Materials and methods
Ethical Statement
All animal studies were approved by the Old Dominion University IACUC and adhere to the principles of animal experimentation as published by the American Physiological Society.
Materials
The PTP4A3 inhibitor KVX-053 was kindly provided by KeViRx, Inc. (Charlottesville, VA). Recombinant SARS-CoV-2 Spike Protein, subunit 1 (S1SP) was purchased from RayBiotech (No. 230–011101-100), radioimmunoprecipitation assay buffer (RIPA) and protease inhibitor cocktail were obtained from Sigma-Aldrich Corporation (St. Louis, MO). Socumb (pentobarbital), Anased (xylazine), and Ketaset (ketamine) were supplied by Henry Schein Animal Health (Pittsburgh, PA). Wright-Giemsa Stain Kit, 10% formaldehyde, 10% SDS, and ammonium persulfate were purchased from Thermo Fisher Scientific (Waltham, MA, USA); the bicinchoninic acid (BCA) protein assay kit was from Pierce Co. (Rockford, IL), and EDTA and Western blot membranes from GE Healthcare (Chicago, IL). ProtoGel (30 % acrylamide mix) and tetramethyl ethylenediamine (TEMED) were from National Diagnostics (Atlanta, GA); Tris–HCl buffer was from Teknova (Hollister, CA), and Protein Dual Color Standards and Tricine Sample Buffer from Bio-Rad Laboratories (Hercules, CA). All antibodies were purchased from reputable commercial sources and have published immunospecificity data. Rabbit total and phosphorylated IκBα (No. 9242 and No. 2859 respectively), STAT3 (No. 4904 and No. 9145 respectively), and NLRP3 inflammasome antibody (No. 15101) were obtained from Cell Signaling Technology, Inc. (Denver, CO); mouse monoclonal anti-β-actin from Sigma-Aldrich Corporation; and IRDye 800CW Goat anti-rabbit and IRDye 680RD Goat anti-mouse were from LI-COR Biosciences (Lincoln, NE). A polyclonal antibody against PTP4A3 (or PRL3) for immunohistochemistry was purchased from Proteintech Group, Inc (Rosemont, IL).
Animal Model and Treatment Groups
Male K18-hACE2 transgenic mice 8–10 -week-old, 24–28 g body weight, were obtained from Jackson Laboratories (Bar Harbor, ME) and were housed under pathogen-free conditions.
S1SP (400 µg/kg in 2 mL/kg body weight) was given intratracheally to K18-hACE2 mice (Fig.1). Briefly, mice were anesthetized with intraperitoneal injection of xylazine (6 mg/kg) and ketamine (60 mg/kg). After cleaning and disinfecting the surgical field, a small neck skin incision (∼1 cm) was made, and the salivary glands were separated to visualize the trachea. Mice were suspended vertically from their incisors and a fine (20-22 G) plastic catheter was advanced from the mouth into the trachea (∼2 cm) in such a way that it could be seen through the walls of the trachea. S1SP was introduced and flushed with 150 µL air. The catheter was withdrawn, neck incision closed by surgical adhesive, and the animal was placed in the ventral position in a small chamber on top of a heating pad, under supplemental oxygen (slowly weaned from 100% to 21% O2) and observed constantly for the next 5 h for signs of respiratory distress before being returned to the regular cage. Mice were monitored daily for abnormal physical appearances. Seventy-two hours later, mice were subjected to bronchoalveolar lavage (Buzhdygan et al.), analysis of lung tissue and bronchoalveolar lavage fluid (BALF) and histological evaluation. The treatment group received PTP4A3 inhibitor KVX-053 (10 mg/kg, i.p.) 1, 24 and 48 h after S1SP instillation. Control included administration of saline to K18-hACE2 mice. All analyses were performed at 72h post intratracheal instillation.
Histopathology and Lung Injury Score
Mice were euthanized and fixed in the upright position. A small transverse incision was made in the middle of the trachea and the lungs were instilled and inflated with 10% formaldehyde solution to a pressure of 15 cm H2O using a 20 G catheter. The trachea was then ligated with suture above the bifurcation; the lungs were removed from the thorax and placed in 10% formaldehyde solution for 72 h. Mid-transverse slices were made from the formalin-fixed lung and embedded in paraffin. Sections 5 µm thick were stained with hematoxylin and eosin (H&E). Some sections were also immunostained with PTP4A3 polyclonal antibody. Twenty randomly selected fields from each slide were examined under ×10 and ×60 (with immersion) magnification and the lung injury score were computed (Matute-Bello et al., 2011).
Measurement of Total Protein, Cell, and Cytokine Concentration in BALF
BALF was centrifuged at 2,500 g for 10 min, and the supernatant was collected for total protein and cytokine analysis. Total protein concentration was determined using the BCA protein assay kit according to manufacturer’s instructions. Cell numbers were measured with a hemocytometer and differential analysis was performed with the Wright-Giemsa stain kit. Quantitative analysis of BALF and serum chemokines and cytokines were performed by the University of Virginia Core Facility, using a MILLIPLEX map murine cytokine/chemokine premixed panel (EMD Millipore, Burlington, MA) and a Luminex MAGPIX system.
