Materials and Methods Materials RPMI 1640 medium was purchased from Biochrom (Berlin, Germany). FCS was from Life Technologies (Carlsbad, CA, USA). Penicillin and streptomycin were from PAA Laboratories (Pasching, Austria). PBS was obtained from PAN-Biotech (Aidenbach, Germany). EBSS was from Sigma-Aldrich (Saint Louis, MO, USA). Bronchial epithelial basal medium (BEBM) and supplementary growth factors were all from Lonza (Walkersville, MD, USA). PureCol (type I bovine collagen solution, 3 mg/ml) was from Advanced BioMatrix (San Diego, CA, USA). Soybean trypsin inhibitor (STI), DNase I, and Protease XIV were from Sigma-Aldrich. Primary antibodies detecting p-STAT1 and actin were all from Cell Signaling Biotechnology (Frankfurt, Germany). IFNλ antibody was from abcam (ab38569, Cambridge, UK). Cell Culture Human bronchial epithelial BEAS-2B cells were cultured in RPMI growth medium, supplemented with 10% FCS, 1% penicillin/streptomycin at 37°C in a humidified incubator at 5% CO2. Human primary bronchial epithelial cell cultures (primary HBE) were bought from Lonza (Visp, Switzerland) or isolated from biopsies from individuals treated by lobectomy because of non-small-cell lung carcinoma at the Thoraxklinik Heidelberg. Ethics approval (S-381/2014) was obtained from the regional Ethics Committee at the University of Heidelberg, and all study participants provided a written informed consent. Two methods of bronchial epithelial cells isolation were used (65, 66): Briefly, the obtained biopsies were washed with cold EBSS and cleaned from any additional connective tissue and mucus. The segments were then cut open and incubated on a shaker at 4°C overnight in 9 ml EBSS including 1 ml digestion solution (DS, 10x: 0.01% DNase I, 1% Protease XIV in sterile PBS). On the next day, 1.1 ml FCS was added to the solution to terminate digestion. The epithelium was scraped into the digestion medium using a sterile scalpel. The biopsies were additionally washed with EBSS. All epithelial cells from scraping were collected by spinning at 4°C at 500 × g for 5 min. Cell pellets were resuspended in 10 ml of warm BEGM (Lonza) and cells were then seeded into collagen-coated (3 mg/ml PureCol, Cellsystems, 1:75 in ddH2O) prewarmed 10 cm culture dishes. Additionally, isolation using an outgrowth method was performed: Bronchial segments were cut into 2–3 mm3 pieces and placed into collagen-coated 6-well-plates. These pieces served as a source of primary cells and grown out epithelial cells were transferred into collagen-coated 10 cm dishes as soon as they reached 70% confluence. Primary cell cultures were maintained at 37°C in a humidified incubator at 5% CO2. Culture medium (BEBM + Supplements) was changed every 2–3 days. Cells were passaged when reaching 70–90% confluency. After 2 washes with PBS, they were trypsinized with 2 ml of 0.05% trypsin/EDTA and incubated 5–10 min at 37°C. Cells were then rinsed twice with PBS, harvested and pooled into a tube containing soybean trypsin inhibitor (STI, 1 mg/ml) on ice. After spinning at 500 × g for 5 min, the pellets were resuspended in 10 ml BEGM and cell suspension was split into two new collagen-coated 10 cm dishes. P. aeruginosa Strains and Virus Infections Various P. aeruginosa strains (Table 1) were cultured in lysogeny broth (LB) broth overnight at 37°C, shaking at 200 rpm. The culture was then diluted 1:50 in RPMI-1640 medium and grown for 5 days at 37°C, shaking at 200 rpm, in order to activate quorum-sensing and virulence factor secretion. On day 5, the medium was centrifuged for 10 min at 4,200 rcf and passed through a 0.2 μm filter. This medium was used as conditioned medium (CM). For each experiment at least two different CM were used. A RPMI-1640 control was cultured and filtered alongside the cultured P. aeruginosa strains. 100,000 BEAS2B or primary cells were seeded in 24-well-plates in BEAS2B medium. The following day, the cell medium was changed to infection medium, which was RPMI-1640 supplemented with 2% FCS, 1% P/S, 25 mM HEPES, and 0.075% NaHCO3. Twenty-four hours later, BEAS2B were pretreated with CM (20% v/v) for 1 h at 37°C, 5% CO2. Cell medium was then removed, and respiratory syncytial virus (RSV) or rhinovirus (RV1B) at a multiplicity of infection of 1 (MOI 1) (stocks prepared in house) in combination with fresh CM was added to BEAS2B cells. After 1 h at room temperature, shaking at 30 rpm, medium was removed, cells washed twice with warm 1X PBS, and infection medium added to the cells. Fresh CM was