Veterinary Science - Theses
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ItemRole of mucosal-associated invariant T (MAIT) cells in Helicobacter pylori infection( 2017)Helicobacter pylori infects the human stomach of approximately half the world’s population and is aetiologically associated with a range of pathologies including gastritis, duodenal ulcers and gastric cancer. The development of severe disease depends on a complex interplay of the host immune system, bacterial virulence factors and environmental factors. A key feature that determines which H. pylori positive individuals develop disease is the severity of inflammation. Mucosal-associated Invariant T (MAIT) cells are innate like T cells that are restricted by the non-classical MHC class I-related molecule, MR1. MAIT cells detect antigens that are derived from microbial vitamin B2 (riboflavin) synthesis, and produce inflammatory cytokines, including IL17, IFNγ and TNF, and cytotoxic granzymes. Although MAIT cells have been shown to be protective against some pulmonary infections, their production of pro-inflammatory cytokines suggest chronic stimulation of MAIT cells could contribute to pathology. The studies presented in this thesis aimed to understand the role of MAIT cells in the development of gastritis during H. pylori infection and identify the mechanisms by which MAIT cells perform this role. Using highly specific MR1 tetramers and mouse models of H. pylori infection, it was shown that MAIT cells accumulate in the stomach and accelerate pathology. This occurred both in MAIT TCR transgenic mice, and in a prime-boost model of C57BL/6 mice where MAIT cells were first primed intranasally either with Salmonella Typhimurium or with TLR agonist plus synthetic antigen. MAIT cells have the ability to recruit other T cells and innate immune cells that then help precipitate disease and exacerbate inflammation leading to atrophic gastritis. The results in this thesis also demonstrate that the riboflavin pathway is essential for MAIT TCR based stimulation and that neither antigen alone nor TLR agonist alone can induce MAIT cell expansion, suggesting a co-stimulus either in the form of bacterial infection or TLR stimulation is essential for MAIT cell expansion and induction of pathology on H. pylori infection. MAIT TCR repertoire analysis revealed that the TCR repertoire does not change dramatically post infection with either S. Typhimurium in the lung or H. pylori infection in the stomach, suggesting shared antigen specificity. In summary, the studies in this thesis demonstrated a pathogenic role for MAIT cells for the first time in Helicobacter-associated inflammation. These findings add to our understanding of MAIT cells and implicate MAIT cells in chronic inflammation, and will help develop therapeutic strategies to target these cells in inflammatory disorders.
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ItemMucin 1 regulation of Helicobacter pylori-induced pathology( 2012)Helicobacter pylori infects the gastric mucosa of approximately half of the world’s population. H. pylori infection causes a range of gastric pathologies, ranging from mild gastritis to clinically significant diseases, the most severe of which is gastric adenocarcinoma. The resistance or susceptibility of individuals to develop pathologies resulting from H. pylori infection is believed to be due to the influence of many environmental, bacterial and host genetic factors. One host genetic factor that has been associated with H. pylori-induced gastritis and gastric adenocarcinoma is the cell surface mucin MUC1. Muc1 has also been experimentally demonstrated to limit colonisation by H. pylori and the associated gastritis resulting from infection. While MUC1 has been identified to limit H. pylori colonisation by acting as a physical barrier and decoy adhesion target when expressed on the apical surface of mucosal epithelial cells, the mechanism by which MUC1 limits H. pylori-associated gastritis and gastric carcinogenesis is unclear. The studies presented in this thesis aimed to experimentally demonstrate a role for Muc1 in regulating further sequelae of H. pylori infection, such as gastric cancer, and to identify the mechanisms by which Muc1 performs this role. Muc1 was shown to be essential in modulating disease susceptibility, as >50% of Muc1-/- mice infected long-term with H. pylori succumbed to infection, while wildtype mice remained asymptomatic. Signs of dysplasia or spasmolytic polypeptide-expressing metaplasia (a precursor) were seen in 33% of these long-term infected Muc1-/- mice compared to only 4% of long-term infected wildtype mice. The immunological mechanism by which Muc1 limits H. pylori-induced pathologies was found to be i) dependent on expression of Muc1 on haematopoietic cells and ii) associated with elevated IL-1β production. H. pylori was demonstrated to induce, and Muc1 to suppress IL-1β secretion. IL-1β is produced as a pro-cytokine, with proteolytic maturation dependent on the formation of inflammatory cytosolic complexes called inflammasomes. The suppressive effect of Muc1 was narrowed down to the Nlrp3 inflammasome, and the capability of Muc1 to limit Nlrp3 expression identified as a possible molecular mechanism. In summary, the studies in this thesis have not only experimentally demonstrated the crucial role of Muc1 in further sequelae of H. pylori infection and identified an immunological and molecular mechanism by which Muc1 limits H. pylori-induced pathology, but have also identified a completely novel interaction between Muc1 and the Nlrp3 inflammasome. These findings add to our understanding of MUC1 regulation of H. pylori-induced pathologies, and have broad implications for the role of MUC1 in regulating other inflammatory diseases.
