Cigarette smoke alters primary human bronchial epithelial cell differentiation at the air-liquid interface

The differentiated human airway epithelium consists of different cell types forming a polarized and pseudostratified epithelium. This is dramatically altered in chronic obstructive pulmonary disease (COPD), characterized by basal and goblet cell hyperplasia, and squamous cell metaplasia. The effect of cigarette smoke on human bronchial epithelial cell (HBEC) differentiation remains to be elucidated. We analysed whether cigarette smoke extract (CSE) affected primary (p)HBEC differentiation and function. pHBEC were differentiated at the air-liquid interface (ALI) and differentiation was quantified after 7, 14, 21, or 28 days by assessing acetylated tubulin, CC10, or MUC5AC for ciliated, Clara, or goblet cells, respectively. Exposure of differentiating pHBEC to CSE impaired epithelial barrier formation, as assessed by resistance measurements (TEER). Importantly, CSE exposure significantly reduced the number of ciliated cells, while it increased the number of Clara and goblet cells. CSE-dependent cell number changes were reflected by a reduction of acetylated tubulin levels, an increased expression of the basal cell marker KRT14, and increased secretion of CC10, but not by changes in transcript levels of CC10, MUC5AC, or FOXJ1. Our data demonstrate that cigarette smoke specifically alters the cellular composition of the airway epithelium by affecting basal cell differentiation in a post-transcriptional manner.


Cultivation, differentiation and CSE exposure of primary human bronchial epithelial cells
Normal primary human bronchial epithelial cells in passage 1 (pHBECs) (Lonza; Wokingham, UK) were cultured in BEGM medium (Lonza) at 37°C in a humidified cell incubator with 95% air and 5% CO 2 1 . When cell density reached 80%, cells were detached using the Clonetics™ ReagentPack™ (Lonza) for subculturing.
For differentiation of HBECs, 80,000 -90,000 cells at passage 2 were seeded onto pre-warmed human placental collagen type IV-coated (Sigma-Aldrich; St. Louis, MO) 12-well transwell inserts (transparent, 0.4 µm; Greiner; Solingen, Germany or clear, 0.4 µm; Corning; New York, NY) in pre-warmed BEGM medium. Cells were lifted to the air-liquid interface (ALI) when confluent, referred to as day 0 of ALI culture. For this, the apical medium was aspirated and the basolateral medium substituted with PneumaCult™-ALI medium (Stemcell Technologies; Köln, Germany) containing 1% penicillin/streptomycin. Medium was changed every 2 -3 days. pHBEC cultures were left to differentiate up to 28 days after air-lift. For CSE treatment during differentiation, cells were chronically exposed to 2.5 and 5% CSE between day 0 and day 28 of ALI culture from the basolateral side of the transwell. CSE treatment was renewed every 2 -3 days, i.e. each time the growth medium was changed. The apical surface was washed weekly with 0.5 ml pre-warmed HBSS to remove mucus. This solution (further referred to as "cell supernatant"), as well as the basal medium, was frozen at -80°C for analysis of secreted proteins.

Cytotoxicity Assays
To assess cytotoxicity of CSE on cells, lactate dehydrogenase (LDH) levels were quantified in the cell supernatant and basolateral culture medium using the Cytotoxicity Detection Kit (LDH) (Roche; Mannheim, Germany) according to the manufacturer's instructions. Briefly, supernatant and medium of untreated (= low control) and treated cells were collected. To control for maximal LDH release of the cells used (= high control), untreated cells were lysed with 2% Triton X-100/ media/0% FCS for 15 minutes at room temperature. Cell debris were pelleted with 250 x g for 10 minutes at 4°C and the cell-free liquids were stored at 4°C until analysis. Samples were mixed with equal amounts of substrate mixture and incubated for 30 minutes at room temperature in the dark. Absorbance (490 nm) was measured using the Sunrise multiplate reader, background values were subtracted, and cytotoxicity calculated using the formula:

