Th1/17-Biased Inflammatory Environment Associated with COPD Alters the Response of Airway Epithelial Cells to Viral and Bacterial Stimuli

Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation associated with a Th1/17-biased cytokine environment. Acute exacerbations of COPD (AECOPD) are most often triggered by respiratory infections, which elicit an exaggerated inflammatory response in these patients, via poorly defined mechanisms. We investigated the responses of airway epithelial cells (AECs) to infective stimuli in COPD and the effects of the Th1/17-biased environment on these responses. Cytokine expression was assessed following exposure to virus-like stimuli (poly I:C or imiquimod) or bacterial LPS. The effects of pretreatment with Th1/17 cytokines were evaluated in both primary AECs and the Calu-3 AEC cell line. We found that poly I:C induced increased expression of the proinflammatory cytokines IL1β, IL6, CXCL8, and TNF and IFN-β1 in AECs from both control subjects and COPD patients. Expression of IL1β in response to all 3 stimuli was significantly enhanced in COPD AECs. Primary AECs pretreated with Th1/17 cytokines exhibited enhanced expression of mRNA for proinflammatory cytokines in response to poly I:C. Similarly, Calu-3 cells responded to virus-like/bacterial stimuli with increased expression of proinflammatory cytokines, and a Th1/17 environment significantly enhanced their expression. Furthermore, increased expression of pattern recognition receptors for viruses (TLR3, TLR7, IFIH1, and DDX58) was induced by Th1/17 cytokines, in both primary AECs and Calu-3 cells. These findings suggest that the Th1/17-biased environment associated with COPD may enhance the proinflammatory cytokine response of AECs to viral and bacterial infections and that increased signaling via upregulated receptors may contribute to exaggerated inflammation in virus-induced AECOPD.


Determination of the concentration of stimuli for experiments with primary AECs
In primary AEC, a concentration of 1 µg/ml of poly I:C was selected for stimulation experiments. This concentration was the lowest concentration tested that consistently resulted in significant increases in mRNA for pro-inflammatory cytokines (IL-6, IL-8 and TNF-α) after stimulation for 4 hours relative to cells in media alone (Supplementary Figure 2).

Determination of the concentration of stimuli for experiments with Calu-3 cells
The concentration of poly I:C and imiquimod used to stimulate Calu-3 cells was determined in previously published work from our laboratory [1]. Samples were kept at room temperature for 15 minutes and then centrifuged at 14,000 g for 15 minutes. The upper aqueous layer was carefully removed and transferred this to a new tube. The bottom layer was discarded as chemical waste. 500 µL isopropanol (Sigma-Aldrich) and 8 µL of 5 mg/ml glycogen (Sigma-Aldrich) were added to the same tube, after which the samples were vortexed and left at room temperature for 10 minutes. Samples were then, centrifuged at 14,000 g and 4 °C for 10 minutes.
The supernatant was discarded (taking care not to lose the pellet), and 1 ml of 80% ethanol in DEPC water was added to the pellet and vortexed briefly to wash. Samples were centrifuged at 7500 g for 5 minutes (room temperature), and the supernatant and all residual ethanol was removed and discarded. After drying the pellets briefly using a vacuum pump, the pellets were resuspended in a small volume of DEPC-treated water (20 µL). The concentration and A260/280 ratio of the RNA samples was measured using a Nano-drop spectrophotometer. RNA samples with a purity ratio (A260/A280) of 1.8~2 were considered as acceptable. Lastly, the tubes were labelled and stored them at -20 °C (short-term) or -80 °C (longer-term).

DNase Treatment and Reverse Transcription
To perform the DNase treatment, 1 µL Turbo DNase (Life Technologies) and 0.5 µL 10X Turbo DNase buffer (Life Technologies) were added to each 200 µL tube (Life Technologies), followed by adding 8.5 µL total RNA sample in the same tube. Tubes were vortexed and briefly spun down. Tubes were then, incubated at 37 °C for 30 minutes. After incubation, 1 µL of 50 mM EDTA was added to each tube. Tubes were vortexed and spun down before being incubated at 75 °C for 10 minutes. 8 In a new 200 µL tube, 10 µL DNase-treated RNA, 1 µL Oligo dT primer (Rocha, NSW, Australia) and 2 µL Random hexamer primers (Roche) were mixed well and heated to 65 °C for 5 min, then immediately transferred to ice for 5 min.
RT master mix was prepared by mixing 4 µL 5X RT buffer (Roche), 0.5 µL RNase inhibitor (Roche), 2 µL dNTP mix (Roche) and 0.5 µL Transcriptor enzyme (Roche) for each sample. 7 µL of RT master mix was added to each sample tube, mixed well and spun down. Samples were incubated at 25 °C for 10 min, 55 °C for 30 min, then inactivated by heating to 85 °C for 5 min, then placed the samples on ice. RT products were diluted with distilled water and stored at -20 °C until use.

Quantitative real-time RT-PCR
Expression of mRNA was assessed using qRT-PCR with SensiFast SYBR green (BioLine, Data were expressed relative to HPRT using the ∆Ct method. The following equation, which is recommended by Applied Biosystems, was used to calculate relative gene expression: Target A B