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\begin{document}

\title{Novel proteinase-activated receptor-2 (PAR2) antagonist C391 blocks
Alternaria-induced human airway epithelial signaling and asthma
indicators in murine models}



\author[1]{Candy Rivas}%
\author[2]{Michael Yee}%
\author[1]{Kenneth Addison}%
\author[1]{Marissa Lovett}%
\author[2]{Kasturi Pal}%
\author[3]{Julie Ledford}%
\author[4]{Greg Dussor}%
\author[4]{Theodore  Price }%
\author[5]{Josef Vagner}%
\author[2]{Kathryn DeFea}%
\author[1]{Scott Boitano}%
\affil[1]{The University of Arizona Arizona Health Sciences Center}%
\affil[2]{University of California Riverside School of Medicine}%
\affil[3]{University of Arizona Medical Center - University Campus}%
\affil[4]{The University of Texas at Dallas School of Behavioral and Brain Sciences}%
\affil[5]{The University of Arizona BIO5 Institute}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Despite 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 pre-clinical animal models as important to asthma
initiation and progression following allergen exposure. Proteinase
activation of PAR2 induces intracellular Ca2+, mitogen activated protein
kinase (MAPK) and ?-arrestin signaling the airway, leading to both
inflammatory and protective effects. We have developed C391, a potent
PAR2 antagonist effective in blocking peptidomimetic- and
trypsin-induced PAR2 signaling in vitro as well as reducing inflammatory
PAR2-associated pain in vivo. We hypothesized that PAR2 reduction with
C391 would attenuate allergen-induced asthma indicators in murine
models. Experimental Approach: We evaluated the ability for C391 to
alter Alternaria alternata-induced PAR2 signaling 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 asthma
indicators in vivo in two murine strains. Key Results: C391 blocked A.
alternata-induced, PAR2-dependent Ca2+ and MAPK signaling in 16HBE14o-
cells, as well as ?-arrestin recruitment in HEK 293 cells. C391
effectively attenuated A. alternata-induced inflammation, mucus
production, mucus cell hyperplasia and airway hyperresponsiveness in
acute asthma murine models. Conclusions and Implications: To our
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.%
\end{abstract}%



\sloppy


\textbf{Title:} Novel proteinase-activated receptor-2 (PAR2) antagonist
C391 inhibits \emph{Alternaria} -induced human airway epithelial
signaling and asthma indicators in murine models

\textbf{Running Title:} Inhibition of PAR2 signaling in allergen-induced
asthma

\textbf{Authors:} Candy M. Rivas\textsuperscript{1,2}, Michael C.
Yee\textsuperscript{3}, Kenneth J. Addison\textsuperscript{2,4}, Marissa
Lovett\textsuperscript{1}, Kasturi Pal\textsuperscript{3}, Julie G.
Ledford\textsuperscript{2,4}, Gregory Dussor\textsuperscript{5},
Theodore J. Price\textsuperscript{5}, Josef Vagner\textsuperscript{6},
Kathryn A. DeFea\textsuperscript{3},\textsuperscript{7} and Scott
Boitano*\textsuperscript{1,2,4,6, 8}

\textsuperscript{1}University of Arizona Physiological Sciences Graduate
Interdisciplinary Program

\textsuperscript{2}University of Arizona Heath Sciences, Asthma and
Airway Disease Research Center

\textsuperscript{3}University of California Riverside, Biomedical
Sciences

\textsuperscript{4}University of Arizona Heath Sciences, Department of
Cellular and Molecular Medicine

\textsuperscript{5}University of Texas at Dallas, School of Behavioral
and Brain Sciences and Center for Advanced Pain Studies

\textsuperscript{6}University of Arizona Bio5 Collaborative Research
Institute

\textsuperscript{7}PARMedics, Inc.

\textsuperscript{8}University of Arizona Heath Sciences, Department of
Physiology

*\textbf{Corresponding Author} : Kathryn A. DeFea, Ph.D.; CEO, PARMedics
Inc.; 41593 Winchester Rd. Suite 228, Temecula, CA, 92590 and Professor
Emeritus, Biomedical Sciences, University of California Riverside;
Riverside, CA, 92521 kdefea@parmedics.com, +1-925-967-2363

\textbf{Author Contributions:} CMR: experimental design, experiments,
manuscript writing and editing; MCY: conducted experiments and data
analysis, manuscript editing; KJA: experiments and data analysis; ML:
conducted experiments and data analysis; KP: conducted experiments and
data analysis; JGL; experimental design, manuscript editing; GD:
experimental design, manuscript editing; TJP: experimental design, data
analysis, manuscript writing and editing; JV: experimental design, data
analysis and manuscript editing; KAD: experimental design, data
analysis, manuscript writing and editing; SB: experimental design,
experiments, data analysis, manuscript writing, and editing

\textbf{Word Count} : 6187

\textbf{Acknowledgements} : We would like to thank Dr. Kim Barrett for
her guidance in helping to prepare the manuscript. This work was funded
in part through grants from the National Institute of Health grants
(NINDS NS098826 SB, TJP, JV, GD; NIAID AI140257 SB). CMR was an American
Physiological Society William H. Townsend Porter Pre-doctoral Fellow.

\textbf{Competing Interests} : There are no competing interests.



\emph{Abbreviations:}

16HBE14o- cells - an SV40-transformed human bronchial epithelial cell
line

2at-LIGRL-NH\textsubscript{2} -
2-aminothiazole-LIGR-NH\textsubscript{2}, a PAR2 agonist

2f-LIGRLO-NH\textsubscript{2} - 2-furoyl-LIGRLO-NH\textsubscript{2}, a
PAR2 agonist

AEBSF - 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, a serine
proteinase inhibitor

AC-55541 - PAR2 antagonist

AC-264613 - PAR2 antagonist

AZ8838 - PAR2 antagonist

AZ3451 - PAR2 antagonist

BRET - bioluminescence resonance energy transfer

C391 - compound 391, a PAR2 antagonist

{[}Ca\textsuperscript{2+}{]}\textsubscript{i} - Intracellular
Ca\textsuperscript{2+} concentration

CPMCRI - California Pacific Medical Center Research Institute

E64 - (1\emph{S} ,2\emph{S} )-2-(((\emph{S}
)-1-((4-Guanidinobutyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)cyclopropanecarboxylic
acid, a cysteine proteinase inhibitor

GB88 - PAR2 antagonist

H\&E - haematoxylin and eosin stain

HEK 293 - human embryonic kidney cell line

ICW - In Cell Western

K-14585 - PAR2 antagonist

LHC - Lechner and LaVeck basal medium

Luc - Luciferase

MAPK - mitogen activated protein kinase

Max Rrs - maximum total airway resistance

MCS - Matrix coating solution

MEM - Minimal essential medium

p-MAPK - phosphorylated MAPK

PAR2 - proteinase-activated receptor-2

PAS - Periodic Acid Schiff

PAR2\textsuperscript{-/-}- PAR2 knockout animals

YFP - Yellow fluorescent protein

\emph{}

\emph{Bullet Point Summary}

\textbf{What is already known:}

Asthma is a prevalent and growing disease with limited pharmacological
treatments that focus on symptom relief

Proteinase activated receptor-2 (PAR2) knockout models suggest that PAR2
is a novel target for asthma treatment

\textbf{What this study adds}

C391 is a potent PAR2 antagonist that limits asthma allergen
(\emph{Alternaria alternata} ) -induced Ca\textsuperscript{2+}, MAPK and
\selectlanguage{greek}β-\selectlanguage{english}arrestin cellular signaling in vitro

C391 prophylactically limits \emph{A. alternata} -induced asthma
indicators in acute exposure murine models of asthma

\textbf{Clinical Significance}

This is the first indication of pharmacological control of
allergen-induced asthma response via PAR2 small molecule antagonism in
pre-clinical models

Further development of PAR2 antagonists may provide novel treatments for
allergic asthma

\textbf{Abstract and Keywords}

\textbf{Background and Purpose} : Despite 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 pre-clinical animal models as important to asthma
initiation and progression following allergen exposure. Proteinase
activation of PAR2 induces intracellular Ca\textsuperscript{2+}, mitogen
activated protein kinase (MAPK) and \selectlanguage{greek}β-\selectlanguage{english}arrestin signaling the airway,
leading to both inflammatory and protective effects. We have developed
C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and
trypsin-induced PAR2 signaling \emph{in vitro} as well as reducing
inflammatory PAR2-associated pain \emph{in vivo} . We hypothesized that
PAR2 reduction with C391 would attenuate allergen-induced asthma
indicators in murine models.

