Study design and participants enrollment
To obtain the AMs from clinical patients, we prospectively enrolled participants who receiving scheduled bronchoscopy for clinical purposes at the National Cheng Kung University Hospital (NCKUH) between June 2020 and January 2022. The Institutional Review Board of NCKUH approved this study (B-ER-109-016) prior to commencement. Written informed consent was obtained from all subjects. Exclusion criteria were as follows: (1) age < 20 years; (2) pulmonary infection or exacerbation of inflammatory airway disease within one month; (3) had diffuse pulmonary lesion or interstitial lung disease; (4) received inhaled or systemic immunosuppressive therapy; (5) had been infected with COVID-19; (6) received NAC. Baseline information of the participants, including demographics, smoking status, comorbidities, concurrently used medication, pulmonary function test, and chest computed tomography (CT) images were collected from electronic medical records in the NCKUH database. Participants who had persistently stopped smoking for more than one month were defined as ex-smokers. Participants were defined as having COPD if the forced expiratory volume in one second (FEV1) to forced vital capacity (FVC) ratio was < 70%. For participants who had not received pulmonary function test, they were defined as COPD if they had been smoking for more than 10 pack-years in combination with diffuse pulmonary emphysema on chest CT43.
Collection of pulmonary immune cells
We used BAL to collect pulmonary immune cells from the participants. The procedures were performed in accordance with the recommended practice guidelines56. Briefly, BAL is performed with the fiberoptic bronchoscope after wedging in the targeted segmental bronchus. For participants with unilateral lung lesions, BAL was performed in the unaffected side of lung. The first instilled aliquot of 25 mL isotonic sodium chloride solution was discarded to avoid contamination of bronchial secretions. The following instilled aliquots were withdrawn, and the total installed and retrieved volume ranged from 100–300 mL to 30–50 ml, respectively. The retrieved BAL fluid immediately stored in 4 degrees Celsius refrigerator and filtered using a 70 μm strainer and centrifuged at 1500 RPM for 10 min later. The pellets of BAL cells were washed and re-expanded using PBS for flow cytometry or culture.
Classification of pulmonary immune cells, and quantification of ACE2 and cytokine production
We used flow cytometry (FACS Canto II (BD Biosciences)) to classify pulmonary immune cells from BAL and quantify the ACE2 expression. Antibodies were purchased from BD Biosciences (NJ, USA), and Biolegend (CA, USA). To block non-specific antibody binding of immune cells, BAL cells were inoculated in staining buffer with Human BD Fc Block for 30 min. Then, the following antibodies were used: FITC-conjugated anti-CD45 (2D1), PE-conjugated anti-CD11b (ICRF44), PerCP-conjugated anti-CD15 (W6D3), APC-cy7-conjugated anti-CD16 (3G8), BV510-conjugated anti-CD169 (3G8), BB700-conjugated anti-CD206 (19.2), PE-conjugated anti-IL-1β (AS10), APC-conjugated anti-IL-10 (JES3-19F1), PE-cy7-conjugated anti-IFN-γ (B27), PE-conjugated anti-ACE1 (BB9) and AF647-conjugated anti-ACE2(A20069I). AM, non-AM macrophages, neutrophil and lymphocytes were defined as follow: AM (CD11b+CD16+CD45+CD169+ CD206+), non-AM macrophages (CD11b+CD15–CD16+CD45+CD169–CD206+), Neutrophil (CD11b+CD15+CD16+CD45+CD169–) and lymphocytes (CD45+CD11b–)22,57. Quantitative flow cytometry was performed to quantify ACE2, IL-1β, IL-6, IL-10, and IFN-γ in cells by calculating the MFI of each substance. MFI can be used as a quantitative indicator for the study materials58. Fluorochrome minus one control with corresponding isotype control antibodies (IgG1-PE, IgG1-FITC, IgG1-PE-cy7, IgG1-APC-cy7, IgG1-BB700, IgG1-PerCP, IgG1-BV510, IgG2A-AF647, IgG2A-APC BD Biosciences) was performed to eliminate the interference of background fluorescence. The flow cytometry data were acquired on a FACSCanto II instrument (BD Biosciences) and analyzed using FlowJo software (TreeStar).
SARS–CoV–2 spike protein stimulation and pseudovirus infection assay
SARS-CoV-2 spike protein (CoV-2 Sp; Leadgene Biomedical, Inc, Taiwan) and lentivirus–based spike protein expressing pseudovirus (CoV-2 PsV; ACE Biolabs CO., Taiwan) were used to test the susceptibility of AMs to SARS-CoV-2 infection. First, ex-vivo human AMs were inoculated in 96-well cell culture plates at a volume of 4 × 104 cells/well. For the CoV-2 Sp assay, 5 μg/ml of CoV-2 Sp was placed in each well and inoculated for 24 h. Then, ACE2, IL-1β, IL-6, and IFN-γ expression levels in AMs were measured using flow cytometry. For the pseudovirus infection assay, 6 μL of CoV-2 PsV was added and infected for 72 h. Then, the infection efficiency was determined using green fluorescent’s mean fluorescence intensity in flow cytometry.
