IL-1b Production in Cystic Fibrosis NF-kB activation is required for IL-1b and IL-8 responses to P. aeruginosa We confirmed the dependence of PAO1-induced IL-1b and IL- 8 production on NF-kB activation using THP-1 cells expressing a reporter driven by NF-kB and AP-1 response elements. We found that stimulation of primed THP-1 reporter cells with heat-killed PAO1 produced the highest levels of NF-kB/AP-1 activation (Fig. 6a) and this correlated with IL-8 secretion (Fig. 6b) but negligible amounts of IL-1b were secreted (Fig. 6c). Stimulation of primed THP-1 reporter cells with live PAO1 did not significantly increase NF-kB/AP-1 activity (Fig. 6a) or IL-8 secretion (Fig. 6b) over priming alone. However, IL-1b production was greatly augmented over primed cells stimulated with heat-killed PAO1 or unprimed cells stimulated with live PAO1 (Fig. 6c). This confirmed that NF-kB activation alone is not sufficient for maximal IL-1b secretion, but increased priming of NF-kB is capable of augmenting IL-1b production and secretion upon inflammasome stimulation. Dependency of these responses on NF- kB was confirmed by pharmacologic inhibition of NF-kB using the Bay11-7082 inhibitor of IkBa phosphorylation, which significantly reduced NF-kB/AP-1 activation (P,0.001) (Fig. 6d) and the subsequent production of IL-8 (P,0.01) (Fig. 6e) and IL-1b (P,0.001) (Fig. 6f) in response to both heat-killed and live PAO1. These results were also verified in CF and control PBMCs for each inflammasome examined (P,0.001) (Fig. 6g–h). Overall these results confirm that NF-kB is an important modulator of IL-1b production and that increased activation of NF-kB augments inflammasome-mediated production of IL-1b. Bay11-7082 inhibits pro-IL-1b production in response to P. aeruginosa In addition to inhibition of NF-kB activity, Bay11-7082 can also directly inhibit the NLRP3 inflammasome [38]. To validate its use in this study as an NF-kB inhibitor, western blots for pro-IL-1b Figure 3. Airway cells do not strongly upregulate caspase-1 activity in response to inflammasome stimuli. S9 and IB3-1 cells were examined for caspase-1 activation following inflammasome stimulation with P. aeruginosa (MOI = 10) and ATP (5 mM). Cells were primed with LPS for 5 hours where appropriate. A representative histogram of % caspase-1-active cells is shown in (A) and the averaged values are shown in (B) (n = 3 separate experiments). (C) IB3-1 cells (56104 cells/well) were pre-treated for 1 hour with increasing concentrations of z-YVAD-fmk (10–30 mM) prior to stimulation with P. aeruginosa (MOI = 50). Cell culture supernatants were collected after 6 hours and assayed for IL-6 by ELISA (n = 3). doi:10.1371/journal.pone.0037689.g003 PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37689