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ABSENCE OF FUNCTIONAL PROTEINASE-ACTIVATED RECEPTOR-2 (PAR₂) MODULATES INFLAMMATORY RESPONSES IN MOUSE COLON INDEPENDENT OF TH CYTOKINE PROFILE

E Hyun¹, P Andrade-Gordon², N Vergnolle¹
¹Mucosal Inflammation Research Group, University of Calgary, Calgary, Alberta; ²Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania, USA

Trypsin and tryptase can act as inflammatory mediators especially via cleaving and activating the second member of proteinase-activated receptors (PAR₂). The aim of this study was to examine the effects of PAR₂ deficiency (via use of PAR₂-deficient mice: PAR₂^-/-) in various models of colonic inflammation in order to elucidate the role of endogenous PAR₂ during the course of inflammation in the gut. Colonic inflammation in C57BL6 wild type and PAR₂^-/- mice was induced by treatment with 2.5% DSS (in drinking water) or TNBS (1mg or 2mg in 100ul of 40% ethanol, intracolonically) or pre-sensitizing mice with 3% oxazolone (dissolved in olive oil) applied to the skin of the abdomen, and 7 days later, a single intracolonic injection of 1% oxazolone (dissolved in 50% ethanol). Each models of colitis exhibits different Th cytokine profiles; TNBS with Th1 cytokines (IL-2 and INF-gamma), oxazolone with Th2 cytokines (IL-4 and IL-10), and oxazolone with both Th1 and Th2 cytokines (IL-2 and IL-4). Intravital microscopy was performed, 7 days (TNBS/DSS) or 4 days (oxazolone) after induction of colitis on selected venules of the colon to assess changes in leukocyte rolling, adhesion and vessel diameter. Macroscopic/microscopic damage scores, bowel thickness and myeloperoxidase (MPO) activity were also measured. PAR₂^-/- mice showed significantly lower leukocyte adherence and vessel dilation compared to the wild type mice in DSS, and TNBS challenge. Treatment with DSS, TNBS, and oxazolone resulted in significantly lower MPO activity in PAR₂^-/- compared to wild type. In all three challenges, macroscopic damage score and bowel thickness in wild type were significantly higher than that of PAR₂^-/- mice. Lastly, PAR₂^-/- treated with TNBS and oxazolone showed significantly lower amount of weight loss as compared to the wildtype mice. Therefore, our evidences indicated that the absence of PAR₂ attenuates the inflammatory responses of the gut potentially by modulation of the leukocyte recruitment, regardless of the Th cytokine profile of colitis. The reductions in various inflammatory parameters in PAR₂^-/- mice in three different gastrointestinal inflammatory models indicate a pro-inflammatory role of PAR₂ in the mice colon. This research is a first major attempt to determine the role of endogenous PAR₂ during intestinal inflammation thereby assessing the potential therapeutic effects of PAR₂ antagonism.

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