Roflumilast Partially Reverses Smoke-induced Mucociliary Dysfunction

Andreas Schmid; Nathalie Baumlin; Pedro Ivonnet; John S. Dennis; Michael Campos; Stefanie Krick; Matthias Salathe


Respiratory Research. 2015;16(135) 

In This Article



Roflumilast was provided by Forest Laboratories Inc. (New York, NY, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA), unless otherwise specified.

Cell Cultures

Normal human bronchial epithelial (NHBE) cells from non-smokers were isolated from lungs unsuited for transplant and generously donated for research by organ donors. These lungs were recovered with IRB approved consents by the LifeCenter Northwest in Washington State and the Life Alliance Organ Recovery Agency of the University of Miami. After isolation as previously described,[12–15] NHBE cells were dedifferentiated through expansion on plastic and redifferentiated at an air-liquid interface (ALI) on collagen-coated Transwell permeable supports (12 mm, 24 mm and Snapwell, Costar Corning, Tewksbury, MA, USA). NHBE cells were considered fully differentiated after 3–4 weeks at the ALI as assessed by beating cilia and mucus transport.

Smoke Exposure of NHBE ALI Cultures

Fully differentiated NHBE cells were exposed to cigarette smoke using a Vitrocell VC-10 smoking robot (Vitrocell Systems GMBH, Waldkirch, Germany). Four cigarettes (University of Kentucky 3R4F) were smoked according to ISO standard 3308: six puffs per cigarette with a 35 mL volume per puff and a waiting time between each puff of 60 s. Roflumilast was added 1 h before and re-added after smoking. CBF, FRET, and CFTR conductance were measured 3 h after smoke exposure. After these measurements, NHBE cells were lysed and lysate stored at −20 °C for quantitative PCR.

Pseudotyped Lentivirus Vectors and Infection of Airway Epithelial Cells

Third generation, propagation-deficient, HIV pseudotyped lentiviruses encoding fluorescently tagged PKA subunits (RII-CFP and CAT-YFP) under the transcriptional control of the ciliated cell-specific foxj1 promoter were used for FRET as described previously.[13] Briefly, recombinant lentiviruses were constructed using the pRRLsinPPT.CMV.MCS.Wpre vector.[16] For the initial constructs, genes encoding the catalytic PKA subunit CAT and the regulatory PKA subunit RII, fused to the fluorescent proteins YFP and CFP, respectively,[17] were cloned into the multiple cloning site downstream of the ciliated cell-specific foxj1 promoter for sole expression in ciliated cells.[18] Using calcium phosphate co-precipitation (Clontech Laboratories, Inc., Mountain View, CA, USA), lentiviruses were prepared by co-transfecting HEK 293 T cells with vector and packaging DNAs plasmids. Virus-containing medium was collected 48 h and 72 h later, concentrated by polyethylene glycol (11 %) precipitation, and stored at −80 °C. An estimation of the virus titer was performed using the p24 HIV Antigen ELISA kit (PerkinElmer, Wellesley, MA, USA). Dedifferentiated cells were used for co-infection with both viral constructs. At the time of plating the cells on Transwells, virus was added at a ratio of 100 ng per 500,000 cells in bronchial epithelial growth medium (BEGM) containing polybrene (2 μg/ml final concentration). The infection was done overnight, at 37 °C in 5 % CO2. The following day, virus was removed, and BEGM was changed to ALI medium top and bottom until cells reached confluence, when an air liquid interface was created. Expression of the fluorescently tagged proteins was monitored using an inverted fluorescence microscope.

Measurement of CBF and FRET in Airway Epithelial Cells

Fully differentiated NHBE cells cultured on 24 mm Transwell supports were placed in a customized, fully enclosed chamber, allowing independent perfusion of the apical and basolateral compartments. The chamber was mounted at room temperature on the stage of an upright Nikon E600fn microscope. Water was added on top of the closed chamber for use of a 63× water immersion objective with a numerical aperture of 1.0. FRET was measured as described previously,[13] with images acquired every 10s. CBF was recorded according to published methods,[13,19] using infrared differential interference contrast video microscopy. CBF and FRET were measured in real time and simultaneously in ciliated cells that expressed both fusion proteins. In addition, CBF was also recorded on an inverted Zeiss Axiovert without apical perfusion before and after apical DPBS supplementation.