\textbf{Experimental Approach} : We evaluated the ability for C391 to
alter \emph{Alternaria alternata} -induced PAR2 signaling
pathways\emph{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
\emph{A. alternata} -induced asthma indicators \emph{in vivo} in two
murine strains.

\textbf{Key Results} : C391 blocked \emph{A. alternata} -induced,
PAR2-dependent Ca\textsuperscript{2+} and MAPK signaling in 16HBE14o-
cells, as well as \selectlanguage{greek}β-\selectlanguage{english}arrestin recruitment in HEK 293 cells. C391
effectively attenuated \emph{A. alternata} -induced inflammation, mucus
production, mucus cell hyperplasia and airway hyperresponsiveness in
acute asthma murine models.

\textbf{Conclusions and Implications} : To our knowledge, this is the
first demonstration of pharmacological intervention of PAR2 to reduce
allergen-induced asthma indicators \emph{in vivo} . These data support
further development of PAR2 antagonists as potential first-in-class
allergic asthma drugs.

\textbf{Key Words} : Airway Inflammation; Airway Hyperresponsiveness;
Allergen-induced asthma; \emph{Alternaria alternata} ; C391; Mucus cell
hyperplasia; PAR2;

\textbf{Main Text}

\textbf{Introduction:}

Asthma is a common and growing lung disease afflicting the world
population (Martinez \& Vercelli, 2013; To et al., 2012). While there is
no cure, many therapeutic options have been successfully used to control
mild-to-moderate asthma symptoms for the past 60 years. The most
commonly-prescribed asthma medications include bronchodilators composed
of aerosolized \selectlanguage{greek}β-\selectlanguage{english}adrenergic agonists or cholinergic antagonists, and
anti-inflammatory medicines such as corticosteroids, anti-leukotrienes,
and monoclonal antibodies (Stephenson et al., 2016). Despite the seeming
plethora of treatment options to control asthma, approximately 50\% of
asthmatics with moderate to severe disease do not have adequate control
of their symptoms (Martinez \& Vercelli, 2013).

Allergic asthma can be triggered by a variety of environmental factors
including exposure to the ubiquitous fungus \emph{Alternaria alternata}
.\emph{A. alternata} is a prominent fungal allergen that has been
associated with increased asthma-related hospitalization in children,
and homes with higher levels of \emph{A. Alternata} reported a higher
incidence of moderate to severe asthma (Batra et al., 2021; Bush \&
Prochnau, 2004; Halonen, Stern, Wright, Taussig \& Martinez, 1997;
Lombardi, Savi, Ridolo, Passalacqua \& Canonica, 2017; Mendy, Wilkerson,
Salo, Zeldin \& Thorne, 2020; Pulimood, Corden, Bryden, Sharples \&
Nasser, 2007). When found in semi-arid regions, \emph{A}
.\emph{alternata} contains a proteinase, \emph{Alternaria} alkaline
Serine Proteinase (AASP), that acts through the G-protein-coupled
receptor (GPCR), proteinase-activated-receptor-2 (PAR2), to induce
airway inflammation, mucus production and bronchoconstriction (Boitano
et al., 2011; Snelgrove et al., 2014; Yee et al., 2018). Trypsin-like
serine proteinases associated with other allergy-inducing pathogens such
as cockroach and house dust mite also activate PAR2 to promote airway
inflammation {[}e.g., (Adam et al., 2006; Page, Ledford, Zhou, Dienger
\& Wills-Karp, 2010; Polley et al., 2017){]}. Studies using PAR2
knockout (PAR2\textsuperscript{-/-}) animals have demonstrated that PAR2
expression is critical for allergen-induced asthma indicators (Arizmendi
et al., 2011; Davidson et al., 2013; Day, Zhou, Ledford \& Page, 2010;
Page, Ledford, Zhou, Dienger \& Wills-Karp, 2010; Yee et al., 2018).
Further, studies using PAR2-neutralizing antibodies have demonstrated a
reduction in allergen-induced asthma indicators in both acute and
chronic allergen-exposed animal models (Asaduzzaman, Davidson, Nahirney,
Fiteih, Puttagunta \& Vliagoftis, 2018; Asaduzzaman et al., 2015).
Collectively, PAR2 has emerged as a viable drug target for the treatment
of asthma (Ramachandran, Noorbakhsh, Defea \& Hollenberg, 2012).

A common approach to developing novel GPCR drugs is via screening of
large libraries, however, this approach has had limited success with
PAR2 ligands (Cheng et al., 2017; Gardell et al., 2008). An alternative
approach to developing PAR2 ligands is the use of small peptides
{[}e.g., SLIGRL-NH\textsubscript{2} (Hollenberg, Saifeddine \& al-Ani,
1996){]} or peptidomimetics {[}e.g.,
2-furoyl-LIGRLO-NH\textsubscript{2}; (McGuire, Saifeddine, Triggle, Sun
\& Hollenberg, 2004){]} that mimic the activating peptide sequence
following proteolytic cleavage of the receptor (Ramachandran \&
Hollenberg, 2008; Yau, Liu \& Fairlie, 2013). A more recent advance from
our laboratory is the modification of the peptide sequence of the high
potency peptidomimetic PAR2 agonist,
2-furoyl-LIGRLO-NH\textsubscript{2}, to form a novel PAR2 antagonist
pharmacophore, compound 391 {[}C391; (Boitano et al., 2015){]}. C391 is
one of a handful of recently proposed PAR2 antagonists (Avet et al.,
2020; Cheng et al., 2017; Goh, Ng, Nilsson, Kanke \& Plevin, 2009;
Hollenberg et al., 2014; Huang, Ni, Xi, Chu, Zhang \& You, 2019; Kanke
et al., 2005; Suen et al., 2012; Suen et al., 2014; Yau, Lim, Liu \&
Fairlie, 2016). It is distinct from other reported antagonists in that
it requires minimal pre-incubation for inhibition of peptide activation
of PAR2-dependent Ca\textsuperscript{2+} signaling and it acts as an
antagonist for mitogen activating protein kinase (MAPK) signaling
pathways. Further, C391 is one of the only known antagonists shown to be
effective in blocking both natural proteinase and peptidomimetic
activation of PAR2 \emph{in vitro} and to be effective for blocking pain
response \emph{in vivo} (Boitano et al., 2015).

In this study we sought to evaluate the ability of C391 to inhibit
allergen-induced, PAR2-dependent signaling in airway epithelial cells,
and asthma indicators in murine models. We hypothesized that C391 would
be effective at protecting against the development of cellular
inflammation and broncho-constriction in pre-clinical asthma models. To
this endeavor we first used established cellular models to demonstrate
that C391 reduced or eliminated \emph{A. alternata} -induced
Ca\textsuperscript{2+} mobilization, \selectlanguage{greek}β-\selectlanguage{english}arrestin recruitment and MAPK
activation. Finally, we demonstrated that C391 was able to reduce or
eliminate \emph{A. alternata} -induced asthma indicators (inflammation,
mucus production and airway hyperresponsiveness) in acutely-exposed
murine models. These experiments demonstrate the ability of C391 to
alter multiple allergen-induced and PAR2-dependent signaling
pathways\emph{in vitro and} demonstrate the first successful
pharmacological reduction of asthma indicators in a pre-clinical model.
Taken together, these data further support PAR2 as a druggable target to
control allergen-induced asthma.

\textbf{Materials and Methods}

Materials

\emph{Alternaria alternata} filtrate was purchased from Stallergenes
Greer Laboratories (Lenoir, NC). PAR2 agonists
{[}2-aminothiazole-LIGR-NH\textsubscript{2}(2at-LIGRL-NH\textsubscript{2})
and
2-furoyl-LIGRLO-NH\textsubscript{2}(2f-LIGRLO-NH\textsubscript{2}){]},
and the PAR2 antagonist C391 were manufactured in our laboratory as
described (Boitano et al., 2011; Boitano et al., 2015; Flynn et al.,
2011; Hoffman et al., 2012). Unless listed below, all other
chemicals/components were of Molecular Biology or higher grades and
purchased from Fisher Scientific (Pittsburgh, PA), ThermoFisher
Scientific (Watham, MA), Sigma-Aldrich (Burlington, MA), or VWR (West
Chester, PA).

