Pseudomonas aeruginosa Exotoxin Pyocyanin Causes Cystic Fibrosis Airway Pathogenesis

The cystic fibrosis (CF) airway bacterial pathogen Pseudomonas aeruginosa secretes multiple virulence factors. Among these, the redox active exotoxin pyocyanin (PCN) is produced in concentrations up to 100 μmol/L during infection of CF and other bronchiectatic airways. However, the contributions of PCN during infection of bronchiectatic airways are not appreciated. In this study, we demonstrate that PCN is critical for chronic infection in mouse airways and orchestrates adaptive immune responses that mediate lung damage. Wild-type FVBN mice chronically exposed to PCN developed goblet cell hyperplasia and metaplasia, airway fibrosis, and alveolar airspace destruction. Furthermore, after 12 weeks of exposure to PCN, mouse lungs down-regulated the expression of T helper (Th) type 1 cytokines and polarized toward a Th2 response. Cellular analyses indicated that chronic exposure to PCN profoundly increased the lung population of recruited macrophages, CD4⁺ T cells, and neutrophils responsible for the secretion of these cytokines. PCN-mediated goblet cell hyperplasia and metaplasia required Th2 cytokine signaling through the Stat6 pathway. In summary, this study establishes that PCN is an important P. aeruginosa virulence factor capable of directly inducing pulmonary pathophysiology in mice, consistent with changes observed in CF and other bronchiectasis lungs.Cystic fibrosis (CF) is one of the most common fatal genetic disorders among the Caucasian population, affecting ∼30,000 individuals in the United States alone. CF is caused by mutations in the gene encoding the CF transmembrane regulator, which mediates anion (predominantly chloride, Cl⁻) conductance. Because of temperature-dependent misfolding and misprocessing in the cytoplasm, the most common CF transmembrane regulator mutation, ΔF508, exhibits reduced levels of CF transmembrane regulator localization to the apical membrane of lung epithelial cells, causing reduced levels of Cl⁻ secretion.^1,2 The main pathological feature of CF airways is the accumulation of thick, inspissated mucus, which has been attributed to mechanisms including excessive airway water and sodium absorption by airway epithelia leading to airway surface liquid volume depletion, increased mucus concentration, mucus adhesion to airway surfaces, and delayed mucus transport.^1,2 Defective mucociliary clearance has severe consequences in the lung as patients develop mucus obstruction of large and small airways, goblet cell hyperplasia, neutrophilic infiltration, and poor bacterial clearance, ultimately leading to scarring and airway fibrosis.^1,2 The major clinical problem for CF patients is a progressive loss of lung function caused by chronic lung infection with mucoid Pseudomonas aeruginosa, resulting in the death of >80% of patients.^1,2 The repeated cycles of pro- and anti- inflammatory responses triggered by P. aeruginosa-associated surface antigens, as well as secreted exoproducts, progressively compile the damage on CF lungs.^1,2,3,4Lung damage in P. aeruginosa-infected CF airways has been proposed to be partially because of imbalances between oxidants and antioxidants and between protease and antiprotease activities.^1,2,3,4 In normal airways, the antioxidant capacity exceeds the level of oxidant formation because of the presence of a variety of antioxidants including enzymes, vitamins, metal chelators and thiols. Collectively, these antioxidants protect cellular components from oxidative damage. In P. aeruginosa-infected CF airways, there is an abundant neutrophilic inflammatory response stimulated by both host and bacterial factors. Dysregulated inflammatory responses lead to high levels of cytotoxic phagocyte-derived reactive oxygen species (ROS). ROS are also produced by the redox-cycling activity of pyocyanin (PCN), a blue-colored tricyclic phenazine (Figure 1A) that is produced in concentrations up to 100 μmol/L by P. aeruginosa in CF airways.⁵ Notably, PCN-mediated ROS inhibit catalase activity, deplete cellular antioxidant reduced glutathione, and increase the oxidized reduced glutathione in the bronchiolar epithelial cells.^3,4 Excessive and continuous production of ROS and inhibition of antioxidant mechanisms overwhelm the antioxidant capacity, leading to tissue damage.Open in a separate windowFigure 1Biosynthesis and profiling of PCN in clinical isolates of P. aeruginosa from CF patients. A: PCN is synthesized from phenazine carboxylic acid via enzymatic modification by PhzM and PhzS. B: The majority of P. aeruginosa CF clinical isolates overproduced PCN.As an immunomodulator, PCN inhibits ciliary beating of airway epithelial cells,⁵ nitric oxide production by macrophages and endothelial cells,⁶ prostacyclin production by endothelial cells,⁷ oxidation of leukotriene B4 by neutrophils,⁸ and eicosanoid metabolism by platelets.⁹ PCN also enhances superoxide production,¹⁰ increases apoptosis in neutrophils,^11,12 and inactivates α1-protease inhibitor.¹³ In addition, PCN increases calcium signaling in human airway epithelial cells, stimulates interleukin (IL)-8 release, and inhibits regulated on activation normal T cell expressed and secreted and monocyte chemoattractant protein-1 release in human epithelial cells.^14,15,16,17 PCN furthermore inhibits the expression of IL-2 and its receptor.¹⁸ In animal models, PCN stimulates IL-8 release, neutrophil influx, and bronchoconstriction in sheep and decreases tracheal mucus velocity in sheep, guinea pigs, and baboons.^19,20,21,22Recently, we provided direct evidence that PCN participates in P. aeruginosa virulence using PCN-deficient mutants that were found to be attenuated in their ability to infect mouse lungs in an acute pneumonia model of infection when compared with isogenic wild-type bacteria.²³ These mutants also were less competitive than isogenic parental wild-type bacteria during competitive mixed infection using the agar bead model of chronic lung infections.²³ Thus, the production of PCN appears to confer a growth and/or survival advantage in mixed culture settings. These studies provide the most direct evidence for the importance of PCN in the P. aeruginosa-infected airway.PCN biosynthesis is regulated by the intercellular process of bacterial communication known as quorum sensing,^3,4 a process that is also critical for biofilm formation and full virulence.²⁴ PCN biosynthesis is increased during P. aeruginosa growth in biofilms in vitro.^25,26 As biofilms are the predominant mode of P. aeruginosa growth within CF airways,^1,2,4,27 not surprisingly, PCN concentrations of up to 100 μmol/L have been detected in pulmonary secretions of CF patients.⁵ Because the major CF pathogen is P. aeruginosa, lung epithelia and macrophages are constantly being exposed to PCN, which contribute to oxidative stress and immunomodulation. In this study, we examined whether continuous exposure of mouse lungs to PCN could result in some pathological features commonly found in CF airways of humans.