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The Role of Visceral Peritoneum in LPS Mediated Inflammatory Responses
[Thesis]. Manchester, UK: The University of Manchester; 2017.
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Abstract
Peritoneal adhesions pose a considerable postoperative burden. The mechanisms underlying adhesion formation remain poorly understood but relate principally to inflammation and fibrinolysis. The peritoneum may coordinate the inflammatory response that leads to adhesion formation but human adhesion formation cannot be studied directly so most research has employed animal models and individual transformed cell lines. Furthermore, the specific roles of the visceral or parietal peritoneum in adhesion formation are unclear. The aims of the studies undertaken in this thesis are to investigate the regulation of a gut bacteria-driven inflammatory response in human visceral peritoneum. A novel ex vivo model was designed using human small bowel visceral peritoneum, cultured with E. coli derived LPS, for 18 to 48 hours. ELISA and rtPCR were used to analyse supernatant protein concentration and gene expression respectively for key inflammatory mediators associated with adhesion formation including cytokines (IL-6, IL-10, TNFα), transforming growth factor-β1 (TGF-β1) and fibrinolytic factors (tPA and PAI-1) as well as the signalling receptors thought to be involved in LPS-mediated inflammation (TLR2 and 4). LPS exposure resulted in stimulation of IL-6, IL-10 and TNFα production from human visceral peritoneum at 18 hours. These increases correlated with increased gene expression for IL-6 and TNFα but not IL-10. TGF-β1 production and gene expression were unchanged at 18 & 24 hours but increased at 48 hours when co-incubated with IL-1β. PAI-1 was increased but tPA was unchanged following LPS stimulation, suggesting a reduction in peritoneal fibrinolytic activity. Peritoneal tissue expressed the signalling receptors TLR 2 and 4. However, inhibition of TLR 2 or 4 failed to abrogate production of cytokines in cultured primary mesothelial cells or peritoneal tissue post-LPS exposure. TLR 4 inhibition did reduce mesothelial cell production of chemokines, CCL2 and CXCL1. These novel findings demonstrated that human visceral peritoneum actively participates in LPS-induced peritoneal inflammatory responses and reduced fibrinolysis which may account for the visceral peritoneum’s affinity to form adhesions. Ex vivo human peritoneal culture is a novel, feasible and reproducible method of investigating the role of the peritoneum in inflammation.