The CSE preparation protocol was adapted from previous study with modification59. Briefly, five cigarettes (Marlboro red; tar: 10 mg, nicotine, 0.8 mg; Philip Morris USA Inc) was burned. The smoke was sequentially bobbled into 10 ml vessel containing phosphate-buffered saline using a syringe pump at a flow rate of 300 ml/min. The CSE was sterilized by filtrated via a 0.2-μm Millipore filter before administration. The CSE was prepared freshly and diluted by phosphate-buffered saline to a 1% solution for each utilization. The results of the dose and time response of ACE2 in human AMs to CSE administration, as well as the positive control with metformin60, are presented in Supplementary Fig. 3. Based on the test results, we treated AMs with 1% CSE for 24 h in our experiments.
Measurement of intracellular ROS levels
Ex-vivo AMs were washed with suspension buffer (1X phosphate buffered saline and 2% fetal bovine serum) three times and incubated with 1% CSE and NAC (10 mM; Sigma-Aldrich) at 37 °C for 30 min in the presence of 20 mM DCF-DA (abcam) at 37 °C. After stimulation with DCF-DA for the desired time, the cells were washed and followed by fluorescence at Ex/Em = 485/535 nm and analysis with flow cytometry. Data were analyzed with FlowJo software (TreeStar).
Preparation of AMs from wild type and Cybb–/– mice
AMs were obtained from C57BL/6 wild type and Cybb–/– mice aged 8–12 weeks. The protocol for lung extraction and digestion was adapted from a previous study61. Briefly, the mice’s lungs were extracted under anesthesia induced by an intraperitoneal injection of Zoletil (50 mg/kg body weight) and Rompun (2.332 mg/kg body weight) cocktail. The cardiac ventricles were dissected and flushed with 2 ml of phosphate-buffered saline (PBS) with 0.1% heparin to remove blood in the pulmonary circulation. Then, both lungs were removed separately and minced with scissors into 2–3 mm pieces, which were digested in medium (RPMI 1640, 1% penicillin/streptomycin; Gibco, Waltham, MA, USA) with Liberase thermolysin medium (100 μg/mL; Roche, Basel) and DNase I (1 μg/mL; Sigma, Saint Louis, MO, USA) solution at 37 °C for 30 min in a shaker. After digestion, the fluid was filtered using a 70-μm nylon mesh (BD Biosciences, San Jose, CA, USA) and re-suspended over a 50 ml conical tube. Then, the cell pellets were obtained through centrifugation at 1200 rpm for 10 min at 4 °C. The protocol for isolation and culture of AMs was adapted from previous studies62,63. AMs were processed after acquisition using the MACS cell separation (Miltenyi, Auburn, CA, USA) and a CD11C, Siglec-F positive selection on a magnetic column. Cell suspensions were filtered using a 40 μm nylon mesh. Cell viability, CD45, CD11C, and Siglec-F positive cell purity and characterization of surface marker expression were assessed using a flow cytometer. All experiments were conducted according to the National Institute of Health guidelines for animal care and were approved by the Institutional Animal Care and Use Committee (IACUC) at National Cheng Kung University (IACUC Approval Number: 111258). Study details are in accordance with ARRIVE guidelines.
Measurement of ACE2 expression in mice AMs using ELISA
CD45+Siglec-F+ cells were selected from the cultured CD11C+Siglec-F+ population using magnetic–activated cell sorting to enhance the purity of AMs. Then, the AM lysates were harvested with 100 μl of RIPA lysis buffer (Sigma, Saint Louis, MO, USA) with a 50X protease inhibitor mixture. Samples were centrifuged at 12,000 rpm for 20 min at 4 °C to pellet cell debris. ACE2 expression in the whole lung AM culture samples were measured using a duplicate CUSABIO mouse ACE2 enzyme-linked immunosorbent assay (ELISA) Kit (CSB-E17204m, CUSABIO, Houston, TX, USA), according to the manufacturer’s instructions.
In the clinical cohort study, we utilized the MFI of ACE2 as an indicator for ACE2 expression on AMs. The ACE2 MFI was transformed using log2 to establish linear data. The Student’s t test was used to compare the mean ACE2 between different subgroups. Spearman’s correlation tests were conducted to analyze the correlations between ACE2 and clinical factors. Univariate and multivariate linear regression analyses were performed to investigate the association between ACE2 expression and clinical factors. Age, sex, body mass index (BMI), and other clinical factors, such as smoking status, COPD, lung cancer, DM, CVD, use of RAAS inhibitor, all of which are known to increase ACE2 expression, were entered into the multivariate linear regression model as covariates43,44,64,65,66. Among the covariates, age and BMI were entered into the model as continuous variates, whereas sex (male vs. female), smoking status (active smoker vs. ex– or never–smoker), COPD (with vs. without), lung cancer (with vs. without), and RAAS inhibitor (use vs. no use) were entered as categorical variates. For in vitro laboratory research, the data were expressed as mean and Standard Error of the Mean (SEM). A paired t test was performed to compare the ACE2 expression between the controls and investigating samples. Statistical significance was set at two–sided P < 0.05. SAS software (version 9.4, Cary, NC: SAS Institute Inc; 2014) and GraphPad Prism 7 (GraphPad Software, San Diego, California USA) were used for statistical analysis.
This study was approved by the Institutional Review Board of National Cheng Kung University Hospital before commencement (B-ER-109-016). Written informed consent was obtained from all subjects. All experiments of animals were conducted according to the National Institute of Health guidelines for animal care and were approved by the Institutional Animal Care and Use Committee (IACUC) at National Cheng Kung University (IACUC Approval Number: 111258). Study details are in accordance with ARRIVE guidelines.