Cellular Studies

16HBE14o- cells. 16HBE14o- cells are a SV40-transformed human bronchial
epithelial cell line (Gruenert, Finkbeiner \& Widdicombe, 1995) obtained
from the California Pacific Medical Center Research Institute (CPMCRI;
San Francisco, CA, USA) that naturally express PAR2 and thus provide an
experimental cellular model for evaluating PAR2 agonism and antagonism.
16HBE14o- cells were passaged as suggested by CPMCRI. Cells were
expanded in tissue culture flasks prior to transfer to cultureware for
specific experiments. Flasks were coated initially with a matrix coating
solution {[}MCS; 88\% LHC (Lechner and LaVeck) basal medium, 10\% bovine
serum albumin (BSA; from 1 mg/ml stock), 1\% bovine collagen type I
(from 2.9 mg/ml stock), and 1\% human fibronectin (from 1 mg/ml stock
solution){]} and incubated for two hrs at 37\selectlanguage{ngerman}°C, after which the MCS was
removed and the flasks allowed to dry for at least one hr. 16HBE14o-
cells were plated onto the MCS-coated cultureware at a density of
1x10\textsuperscript{5}cells/cm\textsuperscript{2}. 16HBE14o- cells were
cultured in Eagles minimal essential medium with Earle's salts
supplemented with 10\% FBS, 2 mM glutamax, penicillin and streptomycin
(MEM) at 37°C in a 5\% CO\textsubscript{2} atmosphere. Medium was
replaced every other day until the cells reached confluence (5 - 7
days). Cells were then transferred to appropriate MCS-coated substrate
for analyses.

\textbf{Alternaria alternata \emph{filtrate.}} \emph{A.
alternata}filtrate was purchased as a lyophilized powder (Stallergenes
Greer \#XPM1D3A), resuspended in HBSS, vortexed and stored at 2.5 mg/mL
protein at -20°C until experimental use. Proteinase content for each lot
was determined prior to use; activity of the filtrate used in this study
was comparable to 6.5 U /mL trypsin activity (Sigma \#T6567; 13,190
U/mg) as determined with a commercial proteinase assay kit (Sigma
\#PF-0100).

Ca\textsuperscript{2+} signaling measurements . Intracellular
Ca\textsuperscript{2+} concentrations
({[}Ca\textsuperscript{2+}{]}\textsubscript{i}) were measured using
digital imaging microscopy as described (Boitano et al., 2011; Boitano
et al., 2015). 16HBE14o- cells were plated onto MCS-coated 15 mm glass
coverslips and grown to confluence. Coverslip cultures were washed with
HBSS and loaded for 45 min with 5 \selectlanguage{greek}µ\selectlanguage{english}M fura 2-acetomethoxy ester (fura
2-AM) in HBSS and then transferred to HBSS for 30 min before imaging.
Fura 2 fluorescence was observed on an Olympus IX70 microscope with a
40\selectlanguage{ngerman}× oil immersion objective (corrected for UV light) after alternating
excitation between 340 nm and 380 nm by a 75 W Xenon lamp linked to a
Delta Ram V illuminator (PTI, London, Ontario, Canada) via a gel optic
line. {[}Ca\textsuperscript{2+}{]}\textsubscript{i} for each individual
cell in the field of view was calculated by ratiometric analysis of fura
2 fluorescence using equations originally published in (Grynkiewicz,
Poenie \& Tsien, 1985). For verification of \emph{A. alternata} , ATP
(Sigma A2383) or Thrombin (Sigma 10602400001; 20 U/22.23 mg protein)
agonist activity, fura 2-loaded cells were monitored for 20 sec in HBSS
to record baseline {[}Ca\textsuperscript{2+}{]}\textsubscript{i}followed
by a 10 sec wash to introduce agonist and an additional 2 min 30 sec of
monitoring for agonist-induced changes in
{[}Ca\textsuperscript{2+}{]}\textsubscript{i}. For C391 antagonism
(\emph{A. alternata} ) or control (ATP or Thrombin) experiments, a
similar protocol was used with a 2 min incubation with C391 dispensed
after the 20 sec baseline measurement and with the agonist solution
containing an identical concentration of C391. Individual ratios
(\textasciitilde{}1/sec) were calculated throughout the 3- or 5-min
experiments. {[}Ca\textsuperscript{2+}{]}\textsubscript{i} traces over
time were represented as average
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} of all cells within a
field of view (\textasciitilde{}80 - 100) and are representative of at
least 4 experiments. Color maps of \emph{A. alternaria} responses (+/-
C391) were constructed using Adobe Photoshop (Cupertino, CA) and
indicate approximate {[}Ca\textsuperscript{2+}{]}\textsubscript{i} of
individual cells at given time in the experiment.

\emph{Detection of MAPK Signaling/In-Cell Western (ICW)}\textbf{.} ICW
studies were adapted from methods described in (Flynn et al., 2011).
16HBE14o- cells were plated onto MCS-treated, clear bottom, black-walled
96-well plates at a density of 2 x 10\textsuperscript{4} cells per well.
Cells were allowed to reach confluence (3 - 4 days of growth) and were
subsequently serum-starved overnight in MEM supplemented with 10 \selectlanguage{greek}μ\selectlanguage{english}M
AEBSF and 10 \selectlanguage{greek}μ\selectlanguage{english}M E64. On the following day, cells were treated with C391
suspended in MEM to a final concentration of 1\selectlanguage{ngerman}× (final concentration
range of 100 nM - 100 \selectlanguage{greek}µ\selectlanguage{english}M C391) and cells were incubated for 15 min. This
was followed by a treatment of equal volumes of \emph{A.
alternata}(final concentration of 20 \selectlanguage{greek}µ\selectlanguage{english}g/mL; 2 U/mL trypsin activity)
mixed with 1\selectlanguage{ngerman}× final C391 concentration. After 5 min, cells were fixed
with 4\% paraformaldehyde and permeabilized with 100\% methyl alcohol.
Fixed cells were incubated with blocking buffer (5\% goat serum, 0.3\%
Triton X-100) prior to incubation with primary phosphorylated MAPK
antibody (p-MAPK; rabbit anti-mouse p-ERK; \#9101 Cell Signaling
Technologies, Grand Island, NY), secondary antibody (goat anti-rabbit
DyLight; \#5151 Cell Signaling Technologies) and nuclear stain DRAQ5
(\#4804, Cell Signaling Technologies). Plates were imaged on an Odyssey
Scanner (LI-COR, Lincoln, NE) following the manufacturer's protocol.
p-MAPK levels were normalized to DRAQ5 content and data are presented as
percent above the MEM-treated (baseline) control. IC\textsubscript{50}s
were determined using a 4-parameter curve fit, with hill slopes
constrained to one, with GraphPad Prizm software (San Diego, CA).

\selectlanguage{greek}\emph{\textbf{Bioluminescence resonance energy transfer (BRET)
measurements of recruitment of β-arrestin-2}.}\selectlanguage{english} Competitive BRET
measurements with \emph{A. alternata} (65 \selectlanguage{greek}µ\selectlanguage{english}g/mL; 6.5 trypsin U/mL) and
C391 (concentration range; 100 pM - 100 \selectlanguage{greek}µ\selectlanguage{english}M) were adapted from (Yee et
al., 2018). Briefly, HEK 293 cells transfected with PAR2-YFP and
Luciferase (Luc) tagged \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 were plated in 96-well microplates
and \emph{A. alternata} (w/without C391) was added along with the Luc
substrate coelenterazine. Readings were collected using a Multilabel
Reader Tristar9640 (Berthold, Bad Wildbad, Germany) and emission was
detected at 480 nm (Luc) and 535 nm (YFP). Net BRET was determined from
the ratio of emission in the YFP channel to the emission in the
luciferase channel (E535/E488) minus the donor-only (\selectlanguage{greek}β-\selectlanguage{english}arrestin-Luc)
control values. To eliminate possibility that observed signals
represented non-specific interactions, we included a \selectlanguage{greek}β-\selectlanguage{english}arrestin-Luc +
YFP in every experimental condition, which gave values similar to donor
only controls. Data points were fitted to a curve and
IC\textsubscript{50} calculated using a 4-parameter curve fit, with hill
slopes constrained to one, with GraphPad Prizm (San Diego, CA).

\emph{Animal Studies}\textbf{:} All animal procedures were in accordance
with the guidelines on the use and care of laboratory animals set by the
National Institutes of Health and approved by the Institutional Animal
Care and Use Committees at the University of California, Riverside, or
the University of Arizona and in accordance with ARRIVE and\emph{British
Journal of Pharmacology} guidelines. Wild type C57Bl/6J (\#000664) and
BALB/c mice (\#000651) were purchased from The Jackson Laboratory (Bar
Harbor, ME). C57Bl/6 animals were bred in-house. Animals used in these
studies were male, between 6 and 10 weeks of age. Male mice were used
due to the unknown effects of cycling estradiol on airway inflammation
and asthma. On days 0, 3 and 7, C57Bl/6 mice (8 - 10 weeks old at day
0), were sedated under brief ketamine anesthesia and intranasally
exposed to one of the following: 25 \selectlanguage{greek}μ\selectlanguage{english}L of HBSS control, 8 \selectlanguage{greek}µ\selectlanguage{english}g of \emph{A.
alternata} filtrate in HBSS; or 8 \selectlanguage{greek}µ\selectlanguage{english}g of \emph{A. alternata} filtrate
supplemented with 0.25 nmoles or 2.5 nmoles of C391. For BALB/c mice, 50
\selectlanguage{greek}μ\selectlanguage{english}L of HBSS control; 5 \selectlanguage{greek}µ\selectlanguage{english}g of \emph{A. alternata}filtrate in HBSS; or 5 \selectlanguage{greek}µ\selectlanguage{english}g
of \emph{A. alternata} filtrate supplemented with 5 nmoles C391 was
administered on days 0, 3 and 7 under isoflurane anesthesia. Assessment
of asthma indicators was performed on day 8 for both murine models. Each
experiment contained a minimum of 2 repeats with 3 animals in each
group. Mice were assigned randomly to groups. Specific numbers for each
experiment are listed in the text and/or Figure Legends.

