RNA-seq of human bronchial epithelial cells treated with cigarette smoke and mycobacteria
RNA-seq of human bronchial epithelial cells treated with cigarette smoke and mycobacteria
Source of bronchial epithelial cells Primary bronchial epithelial cells (PBEC) were isolated from macroscopically normal lung tissue obtained from patients undergoing resection surgery for lung cancer at the Leiden University Medical Center (LUMC), the Netherlands. Patients from which this lung tissue was derived were enrolled in the biobank via a no-objection system for coded anonymous further use of such tissue (www.coreon.org). Samples from this Biobank were approved for research use by the institutional medical ethical committee (BB22.006/AB/ab). Since 01-09-2022, patients are enrolled in the biobank using written informed consent in accordance with local regulations from the LUMC biobank with approval by the institutional medical ethical committee (B20.042/KB/kb). All experiments were performed in accordance with relevant guidelines and regulations. The study population included 8 individual donors, of whom 4 were female. Median age was 66.5 years (range 56-70). Of these donors, none were current smokers, 5 were ex-smokers (62.5%), 2 were non-smokers (25%) and no information was available for 1 donor (12.5%). None of the donors were diagnosed with chronic obstructive pulmonary disease. Donors were not tested for Mycobacterium infection prior to donating tissue. Given that we are based in a low-endemic setting, it is unlikely that the donors had active TB at the time of surgery.  Bronchial epithelial cell culture PBEC were cultured as previously described (18, 19). In brief, PBEC (passage 1) of individual donors were expanded for 5 days in T75 culture flasks pre-coated with a mixture of 30 μg/ml Purecol (Advanced BioMatrix, Carlsbad, CA, USA), 5 μg/ml human fibronectin (PromoCell, Heidelberg, Germany) and 10 μg/ml BSA (Fraction V; Thermo Fisher Scientific, Carlsbad, CA, USA). Per experiment, cells from 2 unique donors were seeded at a density of 40.000 cells per donor on pre-coated 0.4 µm membrane pore size polyethylene terephthalate (PET) inserts (cellQART, Northeim, Germany) in 12-well plates. Cells were cultured in medium consisting of one part Bronchial Epithelial Cell Medium-basal (BEpiCM-b (ScienCell, Carlsbad, CA, USA)) and one part Dulbecco's modified Eagle's medium (DMEM (Gibco, Thermo Fisher Scientific)) supplemented with Bronchial Epithelial Cell Growth Supplement (BEpiCGS; ScienCell), 12.5 mM HEPES (Gibco, Thermo Fisher Scientific), 100 U/ml penicillin, 100 ug/ml streptomycin (all from ScienCell), 2 mM glutaMAX (Thermo Fisher Scientific), further referred to as complete BD-medium (cBD). In the submerged stage, cBD was supplemented with 1 nM EC 23 (Tocris, Bristol, UK). After reaching 100% confluence, apical medium was removed and cells were differentiated at the air-liquid interface (ALI) in cBD supplemented with 50 nM EC 23. Culture medium was changed 3 times per week during which the apical side of the culture was carefully washed with 200 µl warm PBS for 10 min at 37 °C to wash away mucus and cell debris. After 14 days of mucociliary differentiation at ALI, all dominant cell-types were present and ciliary activity was observed. Cultures were subsequently used for experiments.  Exposure of epithelial cell cultures to cigarette smoke Medium of ALI-PBEC was replaced with cBD medium containing 50 nM EC 23 and 5 µg/ml gentamicin directly before smoke exposure. Cigarette smoke (CS) exposure of ALI-PBEC was performed as described previously (20, 21). In brief, CS was derived from 3R4F Research Cigarettes (University of Kentucky, USA) which were burned until the filter paper line. ALI-PBEC were placed in chambers and exposed to either whole CS from one cigarette or room air, for 4-5 min. After exposure, the exposure chambers were ventilated for 10 min to remove CS. The chambers received on average 2.03 (±0.32) mg of CS particles, as calculated from the weight of the outlet filter before and after CS exposure. For one experiment, the filter weight could not be measured reliably due to misalignment of the filter. Following CS exposure, ALI-PBEC were incubated for 2 h at 37 °C, after which the medium was replaced by antibiotics-free cBD medium containing 50 nM EC 23. PBEC were incubated another 2 h before infection with mycobacteria.  Infection of airway epithelial cell cultures ALI-PBEC were CS-exposed prior to infection as described above, and infected as previously described (18). In brief, Mtb Venus and Mav Wasabi were cultured as described above, and OD600 of bacterial cultures was measured. A multiplicity of infection (MOI) of 100 was prepared accordingly in antibiotics-free cBD containing 50 nM EC 23. For this calculation, the cell density per insert was estimated at 1x106 cells. To remove mucus from the apical surface of ALI-PBEC cultures, they were washed with 200 µl warm PBS for 10 min. Immediately following mucus removal, 50 µl bacterial suspension was added to the apical side of the cells. Control cultures received 50 µl medium without bacteria. Next, cultures were centrifuged briefly at 300 x g for 2 min to spin bacteria down onto the cells, and incubated at 37 °C for 24 h. Then, ALI-PBEC were washed with 200 µl PBS before 50 µl gentamicin (30 µg/ml) was added for 15 min at 37 °C to eliminate extracellular bacteria. ALI-PBEC were washed again with 200 µl PBS, and then incubated for an additional 6 days at 37 °C so the total intracellular infection lasted 7 days. ALI-PBEC cultures to be used in crystal violet assays were not washed or treated with gentamicin, and instead were incubated a total of 7 days with mycobacteria. During infection, all PBEC were supplied basally with 1 ml new cBD containing 50 nM EC 23 every 3 days.  Before sample collection, ALI-PBEC were washed with 200 µl PBS apically for 10 min at 37 °C to remove mucus and cell debris. Then, the cell layer was lysed with 350 µl TRIzol (Ambion, Foster City, CA, USA) for 5 min at RT. All liquid was collected into microtubes and stored at -70 °C awaiting processing. RNA was isolated using the Direct-zol RNA Isolation Miniprep kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions.  Protocol available upon request from GenomeScan, The Netherlands. (SOP 70 RNA-Seq Analysis)