Proteinase-Activated rreceptor-2 (PAR 2 ), a G-protein–coupled Receptor, activated by serine proteinases, is reported to have both protective and proinflammatory effects in the airway. Given these opposing actions, both inhibitors and activators of PAR 2 have been proposed for treating asthma. PAR 2 can signal through two independent pathways: a β-arrestin–dependent one that promotes leukocyte migration, and a G-protein/Ca 2+ one that is required for prostaglandin E 2 (PGE 2 ) production and bronchiolar smooth muscle relaxation. We hypothesized that the proinflammatory responses to PAR 2 activation are mediated by β-arrestins, whereas the protective effects are not. Using a mouse ovalbumin model for PAR 2 -modulated airway inflammation, we observed decreased leukocyte recruitment, cytokine production, and mucin production in β-arrestin-2 −/− mice. In contrast, PAR 2 -mediated PGE 2 production, smooth muscle relaxation, and decreased baseline airway resistance (measures of putative PAR 2 “protective” effects) were independent of β-arrestin-2. Flow cytometry and cytospins reveal that lung eosinophil and CD4 T-cell infiltration, and production of IL-4, IL-6, IL-13, and TNFα, were enhanced in wild-type but not β-arrestin-2 −/− mice. Using the forced oscillation technique to measure airway resistance reveals that PAR 2 activation protects against airway hyperresponsiveness by an unknown mechanism, possibly involving smooth muscle relaxation. Our data suggest that the PAR 2 -enhanced inflammatory process is β-arrestin-2 dependent, whereas the protective anticonstrictor effect of bronchial epithelial PAR 2 may be β-arrestin independent.

Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR2) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2/β-arrestin signaling axis, identify the protease activity responsible for PAR2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2/β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2−/− and β-arrestin-2−/− mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR2−/− or β-arrestin-2−/− mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR2 signaling and induce β-arrestin-2−/−-dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2/β-arrestin signaling axis. Thus, β-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.

Functional blockade of PAR2 in the airways during allergen challenge improves allergen-induced AHR and inflammation in mice. Therefore, topical PAR2 blockade in the airways, through anti-PAR2 antibodies or molecules that interrupt PAR2 signalling, has the potential to be used as a therapeutic option in allergic asthma.

As asthma prevalence, health care use, and mortality increase therapeutics with a distinct target are needed. One attractive target is a G Protein Coupled Receptor (GPCR) known to play both protective and pro‐inflammatory roles in the airways. Namely, Proteinase Activated Receptor‐2 (PAR2) is activated by serine‐like proteases such as trypsin, mast cell tryptase, and those found in cockroach frass and Alternaria alternata. Given the dual and apparently opposing actions, both inhibitors and activators of PAR2 have already been proposed as therapeutics for asthma. PAR2 can signal through two independent pathways: a G‐protein‐dependent and a β‐arrestin‐dependent/G‐protein‐independent one. The β‐arrestin‐dependent pathway promotes leukocyte migration, while bronchiolar smooth muscle relaxation requires G‐protein signaling intermediates. We use a mouse ovalbumin model for PAR2‐modulated airway inflammation to address the hypothesis that inflammatory responses to PAR2 activation are β‐arrestin dependent while ‘protective’ anti‐constrictor effect of bronchial epithelial PAR2 are β‐arrestin‐independent. We show that PAR2‐ induced lung inflammation, mucus production, Th2 cytokines, airway responsiveness and cellular inflammation were abolished in β‐arrestin‐2−/− mice while tracheal smooth muscle relaxation and PGE2 production were equivalent in wild type and β‐arrestin‐2−/− mice.

A common cause of asthma is the exposure to allergens such as the household fungus, Alternaria alternata (AltA), and the feces of the common cockroach, Blattella germanica (BG), which contain proteases that activate Protease‐activated‐receptor‐2 (PAR2). In several murine models of asthma, PAR2 activation has been shown to be pro‐ and anti‐inflammatory. Recently, our lab has shown that the pro‐inflammatory pathway is mediated by beta‐arrestins, which act as both terminators of GPCR signaling through G‐proteins, and as scaffolding proteins that signal independently of G‐proteins. In contrast, the anti‐inflammatory pathway requires G‐protein signaling. We hypothesize that proteases from AltA and BG extracts will promote activation of the beta‐arrestin‐2 dependent inflammatory pathway in murine models that we have previously observed with intranasal administration of Par2 peptidomimetic agonists. Here, we have performed flow cytometric analyses of immune cell populations and analyzed histological samples in physiological mouse models of AltA and BG‐induced allergic asthma. Our results show that airway inflammation induced by AltA and BG are abolished in beta‐arresitn‐2‐/‐ and PAR2‐/‐ mice. Furthermore, AltA and BG proteases activate downstream signaling mechanisms in a beta‐arrestin‐2‐dependent way. This suggests that the proteases can activate the PAR2‐beta‐arrestin signaling axis, which in turn regulates immunological responses in the airway.