The study protocol was approved by the animal research and ethics committees of the Montreal Heart Institute and all experiments conducted in accordance with the Canadian guidelines for the care of laboratory animals.
Animal model and colchicine administration. Male Wistar rats weighing 250–300 g were purchased from Charles River, St. Constant, Québec. They were divided in three groups: Sham + placebo (n=8), oleic acid + placebo (n=8) and oleic acid + colchicine (n=8). Oleic acid (150 mg/kg in 0.3 cc) was prepared daily as a mixture with 0.1% bovine serum albumin (BSA) dissolved in distilled water, stored and protected from light at room temperature. Colchicine (1mg/kg) or distilled water (placebo) was administered by daily gavage (in a volume up to 2 ml/kg) for three days before the induction of the ALI model. On the beginning of the fourth day a last forth dose of colchicine or placebo was administered and rats immediately injected via a jugular vein cannula with oleic acid or with 0.1% BSA. There was no death in the sham group. There was one death in the oleic acid + placebo group 3 hours after injection of oleic acid and prior to any measurement. There was one surgical death in oleic acid + colchicine treatment by laceration of the jugular vein, prior to injection of oleic acid.
Oleic acid was administered under ketamine/xylazine anesthesia by slow bolus injection over 30 seconds and the catheter was flushed with 0.3 ml of 0.1% BSA before and after injection. Four hours later, the animals were studied. Conscious unrestrained respiratory parameters were obtained by whole body plethysmography (Emka technologies). Rats were then anesthetized with 2.5% isoflurance and 100% oxygen (1L/min) administered with a nose cone/face mask for a duration of 5 minutes before terminal exsanguination. 2ml of arterial blood was collected through the thoracic aorta into a syringe containing lyophilized heparin for arterial blood gas assessment. The remaining of blood was collected into EDTA Lavendar tubes for complete blood count and flow cytometry. Cytokines measurements were assessed with serum isolated with serum clot activator tubes.
The lungs were excised for analysis: the left lung was cannulated and perfusion-fixed with 10% buffered formalin for histology and immunohistochemistry. The right superior and middle lobes were used to measure the lung weight and edema. The inferior lobes of the right lung were snap-frozen and stored at -80 °C for gene expression analysis. Pulmonary edema was measured from the ratio of total divided by dry weight of the right lung. Total weight, including water, was measured and lung tissue was dried in an oven at 60 °C for 5 days and reweighted as dry weight.
Lung histology and immunohistology. All histological and immunohistological procedures were performed by the same person blinded to treatment assignment. The left lung was cannulated and perfused with 10% formalin PBS-buffered solution and fixed for 5 days. Tissues were dehydrated by incubating in a series of solutions with an increased ethanol content (70, 95 and 100%), followed by xylene, and embedded in paraffin. The specimens were cut into 6-µm sections, mounted on charged slides and processed with hematoxylin phloxine saffron (HPS) staining.
Immunohistological procedures were initiated by incubating the slides in citrate antigen retrieval (pH 6.0) and endogenous peroxidase blocking (3% hydrogen peroxide). Sections were then blocked by incubating in PBS containing 10% normal goat serum (same species as secondary antibody) for 60 minutes. Slides were incubated with a rabbit polyclonal anti-myeloperoxidase (MPO, Pa5-16672; ThermoFisher, MA USA) for neutrophil detection, with a rabbit polyclonal anti-histone H3 citrulline R2 + R8 + R17, (Cit-H3, ab5103; ABCAM, Cambridge, United Kingdom) for NETosis and primary antibodies were omitted for negative controls. After washing, sections were incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) for 30 minutes, washed, then incubated with avidin-biotin complex (ABC kit) and visualized using diaminobenzidine substrate (Vector Laboratories) (27-29).
The HPS slides were scanned to get a picture of the whole left lung (Super coolscan 5000; Nikon, Tokyo Japan). Using a brightfield microscope (BX45, Olympus, Richmond Hill, ON, Canada), images were acquired under 200× magnification on the most damaged/altered regions, acquiring 5 fields per slide for the HPS staining and 10 fields per slide for IHC (MPO and Cit-H3) staining. For the HPS staining, the following analyses were performed: 1) thickness of alveolar membranes, 2) percentage (%) of altered lung tissue, 3) injury score of the lungs.
To assess the thickness of alveolar membranes, 20 measurements per field were performed (corresponding to 100 measurements per slide) and were expressed as the mean thickness (µm) of the alveolar membranes. A morphometric analysis has been performed to assess the percentage of altered lung tissue over total lung area (excluding trachea, major bronchi and blood vessels >700 µm diameter). To evaluate lung injury, we used an adapted version of the standardized histology score from the American Thoracic Society Documents (30). The histology scores (0, 1 or 2), were given for: 1) neutrophils in the alveolar space, 2) neutrophils in the interstitial space, 3) proteinaceous debris, 4) alveolar septal thickening, 5) alveolar hemorrhage, 6) interstitial space/membrane hemorrhage and 7) alveolar necrosis. For each slide the maximal injury score, corresponding to the sum of the score (score 0 to 2) of the 7 parameters x 5 fields per slide, is 70 points (2x7x5).
