We are using mouse models to study H. pylori infection, pathogenic mechanism(s) and mucosal-immune responses. We selected wild type (wt) C57BL/6 and C57BL/6 IL-10 knockout mice to evaluate the role of endogenous IL-10 on the regulation of mucosal immune response to H. pylori infection. The IL-10 knockout (IL-10-/-) mice allowed us to examine the in vivo role of IL-10 response as it relates to bacteria load per gram of stomach tissue and pathology. We monitored histological changes by pathology scoring. In collaboration with Dr. Richard DiPaolo, Saint Louis University Medical School, we developed a new technique that allows us to isolate the inflammatory cells from the stomach mucosa of mice. Using this technique, we identify, quantify, and determine the functions and specificities of cells infiltrating the stomach after infection. We observed a 7-fold increase of CD4+ and a 6-fold increase of CD8+ T cells infiltrating the stomach tissue of wt infected mice. The functional assays allowed us to examine the CD4+ and CD8+ T cell responses in the stomach after H. pylori infection. For example, using this technique we confirmed a previously reported observation that T cells infiltrating the stomach produce IFN (a T-helper type 1 response). We also made a new observation that there are T cells producing IL-17, and T cells producing both IL-17 and IFN. The discovery of IL-17 producing T cells suggests an additional T-helper type 17 response to H. pylori infection. This is an important finding because IL-17 production has been associated with the recruitment of neutrophils, causing and sustaining tissue damage related to various autoimmune disorders. This finding provides a link between H. pylori infection and autoimmune gastritis. We are currently characterizing the immune response in H. pylori infected IL10-/- mice, and comparing the response with wild type mice. Thus far, we found that despite having several orders of magnitude fewer H. pylori per gram of stomach tissue, the IL-10-/- mice have more cytokine producing (IFNg/IL17/TNFa) CD4+ and CD8+ T cells (5- and 4-fold, respectively than wt infected mice) in the stomach and a more severe gastritis. We are characterizing these responses in greater detail to determine whether the CD4+ and CD8+ T cells infiltrating the stomachs in IL10-/- mice can transfer the disease in non-infected mice. If so, this may indicate that lacking IL10 is a double-edged sword in H. pylori infection, i.e, greater immunity to the pathogen (lower colony counts) but higher susceptibility to autoimmunity. We are also studying innate and adaptive immunity as it relates to H. pylori infection. The cellular and molecular mechanisms that initiate H. pylori adaptive immunity and T-cell response are poorly understood. Dendritic cells are central to the initiation of adaptive immunity. We initiated in vitro studies to compare immune responses to H. pylori infection involving a common adaptor molecule, myeloid differentiation protein (MyD88) and selected Toll-like receptors. Our goal is to assess the relative contribution of MyD88 and Toll-like receptors 2 and 4 in host response to H. pylori infection and to monitor T-cell responses. An in vitro approach was used to demonstrate that naive T cells could be induced to produce IL-17 when cocultured with DC exposed to H. pylori. Purified splenic T cells or a T cell subset (CD8+ or CD4+ cells) from wt C57BL/6 mice were cocultured in vitro with H. pylori exposed DC from MyD88-/-, TLR2-/-, TLR4-/-, TLR2-/-/TLR4-/- (double knockout animals) or wt C57BL/6 mice. Significant amount of IL-17 (4500 pg/ml) was detected in the supernatants of DC/lymphocyte cocultures by ELISA when the primed DC were from wt C57BL/6 mice. IL-17 production was significantly reduced (600 pg/ml) in supernatants from cocultures containing DC from MyD88 knockout mice or TLR2/TLR4 double knockout mice. We conducted flow cytometric analysis to characterize and quantify the cellular components of the cocultures at the single cell level. Similarly, we found that after H. pylori stimulation, wt DCs induced IL-17 production in T cells (8.09% of CD4+ cells and 22.3% of CD8+ cells). In contrast, less than 1% of the T cells produced IL-17 when cocultured with DC from MyD88 KO mice or TLR2/TLR4 DKO mice. These findings strongly demonstrated that the induction of T cells to produce IL-17 by H. pylori primed-DC is MyD88 dependent and partially dependent on TLR2 and TLR4. The differentiation of naive T cells to Th-17 subset requires IL-6, TGF-βand IL23. We used ELISA and the supernatants of wt DC cultures exposed to H. pylori and detected the secretion of these differentiating cytokines. mRNA expression levels for these cytokines increased in DC from wt mice 3 hours post-infection. This increase in differentiating cytokine expressions necessary for TH-17 response is MyD88 dependent. Animal studies confirmed an IL-17 local immune response and a significant increase in infiltration of CD4+ and CD8+ T-cells to H. pylori infection of gastric tissue. Flow cytometric studies of stomach infiltrates indicated that CD4+ T-cells were the primary T-cell subset involved in the IL-17 immune response. This observation is in contrast to a more prominent role for CD8+ T cells found in in vitro studies. Immunohistochemistry analysis of stomach infiltrates in gastric tissues also provided evidence that CD4+ were the primary IL-17 producer in vivo. Real-time PCR analyses of mouse stomach tissues, three months post-infection, indicated an up-regulation of IL-17 and interferon gamma expression. IL-17 and interferon gamma expression was essentially abrogated in the stomach tissue of MyD88 and TLR2/TLR4 knockout mice. GM-CSF expression, stimulated by IL-17, was also increased in stomach tissues of mice infected with H. pylori. Our results clearly indicate that Helicobacter pylori induces IL-17 signaling in murine models. In collaboration with Dr. Griffin Rodgers research group, Molecular and Clinical Hematology Branch, NHLBI, we showed that olfactomedin 4 (OLFM4), a novel member of the olfactomedin-related glycoprotein superfamily, plays a role in the host immune response to H. pylori infection. OIFM4 is constitutively expressed in neutrophils and the gastrointestinal tract. We demonstrated that OLFM4 acts as anti-inflammatory agents after H. pylori infection. We generated OLFM4-/- mice to investigate potential role(s) of OLFM4 in gastric mucosal responses to H. pylori infection. Histological examination of tissues including bone marrow, esophagus, small intestine and colon did not reveal any discernable abnormalities in OLFM4-/- mice. H. pylori colonization in the gastric mucosa of OLFM4-/- mice was significantly lower compared with wild-type littermates. Production and expression of proinflammatory cytokines/chemokines such as IL-1β, IL-5, IL-12 p70, and MIP-1αwas increased in OLFM4-/- mice compared with infected controls. Dipeptidyl peptidase I (DPPI) was identified as a binding partner for OLFM4. Proteolytic activity of DPPI, cathepsin G and neutrophil elastase was significantly higher in neutrophils of OLFM4-/- infected mice compared with wild-type mice. Furthermore, OLFM4 appeared to negatively affect NF-κB activation during H. pylori infection via a direct interaction with nucleotide-binding oligomerization domain-2 (NOD2). These data suggest that OLFM4 may limit host responses against H. pylori infection and contribute to persistent H. pylori infection.
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