| Background/Objective:Helicobacter pylori (H. pylori) can escape from the host immune elimination, and colonize in the gastric mucosa lifelong,thus cause chronic and persistent infection of pathogens. But the mechanism is not clear until recently. Regulatory T Cells(Treg) is a population of T cells with the function of immunosuppression, which play an important regulatory role in many immune diseases. Recently, research showed Treg played a certain role in the process that make H. pylori escaping the host immune elimination and regulate the inflammation-induced H. pylori infection. Therefore, this study observe the influence of H. pylori on CD4+CD25+Foxp3+Treg in vivo and vitro, discuss the pathogenic mechanism of H. pylori through CD4+CD25+Treg, provide theoretical and experimental bases for treatment target of CD4+CD25+Treg on prevention H. pylori infection and H. pylori associated diseases in future.Methods:⑴Patients and Samples: 239 biopsy gastric mucosa samples included: Chronic Chronic Non-Atrophic Gastritis (CNAG) 75 (H. pylori–positive 50, H. pylori-negative 25), Chronic Atrophic Gastritis (CAG) 24 (H. pylori–positive 13, H. pylori-negative 11), Intestinal Metaplasia and Dysplasia 90 (H. pylori-positive 45, H. pylori-negative 45), Gastric carcinoma 50 (H. pylori–positive 15, H. pylori-negative 35).⑵H. 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 four groups:①H. pylori infection group: drug excipient 5% gum arabic solution only;②H. pylori treatment group 1: amoxicillin (AM) and 5% gum arabic solution;③H. pylori treatment group 2: AM and omeprazole (OM) plus 5% gum arabic solution;④H. pylori treatment group 3: AM and OM plus chitosan and 5% gum arabic solution.; 10 mice in each group; Drugs were administered 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.⑶Coculture 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, 0.50×107CFU/ml, 1.00×107CFU/ml, 2.00×107CFU/ml and 4.00×107CFU/ml), and cells were collected at 3h, 6h, 12h and 24h.⑷Assessment:①H. pylori colonization in gastric mucosa were determined by improved Giemsa staining and H. pylori quantitatively culture;②The levels of H. pylori-specific antibodies (CagA IgG) in serum were determined by indirect ELISA;③The expression of Foxp3 mRNA in gastric mucosa and lymphocytes of mice was determined by RT-PCR;④The expression of Foxp3 protein in gastric mucosa was determined by two-step immunohistochemistry;⑤The percentage of CD4+CD25+Treg cells were determined by flow cytometry.Results:⑴The influence of H. pylori infection and eradication on Foxp3 in gastric mucosa:①The expression of Foxp3 mRNA in gastric mucosa of H. pylori positive patientes was higher than that of H. pylori negative patients(P<0.001, 0.01), but the expression of Foxp3 mRNA in gastric carcinoma group was significantly higher than that in the CNAG, CAG and IM+DYS groups(P<0.001,0.005); the percentage of Foxp3+ cells in gastric mucosa of patientes infected with CagA+ or CagA- H. pylori strain were not significantly different (P>0.05).②The expression of Foxp3 mRNA and the percentage of Foxp3+ cells in gastric mucosa of H. pylori infected mice were significantly higher than that of non-infected mice(P<0.001); But the expression of Foxp3 mRNA and the percentage of Foxp3+ cells in gastric mucosa of H. pylori eradicated mice significantly decreased, and were significantly lower than that of H. pylori-infected mice and non-eradicated mice(P<0.001), but with no significant difference with normal mice(P>0.05). In the same treatment group, the expression of Foxp3 mRNA and the percentage of Foxp3+ cells in gastric mucosa of H. pylori eradicated mice were significantly lower than that of non-eradicated mice.⑵The influence of H. pylori on CD4+CD25+Treg in lymphocytes of mice:①The expression of Foxp3 mRNA in lymphocytes of mice was not significantly different when cocultured with different concentration of alive H. pylori at 3h, 6h and 24h (P>0.05); nevertheless, the expression of Foxp3 mRNA in lymphocytes of mice was significantly decreased at 24h in a concentration- dependent manner (P<0.001), with the concentration increase of H. pylori, the expression of Foxp3 mRNA decreases gradually, the expression of Foxp3 mRNA in various concentration groups was significantly lower than that of the control groups (P<0.001, 0.05). the expression of Foxp3 mRNA in 2.00×107CFU/ml group was significantly lower than in 0.50×107CFU/ml groups (P<0.05); the expression of Foxp3 mRNA in 4.00×107CFU/ml groups was significantly lower than other various concentration groups (P<0.001).②Flow cytometric analysis showed that, the percentage of CD4+CD25+Treg cells in lymphocytes of mice was not significantly different when cocultured with different concentration of alive H. pylori at 6h and 12h (P>0.05), nevertheless, it was significantly decreased at 24h in a concentration-dependent manner. with the concentration increase of H. pylori, the percentage of CD4+CD25+Treg cells decreases gradually, the percentage of CD4+CD25+Treg cells in 4.00×107CFU/ml groups was significantly lower than in the control groups(P<0.01), 0.50×107CFU/ml groups(P<0.05) and 1.00×107CFU/ml groups(P<0.01).Conclusion:(1) Colonization of H. pylori in gastric mucosa of mice could increase the number of Foxp3+Treg cells in gastric mucosa, which might take part in escaping from host immune clearance. This may be one of the important reasons that H. pylori cause chronic and persistent infection in vivo.(2) In gastric carcinoma subjects, no matter H. pylori negative or positive, the number of Foxp3 positive cells in gastric mucosa was significantly increased, which indicated that there was immunosuppression in gastric carcinoma patients.(3) The number of CD4+CD25+Foxp3+Treg cells in gastric mucosa of H. pylori eradicated mice significantly decreased, it is secondary to H. pylori eradication, or it is made for H. pylori eradication that the number of CD4+CD25+Foxp3+Treg cells in gastric mucosa decreased, it remains to be further studied.(4) H.pylori down-regulate the expression of Foxp3 mRNA and decrease the number of CD4+CD25+Treg cells in lymphocytes of mice in vitro, indicating that H. pylori could inhibit the differentiation of lymphocytes towards Treg cells, which is different to the influence of H. pylori in vivo. And this may be one of the mechanisms of different immune responses induced after natural infection of H. pylori and immunization of H. pylori vaccine.
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