\emph{Airway hyperresponsiveness in} \textbf{A. \emph{alternata-treated
BALB/c mice}:} On day 8 direct measurements of respiratory mechanics in
response to methacholine were obtained with the use of the Flexivent
system (SCIREQ Inc., Montreal, Qc Canada) adapted from procedures
previously used in our laboratory (Addison, Morse, Robichaud, Daines \&
Ledford, 2017). Briefly, mice were anesthetized with an intraperitoneal
injection of urethane in sterile H\textsubscript{2}O (125 mg/mL) at a
final dose of 16 \selectlanguage{greek}µ\selectlanguage{english}L/gram of body weight. The average mouse weighed 25 g
and thus received 400 \selectlanguage{greek}µ\selectlanguage{english}L of urethane. Once a surgical plane of
anesthesia was reached, the trachea of the mouse was fit with a 19-gauge
metal cannula and subsequently ventilated with a computer-controlled
small animal ventilator with a tidal volume of 7.5 mg/kg and a positive
end-expiratory pressure of 3 cm H\textsubscript{2}O. The mice were given
a muscular paralytic (pancuronium bromide; Sigma \#P1918) solubilized in
saline (0.8 mg/mL) and administered at a final dose of 10 \selectlanguage{greek}µ\selectlanguage{english}L/gram of
body weight to eliminate any breathing interference in the recordings. A
25 g mouse would receive 250 \selectlanguage{greek}µ\selectlanguage{english}L of pancuronium bromide. Respiratory
mechanics were obtained by the forced oscillation technique; values were
reported as total pulmonary resistance (RT) cm H\textsubscript{2}O/mL/s.
Mice were sacrificed after completion of these experiments for
examination of cellular inflammation.

\emph{Histochemical and inflammation analyses in} \textbf{A.
alternata\emph{-treated mice:}} Bronchioalveolar lavage fluid (BALF) was
collected in 1-5 mLs of PBS after which lungs were removed, fixed in
10\% formalin and stored in 70\% ethanol until paraffin embedding.
Paraffin embedded C57Bl/6 lung sections were stained with hematoxylin
and eosin (H\&E) or Alcian Blue {[}to stain acidic mucins; (\#AFR-1-IFU
Scytek Laboratories, Logan, UT){]}. Sections were imaged using a Nikkon
Eclipse E600 microscope with 10X air or 40X oil immersion objectives,
using a PAXcam microscope camera and PAXIT! Software (Villa Park, IL).
Ethanol-preserved Balb/c lungs were sent to AML Laboratories (St.
Augustine, FL) for processing (paraffin embedding; 4 \selectlanguage{greek}µ\selectlanguage{english}m sections) and
stained (H\&E or Periodic Acid Schiff (PAS) as the mucin stain).
Sections were imaged using an Olympus IX70 inverted microscope and 20\selectlanguage{ngerman}×
air or 60× oil immersion objectives. Images were captured using a
MicroPublisher 6 camera (QImaging, Tucson). \emph{A. alternata} includes
a variety of allergenic proteins (e.g., Alt a 10, chitin, particulates
and proteinases), but our previous studies indicate that PAR2-activating
serine-proteinase(s) from \emph{Alternaria} are essential for the
observed airway inflammation, goblet cell hyperplasia and mucus
overproduction (Boitano et al., 2011; Yee et al., 2018)., AZ).
Semi-quantitative scoring of inflammation and mucin staining was
performed by histological grading as previously described (Nichols et
al., 2012), based on infiltration of white blood cells and perivascular
thickness. using a scoring scale of 1 - 4, with 1 being no inflammation
present, 2 being 1 ring of leukocytes around the vasculature and mild
infiltration, 3 being 2 - 3 rings and moderate infiltration, and 4 being
4 or more rings and numerous leukocytes throughout the epithelial tissue
(Zeldin et al., 2001). Epithelial thickness was quantified using NIH
ImageJ to measure the distance from the basolateral surface of the
epithelial cell to the muscular layer. Mucin positive cells were
quantified in Alcian Blue sections by calculating the percentage of
total epithelial cells that stained positive, and mucin staining
determined using a user-defined NIH ImageJ macro to calculate red
channel density per 100 cells after image inversion. Because Alcian Blue
can also stain basal lamina matrix proteins, staining at the basal
lamina was not considered positive. Evaluation of mucus production using
Periodic-Acid Schiff (PAS) staining was performed by scoring sections on
a scale of 1 -- 5, based on extent of staining of epithelial layer and
bronchiole lumen indicative of mucus plugs as described (Ledford et al.,
2016). In these samples, a score of 1 represented diffuse mucus staining
in the epithelial layer; a score of 2 - 3 indicated increased mucus
staining in the epithelial layer; a score of 4 reflected complete
epithelial staining and additional mucus staining in the lumen; a score
of 5 reflected complete lumen staining of mucus and full mucus plug of
large lumens or multiple smaller lumens as described previously. A
minimum of three sections were evaluated per mouse and reported scores
reflect evaluation by at least 4 blinded evaluators.

To determine inflammatory cell counts, bronchoalveolar lavage fluid
(BALF; 200 \selectlanguage{greek}µ\selectlanguage{english}l) was spun onto glass slides using a Shandon Cytospin and
cells stained using Hema 3 stain kit (Fisher Scientific, \#22122911)
following the manufacturer protocol. Differential counts were obtained
by counting 200 cells per slide and categorized based on morphological
criteria.

\emph{Data analysis}\textbf{:} Sample sizes, n, for all experiments
reflect the number of independent values (either replicates for cell
assays or individual mice). All statistical analyses were evaluated with
GraphPad Prism software (San Diego, CA) on independent values.
Multivariate comparisons were done with a two-way ANOVA with Tukey's
multiple comparison post-test for individual experiments where the
F-statistic exceeded the threshold for statistical significance.
Pair-wise comparisons were conducted with a two-tailed Student's\emph{t}
-test. A value of p \textless{} 0.05 was used to establish a significant
difference between samples. Data in Figures are graphed \selectlanguage{ngerman}± Standard Error
of the Mean (SEM) unless otherwise noted. This manuscript complies with
the recommendations and requirements on experimental design and analysis
set forth by the British Journal of Pharmacology (Curtis et al., 2018).



\textbf{RESULTS}

\emph{C391 inhibits} \textbf{Alternaria alternata\emph{-induced
signaling in airway epithelial and HEK293 cells}}

Proteinases contained in \emph{A.} \emph{alternata} activate PAR2 to
promote Ca\textsuperscript{2+} mobilization, \selectlanguage{greek}β-\selectlanguage{english}arrestin recruitment and
activation of downstream signaling events (Boitano et al., 2011; Yee et
al., 2018). We have shown that \emph{A. alternata} -induced
Ca\textsuperscript{2+} mobilization in human airway epithelial
(16HBE14o-) cells requires PAR2 (Boitano et al., 2011). To determine
whether C391, which can block both peptidomimetic-induced and
trypsin-induced PAR2 signaling (Boitano et al., 2015), can effectively
block \emph{A. alternata} -induced signals, we examined
Ca\textsuperscript{2+} mobilization, MAPK phosphorylation and
\selectlanguage{greek}β-\selectlanguage{english}arrestin-2 recruitment. Treatment of 16HBE14o- cells with \emph{A.
alternata} (\textasciitilde{}0.65 U/mL trypsin activity) raised
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} from an average resting
concentration of \textasciitilde{}35 nM to [?] 150 nM, within the 3 min
experimental protocol, with 59.0 \selectlanguage{ngerman}± 24.0\% of cells showing a
Ca\textsuperscript{2+} response
({[}Ca\textsuperscript{2+}{]}\textsubscript{i} \textgreater{}100nM)
(\textbf{Figure 1A, C, E - H} ; n = 3). Pre-incubation of cells with 3
\selectlanguage{greek}µ\selectlanguage{english}M C391 for 2 min prior to addition of \emph{A. alternata} significantly
reduced the percentage of cells exhibiting a Ca\textsuperscript{2+} (
6.4 \selectlanguage{ngerman}± 2.7\%), similar to that observed with 3 \selectlanguage{greek}µ\selectlanguage{english}M C391 alone (4.2 \selectlanguage{ngerman}±
1.3\%), and resulted in an average
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} of less than 75 nM
(\textbf{Figure 1A, D, I-L} ; n = 3, p \textless{} 0.05).

