Redox-independent activation of NF-κB by Pseudomonas aeruginosa pyocyanin in a cystic fibrosis airway epithelial cell line

Abstract

The roles of the Pseudomonas aeruginosa-derived pigment pyocyanin (PYO) as an oxidant and activator of the proinflammatory transcription factor NF-κB were tested in a cystic fibrosis (CF) airway epithelial cell line, CF15. 100 μM PYO on its own had no effect or only small effects to activate NF-κB (<1.5-fold), but PYO synergized with the TLR5 agonist flagellin. Flagellin activated NF-κB 4-20-fold, and PYO increased these activations >2.5-fold. PYO could have synergized with flagellin to activate NF-κB by redox cycling with NADPH, generating superoxide (O2.-), hydrogen peroxide (H2O2), and hydroxyl radical (HO .). Cytosol-targeted, redox-sensitive roGFP1 and imaging microscopy showed that 1-100 μM PYO oxidized CF15 cytosol redox potential (Ψcyto) from -325 mV (control) to -285 mV. O2.- (derived from KO2.- or xanthine + xanthine oxidase) or H2O2 oxidized Ψcyto dose-dependently but did not activate NF-κB, even in the presence of flagellin, and 400 μM H2O2 inhibited NF-κB. Overexpressing intracellular catalase decreased effects of PYO and H 2O2 on Ψcyto but did not affect flagellin + PYO-activated NF-κB. Catalase also reversed the inhibitory effects of H2O2 on NF-κB. The HO. scavenger DMSO did not alter the effects of PYO on Ψcyto and NF-κB. The synergistic NF-κB activation was calcium-independent. Thus, in the presence of flagellin, PYO activated NF-κB through a redox- and calcium-independent effect. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.