Development and Testing of a Novel Humanized Protease-Activated-Receptor-2 Mouse as a Model for Allergic Asthma

Abstract

Allergic asthma, the most common form of asthma, is characterized by airway inflammation, mucus overproduction, leukocyte infiltration, and airway hyperresponsiveness (AHR). Allergic asthma can be triggered by allergen exposure, which includes house dust mites (HDM) and Alternaria alternata (A.alternara). Protease-activated-receptor-2 (PAR2) activation by HDM and A. alternata has been linked to the development of asthma indicators in preclinical models. Despite the critical role of PAR2 in allergic asthma, limited research has been conducted on targeting PAR2 in treating allergic asthma, all of which has been performed on mice expressing the murine receptor (mPAR2). In this thesis, I address this gap in research with the development of a transgenic mouse model expressing only the human form of PAR2 (hTgPAR2) devoid of mouse PAR2 in a C57Bl/6 background. Both hTgPAR2 and C57Bl/6 mice responded to acute HDM challenge with increased airway inflammation, mucus production, and leukocyte migration with limited sex differences. However, the hTgPAR2 mouse presented with significantly increased AHR in response to HDM exposure in both male and female mice, whereas the C57Bl/6 mice displayed no difference from control. We have shown that A. alternata can induce allergen indicators in pre-clinical mouse models and that these indicators can be pharmacologically controlled by a full (e.g., Gq/Ca2+ and β-arrestin-2/MAPK signaling) PAR2 antagonist (C391) or a biased β-arrestin-2/MAPK signaling (C781) PAR2 antagonist. As with C57Bl/6 and BALB/c mouse strains, acute A. alternata exposure induced AHR, increased inflammation, and mucus production in hTgPAR2 mice. However, clear sex differences were observed with the male hTgPAR2 mice driving the increased AHR and female hTgPAR2 mice driving heightened inflammation and mucus production. When tested in hTgPAR2 mice, the biased PAR2 antagonist, C781, demonstrated greater efficacy in controlling AHR induced by A. alternata than C391. Lastly, two new PAR2 antagonists, C937 and C938 possess improved pharmacokinetics and are shown to be biased PAR2 antagonists in in vitro studies, selectively limiting the β-arrestin-2/MAPK signaling pathway. Overall, my findings suggest that the hTgPAR2 mouse may serve as a model for allergic asthma and as an in vivo tool for evaluating PAR2 antagonists in the treatment of allergic asthma, with biased PAR2-β-arrestin-2/MAPK antagonists such as C781 appearing to be a promising therapeutic approach.