Animals and experimental protocol
Female C57BL/6J mice were purchased from Charles River Japan, Inc. (Yokohama, Japan). IL33−/− mice were generated as described by Yasuda et al. . All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Okayama University Medical School (Okayama, Japan).
Elastase-induced emphysema model
To generate an elastase-induced emphysema model, female IL33−/− mice (8 – 10 weeks old) and wild-type (WT) mice received 2.0 or 3.75 U of PPE by intratracheal instillation on day 0 . Control mice received 40 µl of phosphate-buffered saline (PBS) at the same time point. The mice were sacrificed on days 0, 2, 4, 7, 14, and 21 to evaluate lung histology, lung function, airway inflammation, and the levels of cytokines and chemokines.
Determination of static lung compliance
A flexiVent small-animal ventilator (SCIREQ, Montreal, QC, Canada) was used to assess static lung compliance (Cst), which is a measure of the elasticity of the lung. Cst was calculated from the pressure volume curves using flexiVent software (version 5.0; SCIREQ), as described previously . Briefly, the mice were anesthetized by intraperitoneal injection of 150 mg/kg of ketamine and 10 mg/kg of xylazine (Kyoritsu Seiyaku, Tokyo, Japan). The mice were tracheostomized with a 5-mm section of metallic tubing (18 G cannula) and ventilated at 150 breaths/min with a tidal volume of 10 ml/kg and positive end-expiratory pressure of 3 cmH2O. The Cst was then measured.
Bronchoalveolar lavage (BAL) fluid
Lungs were lavaged with Hanks’ balanced salt solution via the tracheal tube (2 × 1 ml, 37°C), and the cells in BAL fluid were counted. Cytospin slides were stained and differentiated in a blinded manner by counting at least 200 cells under a light microscope, as described previously .
Lung histology and morphometric measurements of airspace size
The left lung was inflated by intratracheal instillation of 10% formalin at a static pressure of 20 cmH2O and fixed in 10% formalin. The tissue was then embedded in paraffin and 2-µm-thick sections were stained with hematoxylin and eosin (H&E). The mean linear intercept (Lm), which represents the average size of alveoli, was calculated by counting lines of a defined length, as described previously  [17, 18].
Measurement of cytokines and chemokines
The cytokine levels in the BAL fluid and supernatants of homogenized lungs were measured by enzyme-linked immunosorbent assay (ELISA), as described previously . For preparation of lung homogenates, lung tissue was frozen at −70°C immediately after euthanasia. The lung tissue was mixed with 0.1% Triton-X100 solution in PBS containing proteinase inhibitors at a 1:2.5 ratio of weight per volume (Sigma-Aldrich, St. Louis, MO, USA). The specimens were homogenized and then centrifuged at 21480 × g for 30 minutes. The supernatants were frozen at −80°C until analysis. The limits of detection were 2 pg/ml for keratinocyte-derived chemokine (KC), 2.0 pg/ml for monocyte chemoattractant protein-1 (MCP-1), 1.5 pg/ml for macrophage inflammatory protein-2 (MIP-2), 12.1 pg/ml for HGF, 3.0 pg/ml for VEGF, and 0.014 ng/ml for matrix metalloproteinase-9 (MMP-9) (R&D Systems, Minneapolis, MN, USA).
Lung cell isolation
Lungs of PPE-treated mice were placed in PBS containing heat-inactivated 10% FCS. Lung tissue was minced and incubated for 1 hour at 37°C in 5 ml of PBS containing 0.05% collagenase I (Sigma-Aldrich), and then dispersed by passing through a 20 G needle several. The suspensions were strained through a cell strainer. The pulmonary mononuclear cells were isolated by density gradient cell centrifugation over Histopaque (Sigma-Aldrich) .
Analyses of ILC2s. The cells isolated from digested lungs were stained with biotin-conjugated antibody mixtures for lineage markers (CD4, CD5, CD8, CD11c, CD11b, CD19, NK1.1, Gr-1, TER119, FcεRI, and B220), Pacific blue-conjugated anti-Sca-1, PECy7-conjugated c-Kit (CD117), APC-conjugated anti-IL-7Rα (CD127), FITC conjugated anti-T1/ST2, APC-Cy7-conjugated anti-CD25, and PE conjugated anti-streptavidin, and analyzed using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin−Sca+c-Kit+IL-7R+CD25+ST2dim cells were identified as lung ILC2s  . The data were analyzed using FlowJo (TreeStar, Ashland, OR, USA). APC-Cy7-conjugated anti-CD25, Pacific blue-conjugated anti-Sca-1, biotin-conjugated anti-CD4, anti-CD5, anti-CD8, anti-CD11b, anti-NK1.1, anti-Gr-1, anti-TER119, and anti-B220, and PE-conjugated anti-streptavidin antibodies were obtained from BD Biosciences (Franklin Lakes, NJ, USA). FITC-conjugated anti-T1/ST2 was from MD Bioscience (St Paul, MN, USA). APC-conjugated anti-IL-7Rα and biotin-conjugated anti-FcεRI antibodies were from BioLegend (San Diego, CA, USA). PECy7-conjugated c-Kit was from eBioscience (La Jolla, CA, USA). Biotin-conjugated anti-CD11c and anti-CD19 were from TONBO Biosciences (San Diego, CA, USA).
Administration of recombinant IL-33 (rIL-33)
WT mice received PPE or PBS by intratracheal instillation on day 0. The mice received an intraperitoneal injection of 1,000 ng of rIL-33 (R&D Systems) on days 0 and 3. Control mice received PBS at the same time points. Lung function and lung histology were evaluated on day 21.
CSE-induced emphysema model
CSE was prepared using the 3R4F Kentucky reference cigarette. One non-filtered cigarette was burned; the smoke was passed through 8 ml of PBS using a vacuum pump with a constant air flow of 2 L/min. The extract was used when pH was between 6.2 and 6.4 and the optical density at 320 nm (OD320) was between 1.600 and 1.900. The extract was freshly prepared for each experiment and administered after the pH had been adjusted between 7.00 and 7.40 and the solution had been filtered through a 0.2-μm pore-size filter to remove particles and bacteria .
The experimental mice received 40 µl of CSE intratracheally on days 0, 7, and 14. Control mice received 40 µl of PBS at the same time points. The mice were sacrificed on day 21 to evaluate lung histology. In some experiments, the mice received 400 µl of CSE intraperitoneally on days 0, 7, 14, and 21. The control mice received 400 µl of PBS at the same points. The mice were sacrificed on day 28 to evaluate lung histology .
All results are presented as the mean ± standard error of the mean (SEM). Groups were compared by one-way analysis of variance (ANOVA). Pairs of samples with a parametric distribution were compared by unpaired two-tailed Student’s t test, and samples with a nonparametric distribution were compared by the Mann–Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.