Cell stimulation and cytokine quantification
Bronchial epithelial cells were plated and allowed to adhere overnight prior to stimulation. Bronchial epithelial cells were rested or primed with LPS for 5 hours and stimulated with live P. aeruginosa PAO1 or ATP for the times indicated. PBMCs and THP-1 reporter cells were either rested or primed with LPS (Invivogen) or heat-killed PAO1 overnight (16 hours). The next day the cells were challenged with live PAO1, PAO1ΔexsA, ATP (Invivogen), or Poly(dA:dT) (Sigma Aldrich) for the times indicated (see Fig. 1). For stimulations with Poly(dA:dT), lipofectamine LTX was used at a 1∶1 (w∶v) ratio of µg of DNA to µl of lipofectamine and was mixed 30 minutes prior to stimulation. The NF-κB inhibitor Bay11-7082 (Invivogen) was added to cultures 1 hour prior to priming. If no priming was involved, inhibitor was added 1 hour prior to inflammasome stimulation. The CFTR inhibitor CFTRinh172 (Sigma Aldrich) was added to cultures 18 hours prior to inflammasome stimulation. The caspase-1 inhibitor z-YVAD-fmk (Biovision) was added to cultures 1 hour prior to inflammasome stimulation. Supernatants were collected and stored at −20°C. Cytokines released into supernatants from PBMCs stimulated with inflammasome activators were quantified using sandwich ELISA (eBioscience).