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).