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°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 1x105cells/cm2. 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%
CO2 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.
Alternaria alternata filtrate. A. alternatafiltrate 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).
Ca2+ signaling measurements .
Intracellular Ca2+ concentrations
([Ca2+]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 µM 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× 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. [Ca2+]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 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 [Ca2+]ifollowed by a 10 sec wash to introduce agonist and an additional 2 min
30 sec of monitoring for agonist-induced changes in
[Ca2+]i. For C391 antagonism
(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
(~1/sec) were calculated throughout the 3- or 5-min
experiments. [Ca2+]i traces over
time were represented as average
[Ca2+]i of all cells within a
field of view (~80 - 100) and are representative of at
least 4 experiments. Color maps of A. alternaria responses (+/-
C391) were constructed using Adobe Photoshop (Cupertino, CA) and
indicate approximate [Ca2+]i of
individual cells at given time in the experiment.
Detection of MAPK Signaling/In-Cell Western (ICW). 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 104 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 μM
AEBSF and 10 μM E64. On the following day, cells were treated with C391
suspended in MEM to a final concentration of 1× (final concentration
range of 100 nM - 100 µM C391) and cells were incubated for 15 min. This
was followed by a treatment of equal volumes of A. alternata(final concentration of 20 µg/mL; 2 U/mL trypsin activity) mixed with 1×
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. IC50s
were determined using a 4-parameter curve fit, with hill slopes
constrained to one, with GraphPad Prizm software (San Diego, CA).
Bioluminescence resonance energy transfer (BRET)
measurements of recruitment of β-arrestin-2. Competitive BRET
measurements with A. alternata (65 µg/mL; 6.5 trypsin U/mL) and
C391 (concentration range; 100 pM - 100 µM) were adapted from (Yee et
al., 2018). Briefly, HEK 293 cells transfected with PAR2-YFP and
Luciferase (Luc) tagged β-arrestin-2 were plated in 96-well microplates
and 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 (β-arrestin-Luc)
control values. To eliminate possibility that observed signals
represented non-specific interactions, we included a β-arrestin-Luc +
YFP in every experimental condition, which gave values similar to donor
only controls. Data points were fitted to a curve and
IC50 calculated using a 4-parameter curve fit, with hill
slopes constrained to one, with GraphPad Prizm (San Diego, CA).
Animal Studies: 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 andBritish 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 μL of HBSS control, 8
µg of A. alternata filtrate in HBSS; or 8 µg of A.
alternata filtrate supplemented with 0.25 nmoles or 2.5 nmoles of C391.
For BALB/c mice, 50 μL of HBSS control; 5 µg of A. alternatafiltrate in HBSS; or 5 µg of 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.
Airway hyperresponsiveness in A. 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 H2O (125 mg/mL) at a
final dose of 16 µL/gram of body weight. The average mouse weighed 25 g
and thus received 400 µL 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 H2O. 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 µL/gram of
body weight to eliminate any breathing interference in the recordings. A
25 g mouse would receive 250 µL of pancuronium bromide. Respiratory
mechanics were obtained by the forced oscillation technique; values were
reported as total pulmonary resistance (RT) cm H2O/mL/s.
Mice were sacrificed after completion of these experiments for
examination of cellular inflammation.
Histochemical and inflammation analyses in A.
alternata-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 µm 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×
air or 60× oil immersion objectives. Images were captured using a
MicroPublisher 6 camera (QImaging, Tucson). 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 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 µl) 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.
Data analysis: 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’st -test. A value of p < 0.05 was used to establish a
significant difference between samples. Data in Figures are graphed ±
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).