Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.

As part of the innate immune system, epithelial cells represent a first line of defense against pathogens, a phenomenon critical for host survival. Epithelial cells form a tight barrier by means of expression of the junctional complex and mediate inflammatory responses by secreting eicosanoids, nucleotides, cytokines, and chemokines (1). Alterations in the integrity of the host epithelium during infection may cause life-threatening conditions. In cystic fibrosis (CF), chronic infection of the airways by Pseudomonas aeruginosa leads to severe pulmonary damage and is responsible for mortality and morbidity in the CF population (2). CF is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that codes for a cAMP- and ATP-regulated chloride channel that is located in glandular and surface airway epithelial cells. Activation of CFTR contributes to the regulation of airway surface liquid height and viscosity, and thus to efficient mucociliary clearance (3). It is believed that, in CF, absence of functional CFTR impairs hydration of the mucus and impairs bacterial clearance by ciliated cells, thus providing a favorable environment for infection and colonization of the airways and lung by P. aeruginosa. This Gram-negative rod is considered an opportunistic pathogen in patients with compromised innate immune responses, as also observed in individuals suffering of cancer and burns (2).

P. aeruginosa secretes virulence factors and injects cytotoxins into airway epithelial cells, thereby counteracting the innate immune response activated by complex mechanisms involving host membrane receptors (4). TLRs have been shown to stimulate the production of cytokines and nucleotides by airway epithelial cells. Recognition of bacterial pilin, LPS, and flagellin by TLR2, TLR4, and TLR5 has been reported to trigger MyD88-dependent signaling cascades involving NF-κB, ERK, JNK, and p38 to control translational and posttranslational production of proinflammatory chemokines, such as IL-8 (5). Collectively, the release of cytokines and nucleotides by airway epithelial cells is needed for the recruitment of inflammatory cells to infected sites and to facilitate CFTR-dependent chloride fluxes, respectively, thereby optimizing pathogen clearance. In CF, the secretory response would be absent, but activation of inflammatory genes would persist. Indeed, IL-8, a potent chemotactic factor for neutrophils, is the most abundant chemokine detected in the airway secretions of CF patients in both early and advanced stages of the disease (6).

The homeostasis of the epithelium is severely challenged upon infection, a process that might affect the junctional complex (1). The junctional complex consists of anchoring junctions (desmosomes and adherens junctions), occluding junctions (tight junctions), and communicating junctions (gap junctions). Gap junctions have been recently involved in bacterial infections and several infectious diseases, including CF (7). Gap junctions are made by docking of two hexameric hemichannels (connexons) of two apposed cells, thus composed of 12 proteins called connexins. Hemichannels function as transmembrane channels involved in the release to the extracellular space of nucleotides, glutathione, and PGs. Gap junctions ensure the direct cell-to-cell transfer of ions, sugars, nucleotides, short peptides, and second messengers (8). Bacterial contact, bacterial internalization, and/or bacterial cytotoxins have been found to modulate hemichannel activity and gap junctional communication in the host epithelium, resulting in imbalanced homeostasis (7, 9, 10). It was recently reported that gap junction channels favor the cell-to-cell spread of an inflammatory response dominated by IL-8 production upon Shigella flexneri internalization in an intestinal epithelial cell line (11). Less is known, however, regarding the effects of P. aeruginosa on gap junction proteins within the airway epithelium.

We have recently shown in the human airway epithelial Calu-3 cell line grown under polarized conditions that Cx43-made gap junction channels coordinate a signaling network to activate CFTR and modulate airway surface liquid volume (12). Because airway surface liquid homeostasis is a defense mechanism against infection, we sought to investigate innate immune recognition and epithelial responses at the level of Cx43 in polarized Calu-3 cells infected with PAO1, a laboratory strain of P. aeruginosa. We report in this study new findings showing that PAO1-triggered signaling regulates the functional expression of Cx43 in a CFTR-dependent manner. These results imply that CFTR deficiency has important consequences on this mechanism, which may contribute to the CF phenotype.