Neutrophils immunoreactivity in the lungs was performed to assess the presence of neutrophils (MPO immunostaining) and neutrophils undergoing NETosis (Cit-H3). The percent area (%) occupied by neutrophils in the lungs was quantified by color segmentation and represented as the MPO area over total lung tissue area. To assess neutrophils undergoing NETosis, we quantified the intensity of Cit-H3 staining (lumen). All analyses were performed using Image Pro Premier version 3.0 software (Media Cybernetics, Rockville, MD, USA).
Serum cytokine quantification. Quantitative determination of serum IFN-γ, IL-1β, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-α was done using the electrochemiluminescence-based Meso Scale Discovery (MSD) platform (Rockville, Maryland, USA). Proteins levels were measured in a multiplex assay using the V-PLEX Proinflammatory Panel 2 Rat kit (MSD). Samples were diluted 1:5 in proprietary buffer (MSD) and measured. Data were acquired using a MESO QuickPlex SQ 120 plate reader (MSD) and protein concentrations were determined using the MSD Discovery Workbench 4.0 analysis software. All values reported are between the lower and the upper limits of quantification of the kit. Quantitative determination of serum Cit-H3 was measured by a commercially available ELISA kit (Cayman Chemical, 501620, Ann Arbor, MI, USA).
In vitro LPS challenge of blood leukocytes. Whole blood samples collected on EDTA-coated tubes were incubated or not with LPS (500 ng/mL) for 30 minutes at 37°C. The samples were then stained using mouse anti-rat CD45 Alexa Fluor 700 (clone OX-1, conjugated to Alexa Fluor 700, Biolegend), mouse anti-rat CD11b antibody (clone WT.5, conjugated to V450, BD Horizon) and 7AAD (Biolegend) for 30 minutes at 4°C. FACS lysing solution (BD Pharm Lyse, BD Biosciences) was added for 15 minutes at room temperature and samples were stored at 4°C until analysis by flow cytometry (LSRII, BD Biosciences). Data were analyzed on Diva software version 8.0.1 (BD Biosciences).
Leukocyte immunophenotyping. Using reverse pipetting, 100 µl of whole blood collected on EDTA-coated tubes were incubated with anti-CD32 to prevent FC-mediated non-specific binding according to the manufacturer’s instructions. The panel of antibodies as well as the gating strategy were inspired by Barnett-Vanes and al. (31). Briefly, a panel of 11 antibodies (Supplemental Table 1) was used to identify the neutrophils (CD45+/ SSChi / His48+), the monocytes (CD45+/ SSClo/ His48hi or lo/CD43hi or lo), the B lymphocytes (CD45+/ SSClo /CD45R-B220+), the T lymphocytes (CD45+/ SSClo / CD3+/CD4+ or CD8+) and the natural killer cells (CD45+/SSClo /CD161a+). The whole blood samples were incubated with the antibodies for 20 minutes at room temperature and the BD Pharm Lyse solution was then added for 10 minutes to lyse the erythrocytes. Samples were stored at 4°C until analysis by flow cytometry (LSRII BD Biosciences). Accucount (Spherotech) were added to establish a cell count per µl of blood. Data were analyzed on Diva software version 8.0.1 (BD Biosciences).
RNA extraction and quantification of mRNA expression by RT-qPCR analysis
Tissue homogenization, RNA extraction, RT-PCR and real-time PCR. About 30 mg of rat lung samples were homogenized in a TissueLyser II (Qiagen, Germany) in 700 µL of RLT buffer from the RNeasy Mini RNA extraction kit (Qiagen, Germany) supplemented with the anti-foam DX Reagent. Total RNA was- extracted according to the kit’s instructions. RNA integrity and quantity were assessed using a 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA). 500 ng of RNA were used for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). For RT-qPCR, reactions were performed in duplicates using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA), 2.5 ng of cDNA and a final concentration of 100 nM of each primer. The cycling protocol started with a denaturation step at 95⁰C for 5 min, followed by 40 cycles of denaturation at 95⁰C for 15 s and primer annealing and extension at 57⁰C for 30 sec. Upon completion of the cycling steps, a melting curve protocol was performed from 60⁰C to 95⁰C and the reaction was stored at 4⁰C. Real-time PCR was carried out using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The geometric mean of housekeeping genes ACTB, GAPDH and XBP1 was used as an internal control to normalize the variability in expression levels which were analyzed using the 2-∆∆CT method. The primer sequences are provided in Supplemental Table 2. Heat maps of normalized expression values for gene expression was generated using Morpheus from Broad Institute (https://software.broadinstitute.org/morpheus). To normalize the data, the log2 of normalized expression values for IL-2, IL-13, CXCR2, E-sel, Arg1, CXCL1, CalCRL, Ramp2 and ATP1B1 were used.
Statistical analysis. Comparisons between groups were performed by one-way ANOVA followed by Tukey’s post-hoc comparisons. Significant differences were considered if p<0.05. All values are presented as mean ± SEM.