The Characteristics And Role Of IL-22 In Helicobacter Pylori Infection

Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium that resides extracellularly in the gastric mucosa and infects more than 50% of the population worldwide. H. pylori-induced chronic inflammation is the cause of gastritis and peptic ulcer and a risk factor for gastric cancer. H. pylori persistent infection causes severe local inflammation in the gastric mucosa but the detailed pathogenic mechanism is not clear now.Cytokine as the message molecule induces specific biological effect. Interaction between pathogen and host results in H. pylori chronic infection and many cytokines play different roles during the infection. It's proved that CD4~+ T cells (Th cells) play important roles in H. pylori persistent infection, so the study of H. pylori inducing cytokines focused on the Th cells produced. To date, it is generally accepted that gastric inflammation largely depends on IFN-?secretion and IL-4 relieves inflammation and reduces H. pylori colonization.Recently, some novel subsets of effector T cell have been identified, such as Th17, Th22 and Th9. The recent studies have shown that novel T cell subsets have played important roles in inflammatory diseases. The study of cytokines produced by novel effector T cells may increase a better understanding of mechanism in chronic inflammation. The currently published data show that IL-22, which is produced by Th17 and Th22 cells, is an important mediator in dermal inflammation and it activates epithelial cells to secrete pro-inflammatory chemokines and cytokines. However, During K. pneumonia infection of the lung, C. rodentium infection of colon, IL-22 pathway have elucidated its essential protective role in mucosal immunity against these bacterial pathogens. It suggests that IL-22 is a dual-natured cytokine, depending on the context of inflammation, it can have either proinflammatory or protective properties. In our incipient study, we firstly found that IL-22 expression increased in H. pylori-infected gastric tissue. However, the characteristics and role of IL-22 in H. pylori infection have not been elucidated.?Objectives? 1. To characterize IL-22 expression in H. pylori infection.2. To elucidate the role of IL-22 in H. pylori infection and explore the mechanism.?Methods?1. The characteristic of IL-22 expression in H. pylori infected gastric tissueThe gastric biopsy specimens were collected in Gastroenterology department of XinQiao Hospital. Cytokines mRNA expression and the colonization of H. pylori were detected by real-time PCR and the protein level of IL-22 was examined by ELISA. The gastric inflammation was assayed by H&E staining. Some patients were treated with Nexium, Amoxicillin and Clavulanate Potassium and furazolidone for one week. The gastric biopsy specimens were collected after treatment and IL-22 expression was detected by real-time PCR and the gastric inflammation was assayed by H&E staining.2. The response of IL-22-producing T cell in H. pylori infected gastric tissueFirstly, the gastric epithelial cells model infected with H. pylori 26695 were established. IL-22 expression in H. pylori 26695 infected cell models was measured by real-time PCR and the major source of IL-22 was further detected by Double Immunofluorescence staining and FCM. Secondly, IL-22-producing T cell responses were examined by FCM in the gastric mucosa. Lastly, the virulence factor of H. pylori inducing IL-22 expression was identified by real-time PCR in H. pylori infected cell models and gastric tissue.3. The role of IL-22 in H. pylori infectionThe IL-22R1 expression was measured by FCM under the stimulation of various factors on AGS cell surface and real-time PCR and Immunofluorescence staining were used to detect the IL-22R1 expression in gastric tissue. Gastric epithelial cells were stimulated with commercial IL-22 for 24h and cells were collected for analysis of proinflammatory factors and MMPs mRNA expression by real-time PCR. Proinflammatory factors and MMPs mRNA expression were also detected by real-time PCR in gastric tissue and IL-22 expression was analyzed in relation to these factors expression. The influence of IL-22 on lymphocyte chemotaxis was detected by Transwell chemotaxis assay.?Results?1. The characteristic of IL-22 expression in H. pylori infected gastric tissue1.1 Gastric mucosal IL-22 mRNA and protein levels were significantly higher in H. pylori-positive patients than uninfected patients (P < 0.05), simultaneously, the IL-17A, IL-17F and IFN-?mRNA expression were also increased in H. pylori-infected patients (P < 0.05).1.2 Gastric mucosal IL-22 mRNA levels showed significant positive correlation with H. pylori copies in H. pylori-infected people (r = 0.404; P < 0.01). IL-22 mRNA expression in moderate and severe inflammation was higher than mild inflammation tissue and IL-22 mRNA in mild inflammation was also expressed at a high level than normal tissue (P < 0.05). These results showed that IL-22 expression was correlated with the degree of inflammation.1.3 IL-22 mRNA levels significantly decreased after a successful eradication treatment (P < 0.05) and H. pylori eradication results in a marked alleviation of gastric inflammation (P < 0.05). These results proved that IL-22 was correlated with H. pylori copies and inflammation.2. The response of IL-22-producing T cell in H. pylori infected gastric tissue2.1 After the incubation of H. pylori 26695 with the