The human airway epithelial cell line Calu-3 was purchased from the American Type Culture Collection. Calu-3 cells were maintained in DMEM:F12 (3:1 v/v), supplemented with 10% FCS, 30 U/ml penicillin, and 30 mg/ml streptomycin (Life Technologies). The human nasal epithelial CF15 cell line, which was derived from a patient homozygous for the F508del mutations of CFTR, was maintained in the same medium supplemented with growth factors, as previously described (13). To obtain a well-polarized cell monolayer, 100,000 Calu-3 cells or 200,000 CF15 cells were seeded onto 0.33-cm2 porous (0.4-μm) Transwell polyester membranes (Transwell 3470; Corning Costar) and cultured for up to 15 d for calu-3 cells and 8 d for CF15 cells. Primary human airway epithelial cells (HAECs) grown on Transwell inserts at the air–liquid interface from at least 30 d (MucilAir) were purchased from Epithelix (Epithelix Sàrl, Plan-Les-Ouates, Switzerland) and maintained, according to the manufacturer’s instructions.

Cells were infected with the well-characterized P. aeruginosa laboratory strain PAO1. PAO1pilA and PAO1fliC mutant strains lacking expression of pilin and flagellin, respectively, were used as controls. The mutated pilA::Tc and and fliC::Gm loci were transduced from strains PAK-NP and PAO1-RR, respectively, into our PAO1 strain, as previously described (14). Both mutants are therefore isogenic to wild-type PAO1. The PAO1 strain that we use does not display a nfxC phenotype and is not affected in quorum-sensing dependent virulence factor production (14). PAO1 (pIApX2) expressing a GFP (PAO1-GFP; gift of I. Attree, CEA, Grenoble, France) was used for confocal microscopy. For cell infection, bacteria were grown overnight in Luria-Bertani medium with shaking (240 rpm) at 37°C. Bacteria were centrifuged (2 min at 6000 rpm) and resuspended in NaCl solution (0.9% NaCl supplemented with 10 mM HEPES and 1.2 mM CaCl2) to obtain 1010 CFU/ml. Calu-3 and CF15 cells were switched at the air–liquid interface prior to the challenge of apical cell membrane with 10 μl bacterial suspension (containing 2 × 106 CFU) corresponding to 5 multiplicity of infection (MOI) or with 10 μl NaCl solution as a control. Heat-killed (HK)-PAO1 was donated by G. Cabrini (University Hospital of Verona, Verona, Italy). In this case, 100 μl bacterial solution at the indicated concentration was added to Calu-3 cells at the apical side.

Protein kinase activity was blocked by using inhibitors for protein kinase C (PKC), Chelerythrin (Chel, 5 μM); p38, SB203580 (SB, 10 μM); JNK, SP600125 (SP, 10 μM); ERK, UO126 (UO, 5 μM); and PI3K, LY294002 (LY, 10 μM). 18α-Glycyrrhethinic acid (αGA, 100 μM) was used to block gap junctional communication. CFTR activity was blocked with 10 μM GlyH-101 (GlyH). All inhibitors were applied to basal and apical compartments 30–45 min before PAO1 infections and maintained during the course of the experiments. All compounds were dissolved in DMSO, and control experiments were performed using appropriate dilutions of the solvents. All reagents were from Calbiochem, except Chel from Sigma-Aldrich. Purified flagellin from P. aeruginosa (InvivoGen, San Diego, CA) was used at 10 μg/ml. TNF-α (Bachem AG, Bubendorf, Switzerland) was used at 100 U/ml.

