosa of mice infected with H. pylori and immuted by H. pylori vaccine. 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 p H, 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 TLRs signal pathway and Treg on pathopoiesis of H. pylori infection and immune protection of H. pylori vaccine and it’s mechanisms.(1)The influence of TLRs signal pathway and Treg 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:(1) Normal control group: PBS solution;(2)Anti-Tim-3 antibody pretreatment + Normal control group;(3)Anti-CD25 antibody pretreatment + Normal control group;(4)H. pylori antigen + Cholera toxin(CT);(5)Anti-TLR4 antibody pretreatment + H. pylori antigen + CT;(6) Anti-CD25 antibody pretreatment + H. pylori antigen + CT;(7) Chitosan microspheres-H. pylori antigen;(8) Anti-TLR4 antibody pretreatment + chitosan microspheres-H. pylori antigen;(9) Anti-CD25 antibody pretreatment + chitosan microspheres-H. pylori antigen; orally respectively once a week for four weeks. At 4 weeks after the last immunization, the mice from(2),(3),(4) and(5)group 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 TLR4 signal pathway and Treg on pathopoiesis of H. pylori infection and it’s mechanisms:(1)Establishing the H. pylori infection model directly;(2) Establishing H. pylori infection model after TLR4 antibody pretreatment.(3) Establishing H. pylori infection model after CD25 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:(1)H. pylori colonization in gastric mucosa were determined by improved Giemsa staining;(2)The degree of inflammation in gastric mucosa were determined by HE staining, using Sakagami scoring;(3)The protein expression of TLR4, My D88, NF-κBp65, Foxp3 in gastric mucosa were determined by immunohistochemistry;(4)The expression of cytokines IFN, IL-12, IL-17, IL-4, IL-10 in gastric mucosa were determined by double antibody sandwich ELISA;(5)The levels of H. pylori-specific Ig G, Ig G1, Ig G2 a in serum and H. pylori-specific Ig A in saliva 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 p H 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 TLRs signal pathway and Treg 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 was significantly higher than those in control group(P<0.001), and in group with anti-TLR4 antibody pretreatment were significantly higher than group without 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 infected by H. pylori was significantly higher than those in control group(P<0.001), and in group with anti-TLR4 antibody pretreatment were significantly higher than group without pretreatment(P<0.05).(3) The colonization density of H. pylori in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants was significantly higher than those in control group(P<0.05), and in group with anti-CD25 antibody pretreatment were significantly lower than group without 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).(4) The colonization density of H. pylori in gastric mucosa of mice infected by H. pylori was significantly higher than those in control group(P<0.001), and in group with anti-CD25 antibody pretreatment were significantly lower than group without pretreatment(P<0.05).(5)The inflammatory degree in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants were higher than those in control group(P<0.05-0.001), and in groups with anti-TLR4 antibody pretreatment were lower than groups without anti-TLR4 antibody pretreatment(P<0.05). The inflammatory degree in gastric mucosa were not significant different among of groups vaccinated by H. pylori vaccines with different adjuvant(P>0.05).(6)The inflammatory degree in gastric mucosa of mice infected by alive H. pylori were significantly higher than those in control group(P<0.05-0.001), and in group with anti-TLR4 antibody pretreatment were significantly lower than group without anti-TLR4 antibody pretreatment(P<0.05).(7) The inflammatory degree in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants were higher than those in control group(P<0.001), and in groups with anti-CD25 antibody pretreatment were higher than groups without anti-CD25 antibody pretreatment(P<0.05); The inflammatory degree in gastric mucosa were not significant different among of groups vaccinated by H. pylori vaccines with different adjuvant(P>0.05).