once again added to the cells as indicated above. Cell culture supernatant was collected at the indicated times and cells were lysed in RNA lysis buffer. Table 1 Strain descriptions. No. Strain Description and characteristics References 1 PAO1 Pseudomonas aeruginosa wild type DSMZ 22644 2 Boston Pseudomonas aeruginosa wild type ATCC 27853 3 PA14 Pseudomonas aeruginosa wild type DSMZ 19882 4 PA7 Pseudomonas aeruginosa wild type DSMZ 24068 5 CF1.1 CF patient isolate (6, 40) 6 CF1.2 CF patient isolate, clonally related to 5 (6, 40) 7 CF2.1 CF patient isolate (6, 40) 8 CF2.2 CF patient isolate, clonally related to 7 (6, 40) 9 CF3.1 CF patient isolate (6, 40) 10 CF3.2 CF patient isolate, clonally related to 9 (6, 40) 11 CF4.1 CF patient isolate (6, 40) 12 CF4.2 CF patient isolate, clonally related to 11 (6, 40) 13 CF1 CF patient isolate (6, 40) 14 CF1ΔlasR CF1 lasR deletion mutant (6, 40) 15 CF2 CF patient isolate (6, 40) 16 CF2ΔlasR CF2 lasR deletion mutant (6, 40) 17 CF3 CF patient isolate (6, 40) 18 CF3ΔlasR CF3 lasR deletion mutant (6, 40) 19 CF4 CF patient isolate (6, 40) 20 CF4ΔlasR CF4 lasR deletion mutant (6, 40) 21 CF5 CF patient isolate (6, 40) 22 CF5ΔlasR CF5 lasR deletion mutant (6, 40) 23 PA14 Pseudomonas aeruginosa wild type parental strain of 24, 25, 26, 27 (43, 67) 24 PA14ΔAprA PA14 transposon insertion mutant, ID23768 (43, 68) 25 PA14ΔLasA PA14 transposon insertion mutant, ID35267 (43, 68) 26 PA14ΔLasB PA14 transposon insertion mutant, ID31938 (43, 68) 27 PA14ΔPrpL PA14 transposon insertion mutant, ID37740 (43, 68) Western Blotting BEAS2B cells were stimulated as indicated, subsequently washed with PBS, and lysed in Laemmli buffer [400 mM Tris–HCl, pH 6.8, 20% (v/v) β-mercaptoethanol, 40% (v/v) glycerol, 8% (w/v) SDS, and 0.4% (v/v) bromophenol blue]. After incubation for 10 min at 98°C, equal amounts of lysates were fractionated by 10% polyacrylamide gel (SDS-PAGE) and electrotransferred to Nitrocellulose membranes by a semidry blotting procedure [buffer: 25 mM Tris, 192 mM Glycin, 10% (v/v) methanol; 2.5 mA/cm2 for 1 h 15 min]. Blocking of unspecific binding was performed using 5% BSA solution in 1× TBST [1× TBS, 0.05% (v/v) Tween-20] for at least 1 h. Membranes were stained with antibodies against pY-STAT1 (Tyr701, #9167), STAT1 (#9172), β-Actin (#4970) (Cell Signaling, Leiden, Netherlands; 1:1,000) overnight at 4°C. After three 10 min washing steps in 1 × TBST at room temperature, blots were incubated with secondary antibodies for 1 h at RT [HRP-linked anti-mouse or anti-rabbit (Cell Signaling, Leiden, Netherlands)], followed by additional three 10 min washing steps in 1× TBST at room temperature. Proteins were detected using an enhanced chemiluminescence system (Western lightning™ plus ECL, Perkin-Elmer, Rodgau, Germany). Gels were imaged digitally, and contrast adjustments were applied to all parts of a figure. The prestained molecular weight marker was imaged separately (using transmitted light) and aligned to the digital images of the blots. The ladder is represented on the blots as black bars. Where indicated, membranes were stripped and reprobed. Densitometry was performed using ImageJ software (National Institutes of Health). RNA Isolation and Quantitative RT-PCR Total cellular RNA was isolated using peqGold Total RNA Kit (peqlab Biotechnology, Erlangen, Germany) according to the manufacturer's standard protocol. RNA isolation included DNase digestion using an RNase-free DNase set (Qiagen, Hilden, Germany). In order to perform quantitative RT-PCR, total RNA was first reverse transcribed into single stranded cDNA using High Capacity cDNA RT Kit (Applied Biosystems, Foster City, CA). For RT-PCR analysis, 2 μl of cDNA (diluted 1:4) was used as a template in a final reaction volume of 15 μl, combined with SYBR®Green PCR Master Mix Fast (Applied Biosystems) and corresponding primers (Table 2). The analysis was performed on a StepOne Plus RT-PCR platform (Applied Biosystems) in 96-well-format. Each gene was measured in duplicates of each cDNA sample. The baseline and threshold values were detected automatically and the Ct values of the endogenous constitutively expressed reference gene (ACTB) were subtracted from the determined Ct values resulting in a –ΔCt for each target gene, which was then used to calculate the relative expression, rE = 2−ΔCt. To control reaction specificity, all measurements included samples without the reverse transcriptase enzyme (noRT). Melting curves were used to prove specific amplification. Fold