Reactive oxygen species mediate inflammatory cytokine release and EGFR-dependent mucin secretion in airway epithelial cells exposed to Pseudomonas pyocyanin

Molecular Defenses Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.
Mucosal Immunology (Impact Factor: 7.37). 10/2010; 4(2):158-71. DOI: 10.1038/mi.2010.62
Source: PubMed


Despite the long-appreciated in vivo role of the redox-active virulence factor pyocyanin in Pseudomonas airway infections and the importance of airway epithelial cells in combating bacterial pathogens, little is known about pyocyanin's effect on airway epithelial cells. We find that exposure of bronchiolar epithelial cells to pyocyanin results in MUC2/MUC5AC induction and mucin secretion through release of inflammatory cytokines and growth factors (interleukin (IL)-1β, IL-6, heparin-bound epidermal growth factor, tissue growth factor-α, tumor necrosis factor-α) that activate the epidermal growth factor receptor pathway. These changes are mediated by reactive oxygen species produced by pyocyanin. Microarray analysis identified 286 pyocyanin-induced genes in airway epithelial cells, including many inflammatory mediators elevated in cystic fibrosis (granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte CSF, chemokine (C-X-C motif) ligand 1 (CXCL1), serum amyloid, IL-23) and several novel pyocyanin-responsive genes of potential importance in the infection process (IL-24, CXCL2, CXCL3, CCL20, CXCR4). This comprehensive study uncovers numerous details of pyocyanin's proinflammatory action and establishes airway epithelial cells as key responders to this microbial toxin.

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    • "Based on these studies, we examined the effect of PCN on IL-8 release in vitro using the human monocyte model (PMA-differentiated human promonocytic cell line U937) in synergy with inflammatory cytokines. The reasons for specific focus on IL-8 and nuclear factor-κB (NF-κB) pathway for IL-8 modulation are that IL-8 is an established enhancer of neutrophil function [5,6,8], while NF-κB is a transcription factor believed to play a key role in IL-8 expression [15]. "
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    ABSTRACT: Pyocyanin (PCN), an extracellular product of Pseudomonas aeruginosa and a blue redox active secondary metabolite, plays an important role in invasive pulmonary infection. However, the detailed inflammatory response triggered by PCN infection in inflammatory cells (particularly macrophages), if present, remains to be clarified. To investigate the effects of PCN on macrophages, the ability of PCN to induce inflammation reaction and the signaling pathway for IL-8 release in PCN-induced differentiated U937 cells were examined. It was found that PCN increased IL-8 release and mRNA expression in Phorbol 12-myristate 13-acetate (PMA) differentiated U937 cells in both a concentration- and time-dependent manner by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). P38 and ERK MAPKs were activated after 10 min of induction with PCN and their levels returned to baselines after 30 min by Western blotting. It was also found that within 10 min of PCN incubation, the level of p-I-kappaBalpha in the cytosol was increased, which returned to baseline level after 60 min. Meanwhile, the level of p-p65 was increased in the nuclear extract and cytosol, and maintained high in total cell lysates. The results were further confirmed by the observation that p38, ERK1/2 and NF-kappaB inhibitors inhibited PCN-induced NF-kappaB activation and attenuated PCN-induced IL-8 expression in U937 cells as a function of their concentrations. Moreover, it was shown that PCN induced oxidative stress in U937 cells and N-acetyl cysteine, an antioxidant, was able to inhibit PCN-induced IL-8 protein expression. It is concluded that PCN induces IL-8 secretion and mRNA expression in PMA-differentiated U937 cells in a concentration- and time- dependent manner. Furthermore, p38 and ERK MAPKs and NF-kappaBeta signaling pathways may be involved in the expression of IL-8 in PCN-incubated PMA-differentiated U937 cells.
    Full-text · Article · Feb 2014 · BMC Microbiology
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    • "Previous studies have shown that hyperoxic conditions activate the EGFR signaling pathway, leading to nuclear translocation of NRF2, and the transcription of ARE-containing antioxidant genes [33], [34]. Most recently, we and others have shown that PCN induces GCHM and the biosynthesis of major airway mucins MUC5AC and MUC5B in mouse airways, in part, by inducing the ROS-dependent EGFR signaling [11], [15]. We compared the levels of phosphorylated EGFR in A549 cells after 12 h of exposure to sterile H2O or various concentrations of PCN. "
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    ABSTRACT: The redox-active pyocyanin (PCN) secreted by the respiratory pathogen Pseudomonas aeruginosa generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.
    Full-text · Article · Aug 2013 · PLoS ONE
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    • "Previously, we have demonstrated that PCN can inhibit the expression of FOXA2 through the activation of IL-4/IL-13/Stat6 and EGFR signaling pathways [33]. Especially relevant is the finding that EGFR, a major pro-GCHM pathway, is inducible by ROS [55], including those generated by PCN [33,56]. Thus, PCN-mediated GCHM and mucus hypersecretion in diseased airways chronically colonized by PA is likely a cumulative effect of ROS/RNS-mediated posttranslational modification of FOXA2 and the activation of IL-4/IL-13-STAT6 and EGFR signaling pathways that inhibit the expression of FOXA2. "
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    ABSTRACT: The 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. We 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). We 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. These 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.
    Full-text · Article · Aug 2013 · Respiratory research
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