Quercetin: For cell culture studies, quercetin was kindly provided by Quercegen Pharmaceuticals (Sudbury, MA). HPLC analysis indicated that quercetin was 99% pure. 100mM quercetin was prepared in sterile DMSO and stored in aliquots at -80°C. On the day of use quercetin was dilute to 1 µM in the cell culture medium, filter sterilized prior to use. Similar volume of DMSO diluted in cell culture medium was used as a vehicle control.
For clinical trial, quercetin and placebo formulations were purchased from Nutravail Technologies (Chantily, VA) as soft chews. Each quercetin chew contained 500 mg of quercetin, 350 mg of ascorbic acid and 10 mg of niacin. Each placebo chew had 350 mg of ascorbic acid and 10 mg of niacin.
Isolation and culturing of airway epithelial cells
Airway basal cells were isolated from bronchial segments of normal donor lungs and explanted lungs from COPD patients at the time of lung transplantation as described previously [6, 19]. The collection of the tissue was approved by the Institutional Review Board of University Michigan, Ann Arbor, MI (HUM00052806) and Temple University, Philadelphia, PA (4407). Patient characteristics are provided in Supplemental Table 1. The basal cells at passage one, were cultured in 6.5 mm collagen-coated transwells as described previously [19, 20]. In some experiments, 80-90% confluent COPD basal cells cultured in 6.5 mm transwells were treated with 0.1, 1 or 5 μM quercetin or equal volume of DMSO (vehicle) for three consecutive days from both apical and basolateral sides. The cells were then harvested for isolation of total RNA or cultured at air/liquid interface (ALI) for up to 4 weeks to promote polarization and mucociliary differentiation of cells.
Collection of bronchial brushings from COPD patients
The collection of bronchial brushings from COPD patients recruited for clinical trial with quercetin (NCT03989271) was approved by Temple IRB #25738. Patient characteristics are presented in Supplemental Table 2. In this clinical trial, patients were treated with a placebo or 2000 mg/day quercetin for 6 months. Post-bronchodilator FEV1 was measured and bronchial brushings collected from 4 placebo and 7 quercetin-treated COPD patients at baseline and at the end of the treatment. The cells from the bronchial brushings were harvested by centrifugation, lysed in TRIZOL and stored at -80° C. The total RNA was isolated from all the samples at the same time. Blood was collected and analyzed for quercetin levels by reverse phase HPLC as described previously [21]. Blood quercetin levels at baseline was comparable between placebo (0.153 ± 0.086 µM) and quercetin group (0.195 ± 0.069 µM). Six months after treatment, while placebo-treated patients showed no significant increase, quercetin-treated patients showed 11fold increase in their blood quercetin levels.
Transepithelial resistance.
Transepithelial resistance (TER) was measured using the EVOM voltmeter equipped with ENDOHM-6 EVOM electrode (World Precision Instruments, Sarasota, FL) and the TER was expressed as Ohms/cm2.
ELISA
After culturing the cells for 4 weeks at air/liquid interface, transwells were transferred to a new receiver plate containing fresh medium and incubated for 24 h. The basolateral medium was collected and IL-6 and IL-8 protein levels were measured by ELISA (R & D systems, Minneapolis, MN).
Total RNA isolation
Total RNA was isolated from TRIZOL lysates of normal basal cells, DMSO and quercetin-treated COPD cells, and bronchial brushings using a Direct-zolTM miniprep kit (Zymo research, Irvine, CA). The integrity of total RNA was determined by Agilent 2100 bioanalyzer and the RNA integrity number was consistently >7.
Microarray processing
Biotinylated cDNAs synthesized from total RNA isolated from DMSO- or quercetin-treated COPD basal cells were subjected to microarray analysis using Human Gene 2.1 ST arrays. To identify statistically significant differential gene regulation (p-value of < 0.05 and up/down regulated by more than 2-fold), we performed pairwise comparison (DMSO versus quercetin) by the LIMMA methodology (Linear Models for Microarray Data). Normalized data and raw data are available in Gene Expression Omnibus (GEO) with accession number GSE137557.
Gene Ontology
Gene Ontology (GO) and KEGG pathways were analyzed with WebGestalt (WEB-based GEne SeT AnaLysis Toolkit) [22] using the Benjamini-Hochberg correction for multiple testing (FDR 5%). For Gene Ontology, only Biological Process terms are discussed because Cellular Component and Molecular Function terms were less relevant.
Flow cytometry
Mucociliary-differentiated cells were dissociated with accutase (ThermoFisher Scientific, Waltham. MA), cells were fixed with 4% paraformaldehyde, and then blocked/permeabilized by incubating in PBS containing 1% BSA and 0.5% saponin for 30 min. Cells were then incubated with acetylated tubulin (cat#T7451, Sigma Aldrich, St. Louis, MO), Muc5AC (cat#ab24071TP63, Abcam, Cambridge, MA) or TP63 (Cat#ab32353, Abcam), and bound antibodies were detected by using Alexafluor-labeled second antibodies as previously described [10]. The cells were then analyzed in FACS Calibur Flow cytometer (BD Biosciences, San Jose, CA), and data was analyzed by FlowJO version 10 (Tree Star, Ashland, OR).
Histology
For histological evaluation, cell cultures were fixed in buffered formalin, embedded in paraffin, 5μ thick sections were deparaffinized and stained with hematoxylin and eosin (H and E) or periodic acid Schiff’s (PAS) reagent.
Real-time PCR
cDNA was synthesized from total RNA (High-capacity first strand synthesis kit, Thermofisher Scientific) and subjected to qPCR to determine the expression of ELF5, ELF3, GRHL1, IVL, WNT5, ZO-3, HOXA1, HOXB2, VGLL1, IL-8, TGF-β and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using gene-specific primers and probes. The expression level of each gene is presented as a fold change over the house-keeping gene, GAPDH.
Statistical analysis
Data are presented as mean ± SD or median with range. Statistical significance was assessed by t test or Mann-Whitney test to compare two groups and ANOVA or ANOVA on Ranks with Kruskal Wallace nonparametric test to compare three groups as appropriate. For pairwise comparison, a signed rank test was used. For clinical samples, due to high variability in the gene expression at baseline, we compared the gene expression at base line and after completion of treatment for each patient and the statistical significance was determined by Shaipiro-Wilk T test. A p-value of ≤0.05 was considered statistically significant.