Airway epithelial cell culture

A549 human lung epithelial cells (American Type Culture Collection (ATCC)) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% (vol/vol) FCS and 2mM L-Glutamine. Cells were used at passage numbers 90–110. Cells from the transformed human bronchial epithelial cell line BEAS-2B were grown in keratinocyte serum-free medium (K-SFM; GIBCO BRL) supplemented with recombinant human epidermal growth factor (rhEGF) and bovine pituitary extract (PE). BEAS-2B cells were obtained from ATCC and were used at passage numbers 60–80. NHBE cells were obtained from Lonza and cultured to a maximum of five passages to limit variable responses. The NHBE cells were grown in bronchial epithelial medium (BEGM; Lonza) containing a mixture of growth factors, cytokines, and supplements (BulletKit; Lonza). All cells were maintained in 150 cm2 (T150) flasks with 5% CO2 at 37 °C. Medium was replaced every second day, and cells were passaged when > 85% confluent by washing with Dulbecco’s Phosphate Buffered Saline (DPBS) and dislodging with 0.5% trypsin. Cell viability was determined microscopically by trypan blue (Sigma-Aldrich) exclusion, and cell numbers counted by haemocytometer.

Inhibitors, antibodies, and cytokine assays

The putative MAP3K8 kinase inhibitor Tpl2-1 was purchased from Santa Cruz Biotechnology Inc., the inhibitor Tpl2-2 was purchased from Calbiochem. GSK2222284A and GSK2222867A, novel MAP3K8 kinase inhibitors, were gift from GlaxoSmithKline PLC. Anti-MAP3K8 was purchased from Santa Cruz Biotechnology Inc. and phospho-MAP3K8 (T290) was from Invitrogen Corp. Anti-MEK1/2, anti-p-MEK-1/2, anti-SEK1/MKK4, anti-phospho-SEK1/MKK4, anti-MKK7, anti-phospho-MKK7, anti-p44/42 MAPK, anti-phospho-p44/42 MAPK, anti-SAPK/JNK, anti-phospho-SAPK/JNK, anti-p38α MAPK, anti-phospho-p38 MAPK antibodies were purchased from Cell Signaling Technology. Other antibodies and horseradish peroxidase (HRP) used for Western blotting were purchased from DAKO. Human IL-6, IL-8, and RANTES were measured by ELISA kits according to manufacturer’s instructions (DuoSet; R&D Systems Europe).

Gene knockdown reagents and protocols

RNA interference (RNAi) was carried using ON-TARGETplus SMARTpool (Dharmacon Research Inc.) and ON-TARGETplus. Non-targeting Pool negatives were used as controls. RNAi transfection of A549 cells was performed using a previously established protocol [15] by using Lipofectamine RNAiMAX (Invitrogen Corp.). Briefly cells were seeded into 24-well plates (Corning Costar Corp.) at 4 × 104 cells/well to ensure 40–60% confluence was established by the day of transfection. RNAi- Lipofectamine RNAiMAX complexes were formed by adding 2 µl/ml of Lipofectamine RNAiMAX and 50nM of RNAi in serum free medium (Sigma-Aldrich) followed by incubation for 15 min at room temperature. RNAi-Lipofectamine RNAiMAX complexes were then added to a single well and the cells were placed in a CO2 incubator at 37 °C for 24 h. After this initial transfection, RNAi transfection was repeated with addition of a second dose of RNAi-Lipofectamine RNAiMAX complex followed by further incubation at 37 °C for 24 h. Following double transfection, cells were starved in serum free medium for 16 h and then stimulated with 1 ng/ml of IL-1β (R&D Systems, Europe Ltd., UK). Cells were collected and proteins extracted for Western blotting analysis whilst supernatants were harvested for cytokine measurement.

Models of IL-1β-induced inflammation

Airway epithelial cells were grown up in T150 flasks (for media details see Airway epithelial cell culture). When greater than 85% confluent, cells were dislodged from the flask surface by addition of trypsin. Wells of a 24-well plates were then seeded with 4 × 104 cells in 1 ml of complete medium/well and incubated overnight in a CO2 incubator at 37oC to allow cells attachment and growth. Prior to addition of the IL-1β stimulus, cells were starved in serum-free media for 16 h in order to achieve cell cycle synchronization. After stimulation with 1 ng/ml IL-1β, cells were harvested for MAP3K8 gene expression at 2, 4, 6, and 8 h, and harvested for MAP3K8 protein measurement at 5, 10, 20, 30, 45 and 60 min. For the MAP3K8 siRNA knockdown experiments, MAP3K8 protein and phosphorylation of the associated MAPK signalling proteins were measured at 20 and 30 min, whilst inflammatory cytokine gene expression (IL-6 and IL-8) was determined at 2, 4, and 6 h, with cytokine protein release measured at 8 and 24 h. All timings are post IL-1β stimulation.