\emph{A. alternata} has been shown to increase
Ca\textsuperscript{2+}signaling, independent of PAR2, via ATP (O'Grady,
Patil, Melkamu, Maniak, Lancto \& Kita, 2013). We thus repeated the C391
experiments following exposure of 16HBE14o- cells to either 1 \selectlanguage{greek}µ\selectlanguage{english}M (data
not shown) or 2.5 \selectlanguage{greek}µ\selectlanguage{english}M ATP (\textbf{Figure 1M - O, n = 3)} , which induced
a Ca\textsuperscript{2+} response in 15.2 \selectlanguage{ngerman}± 3.8\% and 62.3 ± 11.5\% of
cells, respectively. This Ca\textsuperscript{2+} response was unaffected
by pre-treatment with 3 \selectlanguage{greek}µ\selectlanguage{english}M C391 which resulted in a response in 21.3 \selectlanguage{ngerman}±
96.8\% (1 \selectlanguage{greek}µ\selectlanguage{english}M ATP) and 53.8 \selectlanguage{ngerman}± 9.4\% (2.5 \selectlanguage{greek}µ\selectlanguage{english}M ATP) of cells. Due to the
inherent similarities of PAR2 with other PARs, we also tested the
effects of C391 on thrombin-induced Ca\textsuperscript{2+} signaling in
16HBE14o- cells (\textbf{Figure 1M, P-Q} ). High concentrations of
thrombin (100 \selectlanguage{greek}µ\selectlanguage{english}M) induced a low level of Ca\textsuperscript{2+} response
in 16HBE14o- cells (13.3 \selectlanguage{ngerman}± 2.7\%; n = 6) that was not statistically
different when cells were pre-incubated with 3 \selectlanguage{greek}µ\selectlanguage{english}M C391 (11.0 \selectlanguage{ngerman}± 3.6\%; n
= 7).

Activation of PAR2 results in MAPK signalling, in part through a
\selectlanguage{greek}β-\selectlanguage{english}arrestin-dependent pathway, that can be measured via phosphorylation
of the 42kD and 44kD MAPK family members (p-MAPK) and we have
demonstrated previously that C391 inhibits peptidomimetic-induced MAPK
activation (Boitano et al., 2015). Treatment of 16HBE14o- cells with
increasing concentrations of \emph{A. alternata} for 5 minutes resulted
in a 1.4-fold increase in MAPK phosphorylation, with an
EC\textsubscript{50} of 35 \selectlanguage{greek}µ\selectlanguage{english}g/mL (\textbf{Figure 2A} ; n = 4 for all
concentrations), similar to that observed with the peptidomimetic
agonist 2-at-LIGRL-NH\textsubscript{2} (600 nM; \textbf{Figure 2B} ; n =
4 for all concentrations). However, a 95\% confidence interval for pMAPK
could not be constructed with \emph{A.} \emph{alternata} due to
non-proteinase activation of MAPK pathways (O'Grady, Patil, Melkamu,
Maniak, Lancto \& Kita, 2013; Yee et al., 2018). Pre-treatment with
increasing concentrations of C391 (30 nM - 100 \selectlanguage{greek}μ\selectlanguage{english}M) for 15 min
inhibited\emph{A. alternata (} 20 \selectlanguage{greek}µ\selectlanguage{english}g/mL\emph{)} -induced p-MAPK with an
IC\textsubscript{50} of 12.6 \selectlanguage{greek}μ\selectlanguage{english}M (CI: 3.2 - 50.6 \selectlanguage{greek}μ\selectlanguage{english}M). C391 alone did not
alter p-MAPK at any of the concentrations tested (not
shown;\textbf{Figure 2C} ).

We have previously shown that \emph{A. alternata} -induced activation of
PAR2 leads to recruitment of \selectlanguage{greek}β-\selectlanguage{english}arrestin-2, and many of the indicators of
allergic asthma involving PAR2 activation are abolished in
\selectlanguage{greek}β-\selectlanguage{english}arrestin-2\textsuperscript{-/-} mice (Nichols et al., 2012; Yee et
al., 2018). To determine whether C391 inhibits \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 recruitment
to PAR-2 in response to \emph{A. alternata} , we used bioluminescence
resonance energy transfer (BRET) between PAR2-YFP and \selectlanguage{greek}β-\selectlanguage{english}arrestin2-RLuc.
We have previously used this technique to monitor recruitment of
\selectlanguage{greek}β-\selectlanguage{english}arrestin-1 and 2 to PAR2 in response to trypsin, the potent
peptidomimetic agonist 2-f-LIGRLO-NH\textsubscript{2} (which is an
equivalent agonist to 2-at-LIGRL-NH\textsubscript{2} used in the
Ca\textsuperscript{2+} and MAPK signaling assays), and
\emph{A.alternata} (Flynn et al., 2011; McGuire, Saifeddine, Triggle,
Sun \& Hollenberg, 2004; Nichols et al., 2012; Yee et al., 2018). C391
blocked \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 recruitment in response to
2-f-LIGRLO-NH\textsubscript{2} (Imax = 88\selectlanguage{ngerman}±4\%; Log
IC\textsubscript{50}=-7.8±0.07, CI: 7-21nM, n=6; \textbf{Figure 3A} ),
or \emph{A. alternata}(Imax = 88±5\%; Log IC\textsubscript{50}
=-7.9±0.17, CI 1 -- 27 nM; n=3\textbf{; Figure 3B)} . We conclude that
C391 can inhibit \emph{A. alternata} -induced Ca\textsuperscript{2+}
mobilization, MAPK phosphorylation and \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 recruitment, with
similar efficacy to that seen when PAR2 is activated by peptidomimetic
agonists or trypsin.

\textbf{Alternaria alternata-\emph{induced inflammation and mucus
overproduction in C57Bl/6 and BALB/c mice is limited by C391}}

We have reported that intranasal administration \emph{A.
alternata}increased PAR2-dependent inflammation, mucus production and
mucus cell hyperplasia in wild type but not
\selectlanguage{greek}β-\selectlanguage{english}arrestin-2\textsuperscript{-/-}C57Bl/6 mice (Yee et al., 2018). To
determine if \emph{A. alternata}-induced inflammation could be
controlled pharmacologically with C391, we examined hallmarks of
cellular inflammation after the same exposure to 8 \selectlanguage{greek}µ\selectlanguage{english}g \emph{A.
alternata} filtrate (3 times over 8 days), with or without C391. As
previously reported, histological grading of Hematoxylin and Eosin (H \&
E) -stained lungs \textbf{(Figure 4A-F; K-L, n = 3)} , revealed that
\emph{A. alternata} sensitization and challenge increased inflammation
(histological score 3.3 \selectlanguage{ngerman}± 0.2 compared to 1.3 ± 0.3 in HBSS-treated
controls) and epithelial thickening (2.4 ± 0.28-fold over saline
controls). Concurrent administration of 2.5 nmoles C391 with\emph{A.
alternata} significantly reduced inflammation and epithelial thickening
(histological scores of 1.2 ± 0.1, p\textless{}001 and 0.98 ± 0.0.9,
p=.0012, respectively). A lower dose (0.25 nmoles) of C391 reduced
inflammation to a lesser extent (histological score of 2.3 ± 0.25) but
did not significantly reduce epithelial thickening. A second hallmark of
asthma is the increased production of mucus and \emph{A. alternata}
filtrate increased the number of epithelial cells staining positive for
mucus (80.2 ± 3.1\% compared to 13.7 ± 4.2\% in saline controls), which
was reduced by administration of C391 (42.8 ± 6.4\% with 0.25 nM and
33.9 ± 2.3\% with 2.5 nM C391, p\textless{}.001 for both concentrations;
\textbf{Figure 4G - J, M,} n = 3).