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ItemThe regulatory effects of protease activated receptor 1 on Helicobacter pylori-induced inflammation( 2010)Helicobacter pylori, one of the world’s most prevalent pathogens, infects the gastric mucosa of approximately half the human population. These infections cause a wide range of pathologies from asymptomatic gastritis to gastric cancer due to host genetic variation and a multitude of diverse factors that govern host-pathogen interactions. One host factor hypothesised to be the key regulator of H. pylori-induced gastritis is G protein-coupled Protease-Activated Receptor 1 (PAR1). PAR1, is the downstream signaller of thrombin, a crucial protease generated during inflammation and the main effector of the blood coagulation cascade. It is also known that H. pylori infected patients have higher PAR1 expression in the stomach compared to healthy individuals. Given this knowledge, this thesis was directed towards obtaining a greater understanding of the role of PAR1 in H. pylori-induced gastritis. The role of PAR1 in H. pylori inflammation was examined using PAR1-/- and wild-type mice. H. pylori infection of PAR1-/- mice induced significantly more severe gastritis compared to wild-type controls. H. pylori infected PAR1-/- mice also expressed more pro-inflammatory cytokines (MIP-2, IFNγ and IL-17) in the gastric tissues. These results demonstrated that PAR1 has a protective function against H. pylori induced gastritis. H. pylori stimulation of primary gastric epithelial cells deficient in PAR1 induced increased levels of NF-κB and the pro-inflammatory cytokine, MIP-2 production which is consistent with observations made in vivo. This study suggested that gastric epithelial cells could contribute to the regulation of inflammation. Subsequently, through the use of bone marrow chimeras, it was demonstrated that haematopoietic cells and not epithelial cells play a major role in PAR1 regulation of gastritis. To investigate if PAR1 has a role protective immunity against H. pylori, PAR1-/- and wild-type mice were vaccinated with formalin fixed H. pylori. Immunisation of PAR1-/- and wild-type mice induced a significant increase in antibody titres (including IgA, IgG1 and IgG2 subclasses) in both PAR1-/- and wild-type mice. However, vaccination of PAR1-/- and wild-type mice produced similar reductions in H. pylori colonisation, suggesting that PAR1 does not play an important role in vaccine-mediated protection against this infection. To further indentify the cells types involved in regulating H. pylori-induced inflammation, splenocytes, macrophages and dendritic cells were obtained from PAR1-/- and wild-type mice. No differences were observed in MIP-2 production from PAR1-/- and wild-type macrophages and dendritic cells suggesting that macrophages and dendritic cells are not involved in the PAR1 regulatory process. In contrast, splenocytes stimulated by PAR1 activating peptide and H. pylori expressed significantly less MIP-2 and IFNγ than splenocytes stimulated by H. pylori alone supporting the in vivo findings. Furthermore, it was also observed that PAR1 expression plays a positive role in the regulation of PAR2 expression in splenocytes. PAR2 unlike PAR1 is activated by trypsin and has a pro-inflammatory role in H. pylori infections, as PAR2-/- mice develop reduced severity of H. pylori-induced gastritis. This suggested while PAR2 has an opposing role to PAR1, during inflammation, the expression PAR1 and PAR2 may work in a positive feedback loop mechanism. In summary, this thesis demonstrated that PAR1 is an important host factor in H. pylori pathogenesis. This effect is mediated by haematopoietic cells, where PAR1 modulates pro-inflammatory cytokines production. In vitro studies supported this novel in vivo finding and also demonstrated that PAR1 can modulate PAR2, presumably via a feedback loop mechanism. Further investigations into the specific cell(s) involved in the protective effect of PAR1 in H. pylori-induced gastritis would prove invaluable to identify potential signalling mechanisms of PAR1 which would be useful in the treatment of H. pylori pathogenesis and other inflammatory diseases.