Veterinary Science - Theses
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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.