Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A-/- mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitro and in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin.

SummaryThe redox-active exotoxin pyocyanin (PCN) can be recovered in 100 mM concentrations in the sputa of bronchiectasis patients chronically infected with Pseudomonas aeruginosa (PA). However, the importance of PCN within bronchiectatic airways colonized by PA remains unrecognized. Recently, we have shown that PCN is required for chronic PA lung infection in mice, and that chronic instillation of PCN induces goblet cell hyperplasia (GCH), pulmonary fibrosis, emphysema and influx of immune cells in mouse airways. Many of these pathological features are strikingly similar to the mouse airways devoid of functional FoxA2, a transcriptional repressor of GCH and mucus biosynthesis. In this study, we postulate that PCN causes and exacerbates GCH and mucus hypersecretion in bronchiectatic airways chronically infected by PA by inactivating FoxA2. We demonstrate that PCN represses the expression of FoxA2 in mouse airways and in bronchial epithelial cells cultured at an air-liquid interface or conventionally, resulting in GCH, increased MUC5B mucin gene expression and mucus hypersecretion. Immunohistochemical and inhibitor studies indicate that PCN upregulates the expression of Stat6 and EGFR, both of which in turn repress the expression of FoxA2. These studies demonstrate that PCN induces GCH and mucus hypersecretion by inactivating FoxA2.

BackgroundThe redox-active pyocyanin (PCN) is a toxic, secondary metabolite secreted by the respiratory pathogen Pseudomonas aeruginosa (PA). Previously, we have shown that mouse lungs chronically exposed to PCN develop goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion, fibrosis and emphysema. These pathological features are commonly found in the airways of several chronic lung diseases, including cystic fibrosis (CF), as well as in mouse airways deficient in the forkhead box A2 (FOXA2), a transcriptional repressor of goblet GCHM and mucus biosynthesis. Furthermore, PCN inhibits FOXA2 by activating the pro-GCHM signaling pathways Stat6 and EGFR. However, it is not known whether PCN-generated reactive oxygen (ROS) and nitrogen (RNS) species posttranslationally modify and inactivate FOXA2.MethodsWe examined the posttranslational modifications of FOXA2 by PCN using specific antibodies against oxidation, nitrosylation, acetylation and ubiquitination. Electrophoretic mobility shift assay (EMSA) was used to examine the ability of modified FOXA2 to bind the promoter of MUC5B mucin gene. In addition, we used quantitative real time PCR, ELISA, immunofluorescence and mouse lung infection to assess whether the loss of FOXA2 function caused GCHM and mucin overexpression. Finally, we examined the restoration of FOXA2 function by the antioxidant glutathione (GSH).ResultsWe found that PCN-generated ROS/RNS caused nitrosylation, acetylation, ubiquitination and degradation of FOXA2. Modified FOXA2 had reduced ability to bind the promoter of the MUC5B gene. The antioxidant GSH alleviated the modification of FOXA2 by PCN, and inhibited the overexpression of MUC5AC and MUC5B mucins.ConclusionThese results suggest that PCN-mediated posttranslational modifications of FOXA2 are positively correlated with GCHM and overexpression of airway mucins. Furthermore, antioxidant treatment restores the function of FOXA2 to attenuate GCHM and mucus hypersecretion.

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Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.