for 1 hour. For stimulations, live PAO1 was resuspended to an optical density of 0.5 in PBS and further diluted in culture medium prior to stimulation to achieve the desired multiplicity of infection. Heat-killed PAO1 was added in a volume equivalent to that used to achieve an MOI of 1 for live PAO1. Statistics All graphs display the mean 6 SEM and were generated with Prism 5 (Graphpad). Statistical significance was determined by performing one or two-way ANOVA and the Bonferroni post-test where applicable. References 1. O’Sullivan BP, Freedman SD (2009) Cystic fibrosis. The Lancet 373: 1891–1904. 2. Elizur A, Cannon CL, Ferkol TW (2008) Airway Inflammation in Cystic Fibrosis. Chest 133: 489–495. 3. Hamutcu R, Rowland JM, Horn MV, Kaminsky C, MacLaughlin EF, et al. (2002) Clinical Findings and Lung Pathology in Children with Cystic Fibrosis. Am J Respir Crit Care Med 165: 1172–1175. 4. Venkatakrishnan A, Stecenko AA, King G, Blackwell TR, Brigham KL, et al. (2000) Exaggerated Activation of Nuclear Factor-kappa B and Altered Ikappa B- beta Processing in Cystic Fibrosis Bronchial Epithelial Cells. Am J Respir Cell Mol Biol 23: 396–403. 5. Weber AJ, Soong G, Bryan R, Saba S, Prince A (2001) Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function. Am J Physiol Lung Cell Mol Physiol 281: L71–78. 6. Machen TE (2006) Innate immune response in CF airway epithelia: hyperinflammatory? American Journal of Physiology - Cell Physiology 291: C218–C230. 7. Cantin A (1995) Cystic fibrosis lung inflammation: early, sustained, and severe. Am J Respir Crit Care Med 151: 939–941. 8. Koehler DR, Downey GP, Sweezey NB, Tanswell AK, Hu J (2004) Lung inflammation as a therapeutic target in cystic fibrosis. Am J Respir Cell Mol Biol 31: 377–381. 9. Dinarello CA (2009) Immunological and Inflammatory Functions of the Interleukin-1 Family. Annual Review of Immunology 27: 519–550. 10. Weber A, Wasiliew P, Kracht M (2010) Interleukin-1 (IL-1) Pathway. Sci Signal 3: cm1-. 11. Lamkanfi M (2011) Emerging inflammasome effector mechanisms. Nat Rev Immunol 11: 213–220. 12. Martinon F, Burns K, Tschopp J (2002) The Inflammasome: A Molecular Platform Triggering Activation of Inflammatory Caspases and Processing of proIL-b. Molecular Cell 10: 417–426. 13. Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, et al. (2004) NALP3 Forms an IL-1b-Processing Inflammasome with Increased Activity in Muckle-Wells Autoinflammatory Disorder. Immunity 20: 319–325. 14. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, et al. (2006) Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440: 228–232. 15. Mariathasan S, Newton K, Monack DM, Vucic D, French DM, et al. (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218. 16. Miao EA, Alpuche-Aranda CM, Dors M, Clark AE, Bader MW, et al. (2006) Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf. Nat Immunol 7: 569–575. 17. Fernandes-Alnemri T, Yu J-W, Datta P, Wu J, Alnemri ES (2009) AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458: 509–513. 18. Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, et al. (2009) AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458: 514–518. 19. Sutterwala FS, Mijares LA, Li L, Ogura Y, Kazmierczak BI, et al. (2007) Immune recognition of Pseudomonas aeruginosa mediated by the IPAF/ NLRC4 inflammasome. The Journal of Experimental Medicine 204: 3235–3245. 20. Miao EA, Ernst RK, Dors M, Mao DP, Aderem A (2008) Pseudomonas aeruginosa activates caspase 1 through Ipaf. Proceedings of the National Academy of Sciences 105: 2562–2567. 21. Douglas TA, Brennan S, Gard S, Berry L, Gangell C, et al. (2009) Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis. European Respiratory Journal 33: 305–311. 22. Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, et al. (2009) Cutting Edge: NF-kB Activating Pattern Recognition and Cytokine Receptors License NLRP3 Inflammasome Activation by Regulating NLRP3 Expression. The Journal of Immunology 183: 787–791. 