Western blotting was performed using Abs against Cx43 (2 μg/ml, MAB3067; Millipore AG), CFTR (0.4 μg/ml, clone M24.1, MAB25031; R&D Systems), active Caspase-3 (0.5 μg/ml, p17 subunit, AF835; R&D Systems), phospho-JNK (p-JNK, dilution 1:1000; 4668; Cell Signaling), and total JNK (dilution 1:2000; 9258; Cell Signaling). An anti-GAPDH (0.05 μg/ml, MAB374; Millipore AG) was used as control for protein loading. Culture inserts were rinsed with PBS and scraped into an ice-cold solubilization buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, and a mixture of protease inhibitors [Roche]). After 30 min of incubation, the samples were centrifuged at 4°C for 10 min at 10,000 × g. Supernatants were recovered, and total amounts of protein were determined by a bicinchoninic acid quantification assay (Pierce). Equal amount of protein was electrophoresed in SDS-PAGE and electrotransferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore AG). Membranes were then blocked for 2 h in a 5% defatted milk saturation buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween 20). Next, proteins were immunoblotted overnight at 4°C with appropriate Abs. This step was followed by 1-h incubation with goat anti-mouse or anti-rabbit IgG secondary Abs conjugated to peroxidase (Jackson ImmunoResearch Laboratories). Immunoreactivity was detected through the Super Signal West Pico kit (Pierce). Quantification of intensity of protein bands was performed with Image J software.

For immunostaining, cells were fixed in 4% paraformaldehyde for 15 min, rinsed with PBS, and successively treated with 0.3% Triton X-100 for 15 min, 0.5 M NH4Cl for 15 min, and 2% BSA prepared in PBS for 30 min. Fixed cells were then incubated overnight at 4°C with 5 μg/ml rabbit Abs against TLR5 (36-3900; Invitrogen). Cells were next incubated for 2 h at room temperature using Alexa 488–coupled goat anti-rabbit and rhodamine-phalloidin for staining actin cytoskeleton (Molecular Probes). DAPI was used for counterstaining nuclei. After rinsing, cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA) before microscopic examination. Fluorescent cells were viewed on an inverted Zeiss LSM510 laser-scanning confocal microscope (Carl Zeiss). Images acquired through a 40× or a 63× oil immersion objective were further processed using Metafluor version 7.7.4.0 (Universal Imaging).

Apoptosis was analyzed by annexin V–enhanced GFP (EGFP) binding to phosphatidylserine at the apoptotic cell surface, according to the manufacturer’s instructions (R&D Systems). Briefly, Calu-3 cells were infected for 6 or 8 h at a MOI of 5, gently washed once, and stained with annexin V-EGFP. Simultaneous staining with propidium iodide was used to identify necrosis, although it was never detected in our experiments. Images from six different fields were acquired from each culture condition through a 63× oil immersion objective by using an inverted Zeiss LSM510 laser-scanning confocal microscope (Zeiss) and Metafluor software (Universal Imaging). The volume of annexin V-EGFP fluorescence and the number of DAPI-labeled nuclei were calculated from three-dimensional reconstructed images using Imaris 7.3.1 (Bitplane). Data were expressed as μm3/number of nuclei.

Cellular RNA was extracted from Calu-3 cells with NucleoSpin RNA II (Macheley-Nagel) at time 0, 3, and 6 h after incubation with PAO1 or control solution. For each time point, mRNAs isolated from three separate culture inserts were pooled. The reverse transcription was performed by using the Quantitect reverse-transcriptase kit (Qiagen) at 42°C for 14 min in a Biometra thermocycler (Biolabo Scientific Instruments SA, Châtel-St-Denis, Switzerland). PCR were performed on an ABI StepOne Plus detection system with TaqMan gene expression assays and TaqMan Fast master mix (Applied Biosystems), according to the manufacturer’s instructions. Reactions were performed in triplicate, and the relative amount of Cx43 mRNA was calculated using the comparative ΔΔ cycle threshold method to GAPDH mRNA.

