IL-1b Production in Cystic Fibrosis Figure 6. NF-kB activation potentiates the degree of IL-1b production and secretion upon inflammasome activation. THP-1 reporter cells were primed overnight with heat-killed P. aeruginosa and stimulated the next day with live P. aeruginosa or additional heat-killed P. aeruginosa for the times indicated. Cell culture supernatants were assayed for (A) NF-kB/AP-1 activity, (B) IL-8, and (C) IL-1b secretion (n = 3–6 experiments). Using the same stimulation method, THP-1 reporter cells were treated with Bay11-7082 (20 mM) for 1 hour prior to priming with heat-killed PAO1 or live PAO1. Supernatants were assayed at 24 hours for (D) NF-kB/AP-1 activity, (E) IL-8, and (F) IL-1b secretion (n = 3–5). PBMCs from CF patients (n = 11–15) and controls (n = 10–13) were treated with z-YVAD-fmk (20 mM) or Bay11-7082 (10 mM) and stimulated with live PAO1 (MOI = 1), ATP (5 mM), or Poly(dA:dT) (1 mg/ml) according to the schedule in Figure 1. (G) IL-1b and (H) IL-8 levels were measured at 24 hours. Statistical analysis was performed using two way ANOVA with Bonferroni correction for multiple comparisons. *, **, and *** signify P,0.05, 0.01, and 0.001. doi:10.1371/journal.pone.0037689.g006 and PBMCs with the NF-kB inhibitor Bay11-7082 significantly inhibited both IL-1b and IL-8 secretion (Fig. 6d–h). Therefore, the IL-1b and IL-8 responses observed were both dependent upon NF-kB activation. Priming with heat-killed P. aeruginosa, like LPS, is unable to induce a strong IL-1b response as compared to live P. aeruginosa (Fig. 6c), but generated greater NF-kB/AP-1 activation (Fig. 6a) and IL-8 secretion (Fig. 6b) than live bacteria despite stimulation at equivalent MOIs. This may be indicative of the Figure 7. Bay11-7082 inhibits pro-IL-1b production in response to P. aeruginosa. PMA-differentiated THP-1 cells were treated with 10 mM CFTRinh172 or 20 mM Bay11-7082 and harvested after (A) 4 hours (n = 3) or (B) 0.5, 1, and 1.5 hours (n = 3) stimulation with PAO1. One representative blot is shown with a graph of the averaged fluorescence intensity values over 3 experiments. doi:10.1371/journal.pone.0037689.g007 PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e37689