Patients characteristics
All patients were treated in the Faculty of Medicine of the Jagiellonian University Medical College, Krakow, Poland, and were in stable clinical conditions. The experimental group consisted of 4 patients with moderate asthma (Global Initiative for Asthma (GINA) grade 3-4; 4 females, age (years): 50.2 ± 18.7; BMI (kg/m²): 28.9 ± 4.0; FEV1 (% predicted): 107 ± 20.9; FEV1/FVC (%): 76.5 ± 7.15; iGCS (inhaled glucocorticoids; fluticasone) and long-acting beta agonists (LABAs); 4/4). The control group of 4 non-asthmatic patients in whom diagnostic bronchoscopy ruled out any serious airway pathology (4 females, age (years): 40.5 ± 17.21; BMI (kg/m²): 26.6 ± 3.1; FEV1 (% predicted): 106.45 ± 9.42; FEV1/FVC (%): 81.11 ± 4.10). The control subjects were referred for bronchoscopy due to diagnosis of cough, as a part of routine investigation. All controls were non-allergic, had normal lung function tests. Additionally, respiratory tract disorders (e.g. asthma, sarcoidosis, interstitial lung disease, COPD, lung cancer, etc.) were further ruled out during clinical investigation (e.g. no pathology in airway biopsy or CT scan). All study participants were non-smokers. The study was approved by the Jagiellonian University Ethics Committee (Decision No. 122.6120.16.2016; 28 January 2016) and informed written consent was obtained from all participants.
HBFs cultures
Primary cultures of HBFs were isolated from bronchial biopsies derived from patients with diagnosed asthma (AS) and from non-asthmatic donors (NA) according to the protocols described previously (57). HBFs were cultured in complete medium: Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4500mg/L glucose; HG) supplemented by 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in standard conditions (37ºC, humidified atmosphere, 5% CO2). Cells between 5th-10th passages were used in the experiments. HBFs were plated at a density of 5000 cells/cm2 in complete medium for 24 h. Afterwards cells were incubated for the next 24 h in the serum-free medium: DMEM HG supplemented with 0.1% bovine serum albumin (BSA, Sigma-Aldrich) or in the medium dedicated for air-liquid interface (ALI) cultures (ALI medium; 1:1 mixture of Bronchial Epithelial Basal Medium (BEBM) and DMEM supplemented by BEGMTM SingleQuotsTM Supplement Pack, Lonza). When indicated, the human recombinant TGF-β1 (5 ng/mL, stock prepared in 1 mg/mL BSA/PBS) was added.
Mucociliary differentiation of HBECs
After isolation from bronchial biopsies derived from the AS patients and NA donors described previously (29), HBECs were seeded onto collagen-coated flasks and cultured to ca. 90% confluence in Bronchial Epithelial Growth Medium (BEGM; BEBM with supplements). Then, cells were passaged and transferred onto collagen-coated Transwell polycarbonate inserts (0.4-mm pore size; Corning) at a density of 1.3-1.5x105 cells/cm2 in BEGM. Next day, the medium from HBECs was removed and the medium below inserts was replaced by the ALI medium with all-trans-retinoic acid (50 nM; Sigma-Aldrich). The cells were cultured in the air-liquid interface (ALI) for 30 days (Fig.6) to obtain a pseudostratified epithelium containing ciliary (DNAH9-positive) and goblet cells (MUC5AC-positive).
Epithelial-mesenchymal trophic unit model
HBFs were seeded into 12-well plates in a complete medium. Next day the medium was replaced by the fresh ALI medium and inserts with the mucociliary differentiated HBECs were transferred into appropriate wells (in combinations as in Fig.6B). The EMTU cultures were exposed to the TGF-β1 and cultivated by 4 days (Fig.6A). A profibrotic response of HBECs and HBFs on the TGF-β1 was analyzed by RT-qPCR, immunofluorescence and/or in-cell ELISA assay.
Transepithelial electrical resistance (TEER) measurements
A fresh medium was added to the ALI-cultured HBECs to the upper part of inserts and TEER was measured as described previously (33,34) using Millicell ERS-2 Voltohmmeter (Merck Millipore) each in triplicates. Results were presented as a change in TEER values (Ω x cm2) in the mucociliary differentiated HBECs after 4 days of the EMTU as a % of control, where the control was the same well before the EMTU establishment (Ω x cm2 after EMTU/Ω x cm2 before EMTU)*100%.
