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-κB through Toll-like receptor (TLR) signaling [35], we examined whether chemical inhibition of caspase-1 altered NF-κB-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).
10.1371/journal.pone.0037689.g003 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 (5×104 cells/well) were pre-treated for 1 hour with increasing concentrations of z-YVAD-fmk (10–30 µM) 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).

C