NLRP1 [12], NLRP3 [13,14], NLRC4 [15,16], and AIM2 inflammasomes [17,18], which respond to a variety of different microbial signatures and danger signals [11]. P. aeruginosa, one of the most common and clinically relevant pathogens among CF patients, activates the NLRC4 inflamma- some [19,20]. Infection with P. aeruginosa triggers an increase in levels of IL-1b, IL-6, and IL-8 in bronchoalveolar lavage fluid (BALF) from patients with CF [21]. Inflammasome responses depend on NF-kB signaling, where NF-kB is important in both the upregulation of specific inflammasome components [22,23], as well as IL-1b expression [24,25]. Previous studies support a role for IL-1b in the pathogenesis of CF inflammatory lung disease. Levels of IL-1b are increased in BALF from CF patients with infection [21,26,27,28] and this increase has been temporally associated with a clinical response to treatment [21]. Polymorphisms in the IL1B gene have also been associated with varying degrees of disease severity in CF patients [29]. Murine models of CFTR dysfunction have exhibited significant increases in IL-1b expression or secretion in macro- phages [30,31], and support the hypothesis that the loss of CFTR increases NF-kB activation under basal and stimulatory conditions [4,5,32,33]. Finally, replacement of chloride ions with glutamate or gluconate in cell culture media increases secretion of IL-1b in response to NLRP3 stimulation by adenosine triphosphate (ATP) [34], implying an inhibitory role for extracellular chloride in NLRP3 activation. Taken together, these data implicate the involvement of IL-1b and consequently, the inflammasomes, in CF inflammatory disease. Results Airway epithelial cells do not produce significant amounts of IL-1b in response to inflammasome stimulation The inflammasomes and their respective activators examined in this study are listed in Table 1. Cells were stimulated in accordance with the schedule in Figure 1. In CF, airway epithelial cells have been shown to possess a hyper-inflammatory phenotype and produce an exaggerated pro-inflammatory cytokine response [4,5]. To determine if airway epithelial cells contribute to the increased IL-1b production in patients with CF, CF and control bronchial epithelial cell lines were stimulated with the inflamma- some inducers P. aeruginosa strain PAO1 (PAO1) and LPS followed by ATP. IL-1b levels in cell culture supernatants were not greatly increased in either the CF or control cell lines (Fig. 2a–d), although a small increase in IL-1b production was detected in NuLi-1 and CuFi-1 cells, but not in S9 and IB3-1 cells, by 24 hours. In contrast, these airway cells were highly responsive to other inflammatory stimuli, such as recombinant IL-1b, producing large quantities of IL-8 (Fig. 2a–d inserts). IL-1b Production in Cystic Fibrosis Airway epithelial cells do not significantly upregulate caspase-1 activity in response to inflammasome stimulation To examine if inflammasome activation occurs in these airway cells, caspase-1 activity was quantified by flow cytometry. There was no significant increase upon stimulation with live PAO1 or LPS+ATP at the times examined (Fig. 3a–b). Because previous studies have indicated a role for caspase-1 in the activation of NF-kB through Toll-like receptor (TLR) signaling [35], we examined whether chemical inhibition of caspase-1 altered NF-kB-dependent IL-6 production in response to P. aeruginosa. However, treatment with the caspase-1 inhibitor z- YVAD-fmk (YVAD) did not decrease IL-6 secretion by airway epithelial cells (Fig. 3c). CD14 positive monocytes from CF patients and controls show similar increases in caspase-1 activity upon inflammasome stimulation Monocytes were identified in PBMC populations using CD14 as a phenotyping marker. CD14 positive monocytes from CF patients and healthy controls showed a significant increase in caspase-1 activation upon stimulation with LPS+ATP, PAO1, and LPS+Po- ly(dA:dT) (Fig. 4a) but this activation was not different between CF and control subjects (Fig. 4b). PBMCs from CF patients do not produce increased amounts of IL-1b upon inflammasome stimulation Previous studies have shown that the loss of CFTR results in increased NF-kB activity and pro-inflammatory cytokine secretion [4,5,32,36,37]. To further examine this relationship, PBMCs from CF patients and healthy adult controls were stimulated with PAO1, LPS+ATP, and LPS+Poly(dA:dT), to activate the NLRC4, NLRP3, and AIM2 inflammasomes, respectively. By 24 hours of stimulation, CF PBMCs did not produce increased amounts of IL- 1b (Fig. 5a) or IL-8 (Fig. 5b) when compared to healthy controls. However, we did notice a transient decrease (P,0.001) in the amount of IL-1b produced by CF cells in response to LPS+ATP at 6 hours (data not shown). Stimulation of PBMCs with P. aeruginosa that lacks exsA (PAO1DexsA), a key regulator of type III secretion, produced three-fold less IL-1b compared to the parental PAO1 strain by 24 hours (Fig. 5a). Inflammasome stimulation without priming did not result in any IL-1b production in either CF or control PBMCs. Contrary to our hypothesis, these results indicate that PBMCs from CF patients do not display increased production of IL-1b or IL-8 with inflammasome activation nor do they suggest any increased basal or induced NF-kB activity. These results are consistent with our observation that caspase-1 activity is not different between CF and control PBMCs (Fig. 4). Table 1. Inflammasome Activators. INFLAMMASOME NLRP3 NLRC4 AIM2 doi:10.1371/journal.pone.0037689.t001 INFLAMMASOME STIMULUS ATP (Concentration: 5 mM) Pseudomonas aeruginosa strain PAO1. (Multiplicity of Infection (MOI) = 1) Poly(dA:dT) (Concentration: 1 mg/ml) EXOGENOUS PRIMING OF NF-kB REQUIRED? Yes Yes No (primed by live bacterium) PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37689