Real-time quantitative PCR

Total RNA was isolated from A549 cells using the Qiagen RNeasy mini kit following the manufacturer’s instructions. Reverse transcription was performed on 0.5 µg of RNA and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following manufacturer’s instructions. Real-time PCR was performed using the Rotor-Gene 3000™ Real-Time PCR detection system (Corbett Research) with the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Data were normalized by the level of GAPDH expression in individual samples. Calculation of the threshold value, standard curve preparation and quantification of mRNA in the samples were performed using the Rotor-Gene 6.0 software (Corbett Research).

Western blotting

Whole-cell protein extracts were prepared with the Active Motif Nuclear Extract Kit (Active Motif Europe) according to the manufacturer’s protocol. Protein concentrations were determined with the BCA (bicinchoninic acid) protein assay kit (Pierce, Thermo Scientific) with bovine serum albumin (BSA; Pierce) as the standard control. For Western blots, 40 µg of protein for each sample was separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels (Invitrogen Corp.) with transfer to nitrocellulose membranes using the iBlot™ DryBlotting device (Invitrogen Corp.) and iBlot™ Transfer stacks. Antibody labeling was detected using relevant secondary antibodies conjugated to horseradish peroxidase (HRP) (1:4,000; DAKO Cytomation) and enhanced chemi-luminescence solutions (GE Healthcare ECL/ECL Plus (Amersham)). For quantitative analysis, the bands were scanned in a UVP GelDoc-It imaging system, and the band densities measured with Labworks 4.6 software (Bio-Rad Lab.).

Global gene expression profiling

RNA quality was determined using the RNA 6000 Nano AssayTM kit with Agilent 2100 BioanalyserTM and 2100 Expert Software (Agilent Technologies). RNAs with a RNA integrity number (RIN) score of ≥ 9 were converted to single stranded cDNA, using the AmbionTM WT Expression Kit (Ambion) and then fragmented and labelled using the Affymetrix GeneChipTM WT Terminal Labeling Kit (Affymetriz). Samples were hybridized to AffymetrixTM Human Gene 1.1 ST Arrays using the GeneTitanTM Instrument following the manufacturer’s instructions.

Data was processed with the Robust Multichip Analysis (RMA) algorithm [16], as implemented in Affymetrix Power Tools (APT, version 1.15.0), to generate normalized transcript cluster signal values. Systematic variation in expression between time points and conditions was modelled using LIMMA [17]. Three contrast matrices were generated: extracting contrasts between time-points under control conditions; extracting contrasts between time-points under knockdown conditions; and identifying transcript clusters that respond differently over time between the two conditions. P-values were adjusted using Benjamini and Hochberg’s method to control the false discovery rate (FDR) [18]. Universally low expressed transcript clusters (below the median across all 48 arrays) were excluded.

Functional annotation and enrichment testing was performed using the DAVID Gene Functional Classification Tool, version 6.7 [19, 20]. Unique Entrez IDs represented on the HuGene 1.1 ST array were employed as a background, excluding universally low expressed transcript clusters. P-values were adjusted using Benjamini and Hochberg’s method [18].

Investigation of tool MAP3K8 inhibitors

A549 cells were pre-treated with putative MAP3K8 inhibitors for one hour before IL-1β stimulation (1ng/ml) for 24 h. Cytokine measurements were made in supernatants as detailed above. Cells for proteins extraction and Western blots were harvested after 30 min IL-1β stimulation.

Statistical analyses

Statistical comparisons between treatments were carried out using Mann-Whitney U tests for nonparametric data, comparing medians of samples to controls. Means were compared by Student’s t test. Comparisons between more than two groups were carried out using Kruskall-Wallis (ANOVA) tests. A P value of < 0.05 was considered significant. All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 17.0 for windows, SPSS Inc., Chicago, IL, USA) and graphs were drawn using GraphPad Prism Version 5.01 software (GraphPad Software Inc, California, USA).