Proteinase‐activated receptor‐2 antagonist C391 inhibits Alternaria‐induced airway epithelial signalling and asthma indicators in acute exposure mouse models

Abstract

Background and Purpose Despite the availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase‐activated receptor‐2 (PAR2) has been identified in preclinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 raises intracellular Ca2+, inducing MAPK and β‐arrestin signalling in the airway, leading to inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic‐ and trypsin‐induced PAR2 signalling in vitro as well as reducing inflammatory PAR2‐associated pain in vivo. We hypothesized that PAR2 antagonism by C391 would attenuate allergen‐induced acutely expressed asthma indicators in murine models. Experimental Approach We evaluated the ability of C391 to alter Alternaria alternata‐induced PAR2 signalling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o−) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata‐induced acutely expressed asthma indicators in vivo in two murine strains. Key Results C391 blocked A. alternata‐induced, PAR2‐dependent Ca2+ and MAPK signalling in 16HBE14o− cells, as well as β‐arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata‐induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute allergen‐challenged murine models. Conclusions and Implications To our best knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen‐induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first‐in‐class allergic asthma drugs.

A scoping review: What are the cellular mechanisms that drive the allergic inflammatory response to fungal allergens in the lung epithelium?

“…It should be noted, however, that inhibiting PAR2 in HNE and HBE cells did not completely reduce intracellular calcium or ATP release, cytokine or mucin secretion, or inflammation, suggesting additional PAR2‐independent mechanism(s) for allergic responses 45,74,81,91 . This is supported by recent studies showing non‐protease activation of the MAPK pathway, 98 and attenuated not abrogated responses in PAR‐deficient mice 13 …”

“…Fungal proteases and PAR2 were identified as key contributors to fungal allergy in 19 (31%) and 13 (21%) studies, the 1st and joint 3rd most studied molecules respectively. Fungal proteases have been shown to promote mucin production, goblet cell hyperplasia and AHR 98 . Heat inactivation and protease inhibition confirmed that fungal proteases activated the PAR2 receptor in both bronchial and small airway cell lines as well as primary HNE cells 37,43,81 .…”

“…The role of MAPK signalling is further supported by a more recent study in which direct activation of PAR2 by fungal proteases led to eosinophil recruitment and mucus overproduction. 98 Both calcium signalling and ATP release following fungal exposure were also involved in the subsequent secretion of IL33 41,63,81,88 and other cytokines (IL5, IL13). 88 It is possible that such cytokine release is a direct downstream consequence of PAR2 activation, and earlier work has demonstrated that the release of TSLP in a human bronchia cell line (BEAS-2B) exposed to Alternaria extract involved PAR2.…”

Protease allergens as initiators–regulators of allergic inflammation

“…111,112 Alternaria alkaline serine protease activates PAR2 directly and induces allergic airway inflammation in mice. 106 Additionally, various fungal allergen extracts, including Alternaria 113,114 and Aspergillus 115 extracts, have been shown to activate AEC, eosinophils, and induce cytokines through PAR2 but the exact proteases mediating these effects have not been identified. 116 Fungal Pen c 13 can cleave PAR1 and PAR2 at their activation sites and desensitize AECs to increased intracellular calcium induced by PAR1 and PAR2 agonists.…”

β‐Arrestin‐biased proteinase‐activated receptor‐2 antagonist C781 limits allergen‐induced airway hyperresponsiveness and inflammation

“…Male mice were used due to their enhanced response to allergens that allowed for statistical analysis with a reduced amount of animals. At 8 weeks, mice were randomly divided into four treatment groups of six mice each and subjected to allergen sensitization and challenge using A. alternata filtrate as described (Rivas et al, 2021). Mice were first anaesthetised by placing them in a chamber supplied with vaporized isoflurane and oxygen.…”