While the C57Bl/6 mice from above provided comparison to studies that
used genetically manipulated mice (Nichols et al., 2012; Yee et al.,
2018), BALB/c mice represent a strain that is more prone to allergen
response. H \& E stains demonstrated extensive lung inflammation in
response to \emph{A. alternata} in BALB/c mice (\textbf{Figure 5B} )
that was reduced in the presence of C391 (\textbf{Figure 5C} ). \emph{A.
alternata} histology scores showed increased inflammation over control
that was significantly reduced when the PAR2 antagonist C391 was
included in the \emph{A. alternata} filtrate \textbf{(Figure 5D} ; n =
4, p \textless{} 0.05\textbf{)} . Mucus staining and mucus cell
hyperplasia in the BALB/c mice were evaluated via Periodic Acid Schiff
(PAS) staining (\textbf{Figure 5E - G} ). \emph{A. alternata} treatments
significantly increased PAS staining from a limited baseline scoring (0
on a scale of 5; see Methods) to a robust mucus score (4.2 ±
0.2;\textbf{Figure 5F} ). Inclusion of C391 in the \emph{A.
alternata}treatment significantly reduced the PAS score (2.6 ± 0.4, p
\textless{} 0.05; \textbf{Figure 5G} ), although this remained
significantly above control scores \textbf{(Figure 5E, H} , p
\textless{} 0.05\textbf{)} .

\emph{Cellular composition of Alternaria}\textbf{-\emph{induced airway
inflammation}}

\emph{A. alternata} increased the total number of cells in BALF in both
C57Bl/6 mice (\textbf{Figure 6A} , \textbf{B} ; n = 3 mice per group)
and BALB/c mice (\textbf{Figure 6C, D;} n = 4 mice per group).
Differential cell counts demonstrated that the primary inflammation was
due to increased eosinophils (increasing from 1,150 ± 450 cells to
18,000 ± 4,700 cells in C57/Bl-6 and from 240 ± 140 cells to 49,000 ±
15,000 cells in BALB/c), and lymphocytes (increasing from 2400 ± 821 to
16,000 ± 2656 cells in C57Bl/6 and 650 ± 550 to 12,000 ± 3,000 cells in
BALB/c). In C57Bl/6 mice, 2.5 nM C391 reduced eosinophils and
lymphocytes in the BALF by 90\% (2100±750, 1700±500). Treatment with a
lower concentration of C391 (0.25 nmoles) reduced eosinophils by 75\%
(4300±1900) and lymphocytes by 80\% (3300±550). In BALB/c mice,
treatment with 5 nM C391 reduced eosinophils in the BALF 50\% (23,000 ±
756 cells) and lymphocytes by 80\% (2,700 ± 476 cells), but only the
reduction in lymphocytes was statistically significant (p \textless{}
0.05) compared to the \emph{A. alternata} treatment. In BALB/c but not
C57Bl/6 mice, \emph{A. alternata} significantly increased the number of
macrophages in the BALF (38,000 ± 610 cells to 95,000 ± 1,400 cells,
respectively) and this was significantly reduced by C391 (48,000 ± 6,200
cells, p \textless{} 0.05). A minimal neutrophilic response was observed
in both mouse strains and this was not reduced by C391. Taken together,
these data support that \emph{A. alternata} -mediated airway
inflammation (infiltration of eosinophils and lymphocytes, epithelial
thickening, and mucus overproduction) in C57Bl/6 and BALB/c mice, can be
inhibited by the PAR2 antagonist, C391.

\textbf{Alternaria alternata-\emph{induced airway hyperresponsiveness in
BALB/c mice}}

To better evaluate the physiological response of mice to \emph{A.
alternata} and the potential for C391 to limit this response, we used
the forced oscillation technique to measure respiratory resistance in
response to methacholine (MCh) challenge in \emph{A. alternata}
-sensitized/challenged BALB/c mice (Addison, Morse, Robichaud, Daines \&
Ledford, 2017). \emph{A. alternata} treatment resulted in an increase in
airway hyperresponsiveness (AHR) to MCh as reported by total airway
resistance (Max Rrs \% over baseline) that intensified with each dose of
MCh. In comparison, mice treated with HBSS alone responded to MCh
challenges with stable and relatively low total airway
resistance\textbf{(Figure 7)} . In contrast to \emph{A. alternata-}
treated mice, mice receiving both \emph{A. alternata} and C391 exhibited
significantly reduced total airway resistances that were not
significantly different than total airway resistances observed in
HBSS-treated control animals. These results suggest PAR2 inhibition by
C391 is effective at protecting against the development of AHR in an
acute, \emph{A. alternata} -sensitized/challenged murine model.



\textbf{DISCUSSION}

In this study we demonstrate that the potent PAR2 antagonist, C391, can
effectively inhibit allergen (\emph{Alternaria alternata} )-induced
canonical PAR2 signaling (Ca\textsuperscript{2+} and \selectlanguage{greek}β-\selectlanguage{english}arrestin/MAPK) in
human cell lines and significantly diminish \emph{A. alternata} -induced
asthma indicators in pre-clinical murine models. Immortalized human
bronchial epithelial cells, such as 16HBE14o-, are widely used in
pharmacological \emph{in vitro} studies centered around pulmonary
physiology (Forbes, Shah, Martin \& Lansley, 2003). They are ideal for
drug discovery in the space of PAR-2-directed asthma therapeutics
because they naturally express PAR2 and retain many features of
differentiated bronchial epithelial cells including tight junction
formation, barrier function and directional ion transport (Cozens et
al., 1994). C391 was the first PAR-2 antagonist we identified, using
16HBE14o- for initial screening and subsequent assessment of
Ca\textsuperscript{2+} and MAPK activation in response to trypsin and
high-affinity peptidomimetic PAR2 agonists (Boitano et al., 2015; Flynn
et al., 2011). Here, we have expanded the \emph{in vitro} analyses of
C391 to verify that it can effectively inhibit natural asthma-associated
allergen (\emph{A. alternata} )-induced, PAR2-dependent
Ca\textsuperscript{2+} and MAPK activation in airway epithelial cells.
Since PAR-2 can promote MAPK activation through both G-protein and
\selectlanguage{greek}β-\selectlanguage{english}arrestin pathways (Ge, Ly, Hollenberg \& DeFea, 2003), and
\selectlanguage{greek}β-\selectlanguage{english}arrestin-dependent signaling is crucial for the inflammatory effects
of \emph{A. alternata} -induced PAR2 activation in the airway (Nichols
et al., 2012; Yee et al., 2018), we have also confirmed that C391
inhibits \selectlanguage{greek}β-\selectlanguage{english}arrestin recruitment, using BRET in transfected HEK293 cells.

We show that this PAR2 antagonist effectively reduces asthma indicators
(including inflammation, eosinophil infiltration, mucus cell
hyperplasia/mucus overproduction and airway hyperresponsiveness) in
two\emph{Alternaria alternata} -induced murine asthma models.
Asthma-associated allergen exposure in mouse models normally triggers a
cascade of Th2 cytokine producing processes that result in eosinophil
and leukocyte recruitment and residence in the airway (Wardlaw, 1999).
While we cannot conclude as to where in the inflammatory pathway C391 is
altering recruitment (e.g., airway epithelial- or other cell-dependent
cytokine/chemokine release, or in the inflammatory cell response to
increased proteinase), it is clear that concurrent or prophylactic
treatment of C391 is effective in limiting \emph{A. alternata} -induced
inflammatory cell recruitment, mucus overproduction and airway
hyperresponsiveness. More work is needed to clarify if C391 antagonism
of \emph{A. alternata-induced} signaling is sufficient to reduce asthma
indicators (i.e., act as a therapeutic) or, if C391 is additionally
antagonizing downstream, endogenous protease activation of PAR2 on
epithelial cells or other cells in the airway.