Western Blotting
Lung homogenates from frozen lung tissues were prepared by sonication in ice-cold RIPA buffer with protease inhibitor cocktail (1:100) -EMD Millipore). Protein lysates were agitated for 3 h at 4°C, and then centrifuged at 14,000 g for 10 min. The supernatants were aspirated, and total protein concentration was determined with the BCA assay. Equal amounts of protein from the lysates were first mixed with tricine sample buffer 1:1, heat denatured at 95°C for 10 min and separated on a 10% SDS-PAGE gel by electrophoresis. Proteins were then transferred onto a nitrocellulose membrane, incubated with primary and secondary antibodies, and detected by digital fluorescence imaging (LI-COR Odyssey CLx). β-actin was used as a loading control. Densitometric quantification of the bands from the blots was performed using ImageJ software (National Institutes of Health).
Lung Mechanics
The mice were anesthetized with Socumb (pentobarbital 90 mg/kg, i.p.), tracheostomized with a metal 1.2 mm cannula, and connected to a FlexiVent small animal ventilator (SCIREQ Inc., Montreal, QC, Canada), as previously published(Solopov et al., 2021). Ventilation was performed at a tidal volume of 10 mL/kg and a respiratory rate of 150/min. A 15-min stabilization period was allowed before any measurements began. Firstly, following a deep inflation, resting static compliance (Cst) and pressure volume (PV) loops were estimated by stepwise increasing airway pressure to 30 cm H2O and then reversing the process. Both parameters reflect the intrinsic elasticity of the lungs and are either reduced (Cst) or shifted to the right (PV curves) in fibrosis. Secondly, Snapshot-150 and Quick Prime-3 maneuvers were performed. Respiratory system resistance (Rrs) and elastance (Ers), reflecting the behavior of the entire respiratory system (peripheral and conducting airways, chest wall, and parenchyma); Newtonian resistance (Rn) (Ruben M. L. Colunga Biancatelli et al.); tissue damping (G); inspiratory capacity (A); and the curvature of the PV loops (K) reflecting resistance of the large, conducting airways, parenchymal stiffness, and changes in inspiratory gas dynamics, were calculated, and are presented as a mean of 12 recordings.
Endothelial Cell Culture and Barrier Function
In‐house harvested and identified human lung microvascular endothelial cells (HLMVECs) were isolated and maintained in M199 media supplemented with 20% FBS and antibiotics/antimycotics as described previously (23). The barrier function of confluent endothelial cell monolayers was estimated using electric cell‐substrate impedance sensing (ECIS) model 1600R ζθ (Applied BioPhysics, Troy, NY) as previously described (24). Experiments were conducted with cells that had reached a steady‐state resistance of at least 800 Ω. The single time frequency (STF) mode was selected at 4,000 Hz and at an interval time of 600s. Experiments were performed in triplicate and repeated with at least three independent biological replicates. Resistance values were collected and normalized to t=0. Data are presented as means ±SEM.
Immunocytochemistry and Confocal microscopy
Round glass coverslips (Thermo Fisher Scientific) were placed in 12-well plates, soaked in 70% ethanol for 15 min, and dried under a laminar flow hood. The coverslips inside the wells were coated with 1 ml solution of 0.2% gelatin and incubated at 37°C for 30 min. Excess gelatin was aspirated, and then 500 μl of the cell suspension containing 6 × 105 HLMVEC were placed on top. Coverslips with 75-90% confluent HLMVEC were fixed in 4% paraformaldehyde in PBS for 5 min, washed 3 times, then subjected to permeabilization with 0.1% Triton-X 100 in TBS for 10 min, and subsequently washed three times with PBS. Cells were blocked with 5% BSA in 0.1% TWEEN-20 overnight at 4°C. The following day, coverslips were incubated with VE-cadherin antibody (Abcam, 1:50 dilution) in blocking buffer at 4°C for 24 h. After washing 5 times with PBS, secondary incubation was carried out with Alexa Fluor 488 goat anti-rabbit antibody (Thermo Fisher Scientific, dilution 1:500) in the dark at RT for 1 h and then washed 5 times with PBS. F-actin was visualized with Texas Red-X phalloidin (Life Technologies, diluted 1:300). At the counterstaining stage, cells were incubated with 300 μM 4’,6-diamidino-2-phenylindole (DAPI) for 5 min in the dark. One drop of mounting media (ProLong Gold, Thermo Fisher Scientific) was added to pre-cleaned microscope slides (Superfrost Plus, Thermo Fisher Scientific) and placed over the top of the coverslips facing down, extra PBS was removed from the edges, and final slides were placed in the dark at RT for overnight drying. Confocal microscopy was performed using an Olympus FluoView FV10i confocal microscope with a 488-nm excitation filter for VE-Cadherin (Alexa Fluor 488 Ab), 595-nm for F-actin (Texas Red Phalloidin Ab), and 359-nm filter for DAPI (nuclei). Each coverslip was automatically divided into 48 squares, and 10 squares were chosen randomly and analyzed under 60X magnification, as we previously described (Barabutis et al., 2020).