Analyses of α-SMA intracellular localization and content
For immunofluorescent studies, the HBFs growing on coverslips were fixed with 3.7% formaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS and blocked with 3% BSA/PBS. Immunostaining of α-SMA was performed using primary anti-α-SMA antibody (mouse monoclonal IgG, A2547, clone 1A4, 1:400, Sigma-Aldrich) and compatible goat anti-mouse secondary IgG antibody conjugated with Alexa Fluor 488 (1:500, Thermo Fisher) with Hoechst 33258 (1 µg/mL, Sigma-Aldrich) for cell nuclei detection. Slides were mounted in the fluorescence mounting medium (DAKO, Perlan) and visualized by automatic fluorescent microscope Leica DMI6000B (Leica Microsystems, Wetzlar, Germany), equipped with the LAS-X software, all at the same fluorescent time exposure. For the cell-based enzyme-linked immunosorbent (in-cell ELISA) analyses of the α-SMA and fibronectin (using a primary rabbit polyclonal antibody, F3648; 1:2000; Sigma-Aldrich) content, methanol-fixed HBFs were prepared according to the protocol described previously (57). Results were presented as absorbance values measured at 450 nm by Microplate Reader (Thermo Fisher Scientific).
Real-time quantitative PCR
After 4 days of the EMTU (as depicted in Fig.1A-B), both types of cells were collected separately and the total mRNA was isolated according to the manufacturer’s protocol using the RNAx/miRNA GeneMATRIX UNIVERSAL purification kit (E3599-02, EURx). Afterwards, the reverse transcription reaction carried out using the NG dART RT-PCR Kit (E0801-02; EURx) kit, 150 ng cDNA was used for further analyses. Expression of genes was performed using a real-time polymerase chain reaction (RT-qPCR, real-time PCR) with SYBR™ Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific) and designed specific primers (Table 1). Results were presented as the 2-ΔΔCt or 2-ΔCt value ± SEM.
Table 1. Primers sequences
GENE
|
SEQUENCE
|
ACTA2
|
F'
|
CTGTTCCAGCCATCCTTCAT
|
R'
|
CCGTGATCTCCTTCTGCATT
|
DNAH9
|
F'
|
GAGTCTTCCCAGCCACTCTTAC
|
R'
|
ATTCTGCATTCTCCAGAGCTTC
|
GAPDH
|
F'
|
GAAGGTGAAGGTCGGAGT
|
R'
|
GAAGATGGTGATGGGATTTC
|
MUC5A
|
F'
|
TTCCATGCCCGGGTACCTG
|
R'
|
CAGGCTCAGTGTCACGCTCTT
|
CDH2
|
F'
|
CTCCATGTGCCGGATAGC
|
R'
|
CGATTTCACCAGAAGCCTCTAC
|
P63
|
F'
|
CCCGTTTCGTCAGAACACAC
|
R'
|
CATAAGTCTCACGGCCCCTC
|
SNAI1
|
F'
|
GCTGCAGGACTCTAATCCAGA
|
R'
|
ATCTCCGGAGGTGGGATG
|
SNAI2
|
F'
|
TGGTTGCTTCAAGGACACAT
|
R'
|
GTTGCAGTGAGGGCAAGAA
|
FN1
|
F'
|
TGTGGTTGCCTTGCACGAT
|
R'
|
GCTTGTGGGTGTGACCTGAGT
|
TAGLN
|
F'
|
CGTGGAGATCCCAACTGGTT
|
R'
|
AAGGCCAATGACATGCTTTCC
|
TNC
|
F'
|
GGTCCACACCTGGGCATTT
|
R'
|
TTGCTGAATCAAACAACAAAACAGA
|
18S
|
F'
|
GTAACCCGTTGAACCCCAT
|
R'
|
CCATCCAATCGGTAGTAGCG
|
Statistics
Normality of distribution was estimated using the Shapiro-Wilk test and then appropriate parametric or non-parametric test was used. Data are presented as a mean ± SEM. Statistical significances the Student’s t-test; * p < 0.05, ** p < 0.01, *** p < 0.001 (the comparison in one group between only two different experimental conditions), one-way ANOVA with the Bonferroni multiple comparison post hoc test (the comparison in one group between more than two different experimental conditions); * p < 0.05, ** p < 0.01, *** p < 0.001 or two-way ANOVA with the Bonferroni multiple comparison post hoc test (the comparison of more than two different experimental conditions between the tested groups). The statistical analyses were performed using GraphPad Prism 5.0 software.