Serine proteinases that activate PAR2 are a recurring theme among
asthma-associated allergens (Adams et al., 2011), including those
from\emph{Alternaria} and other molds (Boitano et al., 2011; Chiu,
Perng, Yu, Su \& Chow, 2007; Yee et al., 2018), house dust mite
(Stewart, Thompson \& Simpson, 1989; Sun, Stacey, Schmidt, Mori \&
Mattoli, 2001) and cockroach (Ock, Kim, Kim \& Byun, 2005; Polley et
al., 2017; Sudha, Arora, Gaur, Pasha \& Singh, 2008).
PAR2\textsuperscript{-/-} murine models have been used to demonstrate a
key role for PAR2 in the inflammation induced by these allergens
(Davidson et al., 2013; Day, Zhou, Ledford \& Page, 2010; Page, Ledford,
Zhou, Dienger \& Wills-Karp, 2010; Yee et al., 2018). Additionally, mast
cell tryptase and neutrophil elastase represent endogenous
PAR2-activating proteases that contribute to the asthma phenotype
(Andersson, Tufvesson, Diamant \& Bjermer, 2016; Kabashima et al., 2018;
Ogawa et al., 2018). PAR2-neutralizing antibodies were recently shown to
reduce asthma symptoms in acute and chronic allergic asthma mouse models
(Asaduzzaman, Davidson, Nahirney, Fiteih, Puttagunta \& Vliagoftis,
2018; Asaduzzaman et al., 2015), and over-expression of PAR2 or
intranasal administration of PAR-2 activating peptides (SLIGRL or
2-furoyl-LIGRLO-NH\textsubscript{2}) promotes cellular inflammation and
airway hyperresponsiveness(Ebeling, Forsythe, Ng, Gordon, Hollenberg \&
Vliagoftis, 2005; Nichols et al., 2012). Paradoxically, PAR2 activation
also results in a beneficial Ca\textsuperscript{2+}-dependent
PGE\textsubscript{2} secretion from airway epithelial cells, allowing
for smooth muscle relaxation (Chow, Moffatt \& Cocks, 2000; Cocks et
al., 1999; Henry, 2006; Jairaman, Yamashita, Schleimer \& Prakriya,
2015; Nichols et al., 2012) and resolution of neutrophilia (Rayees et
al., 2019). We have demonstrated that the protective and inflammatory
responses are mediated by separate G-protein and \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 signaling
pathways, respectively (Nichols et al., 2012; Yee et al., 2018). While
these studies might suggest that a biased antagonist that effectively
prevents PAR2-dependent \selectlanguage{greek}β-\selectlanguage{english}arrestin signaling, C391 mitigates multiple
asthma indicators, including airway hyperresponsiveness, despite
blocking both pathways.

There are only a handful of PAR2 antagonists that have been developed
{[}reviewed in (Yau, Lim, Liu \& Fairlie, 2016){]}. These include
K-14585 (Goh, Ng, Nilsson, Kanke \& Plevin, 2009; Kanke et al., 2005),
GB88 (Hollenberg et al., 2014; Suen et al., 2012; Suen et al., 2014),
two small molecules isolated from a large screening library (AC-55541
and AC-264613; (Gardell et al., 2008)), two PAR2 allosteric antagonists
AZ8838 and AZ3451 from Astra Zeneca (Cheng et al., 2017; Kennedy et al.,
2020), a group of small molecules: I-343, I-191 and I-287, from Vertex
(Avet et al., 2020; Jiang et al., 2018; Jimenez-Vargas et al., 2018),
and C391 (Boitano et al., 2015). C391 is distinct from these other
antagonists in that it requires minimal pre-incubation for inhibition of
multiple PAR2 signaling pathways. Although we observed differences in
calculated IC\textsubscript{50} values for C391 between the \emph{in
vitro} Ca\textsuperscript{2+} and MAPK signaling (\selectlanguage{greek}µ\selectlanguage{english}M) and \selectlanguage{greek}β-\selectlanguage{english}arrestin
recruitment (nM), these experiments were in different cell types that
include naturally expressed PAR2 (16HBE14o- cells used for
Ca\textsuperscript{2+} and MAPK) vs transfected PAR2 (HEK 293 cells for
\selectlanguage{greek}β-\selectlanguage{english}arrestin), and we cannot conclude that C391 is a biased antagonist.
Other small molecule PAR2 antagonists also show differences in potency
for different signaling pathways. For example, AZ3451 was reported to
have a lower IC\textsubscript{50} for Ca\textsuperscript{2+}mobilization
(nM) than for \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 recruitment (\selectlanguage{greek}µ\selectlanguage{english}M) and AZ8588 was reported to
have a lower IC\textsubscript{50} for \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 recruitment than
Ca\textsuperscript{2+} mobilization. Both compounds inhibited
inflammation to a similar extent in a paw edema model (Kennedy et al.,
2020). The PAR2 antagonists from Vertex all inhibited PAR2-induced
G-protein but not \selectlanguage{greek}β-\selectlanguage{english}arrestin signaling and show partial efficacy in
mouse models of pain and subcutaneous inflammation (Avet et al., 2020;
Jiang et al., 2018; Jimenez-Vargas et al., 2018). While these inhibitors
show efficacy in peripheral inflammation and pain models, the ability to
inhibit \selectlanguage{greek}β-\selectlanguage{english}arrestin signaling appears to be important for reduction of
asthma indicators, making C391 a more promising therapeutic. It is
possible C391 could be further modified to be more selective for
\selectlanguage{greek}β-\selectlanguage{english}arrestin-2 and this might increase its efficacy as an asthma
therapeutic.

Asthma can be triggered by environmental allergens such
as\emph{Alternaria} , pollens, insect droppings and infection. Due to
the heterogeneity of its pathology, it remains difficult to manage in
some patients, particularly those with moderate to severe disease. Many
of the commonly used treatments for asthma address symptoms without
addressing underlying triggers for the disease. Traditional treatments
include corticosteroids and mast cell stabilizers for inflammation
control, short and long acting \selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}-agonists to relax
bronchial smooth muscle and increase airflow; anticholinergics as
supplemental broncho-relaxants and, leukotriene disruptors (receptor
antagonists or leukotriene synthesis inhibitors) used as an
anti-inflammatory therapy (Martinez \& Vercelli, 2013; Pera \& Penn,
2016). Monoclonal antibody treatments that target immunoglobulin E,
IL-13, IL-5 or IL-4-Receptor show some potential for patients with
moderate to severe asthma who remain poorly controlled with steroids,
although clinical trials have had mixed results. Some patients
experienced improved lung function; however, in many cases overall
eosinophilic inflammation was not significantly reduced and clinical
endpoint reduction was limited (Chung, 2018; Hanania et al., 2016;
Stephenson et al., 2016). Despite these varied approaches, asthma
remains uncontrolled in more than half the patients receiving standard
asthma medication (Peters, Jones, Haselkorn, Mink, Valacer \& Weiss,
2007). While a portion of this population includes non-compliant
patients, a large proportion of patients with refractory asthma would
stand to benefit from improved therapeutics that ameliorate allergic
asthma symptoms. Given that PAR2 is upregulated in both human
respiratory epithelium and peripheral blood monocytes in asthmatics
(Knight et al., 2001; Shrestha Palikhe et al., 2015), PAR2 signaling is
of great interest in asthma development and severity.

A primary advance in this work is the first demonstration of
pharmacological inhibition of allergen-induced asthma indicators
(inflammation, mucus secretion and airway hyperresponsiveness) in
pre-clinical animal models. That C391 reduces airway mucus production is
also particularly exciting because of its potential for reducing asthma
mortality. It has been observed that airway mucus plugging due to goblet
cell hyperplasia coupled with peripheral airway mucus accumulation
appears to be more prevalent in patients who eventually succumb to a
severe asthma attack (Aikawa, Shimura, Sasaki, Ebina \& Takishima,
1992). These same patients appear to be hypo-responsive to
bronchodilators and anti-inflammatory medications. Providing a therapy
that could control mucus production would allow for control of disease
through minimizing airway obstruction caused by mucus plugging and
possibly provide improved patient outcomes. C391 also reduced the
development of airway hyperresponsiveness (AHR) in our pre-clinical
models. AHR closely resembles a bronchoconstriction event or asthma
attack in sensitized individuals and represents a measure that is
commonly used in diagnostic procedures in the clinic. This suggests
inhibition of PAR2 would be useful for prophylaxis in vulnerable
individuals and as a potential treatment for airway hyperresponsiveness
in allergic asthmatics.

We acknowledge that the studies here represent a prophylactic treatment
and it is unknown if application of PAR2 pharmacophores could be
effective in a therapeutic setting, e.g., after the establishment of
asthma. However, these data, and the recent report that PAR2-specific
antibodies can be effective in both preventing and controlling asthma
indicators in pre-clinical exposure models (Asaduzzaman, Davidson,
Nahirney, Fiteih, Puttagunta \& Vliagoftis, 2018; Davidson et al., 2013)
support PAR2 as a druggable target for addressing allergen-induced
asthma. Furthermore, C391 specifically presents as a novel lead compound
in the pursuit of novel asthma drugs.