RNA Isolation and Real-Time Quantitative Reverse-Transcriptase PCR (qRT-PCR) Analysis
For RNA extraction from pHBECs, the RNeasy Mini Plus Kit (Quiagen; Venlo, Holland) was used according to the manufacturer's instructions. RNA concentration was determined using the NanoDropTM 1000 spectrophotometer (NanoDrop Tech. Inc; Wilmington, DE) at 260 nm. For analysis of mRNA expression, total RNA was reverse transcribed using MuLV reverse transcriptase (Applied Biosystems; Carlsbad, CA) and random hexamer primers (Applied Biosystems). For this, 1 µg RNA was diluted up to 20 µl with DNase/RNasefree ddH 2 0. RNA secondary structures were denatured at 70°C for 10 minutes, before samples were incubated on ice for 5 minutes. 20 µl of cDNA synthesis master mix (5 mM MgCl 2 , 1x PCR buffer II (10x), 1 mM dGTP, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, 1 U/µl RNase inhibitor, and 2.5 U/µl MuLV reverse transcriptase) was added to each sample and cDNA synthesis was performed: 60 minutes at 37°C, followed by 10 minutes incubation at 75°C. cDNA was diluted up to 200 µl with DNase/RNase-free ddH 2 0 for usage in qRT-PCR analysis. qRT-PCR was performed in 96-well format using the Light Cycler LC480II instrument (Roche) and LightCycler® 480 DNA SYBR Green I Master (Roche). Relative transcript abundance of a gene is expressed as . For specific gene amplification, primers listed in Table S1 were used.
HPRT was used as a housekeeping gene for standardization of relative mRNA expression. All qRT-PCR reactions were performed in technical triplicates and non-template controls were used for quality controls.

Protein Isolation, SDS-Polyacrylamid Gel Electrophoresis (SDS-PAGE) and Immunoblotting
For whole cell lysates, cells were scratched into ice-cold RIPA lysis buffer (

Immunofluorescence Analysis and Quantification
pHBECs were stained directly on the transwell membrane, which was cut into quarters using a scalpel 1 , or as cytospins Following the indicated cell treatment, cells were washed twice in DPBS and fixed in 3.7% (w/v) formaldehyde for 1 hour at room temperature. Cells were washed twice with DPBS and permeabilized with DPBS/0.1% Triton X-100 for 5 minutes at room temperature. 5% (w/v) BSA/0.2% (v/v) Tween-20 in DPBS served as blocking agent for 1 hour at room temperature. Detection of antigens was performed using the appropriate primary antibody in 5% (w/v) BSA/0.2% (v/v) Tween-20 in DPBS for 1 hour at room temperature.
Subsequent to washing in excess DPBS, the cells were incubated with the appropriate secondary antibody conjugated with either Alexa Fluor 488 or Alexa Fluor 568 for 30 minutes at room temperature in the dark. The cellular DNA was stained with 0.5 μg/ml 4,6-diamidino-2-phenylindole (DAPI). Cells were washed in excess DPBS and mounted in fluorescent mounting medium (Dako; Hamburg, Germany) on microscopy slides using glass coverslips. The mounting medium was dried overnight at room temperature and fluorescent microscopy was performed using an upright microscope (Axiovert II; Carl Zeiss AG; Oberkochen, Germany) or confocal microscopy (LSM710 system; Carl Zeiss AG). Images were processed using ZEN 2010 software (Carl Zeiss AG) or Imaris 7.4.0 software (Bitplane; Zurich, Switzerland). Immunofluorescence quantification was performed using Imaris 7.4.0 software (Bitplane). For this, z-stack images of stained transwell membranes were obtained by 5 confocal microscopy and 1500 -4500 cells per image were analyzed for positivity of specific markers in relation to the total number of cells assessed. Of note, also weakly expressing cells for specific markers were counted as positive signals. The following primary antibodies were used: mouse anti-acetylated tubulin (Sigma), rabbit anti-CC10 (Santa Cruz), and mouse anti-MUC5AC (Abcam).

Cytospin Analysis of pHBECs
In order to obtain cytospins from pHBECs, a single cell suspension of the cells was prepared using pre-warmed trypsin/EDTA from the apical side. Cells were pelleted at 250 x g for 5 minutes at room temperature and resuspended in 1 ml HBSS. 30,000 to 100,000 cells in 200 μl HBSS were transferred to a glass slide using cytocentrifugation with 300 rpm for 10 min at room temperature. Cytospins were left to dry overnight at room temperature and processed immediately for immunofluorescent staining as described above.