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

Abstract

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.

Figure 1. Pyocyanin induces mucin synthesis in bronchiolar epithelial cells

(a) Pyocyanin (10 μM, 2 days) induces mucin production in bronchiolar epithelial cells as detected by Periodic acid-Schiff staining (one representative result shown, n=3). (b) Quantitation of released mucins by dot-blot assay reveals significant increase in mucin production in cells exposed to pyocyanin (mean+/−S.E.M., n=3). (c) A survey of mucin transcripts expressed in the human airway epithelium identified MUC5AC and MUC2 as the two major mucins induced by pyocyanin (mean+/− S.E.M., n=4, qPCR). Pyocyanin-triggered induction of MUC5AC and MUC2 gene expression is dose- (d) and time-dependent (e) (mean+/− S.E.M. of at least three independent experiments). Primers used: graphs (qPCR): MUC2-2, MUC5AC-2; gels (RT-PCR): MUC2-1, MUC5AC-1 (Table S5). (f) Dose- and time-dependent release of MUC5AC protein in bronchiolar epithelial cells treated with pyocyanin (ELISA, mean +/−S.E.M., n=3; dose-dependence at 2 days; time-dependence with 10 μM Pyo). (g) Western blotting detects increased MUC5AC protein in supernatant of pyocyanin-exposed cells (another experiment gave similar results). (h) Only mucoid airway epithelial cells respond to pyocyanin by MUC5AC and MUC2 induction (qPCR, mean +/− S.E.M., n=4, primers: MUC2-2, MUC5AC-2). (i) MUC2 and MUC5AC induction in cells treated with pyocyanin (8 μM, 2d) extracted from 24 hr-old Pseudomonas aeruginosa culture supernatants of different wild-type strains (PA14 WT, PA ATCC10145, PAO1 WT). Pyocyanin prepared from pilus-, flagellum- and LPS-deficient strains of PAO1 also triggered MUC5AC and MUC2 induction. Equivalent extracts from pyocyanin-free supernatants of either short-term cultures of wild-type strains (PAO1 WT (4hrs), PA14 WT (4hrs)) or 24 hr-old cultures of pyocyanin-deficient strains (PA14 PhzM, PA14 Phz1/2, PAATCC 15442) failed to induce mucin expression. The +/− signs denote pyocyanin concentrations detected in each culture supernatant (+++ = 20–100 μM; + = 5–20 μM; − = none), (WT = wild type). Primers used: MUC2-1, MUC5AC-1 (Table S5).

Figure 2. Reactive oxygen species mediate pyocyanin-triggered MUC2 and MUC5AC induction

(a) Intracellular superoxide detection by DHE oxidation in pyocyanin-treated bronchial epithelial cells (10 μM, 3 hrs; one representative experiment, n=3). (b) Intracellular hydrogen peroxide detected by DCF-DA oxidation in cells exposed to pyocyanin (3 hrs) is insensitive to extracellular catalase, whereas extracellular hydrogen peroxide release detected by Amplex Red/HRP is sensitive to exogenous catalase (mean, one representative experiment, n=3). Induction of MUC2 and MUC5AC gene expression (c) and MUC5AC release (d) in pyocyanin-treated cells (10 μM, 2 days) is prevented by intracellular antioxidants (10 mM NAC, GSH). Oxidized glutathione (GSSG, 10 mM) and extracellular ROS scavangers (1500 U/mL catalase + 12.5 μg/ml SOD) have no effect (RT-PCR: one representative experiment, n=3; MUC5AC release: mean+/−S.E.M., n=4). (e) Pyocyanin does not increase mRNA transcripts of ROS-producing NADPH oxidases (10 μM Pyo, 2 days, one representative, n=4).

Figure 3. Pyocyanin activates epidermal growth factor receptor pathway and promotes proliferation in airway epithelial cells

Inhibitors of MEK1/2 (PD98059, U0126), EGFR (AG1478) and protein synthesis (cycloheximide, CHXM) blocked pyocyanin-mediated induction of MUC2 and MUC5AC gene expression (a) and MUC5AC release (b) in bronchiolar epithelial cells. The inhibitor of the platelet-derived growth factor receptor (AG1295) or the control analogue (AG9) had no effect. (Mean+/− S.E.M., n=3). (c) Pyocyanin-induced proliferation of bronchiolar epithelial cells is inhibited by AG1478 (detected by EdU-staining of cells treated with 8 μM pyocyanin for 36 hrs). (d) Quantitation of changes in epithelial cell density upon pyocyanin-treatment (DAPI-stained nuclei/unit surface, mean+/−S.E.M, n=3). (e) Pyocyanin exposure results in increase in proportion of actively dividing epithelial cells (EdU-positive cells/all (DAPI); mean+/− S.E.M., n=3). (f) Pyocyanin greatly enhanced phosphorylation of ERK at 24 hrs post-exposure in bronchiolar epithelial cells (another experiment gave similar results, t=total, p=phospho).