Ussing Chamber Experiments

Snapwell filters containing fully differentiated NHBE cells were rinsed with Krebs-Henseleit solution (KH), and then mounted in Ussing chambers (EasyMount Chambers; Physiologic Instruments, San Diego, CA, USA) containing KH in apical and basolateral chambers. Solutions were maintained at 37 °C by heated water jackets, and were continuously bubbled with a 95 % room air/5 % CO2 mixture to maintain the pH at 7.4. To monitor short-circuit current (ISC), the transepithelial membrane potential was clamped at 0 mV with a six-channel voltage clamp (model VCC MC6; Physiologic Instruments) using Ag/AgCl electrodes in agar bridges. Signals were digitized and recorded with DAQplot software (VVI Software, College Station, PA, USA) via a LabJack A/D converter (LabJack Corp., Lakewood, CO, USA). The input resistance of each filter was measured by the application of 1 mV bipolar pulses of 2-s duration. To eliminate any contribution to the Isc by epithelial sodium channels, 10 μM amiloride was added to the apical chamber. Once the Isc stabilized, roflumilast (100 nM) was included in the apical and basolateral perfusate. After a 20 min pre-treatment with roflumilast, 10 μM albuterol or 10 μM forskolin was added to the apical perfusate, and the resulting increase in chloride conductance was measured as Isc. To assure that conductance changes were related to CFTR, Isc decreases upon apical addition of 10 μM CFTRinh 172 were measured as well. To study the combination effect of long acting ß-mimetics and roflumilast, cultures were incubated with 100 nM roflumilast, 100 nM formoterol, both or none for 2 h before exposures to smoke or air. In the Ussing chamber, an additional 10 μM of albuterol was added but to assess the overall influence on CFTR conductance; then Isc decreases upon addition of the CFTR inhibitor CFTRinh 17 were assessed.

Airway Surface Liquid (ASL) Volume Estimates

ASL volumes from NHBE cells were estimated by meniscus scanning as previously described.[20] Briefly, cultures were washed with phosphate buffered saline (PBS) 24 h prior to measurements to clean the filters from mucus accumulation, which can interfere with ASL reading. At the time of measurement, the 12 mm Transwell supports were placed on a commercially available scanner (Epson perfection V500 PHOTO, Epson America Inc., Long Beach, CA). Scanned menisci were used to estimate ASL volume using software generously provided by Dr. Myerburg (University of Pittsburgh). Calibration of the 12 mm Transwell supports (Costar Corning, Tewksbury, MA, USA) was done by measuring ASL changes after apical addition of different volumes of PBS.

Bronchoscopic Sampling of Airway Epithelial Cells

Bronchial epithelial cells from patients were obtained during bronchoscopy under conscious sedation for other clinical indications as approved by our IRB. After sedation, a cytology brush was passed through the bronchoscope to harvest airway epithelial cells. The brushings were done in the left and right main bronchus just below the carina using eight passages with ten strokes each. After each passage, the brush was withdrawn from the bronchoscope and rinsed in 2 ml of PBS. Cells were collected from current smokers (ten pack year history of smoking), ex-smokers (quit more than 1 year ago) and non-smokers.

Quantitative PCR

Total RNA was isolated using the Total RNA E.Z.N.A. kit from OMEGA (VWR International, Radnor, PA, USA). The concentration of RNA was determined using the NanoDrop 1000 spectrophotometer (Thermo Scientific, Rockford, IL, USA). Complementary DNA (cDNA) was made from total RNA using the iScript Synthesis Kit (BioRad, Hercules, CA, USA). Messenger RNAs (mRNA) of interest were quantified by PCR using a BioRad iCycler and TaqMan probes (Life Technologies, Grand Island, NY, USA). The difference in the threshold cycle between the targeted gene and GAPDH (ΔCt) was used as the relative level of expression.


Results were evaluated by one-way ANOVA. If a significant difference was found, a parametric (Newman Keuls) or non-parametric analysis (Dunns) using Prism 5 (GraphPad, La Jolla, CA, USA) was used for comparison of groups. p < 0.05 was accepted as significant. Error bars in all figures represent SEM.