Study population
This study included 51 patients who received surgery for lung cancer in Ishinomaki Red Cross Hospital and Tohoku University Hospital. Patients with respiratory disease other than COPD were excluded. The diagnosis of COPD was determined based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (https://goldcopd.org). The patients were divided into three groups: non-smokers, non-COPD smokers, and COPD patients. We used the Goddard score for the assessment of low attenuation areas (LAA) [22]. This study was approved by the Ethics Committee at Tohoku University School of Medicine (2017-1-352). Written informed consent was obtained from all patients.
Preparation of human lung single-cell suspensions
We prepared human single-cell suspension as previously described with some modification [23-26]. Minced lung tissues were incubated with Hanks Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA, USA) containing 1.5 mg/ml Collagenase A (Sigma-Aldrich, St. Louis, MO) and 2000 KU/ml DNase I (Sigma-Aldrich) at 37 °C for 45 minutes, then minced again with scissors and incubated at 37 °C for 45 minutes. Single-cell suspensions were filtered with a 100 µm cell strainer (BD biosciences) twice and red blood cells were lysed with ammonium-chloride-potassium lysis buffer (Thermo Fisher Scientific). Cells were resuspended in RPMI 1640 medium (containing L-glutamine and 25 mM HEPES; Thermo Fisher Scientific) with 5% fetal bovine serum and 2% penicillin-streptomycin-amphotericin B suspension (100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml, amphotericin B; FUJIFILM Wako Chemicals, Osaka, Japan) and filtered twice with 70 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Cell numbers were calculated using trypan blue staining and analyzed by flow cytometry.
Mice
C57BL/6 mice were purchased from Charles River Laboratories Japan (Yokohama, Japan) and 7 to 10-week-old female mice were used. LILRB4-deficient mice (gp49B-/-) with the B6 background were previously established [27]. All mice were maintained and bred in the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, under specific pathogen-free conditions. All animal protocols were reviewed and approved by the Animal Studies Committee of Tohoku University.
Elastase-induced emphysema in mouse model
A mouse model of elastase-induced emphysema was prepared as previously described [26]. Briefly, mice were anesthetized with isoflurane temporarily and were given an intranasal instillation of 3 units porcine pancreatic elastase (FUJIFILM Wako Chemicals) in 50 µl of PBS or 50 µl of PBS alone. Analysis of macrophages was conducted on day 7 and histological examination and CT scan were conducted on day 21.
Bronchoalveolar lavage (BAL)
Mice were injected intraperitoneally with triple mixed anesthesia of medetomidine hydrochloride (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg). After euthanization by cutting the aorta, we inserted a 20-gauge needle into the trachea. Bronchoalveolar lavage fluid (BALF) was collected by washing the lungs three times with 1 ml of PBS. BALF was centrifuged at 300 rpm for 5 minutes and the supernatant was used for cytokine analysis. Cells were resuspended in PBS and cell counts were determined by a hemocytometer. Cell fractionation was calculated by cytospin slides stained by Diff-Quick method.
Preparation of mouse lung single-cell suspensions
Mouse single-cell suspensions were prepared as previously described with some modification [28]. Lungs chopped with scissors were incubated at 37 °C for 45 minutes in RPMI solution containing 50 µg/ml Liberase TM (Roche, Basel, Switzerland) and 10 µg/ml DNase I (Roche). The lung tissue was passed through a 40 µm cell strainer. After centrifugation, the cell pellets were resuspended in ACK lysis buffer (Thermo-Fischer Scientific) and incubated to remove red blood cells. The samples were washed with PBS and resuspended in the staining buffer for flow cytometric analysis.
Flow cytometry
Flow cytometry was performed as previously described with some modification [24, 29]. LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, CA) was added to single-cell suspensions and incubated at 4 °C for 30 minutes. After resuspension in FACS buffer containing PBS with 0.1% sodium azide and 2% FBS, human FcR Blocking Reagent (Miltenyi Biotec) in the case of human and anti-CD16/32 mouse antibody in the case of mouse were added to prevent non-specific staining, then incubated 4 °C for 5 minutes. Based on previous reports [30-32], we defined human alveolar macrophages as FSChighCD45+CD206+CD14- cells, human interstitial macrophages as FSCmidCD45+CD206+CD14+ cells, mouse alveolar macrophages as CD45+Ly6G-CD64+CD24-CD11bintCD11c+ cells and mouse interstitial macrophages as CD45+Ly6G-CD64+CD24-CD11b+CD11c- cells. Data were collected by LSR Fortessa (BD Bioscience) and analyzed by FCS express 6 software (De Novo Software, Glendale, CA). Cell sorting was performed using FACS Aria II (BD Biosciences). We utilized fluorescence minus one controls to distinguish the positive population from the negative one.