(8) 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-CD25 antibody pretreatment were significantly higher than group without anti-CD25 antibody pretreatment(P<0.05).(9)The expression of My D88、NF-κB p65、Foxp3 in gastric mucosa of mice vaccinated by two H. pylori vaccines with different adjuvants were significantly higher than those in control group(P<0.01-0.001), and the expression of My D88、NF-κB p65 in groups with anti-TLR4 antibody pretreatment were significantly lower than those in groups without anti-TLR4 antibody pretreatment(P<0.05). The expression of Foxp3 in groups with anti-TLR4 antibody pretreatment were significantly higher than those in groups without anti-TLR4 antibody pretreatment by immunohistochemistry(P<0.05),but there were no significant different between these two group by western blot. The expression of My D88、NF-κB p65、Foxp3 in gastric mucosa of mice vaccinated by H. pylori vaccines with different adjuvants were were not significant different(P>0.05).(10)The expression of My D88、NF-κB p65、Foxp3 in gastric mucosa of mice infected by alive H. pylori were significantly higher than those in control group(P<0.05-0.001); and the expression of My D88、NF-κB p65 in groups with anti-TLR4 antibody pretreatment were significantly lower than those in groups without anti-TLR4 antibody pretreatment(P<0.05). The expression of Foxp3 in groups with anti-TLR4 antibody pretreatment were significantly higher than those in groups without anti-TLR4 antibody pretreatment(P<0.05)(11)The expression of My D88、NF-κB p65、Foxp3 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 the expression of My D88、NF-κB p65 in groups with anti-CD25 antibody pretreatment were significantly higher than those in groups without anti-CD25 antibody pretreatment(P<0.05). The expression of Foxp3 in groups with anti-CD25 antibody pretreatment were significantly lower than those in groups without anti-CD25 antibody pretreatment(P<0.05).The expression of My D88、NF-κB p65、Foxp3 in gastric mucosa of mice vaccinated by H. pylori vaccines with different adjuvants were not significant different(P>0.05).(12)The expression of My D88、NF-κB p65、Foxp3 in gastric mucosa of mice infected by alive H. pylori were significantly higher than those in control group(P<0.01-0.001), and the expression of My D88、NF-κB p65 in groups with anti-CD25 antibody pretreatment were significantly higher than those in groups without anti-CD25 antibody pretreatment(P<0.05). The expression of Foxp3 in groups with anti-CD25 antibody pretreatment were significantly lower than those in groups without anti-CD25 antibody pretreatment(P<0.05).(13) The level of Th1 and Th17 cytokine(IL-12, IFN-γ and IL-17) in gastric mucosa of mice vaccined by H. pylori vaccine were significantly higher than that in control group(P<0.05-0.001). The level of Th2 cytokine(IL-4 and IL-10) in gastric mucosa of mice in group with CT as adjuvant was significantly lower than that in control group(P<0.05) and the level of IL-4 in group with chitosan microphere as adjuvant was higher than control group(P<0.05). The level of IL-10 in group with anti-TLR4 antibody pretreatment were significantly higher than those in control groups without pretreatment(P<0.05), but there were no significantl difference between that and control group with TLR4 pretreatment(P>0.05).There were no significantly different between in group with anti-TLR4 antibody pretreatment and control groups(P>0.05). The level of Th1 and Th17 cytokine(IL-12, IFN-γ and IL-17) in gastric mucosa of mice vaccined by H. pylori vaccine with anti-TLR4 antibody pretreatment were significantly lower than that in group without pretreatment(P<0.05). The level of Th2 cytokine(IL-4 and IL-10) in gastric mucosa of mice vaccined by H. pylori vaccine were no significantly different between in group with anti-TLR4 antibody pretreatment and without pretreatment(P>0.05). The level of Th1 and Th17 cytokine(IL-12, IFN-γ and IL-17) in gastric mucosa of mice vaccined by H. pylori vaccine with chitosan microphere as adjuvant were no significantly different with the group with CT as adjuvant(P>0.05).The level of IFN in gastric mucosa of mice vaccined by H. pylori vaccine with CT as adjuvant was higher than that in group with chitosan microphere as adjuvan(P<0.05), but the level of IL-12 and IL-17 were no significantly different between them(P>0.05).