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-B were tested in a cystic fibrosis (CF) airway epithelial cell line, CF15. 100 M PYO on its own had no effect or only small effects to activate NF-B (<1.5-fold), but PYO synergized with the TLR5 agonist flagellin. Flagellin activated NF-B 4 -20-fold, and PYO increased these activations >2. Pseudomonas aeruginosa is commonly present in lungs of cystic fibrosis (CF) 2 and immunocompromised patients (1, 2). The bacterium secretes a large number of products that contribute to virulence. These include type III-secreted exotoxins that disrupt the cytoskeleton and lyse the cells, as well as proteases, phospholipase, rhamnolipids, and hemolysin (3-8). In addition, P. aeruginosa produce and secrete the blue pigment pyocyanin (PYO), which is found in the sputum of patients with CF and bronchiectasis at concentrations up to 100 M; this is responsible for the blue-green color often observed in CF sputum (4, 5, 9, 10). PYO-deficient P. aeruginosa elicits less mortality in P. aeruginosa-mediated burn-sepsis model in mice, and PYO appears to be important for persistence of P. aeruginosa in lungs of CF patients (3, 4, 11). PYO has a multitude of effects on the physiology of epithelial cells, including inhibition or alteration of antioxidant enzymes (12, 13), ciliary function (14), cellular metabolism, and organelle H ϩ v-ATPase (2, 15). A key aspect of PYO pathology may result from its ability to trigger inflammation leading to the influx of neutrophils to the P. aeruginosa-infected region; PYO stimulates ICAM-1 and IL8 production on its own and also synergizes with IL1␤ and TNF␣ in stimulating IL8 production (16 -18). The resulting IL8 production triggers polymorphonuclear leukocyte infiltration. The polymorphonuclear leukocytes are critical for fighting infections through production of reactive oxygen species (ROS) and proteases, but these products also contribute to the tissue destruction characteristic of CF.PYO activation of IL8 production may occur through effects on cellular signaling leading to activation of the transcription factors AP-1, NF-IL6/C-EBP, and/or NF-B, which control IL8 production (19). It is widely assumed that the effects of PYO on signaling are mediated at least in part through its ability to redox cycle with cellular NADPH and/or GSH leading to the production of ROS (9,13,20,21) and oxidation of the cytosol and/or mitochondria (21,22). Experiments using the electron spin resonance method showed that PYO caused the production of superoxide (O 2 . ) but not hydroxyl radical (HO ⅐ ), indicating that O 2 . and, through the action of superoxide dismutase, H 2 O 2 might contribute to proinflammatory signaling by PYO (16). In addition, the antioxidant N-acetylcysteine and the HO ⅐ scavenger DMSO reduced the proinflammatory effects of PYO, consistent with PYO triggering inflammatory processes through its pro-oxidant effects (18). However, none of these experiment...

Clinical Significance of Microbial Infection and Adaptation in Cystic Fibrosis

“…Bacterial antigens, such as pili, flagella, DNA, and quorumsensing autoinducer molecules (137,151), are detected by the host and induce release of proinflammatory factors such as interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-␣), IL-1, IL-6, complement chemoattractants, and leukotriene B 4 (61, 133,492,651). The bacteria themselves are not merely passive bystanders in this process; rather, P. aeruginosa has the potential to synthesize factors that damage host cells, augmenting release of proinflammatory factors (535). For example, P. aeruginosa proteolytic enzymes alter host iron-containing proteins in a way that favors hydroxyl radical formation, which contributes to host tissue injury and inflammation (70).…”

Modulation of inflammatory response of wounds by antimicrobial photodynamic therapy

“…However, with increase in wound healing time point, loss of viability of P.aeruginosa, and inactivation of its virulent factors possibly led to significant down regulation of NF-kB and inflammatory cytokines. It has been shown that virulent factors secreted by P.aeruginosa such as LPS, phospholipase, proteolytic enzymes and pyocyanin induce damage to fibroblast, endothelial cells in the wound milieu and delay the wound healing [4][5][6]31) . Also, a prolonged inflammatory phase induced by infection, causes increased levels of proteases which destroy components of the extracellular matrix and damage the growth factors 31) .…”

“…Management of infections caused by P. aeruginosa is difficult due to the rapid emergence of multi-antibiotic resistant strains. Further, the toxins and cell components of these bacteria such as lipopolysaccharide (LPS) can induce overproduction of local proinflammatory mediators, casue cell death [3][4][5][6][7][8][9] . Proteases produced by these bacteria also degrade host matrix proteins, thereby impairing host tissue integrity [10][11][12] .…”

Paraoxonase 2 Serves a Proapopotic Function in Mouse and Human Cells in Response to the Pseudomonas aeruginosa Quorum-sensing Molecule N-(3-Oxododecanoyl)-homoserine Lactone

“…Treatments with agonists were made by diluting stock solutions into Ringer solution at the concentrations stated in the text. Fluorescence ratio imaging measurements of Ca cyto were performed using equipment as described above and methods reported previously (46,47). Emission (Ͼ510 nm) images were collected during alternate excitation at 350 Ϯ 5 and 380 Ϯ 5 nm.…”