Figure 4. Pyocyanin-induced transcription of inflammatory cytokines and EGFR ligands mediate MUC5AC secretion

(a) Pyocyanin-induced transcriptional changes for genes known as inducers of mucin synthesis or involved in their signaling in bronchiolar epithelial cells assayed by microarray. Transcription of interleukin-1α, IL-1β, IL-6, IL-8, TNFα, HB-EGF and TGFα were strongly induced by pyocyanin. (b) Pyocyanin-induced transcriptional changes of the inflammatory cytokines, IL-1α, IL-1β, IL-6, TNFα were followed over time by real-time RT-PCR (mean+/−S.E.M., n=3–5). (c) Transcriptional changes of the EGFR ligands, TGFα and HB-EGF were confirmed by real-time RT-PCR (mean +/−S.E.M., n=3–5). (d) IL-1β, TGFα, TNFα, EGF and IL-6 protein levels were determined in supernatants of pyocyanin-treated cells (mean+/−S.E.M., n=4). (e) MUC5AC release from cells exposed to a variety of stimuli was measured in the presence of the EGFR inhibitor (AG1478) or its control compound (AG9) (mean+/− S.E.M., n=4). Concentration of cytokines/growth factors: 10 ng/mL, [ATP]= 100 μM. (f) Neutralizing antibodies against IL-1β, IL-6, TNFα and TGFα inhibit MUC5AC release from pyocyanin-treated bronchiolar epithelial cells (mean +/−S.E.M., n=7–12). (g) Pretreatment of H292 cells with TNFα (2 days) increases EGFR levels and further potentiates pyocyanin-stimulated MUC5AC release (mean +/− S.E.M., n=4. [pyocyanin]= 4μM, 2 days).

Figure 5. Functional clustering of pyocyanin-responsive genes in airway epithelial cells

The heat map shows a two-way clustering of genes (vertical axis) and the associated GO FAT functions (horizontal axis). Red cells indicate assignment of an altered gene to a function. (Due to data standardization, genes that participate in many functions, such as TNF, IL1A, IL1B and IL6, produce pinkish cells.) Clusters encompassing at least 5 genes were selected visually and numbered arbitrarily from 1–11. The clustered genes and corresponding functions are listed in Table 1 and Table S3. The data are obtained from H292 cells exposed to 8 μM pyocyanin for 48 hrs.

Figure 6. Pyocyanin induces epithelial transcription of several inflammatory mediators reported as elevated in CF patients

Gene expression changes in pyocyanin-treated epithelial cells (48 hrs, 8 μM) are shown for 24 inflammatory mediators (cytokines, chemokines or acute phase proteins) previously reported as displaying increased levels in CF patients (Table S4). (a) Six of ten genes specifically associated with Pseudomonas infection in CF are induced at least 2 fold by pyocyanin in bronchiolar epithelial cells (microarray data). (b) Nine of fourteen additional CF-associated genes (not studied in relation to Pseudomonas) were also induced by pyocyanin in bronchiolar epithelial cells (microarray data). (c) Real-time PCR confirmation of gene expression changes of CF-associated genes up-regulated by pyocyanin. qPCR results were not only obtained from samples used for microarray but from additional, independent experiments as well (n= 4–9).

Figure 7. Proposed model of pyocyanin’s inflammatory action in airway epithelial cells

The scheme shows proposed mechanisms for pyocyanin-induced mucin release and transcriptional changes. Reactive oxygen species produced in the host cell by pyocyanin result in transcriptional changes of several genes involved in the host’s response to oxidative stress and in the recruitment of white blood cells to the infected mucosa. Inductions of TNFα, IL-1β, TGFα, IL-6 and HB-EGF collectively lead to activation of EGFR, followed by activation of MEK1/2, ERK, MUC2/MUC5AC transcription and cell proliferation. Red lines indicate points of experimental intervention with inhibitors, ROS scavengers and neutralizing antibodies.