(14) The level of Th1 and Th17 cytokine(IL-12, IFN-γ and IL-17) in gastric mucosa of mice infected by H. pylori were significantly higher than those in control group(P<0.05-0.001). The level of Th2 cytokine(IL-4 and IL-10) in gastric mucosa of mice infected by H. pylori were significantly lower than those in control group(P<0.01); The level of Th1 and Th17 cytokine(IL-12, IFN-γ and IL-17) in gastric mucosa of mice infected by H. pylori were significantly lower than those in group with anti-TLR4 antibody pretreatment(P<0.05-0.001), and there were no significant difference between group with anti-TLR4 antibody pretreatment and without pretreatment(P>0.05).(15) The level of Th1 and Th17 cytokine(IL-12, IFN-γ and IL-17) in gastric mucosa of mice vaccined by H. pylori vaccine were significantly higher than that in control group(P<0.001). The level of Th2 cytokine(IL-4 and IL-10) in gastric mucosa of mice in group with CT as adjuvant was significantly lower than that in control group(P<0.05-0.001) and the level of IL-4 in group with chitosan microphere as adjuvant was higher than control group(P<0.05). The level of IL-10 were no significantly different between them(P>0.05). The level of IFN-γ and IL-17) in gastric mucosa of mice vaccined by H. pylori vaccine with anti-CD25 antibody pretreatment were significantly higher than that in group without pretreatment(P<0.05). The level of IL-12 in gastric mucosa of mice vaccined by H. pylori vaccine with chitosan microphere as adjuvant with anti-CD25 antibody pretreatment were significantly higher than that in group without pretreatment(P<0.05). There were no significantl difference between the vaccined group with and without anti-CD25 antibody pretreatment(P>0.05). The level of IL-10 in gastric mucosa of mice vaccined by H. pylori vaccine with anti-CD25 antibody pretreatment were lower than without pretreatment(P<0.01-0.05). The level of IL-12, IL-17 were no significantly different between in group with CT as adjuvant and chitosan microphere as adjuvant with anti- CD25 antibody pretreatment(P>0.05). The level of IFN in the group with CT as adjuvant with anti- CD25 antibody pretreatment were higher than that in the group with chitosan microphere as adjuvant(P<0.05). The level of Th2 cytokine(IL-4 and IL-10) in gastric mucosa of mice vaccined by H. pylori vaccine with chitosan microphere as adjuvant were higher than that in group with CT as adjuvant with antiCD25 antibody pretreatment(P<0.05)..(16) The level of Th1 and Th17 cytokine(IL-12, IFN and IL-17) of mice infected by H. pylori were significantly higher than those in control(P<0.001); The level of Th2 cytokine(IL-4 and IL-10) of mice infected by H. pylori were significantly lower than those in control(P<0.01-0.001). The level of Th1 and Th17 cytokine(IL-12, IFN-γ and IL-17) in gastric mucosa of mice infected by H. pylori were significantly higher than those in group with anti-CD25 antibody pretreatment(P<0.05-0.001). were no significant difference between group with anti-TLR4 antibody pretreatment and without pretreatment(P>0.05). The level of IL-10 in group with anti-CD25 antibody pretreatment were significantly lower than group without pretreatment(P<0.05), but the level of IL-4 were no significantly difference between them(P>0.05).The level of TH1 and TH17 cytocines were no significantly difference between the group with different adjuvant(P>0.05).The level of TH2 cytocines in the group with chitosan microphere as adjuvant was higher than that in the group with CT as adjuvant(P<0.05).(17) The level of anti-H. pylori Ig G、Ig G1、Ig G2 a 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-TLR4 antibody pretreatment were not affect the level of anti-H. pylori Ig G、Ig G1、Ig G2 a in serum(P>0.05). The level of anti-H. pylori 、Ig G1、Ig G2 a in serum were not significant different among of groups vaccinated by H. pylori vaccines with different adjuvant(P>0.05).