AGS cells for 24h, induction of the scattering phenotype and IL-8 release were observed. These results showed that we established a H. pylori 26695 infected cell model. IL-22 mRNA expression was significantly increased in the three gastric epithelial cell lines and T cell line by H. pylori 26695 infection (P < 0.05). IL-22 and cell marker protein expression was also detected in gastric tissue section by Double Immunofluorescence staining, suggesting that IL-22 was produced by epithelial cell and T cells.2.2 lymphocytes from gastric mucosa were isolated and detected by intracellular cytokine staining. The results showed that IL-22 was both produced by CD4 and CD8 T lymphocytes and the phenotype of IL-22-producing T cells displayed almost CD45RO positive, which showed that in the gastric tissue, the IL-22-producing T cells are mainly memory cells. Further, We found that the percentages of IL-22~+ CD4~+, IL-22~+ CD8~+, IL-17~+ CD4~+, IL-17~+ CD8~+, IFN-?~+ CD4~+ T cells were significantly increased in H. pylori-infected patients as compared to H. pylori-negative individuals (P < 0.05), but no difference in IFN-?~+ CD8~+ T cells. Lastly, we analyzed that percentages of IL-22~+IL-17~+ cells and IL-22~+IFN-?~+ cells in the CD4 or CD8 gate were all increased in the H. pylori-infected patients (P < 0.05). These results showed that IL-22-producing T cells were increased in H. pylori-infected gastric mucosa.2.3 Three gastric epithelial cell lines and T cell line were infected with CagA mutant, UreB mutant or initial H. pylori 26695 strain for 24 h, IL-22 induction was reduced from gastric epithelial cells and T cells cocultured with CagA mutant ( P < 0.05 vs H. pylori 26695 strain), whereas IL-22 induction was similar to H. pylori 26695 strain using the UreB mutant. The IL-22 mRNA expression was also significantly higher in CagA positive patients (P < 0.05) and there was no difference between CagA negative and uninfected patients. These results suggested that CagA might be the virulence factor of H. pylori for regulating IL-22 expression.3. The role of IL-22 in H. pylori infection3.1 We detcted IL-22R1 expression on AGS cell surface under the stimulation of various factors by FCM and found that the mean fluorescence intensity (MFI) of IL-22R1 was upregulated with H. pylori 26695 infection, but no difference with cytokines and recombination proteins stimulation. Immunofluorescence staining showed that IL-22R1 expression was positive in gastric tissue and IL-22R1 mRNA expression in the gastric tissue was elevated in H. pylori-infected patients compared to the H. pylori-negative individuals (P < 0.05). These rusults indicated that H. pylori could regulate IL-22R1 expression in gastric epithelial cells.3.2 The gastric epithelial cells were stimulated with IL-22 for 24 h in vitro and the proinflammatory factors and MMPs expressions were detected by real-time PCR. The results showed that IL-22 significantly induced S100A8, S100A9, IL-8, MMP-1 and MMP-10 mRNA expressions in cell lines (P < 0.05). We also detected these gene expressions in the gastric tissue by real-time PCR. The results showed that IL-8, S100A8 and S100A9 were significantly increased in H. pylori-positive individuals (P < 0.05), but no differences in MMP-1 and MMP-10 expressions. We further analyzed the correlation between IL-22 and the proinflammatory factors expressions in the gastric tissue and found that IL-22 mRNA expression was significantly positive correlated with the IL-8, S1008A and S100A9 mRNA expressions, but not correlated with the MMP-1 and MMP-10 expressions. All these results suggested that IL-22 might participate the inflammatory reaction through producing pro-inflammatory factors in H. pylori infection3.3 The effect of IL-22 on lymphocytes was detected by Transwell chemotaxis assay. The results showed that there is no effect on lymphocytes with IL-22 alone, but after coculture with AGS cells for 24 h, IL-22 could induce much more lymphocytes in the lower chamber than AGS cells in chamber alone (P < 0.05). To determine whether this effect was mediated by IL-8, PBMCs were incubated with conditioned medium in the presence or the absence of neutralizing anti-IL-8 Ab. The anti-IL-8 Ab inhibited the chemotaxis of lymphocytes in response to IL-22 stimulated AGS cells (P < 0.05). These results suggested that IL-22 participated the inflammatory reaction by producing IL-8, which could recruit the lymphocytes in gastric tissue.?Conclusion?1. Gastric mucosal IL-22 expression was significantly higher in CagA positive H. pylori-infected patients than uninfected patients and IL-22 mRNA levels showed significant positive correlation with H. pylori colonization and inflammation in gastric tissues.2. The major source of IL-22 was epithelial cell and memory T cell and IL-22-producing T cells were significantly increased in H. pylori-infected patients as compared to H. pylori-negative individuals in the gastric mucosa. CagA was the virulence factor of H. pylori for regulating IL-22 expression.3. IL-22 participated inflammatory reaction by regulating proinflammatory factors expressions and inducing inflammatory cell infiltration during H. pylori infection.?Significance?Further study of the function and regulation of IL-22 after H. pylori infection may help to elucidate the characteristics of immune response and the pathogenic mechanism of H. pylori infection and to explore novel and effective therapies for H. pylori-associated diseases.