induction was calculated of the ratio of rEtreated/rECtr. Table 2 Primer sequences. Name Direction Sequence MX1 Fw CTGCACAGGTTGTTCTCAGC Rev CCAAGGTCCACCGTGATTAAC OAS1 Fw TGTCCAAGGTGGTAAAGGGTG Rev CCGGCGATTTAACTGATCCTG Rv1b Fw CTAGCCTGCGTGG Rev AAACACGGACACCCAAAGT RSV Fw GATATGCCTATAACAAAT Rev GATACTGATCCTGCATT hIFNa1 Fw CAGAGTCACCCATCTCAGCA Rev CACCACCAGGACCATCAG hIFNa2 Fw CTGGCACAAATGGGAAGAAT Rev CTTGAGCCTTCTGGAACTGG hIFNb Fw CGCCGCATTGACCATCTA Rev GACATTAGCCAGGAGGTTCTCA hIFNL1 Fw GGACGCCTTGGAAGAGTCACT Rev AGAAGCCTCAGGTCCCAATTC hIFNL2/3 Fw CTGCCACATAGCCCAGTTCA Rev AGAAGCGACTCTTCTAAGGCATCTT Measurement of Cytokine Secretion Sandwich enzyme-linked immunosorbent assay (ELISA) was performed using commercially available kits (eBioscience, human IL29 ELISA) to determine the amount of secreted human IFNλ in the cell-free supernatants of stimulated cells. Samples were tested in duplicates and the assays were performed according to the manufacturers' instructions. Cytokines were detected by measuring the absorbance at 490 nm with a 650-nm reference in a photometer (Sunrise reader, Tecan, Salzburg, Austria). Cytokine concentrations were calculated according to a standard dilution of the respective recombinant cytokines using Magellan V 5.0 software (Tecan, Salzburg, Austria). Protease Activity Assay, Zymography CM was treated with non-reducing Western blot sample buffer and separated by SDS-PAGE (10% polyacrylamide gel containing 0.1% gelatin). The gels were washed twice with 2.5% Triton-X-100 for 15 min followed by an overnight incubation at 37°C in substrate buffer (10 mM Tris, pH 8.0, 10 mM CaCl2, 1 μM ZnCl, 150 mM NaCl). Subsequently the gel was stained with 0.5% Coomassie blue in acetic acid:isopropanol:dH2O (10:30:60) and pictures were taken after destaining with dH2O. In order to determine total protease activity in CM a sterile 6 mm filter disk was added on skim milk agar (1.5%) and 10 μl of CM was added. Following incubation at 37°C the diameter of clearance (as an indication of protease activity) was measured. In vitro Degradation of IFNλ IFNλ was incubated with CM for the indicated time at 37°C and 5% CO2 in the dark. As a loading control IFNλ was added to RPMI only. Reactions were stopped by adding western blot sample buffer and heat treatment (95°C, 10 min). Subsequently samples were used for western blot analysis. Quantification was performed using ImageJ and samples were normalized to the input control. Patients and Virus Detection Studies including samples from CF patients were approved by the Ethics Committee of the University of Heidelberg (study number S-370/2011). Informed written consent was obtained from the patients, parents, or legal guardians of all subjects. Airway samples from CF patients (sputum, throat swab) were obtained during routine visits at the CF Center at the University Hospital Heidelberg as previously described (54). The diagnosis of CF was based on established diagnostic criteria. Samples were used for isolation of P. aeruginosa strains (n = 51) and sputum, nose or throat swabs were analyzed for virus infection by multiplex PCR (Seegene, Korea, AllplexTM respiratory panels 1–4) or by targeted RT-PCR after RNA extraction using TRIZOL and cDNA synthesis as described above. Infection status with P. aeruginosa (intermittent or chronic) was classified according to the following definition (69): Intermittent infection was defined as positive microbial culture of P. aeruginosa in at least one and <50% of the samples in the last 12 months and no detection of anti-Pseudomonas antibodies (against alkaline protease, elastase, and exotoxin A). Chronic infection was defined as persistent culture presence of P. aeruginosa for at least 6 months, or less when combined with a positive finding (titer >1,250) of two or more antibodies. Statistical Analysis All experiments were repeated three times unless stated otherwise. Data are shown as mean + SD. Statistical significance of comparison between three or more unmatched groups was determined by one-way ANOVA and if multiple comparisons were performed, two-way ANOVA was used (both including Bonferroni post-test). Statistics on quantitative PCR (qPCR) data were performed on previously log-transformed data to achieve a normal distribution. All statistical analyses were done using GraphPad Prism (GraphPad 5.00 and 6.05, San Diego, USA) software. Significant differences were considered at *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to the control condition. n.s., not significant.