(18) The level of anti-H. pylori Ig G、Ig G1、Ig G2 a in serum of mice inoculated with alive H. pylori were significantly higher than those in control group(P<0.001), anti-TLR4 antibody pretreatment were not affect the level of anti-H. pylori Ig G、Ig G1、Ig G2 a in serum(P>0.05). The level of anti-H. pylori Ig G、Ig G1、Ig G2 a in serum of mice vaccinated by H. pylori vaccines were significantly higher than H. pylori infection(P<0.05).(19)The level of anti-H. pylori Ig G、Ig G1、Ig G2 a 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-CD25 antibody pretreatment were not affect the level of anti-H. pylori Ig G、Ig G1、Ig G2 a in serum(P>0.05). The level of anti-H. pylori Ig G 、Ig G1、Ig G2 a in serum were not significant different among of groups vaccinated by H. pylori vaccines with different adjuvant(P>0.05).(20) The level of anti-H. pylori Ig G、Ig G1、Ig G2 a in serum of mice inoculated with alive H. pylori were significantly higher than those in control group(P<0.001), anti-CD25 antibody pretreatment were not affect the level of anti-H. pylori Ig G、Ig G1、Ig G2 a in serum(P>0.05). The level of anti-H. pylori Ig G、Ig G1、Ig G2 a in serum of mice vaccinated by H. pylori vaccines were significantly higher than H. pylori infection(P<0.05).(21) The level of anti- H. pylori Ig A in saliva of mice of Hp vaccine were significantly higher than that in control group(P<0.01), and in group with anti-TLR4 antibody pretreatment were significantly higher than those in groups without pretreatment(P<0.05). The level of anti-H. pylori Ig A in saliva were not significant different among of groups vaccinated by H. pylori vaccines with different adjuvant(P>0.05).(22) The level of anti- H. pylori Ig A in saliva of mice infected with H. pylori were significantly higher than those in control group(P<0.001); and in group with anti-TLR4 antibody pretreatment were significantly higher than group without pretreatment(P<0.05).(23) The level of anti- H. pylori Ig A in saliva of mice of H. pylori vaccine were significantly higher than that in control group(P<0.01), and in group with anti-CD25 antibody pretreatment were significantly higher than those in groups without pretreatment(P<0.05, 0.01).(24) The level of anti- H. pylori Ig A in saliva of mice infected with H. pylori were significantly higher than those in control group(P<0.01); and in group with anti-CD25 antibody pretreatment were significantly higher than group without pretreatment(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 TLR4 signal pathway can depress the H. pylori vaccine protection rate and exacerbate the inflammatory degree in gastric mucosa of mice vaccinated by H. pylori vaccine. Blocking CD25 can improve the H. pylori vaccine protection rate and reduce the inflammatory degree in gastric mucosa of mice vaccinated by H. pylori vaccine3. Blocking TLR4 signal pathway can exacerbate the H. pylori colonization density, reduce the inflammatory degree in gastric mucosa of mice infected with H. pylori. Blocking CD25 can reduce the H. pylori colonization density, exacerbate the inflammatory degree in gastric mucosa of mice infected with H. pylori.4. Blocking TLR4 signal pathway can down-regulate My D88 expression, depress the NF-κB activation and increase the numbers of CD4+CD25+Foxp3+Treg, Blocking CD25 can up-regulate My D88 expression, promote the NF-κB activation and increase the numbers of CD4+CD25+Foxp3+Treg, this could be the mechanism that it reduce the H. pylori colonization density of mice infected with H. pylori.5. Blocking TLR4 signal pathway can depress Th1 and Th17 immune respond and blocking CD25 can promote Th1 and TH17 immune respond and depress Th2 immune respond.6. Blocking TLR4 and CD25 were not affect the level of anti-H. pylori Ig G in serum.Blocking CD25 can up-regulate the level of anti H. pylori Ig G1、Ig G2 a in serum.7. Blocking TLR4 signal pathway can promote secretion of anti- H. pylori Ig A and blocking CD25 can depress secretion of anti- H. pylori Ig A.8.The protection of H. pylori vaccine with chitosan as adjuvant is same as that of with classic adjuvant CT against H. pylori infection.