23. Kahlenberg JM, Lundberg KC, Kertesy SB, Qu Y, Dubyak GR (2005) Potentiation of Caspase-1 Activation by the P2X7 Receptor Is Dependent on IL-1b Production in Cystic Fibrosis Acknowledgments Patients, family, and staff at the BC Children’s Hospital CF Clinic for support of this research. Dr. RE Hancock for P. aeruginosa PAO1 and PAO1DexsA strains. Author Contributions Conceived and designed the experiments: AT AS. Performed the experiments: AT AS RJ. Analyzed the data: AT AS RJ AFH. Contributed reagents/materials/analysis tools: MAC PML SET. Wrote the paper: AT AFH PML SET. TLR Signals and Requires NF-kB-Driven Protein Synthesis. The Journal of Immunology 175: 7611–7622. 24. Cogswell J, Godlevski M, Wisely G, Clay W, Leesnitzer L, et al. (1994) NF- kappa B regulates IL-1 beta transcription through a consensus NF- kappa B binding site and a nonconsensus CRE-like site. The Journal of Immunology 153: 712–723. 25. Hiscott J, Marois J, Garoufalis J, D’Addario M, Roulston A, et al. (1993) Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter: evidence for a positive autoregulatory loop. Mol Cell Biol 13: 6231–6240. 26. Bonfield T, Panuska J, Konstan M, Hilliard K, Hilliard J, et al. (1995) Inflammatory cytokines in cystic fibrosis lungs [published erratum appears in Am J Respir Crit Care Med 1996 Oct;154(4 Pt 1):following 1217]. Am J Respir Crit Care Med 152: 2111–2118. 27. Osika E, Cavaillon JM, Chadelat K, Boule M, Fitting C, et al. (1999) Distinct sputum cytokine profiles in cystic fibrosis and other chronic inflammatory airway disease. European Respiratory Journal 14: 339–346. 28. Armstrong DS, Hook SM, Jamsen KM, Nixon GM, Carzino R, et al. (2005) Lower Airway Inflammation in Infants with Cystic Fibrosis Detected by Newborn Screening. Pediatric Pulmonology 40: 500–510. 29. Levy H, Murphy A, Zou F, Gerard C, Klanderman B, et al. (2009) IL1B polymorphisms modulate cystic fibrosis lung disease. Pediatric Pulmonology 44: 580–593. 30. Meyer M, Huaux F, Gavilanes X, van den Brule S, Lebecque P, et al. (2009) Azithromycin Reduces Exaggerated Cytokine Production by M1 Alveolar Macrophages in Cystic Fibrosis. Am J Respir Cell Mol Biol. pp 2008–0155OC. 31. Kotrange S, Kopp B, Akhter A, Abdelaziz D, Abu Khweek A, et al. Burkholderia cenocepacia O polysaccharide chain contributes to caspase-1- dependent IL-1beta production in macrophages. J Leukoc Biol 89: 481–488. 32. Xu Y, Krause A, Hamai H, Harvey B-G, Worgall TS, et al. Proinflammatory Phenotype and Increased Caveolin-1 in Alveolar Macrophages with Silenced CFTR mRNA. PLoS ONE 5: e11004. 33. Bruscia EM, Zhang P-X, Ferreira E, Caputo C, Emerson JW, et al. (2009) Macrophages Directly Contribute to the Exaggerated Inflammatory Response in Cystic Fibrosis Transmembrane Conductance Regulator2/2 Mice. Am J Respir Cell Mol Biol 40: 295–304. 34. Verhoef PA, Kertesy SB, Lundberg K, Kahlenberg JM, Dubyak GR (2005) Inhibitory Effects of Chloride on the Activation of Caspase-1, IL-1b Secretion, and Cytolysis by the P2X7 Receptor. The Journal of Immunology 175: 7623–7634. 35. Miggin SM, Palsson-McDermott E, Dunne A, Jefferies C, Pinteaux E, et al. (2007) NF-kappaB activation by the Toll-IL-1 receptor domain protein MyD88 adapter-like is regulated by caspase-1. Proc Natl Acad Sci U S A 104: 3372–3377. 36. Vij N, Mazur S, Zeitlin PL (2009) CFTR is a negative regulator of NFkappaB mediated innate immune response. PLoS One 4: e4664. 37. Hunter MJ, Treharne KJ, Winter AK, Cassidy DM, Land S, et al. (2010) Expression of Wild-Type CFTR Suppresses NF-kB-Driven Inflammatory Signalling. PLoS ONE 5: e11598. 38. Juliana C, Fernandes-Alnemri T, Wu J, Datta P, Solorzano L, et al. (2010) Anti- inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome. J Biol Chem 285: 9792–9802. 39. Di A, Brown ME, Deriy LV, Li C, Szeto FL, et al. (2006) CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 8: 933–944. 40. Averna M, Stifanese R, Grosso R, Pedrazzi M, De Tullio R, et al. (2011) Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1813: 50–59. 41. Rzemieniak SE, Hirschfeld AF, Victor RE, Chilvers MA, Zheng D, et al. Acidification-dependent activation of CD1d-restricted natural killer T cells is intact in cystic fibrosis. Immunology 130: 288–295. 42. LeibundGut-Landmann S, Weidner K, Hilbi H, Oxenius A (2011) Nonhema- topoietic Cells Are Key Players in Innate Control of Bacterial Airway Infection. The Journal of Immunology 186: 3130–3137. PLoS ONE | www.plosone.org 11 May 2012 | Volume 7 | Issue 5 | e37689