Neutrophil elastase

Tissues were fixed with 10% formalin, hydrated, and embedded in paraffin. Tissue sections were cut at 4 μm thickness, then deparaffinized following rehydration in a descendant ethanol bath. H&E staining, immunohistochemistry, and immunofluorescent staining followed the previously published protocols [30 (link)]. An upright microscope (Carl Zeiss Axio Imager, Z1) was used to observe the expression of targeted proteins. Primary antibodies cytokeratin 14 and CD163 were purchased from Proteintech (Illinois, United States). Ki67 and neutrophil elastase were purchased from Abcam (Cambridge, United Kingdom). Antibody against COX-2 was purchased from Cayman (Michigan, United States). Antibody against F4/80 was purchased from Biolegend (California, United States). Antibody against iNOS was purchased from BD transduction Laboratories (California, United States).

After incubation with stimuli or controls, neutrophil-coated coverslips were fixed in 4% paraformaldehyde in PBS, washed in PBS, permeabilized in 0.05% Tween20 in TBS for 1 min, and washed with TBS. For mouse derived neutrophils which were not stimulated with control or WoLP-conjugated beads, cells were then stained for neutrophil elastase (1:200, Abcam), incubated 1 h at room temperature, washed three times 5 min with 1xTBS, and then incubated for 1 h with a secondary antibody (Donkey anti-rabbit AlexaFluor488 IgG (Hparticular, macrophages, identified as L), 1:400, Life technologies). For all human or mouse derived neutrophils, DNA was then stained with 1 μg/ml 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Sigma-Aldrich), for 3 min. Cells were washed with TBS and viewed with a confocal laser-scanning microscope (Leica DM2500). Images were captured with a 40x objective.

Macrophages and neutrophils were labelled with specific marker Ibal-1 (Wako, #019-19741, Japan) and Neutrophil elastase (Abcam, #ab310335, UK), respectively, using the standard immunohistochemistry (IHC) staining protocol. For eosinophil-specific staining, sections were stained for 30 min with chromotrope 2R (Sigma-Aldrich, #4197-07-3, USA) solution (1% chromotrope 2R in 5% phenol), by which the eosinophils were specifically stained in red. Another specific staining with 1% toluidine blue (Sigma-Aldrich, #6586-04-5, USA) was utilized to detect mast cells (MCs) cytoplasmic granules. All sections were examined under light microscopy. The numbers of Ibal-1 positive cells, elastase positive cells, eosinophils and mast cells were quantified according to the NIH Image Analysis system (National Institutes of Health, Bethesda, MD). The inflammatory cells around the airways were expressed as cells per millimeter (mm) basement membrane. At least 10 fields in each slide were examined in a blinded manner.

The lungs were excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Histological examination was performed on 4 μm thick sections stained with hematoxylin and eosin. Immunohistochemical staining was conducted by incubating sections with primary antibodies against Ly-6G (neutrophil; 1:500), Mac-2 (macrophage; 1:500; eBioscience, Inc., San Diego, CA, USA), or neutrophil elastase (1:500; Abcam, Cambridge, MA, USA).

WT or Trpm2^−/− bone marrow neutrophils were stimulated with 1 mM of H₂O₂ or L. monocytogenes (MOI of 10), dead cells were identified by staining with 1 μM of SyTOX green (impermeant to live cells). Neutrophils were acquired by flow cytometry and analyzed with FlowJo software. For the analysis of extracellular DNA in a plate, WT, Trpm2^−/− or Nox2^−/− bone marrow neutrophils were stimulated with 100 nM of PMA. Next, 1 μM of SyTOX green was added and the kinetic of extracellular DNA release was measured by a fluorescence plate reader (488/525 nm). The formation of Neutrophil Extracellular Traps (NETs) was evaluated by immunofluorescence. WT or Trpm2^−/− bone marrow neutrophils were seeded in coverslips and stimulated with 100 nM of PMA, 1 mM of H₂O₂ or L. monocytogenes (MOI of 10) for 3 h, cells were fixed and stained with rabbit anti-mouse Neutrophil Elastase (Abcam) 1:100, wheat germ agglutinin (WGA) Oregon 488 (Thermofisher) 1:1,000, Hoechst 33342 (Thermofisher) and goat anti-rabbit Alexa Fluor 594 (Abcam) 1:500. The slides were mounted with fluoroshield mounting medium (Abcam) and visualized by confocal microscopy.