Antibodies
Brilliant Violet 421-conjugated anti-human CD45 (HI30), APC-conjugated anti-human CD3 (OKT3), APC-conjugated anti-human CD19 (HIB19), FITC-conjugated anti-human CD14 (M5E2), CD11c-conjugated anti-human CD11c (3.9), PE-Cy7-conjugated anti-human HLA-DR (LN3), PerCP-Cy5.5-conjugated anti-human CD56 (5.1H11), PE-conjugated anti-human LILRB4 (ZM4.1), Pacific blue-conjugated anti-mouse CD45 (30-F11), Brilliant violet 510-conjugated anti-mouse CD11b (M1/70), PerCP-Cy5.5-conjugated anti-mouse CD11c (N418), FITC-conjugated anti-mouse Ly6G (1A8), PE-Cy7-conjugated anti-mouse CD64 (X54-5/7.1), APC/Fire 750-conjugated anti-mouse CD24 (M1/69), APC-conjugated anti-mouse IA/IE (M5/114.15.2), PE-conjugated anti-mouse LILRB4 (H1.1), PE-conjugated American Hamster IgG control antibody (HKT888) were purchased from Biolegend. APC conjugated anti-human CD206 (19.2), APC-conjugated mouse IgG1κ control antibody (P3.6.2.8.1), PE-conjugated mouse IgG1κ control antibody (P3.6.2.8.1) were purchased from eBiosciences.
Histological analysis
The lung was refluxed by injecting PBS from the right ventricle. The 10% neutral buffered formalin was injected from the trachea at a pressure of 30 cmH2O and the lung was fixed for 24 hours. We entrusted the production of paraffin-embedded sections to Experimental Animal pathology Platform Section, Tohoku University. Emphysematous changes were evaluated by the mean liner intercept (MLI) [33].
Image analysis
Under 2% isoflurane anesthesia, mouse chest CT scans were performed using an X-ray CT system for laboratory animals (LaTheta LCT-200; Hitachi Aloka Medical Ltd., Tokyo, Japan). Calibration was carried out according to the manufacturer’s protocol. The CT value of air was set to -1000 Housefield Units (HU), and water was set to 0 HU. Data were converted to DICOM files and analyzed by LaTheta software (version 3.22) and Image J software (National Institutes of Health, Frederick, MD). The quantitative evaluation of emphysema was performed using the percentage of low attenuation area, which was defined as the area from -871HU to -610 HU [34].
Quantitative Polymerase Chain Reaction (qPCR)
RNA was extracted from sorting cells using RNeasy Micro Kit (Qiagen, Valencia, CA) and from lung tissue using RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was synthesized by the High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). Quantitative PCR was conducted on StepOne Plus (Thermo Fisher Scientific) using SYBR Premix Ex Taq (TaKaRa, Kusatsu, Japan).
The levels of mRNA expression were evaluated by the comparative CT method and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control gene. The reference sample was one of the PBS-treated control samples or wild-type control samples. Primer sets were as follows: for mouse IL-10: forward, 5’-GCCAGAGCCACATGCTCCTA-3’, and reverse, 5’-GATAAGGCTTGGCAACCCAAGTAA-3’; for mouse IL-1b: forward, 5’-TCCAGGATGAGGACATGAGCAC-3’, and reverse, 5’-GAACGTCACACACCAGCAGGTTA-3’; for mouse Mmp12: forward, 5’-CTCTAGCCAGCACATGACTCCAA-3’, and reverse, 5’-CTGATGTGAAATGAGCCACACAAC-3’; for mouse TNF-α: forward, 5’-ACTCCAGGCGGTGCCTATGT-3’, and reverse, 5’-GTGAGGGTCTGGGCCATAGAA-3’; for mouse GAPDH: forward, 5’-TGTGTCCGTCGTGGATCTGA-3’, and reverse, 5’-TTGCTGTTGAAGTCGCAGGAG-3’
Statistical analysis
Data are expressed by a dot plot with median. Comparison between groups was performed using the Mann–Whitney U test. The Steel-Dwass test was used for nonparametric multiple comparisons among groups. To analysis the relationship between variables, Spearman’s rank correlation coefficient was calculated. All statistical analyses were conducted using GraphPad Prism version 7 (GraphPad Software Inc, Sac Diego, California, USA) or JMP Pro version 16 (SAS Institute Inc, Tokyo, Japan). P < 0.05 was considered significant.