Animals and Animal Procedures

Transgenic mice expressing the human (h) ACE2 controlled by the human keratin 18 (K18) promoter (K18-hACE2) were purchased from the Jackson Laboratory and housed in a sterile, ventilated facility at Old Dominion University (ODU) under standard husbandry. Male mice, 8–10 weeks of age, were used in this study. The Institutional Animal Care and Use Committee of ODU approved all the procedures.

S1SP-induced acute lung injury was modeled by intratracheal (i.t.) injection of S1SP (Ray Biotech, 230–01101) into mice at a dose of 0.5 mg/kg (50 µl) using a micro sprayer (Penn Century Inc.) under anesthesia with ketamine and xylazine. The control mice received 50 µl PBS via i.t. injections. CB2R agonist HU308(3088, Tocris Biosciences) was dissolved in a vehicle (1% DMSO + 1% Tween-80 in PBS). To activate CB2R, HU308 (5 mg/kg) was administered to mice via the intraperitoneal (i.p.) route, 1 h after S1SP administration, and every 24 h after that. Mice were distributed into the following groups: (1) vehicle + PBS, (2) HU308 + PBS, (3) vehicle + S1SP, (4) HU308 + S1SP. ALI was assessed at 48 h following S1SP exposure.

Measurement of Lung Mechanics

Lung mechanics were measured using the FlexiVent system and FlexiWare software (SCIREQ, Montreal, Canada). After 48 h following S1SP exposure, anesthetized mice were intubated intratracheally with an 18 1/2 G catheter and ventilated at a lower tidal volume (10 ml/kg) and 150 breaths/minute for 10 min. Tissue dampening (G) and static lung compliance (Cst) were assessed using FlexiWare software, according to manufacturer recommendations.

Analysis of Bronchoalveolar Lavage Fluid (BALF)

After measuring lung mechanics, the lungs of mice were lavaged with 3 ml of PBS (3 × 1 ml). The total cell number in the BALF was determined using an automated cell counter (Countess II FL, ThermoFisher Scientific). For differential cell count analysis, BALF cells were cytocentrifuged onto glass slides and subsequently subjected to differential staining using the Kwik–Diff™ kit (Thermo Fisher Scientific) following the manufacturer’s protocol. The total protein content in the BALF was measured using the BCA Protein Assay Kit (Bio-Rad Laboratories). Cytokine levels, specifically IL-6, TNF-α, and IL-10 in the BALF were quantified by ELISA (R&D Systems).

Lung Tissue Processing

After collecting the BALF, the right mainstem bronchus was tied off with a 4–0 silk suture, and the right lung was cut and snap-frozen in liquid nitrogen and stored at − 80 °C until further use. The left lung was inflated with 4% paraformaldehyde at 20 cm H2O and fixed overnight at 4 °C. The left lobe was processed for paraffin embedding and tissue sectioning.

Isolation of Mouse Primary Alveolar Macrophages (AMs)

Mouse AMs were isolated as previously described [11, 15]. Following S1SP exposure, BALF was collected from the mice by lavaging the lungs with 3 ml of PBS, 1 ml at a time. The collected cell suspension was centrifuged at 1000 rpm for 10 min, at 4 °C, and the resulting pellet was resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% non-heat inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). BALF cells were plated in a complete DMEM medium for 3 h, followed by extensive washing with PBS to remove unattached cells. The attached cells were identified as AMs. These cells were harvested by gentle scraping and subsequently used for immunoblot analysis.

Isolation of Mouse Primary Alveolar Epithelial Cells

Primary alveolar epithelial cells were isolated from mouse lungs using a previously described protocol [16]. Briefly, following BALF collection, lung tissue was digested with dispase (1.8 U/mL) and passed through a 100-µm mesh filter. Leukocytes were depleted by panning on IgG-coated dishes. The resulting nonadherent cells were sequentially incubated with anti-mouse CD90 antibody to remove fibroblasts, followed by anti-mouse CD31 antibody conjugated to Dynabeads to isolate endothelial cells. The CD31-negative fraction, enriched for a mixture of type I and type II alveolar epithelial cells, was collected and processed for immunoblot analysis.

Immunostaining

Paraffin-embedded lung Section (5 μm) were deparaffinized, hydrated, and subjected to antigen retrieval. The sections were stained for neutrophil elastase (NE) using an anti-NE antibody (1:200, ab68672, Abcam) and for H2B using anti-H2B (1:100, ab52484, Abcam). Sections were then incubated with fluorochrome-labeled species-specific secondary antibodies, and the resultant stained sections were imaged using an Olympus IX73 fluorescent microscope. Three to six mice from each group were used in this experiment.

Immunoblot Analysis

Lung tissues or cells were lysed in Pierce™ IP Lysis Buffer (ThermoFisher Scientific). The lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. The protein concentration in the supernatant was determined by the BCA Protein Assay (Bio-Rad Laboratories). For the immunoblot, 40 µg of the total protein was resolved in SDS-PAGE and electroblotted onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% non-fat milk in PBS containing 0.1% Tween-20 for 30 min at room temperature. The membrane was incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies. The membrane was then developed using ECL Western Blot Substrate (ThermoFisher Scientific). The following primary antibodies were used: anti-IκB (4812, 1:1000), anti-P-IκB (9246, 1:500), anti-Stat3 (9139, 1:500), anti-P-Stat3 (9145, 1:500) from Cell Signaling Technology Inc., anti-peptidylarginine deiminase 4 (PAD4) (1:500), anti-CB2R (1:500) from Abcam, and anti-β-actin (1:5000, A5441, Sigma-Aldrich). For analysis of Nrf2 expression, nuclear extracts were prepared from lung tissue using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific) according to the manufacturer’s instructions. The nuclear fractions were subsequently processed for immunoblotting using anti-Nrf2 (1:1000, NBP1-32822, Novus Biologicals). For immunoblot analysis of BALF samples, equal volumes of BALF from each experimental group were subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were probed with the following primary antibodies: anti-Histone H2B (1:500; ab52484, Abcam) and anti-citrullinated histone H3 (CitH3) (1:500; ab5103, Abcam).

Statistical Analysis

All results are presented as the mean ± standard error of the mean (SEM). Statistical comparisons were made using Prism 10.4.2 (GraphPad Inc). The group differences were analyzed using Student’s t-test or one-way ANOVA, followed by a Tukey’s post-hoc multiple comparison tests when appropriate. Differences were considered statistically significant at p < 0.05.