\textbf{Figure Legends}

\textbf{Figure 1: \emph{A. alternata} -induced
Ca\textsuperscript{2+}signaling in airway epithelial cells is blocked by
C391.} \textbf{(A)}Percentage of 16HBE14o- cells responding with
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} increases [?] 150 nM within
the experimental time frame following \emph{A. alternata} (\selectlanguage{ngerman}± C391)
addition. (\textbf{B - D)} Representative population graphs from single
experiments depicting the average change in
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} in 16HBE14o- cells in
response to indicated compound plotted over time and shown with SEM in
the presence of: \textbf{(B)} 3 \selectlanguage{greek}µ\selectlanguage{english}M C391 alone; \textbf{(C)} \emph{A.
alternata} EC\textsubscript{50} alone and
\textbf{(D)}EC\textsubscript{50} of \emph{A. alternata} combined with 3
\selectlanguage{greek}µ\selectlanguage{english}M C391.\textbf{(E - H)} Changes in
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} are shown as color maps
over time in response to \emph{A. alternata} filtrate. Individual panels
are from \textbf{(E)} 20 sec prior to EC\textsubscript{50} of \emph{A.
alternata} filtrate addition and at \textbf{(F - H)} 60 sec intervals.
White lines approximate cell borders, color key for
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} is displayed in lower
right of first panel. \textbf{(I - L)} The same experiment as (\textbf{E
- H} ) excepting a 2 min preincubation with 3 \selectlanguage{greek}µ\selectlanguage{english}M C391 and continued 3 \selectlanguage{greek}µ\selectlanguage{english}M
C391 treatment during \emph{A. alternata} filtrate exposure.\textbf{(M)}
Percentage of 16HBE14o- cells responding with
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} increases [?] 150 nM within
the experimental time frame in response to the P2Y agonist ATP (2.5 \selectlanguage{greek}µ\selectlanguage{english}M)
or the PAR1 agonist Thrombin (100 \selectlanguage{greek}µ\selectlanguage{english}M) \selectlanguage{ngerman}± C391.
\textbf{(N-Q)}Representative population graphs from single experiments
depicting the average change in
{[}Ca\textsuperscript{2+}{]}\textsubscript{i} in 16HBE14o- cells in
response to indicated compound plotted over time and shown with SEM in
the presence of: \textbf{(N)} 2.5 \selectlanguage{greek}µ\selectlanguage{english}M ATP alone;\textbf{(O)} 2.5 \selectlanguage{greek}µ\selectlanguage{english}M ATP
and 3 \selectlanguage{greek}µ\selectlanguage{english}M C391 \textbf{(P)} 100 \selectlanguage{greek}µ\selectlanguage{english}M Thrombin;\textbf{(Q)} 100 \selectlanguage{greek}µ\selectlanguage{english}M Thrombin
and 3 \selectlanguage{greek}µ\selectlanguage{english}M C391.

\textbf{Figure 2: \emph{A. alternata} -induced MAPK signaling in airway
epithelial cells is inhibited by C391.} \textbf{(A)} MAPK
phosphorylation (p-MAPK) undergoes a concentration-dependent increase in
response to the potent and selective PAR2 agonist
2at-LIGRL-NH\textsubscript{2}. \textbf{(B)} MAPK phosphorylation
(p-MAPK) undergoes a concentration-dependent increase in response
to\emph{A. alternata} filtrates. \textbf{(C)} Concentration response
curve for C391 block of \emph{A. alternata} (20 \selectlanguage{greek}µ\selectlanguage{english}g/mL)-induced MAPK
phosphorylation; IC\textsubscript{50} is noted in the text. For all
experiments, 3 [?] n [?] 4. * indicates difference from HBSS-treated
control\textbf{(A, B)} or from \emph{A. alternata} -treated
control\textbf{(C)} , p [?] 0.05.

\selectlanguage{greek}\textbf{Figure 3: Activation-dependent β-arrestin-2 recruitment to PAR2
is inhibited by C391.}\selectlanguage{english} Bioluminescence resonance energy transfer (BRET)
was measured upon addition of: \textbf{(A)} the potent PAR2 agonist
2-furoyl-LIGRLO-NH\textsubscript{2} (1 \selectlanguage{greek}µ\selectlanguage{english}M) or \textbf{(B)} \emph{A.
alternata} filtrate (\emph{Alt} ; 65 \selectlanguage{greek}µ\selectlanguage{english}g/mL) and increasing
concentrations of C391. C391 effectively blocked \selectlanguage{greek}β-\selectlanguage{english}arrestin-2
recruitment following PAR2 activation via peptidomimetic or \emph{A.
alternata} proteinase. n [?] 3 for all experimental time points;
IC\textsubscript{50}s are noted in the text.

\textbf{Figure 4: C391 reduces asthma indicators in a C57Bl/6 murine
model.} \textbf{(A - F)} Typical H\&E staining of C57Bl/6 lungs treated
with: \textbf{(A)} HBSS; \textbf{(B, E)} \emph{A. alternata}
;\textbf{(C)} \emph{A. alternata} + 0.25 nmoles C391 or \textbf{(D,
F)}\emph{A. alternata} + 2.5 nmoles C391. Images A-D were imaged with
10X and E-F were imaged with a 40X objective. \emph{A. alternata}
induces significant inflammation (increased cellular staining) that is
reduced by low concentrations of C391 and mostly eliminated by higher
concentrations of C391. \textbf{(G - J)} Typical Alcian Blue staining of
C57Bl/6 lungs treated with: \textbf{(G)} HBSS vehicle
control;\textbf{(H)} \emph{A. alternata} ; \textbf{(I)} \emph{A.
alternata} + 0.25 nmoles C391 or \textbf{(J)} \emph{A. alternata} + 2.5
nmoles C391.\textbf{(K - L)} Semi-quantitative histological grading of
lung inflammation and Alcian Blue staining shows a significant increase
in cellular infiltrate (\textbf{K} ), thickening of the basement
membrane (\textbf{L} ) \% bronchial epithelial cells displaying positive
mucus staining (\textbf{M} ), that are significantly reduced by either
low (0.25 nmoles) or high (2.5 nmoles) C391 concentrations. n = 3 for
all experiments, * indicates significant difference from HBSS control,
\# indicates significant difference from \emph{A. alternata} -induced
changes; p [?] 0.05.

\textbf{Figure 5: C391 reduces asthma indicators in BALB/c murine
model.} \textbf{(A - D)} Typical H\&E staining of BALB/c lung slices
from animals treated with: \textbf{(A)} HBSS vehicle
control;\textbf{(B)} \emph{A. alternata} ; or \textbf{(C)} \emph{A.
alternata} + 5 nmoles C391. \textbf{(D)} Semi-quantification of H\&E
staining. Typical PAS staining of BALB/c lung slices from animals
treated with:\textbf{(E)} HBSS vehicle control; \textbf{(F)} \emph{A.
alternata} ;\textbf{(G)} or \emph{A. alternata} + 5 nmoles C391.
\textbf{(H)}Semi-quantification of PAS staining. \emph{A. alternata}
treatment induces significant increases in inflammation and mucus
production that is reduced by C391. B denotes bronchioles, M denotes
mucus plug, simple arrows indicate inflammatory cell staining and closed
arrows are directed at goblet cell staining. n [?] 4 for all experiments;
* indicates significant difference from HBSS control; \# indicates
significant difference from \emph{A. alternata} -induced staining; p [?]
0.05.

\textbf{Figure 6: C391 Reduces airway leukocyte recruitment in C57Bl/6
and BALB/c murine asthma models.}

\textbf{(A - D)} Quantification of bronchoalveolar lavage fluid (BALF)
cell counts in C57Bl/6 \textbf{(A,B)} and BALB/c (\textbf{C, D} ) as
follows: (\textbf{A, C} ) total cells; \textbf{(B, D)} neutrophils,
eosinophils, lymphocytes, and macrophages (with macrophage
quantification shown on the right y- axis). \emph{A. alternata}treatment
significantly increased total cell infiltration, and specifically the
recruitment of eosinophils and lymphocytes in both mouse strains, as
well as increased macrophages in BALB/c. Eosinophils and lymphocytes in
C57Bl/6 were significantly attenuated in \emph{A. alternata} exposed
mice treated with C391, and lymphocytes and macrophages were
significantly attenuated in \emph{A. alternata} exposed mice treated
with C391 in BALB/c. * Indicates significant difference between HBSS
treated and \emph{A. alternata} exposed mice; p [?] 0.05 . \# Indicates
significant difference between \emph{A. alternata} exposed mice and
\emph{A. alternata} exposed mice treated with C391; p [?] 0.05.

\textbf{Figure 7: C391 reduces airway hyperresponsiveness in a BALB/c
murine asthma model.}

BALB/c mice were given nasal instillations of treatment (HBSS
control,\emph{A. alternata} or \emph{A. alternata} with C391) on days 0,
3, 6 and forced oscillation technique with methacholine challenge to
measure lung resistance was performed on day 8. The percent change in
maximum total airway resistance (Max Rrs) is reported\textbf{.} \emph{A.
alternata} treatment increases Max Rrs measurements over control.
However, \emph{A. alternata} treatment in the presence of C391 the Max
Rrs are similar to control. n [?] 4 for each treatment. * Indicates
significant difference between HBSS treated and \emph{A.
alternata}exposed mice; p [?] 0.05. \# Indicates significant difference
between\emph{A. alternata} exposed mice and \emph{A. alterna} ta exposed
mice treated with C391; p [?] 0.05.



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