A lentiviral vector was developed to stably express in Calu-3 cells a short hairpin RNA (shRNA) directed against Cx43. Briefly, small interfering RNA (siRNA) sequences were embedded in the following shRNA structure: 5′-sense siRNA-TCAAGAG-antisense siRNA-3′. The siRNA sequence targeting Cx43 mRNA from Invitrogen (5′-GCG CCT TAG GCA AAC TCC TTG ACA A-3′) and a scramble siRNA sequence (5′-GAT AGA AAG GAT TGA CAG TGG TG-3′) were used to synthesize the shRNAs shCx43 and shCTRL, respectively. The two sequences were cloned in a HIV-based lentiviral backbone (pLVTHM; provided by D. Trono, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) downstream of the RNA polymerase III promoter H1. A GFP reporter gene expression cassette driven by the elongation factor 1α promoter was also contained in the vectors. HIV-based lentiviral particles were produced using 293T cells as packaging cells. Lentiviral vector titers were determined by measuring GFP fluorescence by FACS in HeLa cells transduced with a serial dilution of the vectors. Then, Calu-3 cells seeded on Petri dishes at low density were transduced with lentiviral vectors at a MOI of 10 in culture media containing 2 μg/ml polybrene (Sigma-Aldrich). GFP-positive cells were FACS, and the resultant cell lines were maintained in culture for subsequent analyses and experiments.

The chimeric vector HAdV5F35-GFP-CFTR has been previously described (15). HAdV5F35-GFP-CFTR encoded the wild-type allele of the CFTR gene fused to the 39 end of the GFP gene. Vector stocks were produced and titrated on HEK-293 cell monolayers. The HAdV5F35 vector consisted of hexon and penton base capsomers of HAdV5, and of chimeric fibers made up of the shaft and knob domains of serotype 35 fiber (F35) fused to HAdV5 fiber tail. Polarized CF15 cells were apically transduced for 48 h with HAdV5F35-GFP-CFTR at a MOI of 20 in culture medium.

Gap junctional intercellular communication was determined by intracellular microinjection of Lucifer Yellow (Sigma-Aldrich) in Calu-3 cells cultured on Transwell inserts for PAO1 infection experiments or on Petri dishes for verification of Cx43 gene silencing. The medium was changed to a solution containing 136 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 2.5 mM glucose, and buffered to pH 7.4 with 10 mM HEPES-NaOH. Lucifer Yellow was prepared in 150 mM LiCl and 10 mM HEPES (pH 7.2). The tracer was injected using a thin-tip glass microelectrode into one cell for 3 min, and the number of labeled cells was counted at the end of the injection period. Fluorescent cells were viewed on an inverted TMD300 microscope (Nikon AG) equipped with a high sensitivity black and white CCD Visicam camera (Visitron Systems). Images were captured using software Metafluor 4.01 (Universal Imaging) and processed using Adobe Photoshop CS2 version 9.0 (Adobe Systems).

The CFTR activity was studied by Ussing chamber technique. Transepithelial resistance averaged 317 ± 21 Ω × cm2 (n = 5). The apical and basal chambers were filled with a Krebs-Ringer-bicarbonate buffer containing (in mM): 134 NaCl, 4 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 5 NaHCO3, 1 glucose, and 10 HEPES (pH 7.4). The transepithelial potential difference was voltage clamped at zero, and the resulting short-circuit current (Isc) recorded continuously a VCC MC6 amplifier (Physiological Instruments). Data were sampled using the interface DI-720 (DataQ Instruments) and recorded/displayed using the Acquire & Analyze software 2.3 (Physiological Instruments). CFTR-dependent Isc was evoked by basolateral application of 10 μM trypsin (Sigma-Aldrich), as previously reported (12).

IL-8 secretion was measured by ELISA (Sanquin), according to the manufacturer’s instructions. After 6 h of apical exposition to PAO1 or control solution, basolateral medium of polarized Calu-3 cultures was collected for analysis.

GraphPad Prism software (version 4.03) was used to compare experiments using unpaired t tests, one-way ANOVA, and the nonparametric Mann–Whitney U test, where appropriate. Values are expressed as mean ± SEM. A p value <0.05 was considered significant.

Apical infection of polarized Calu-3 cells for 6 h with the P. aeruginosa wild-type strain PAO1 induced Cx43 upregulation (Fig. 1A). Increased expression of Cx43 was also observed with a PAO1 mutant strain lacking pilin (pilA) but not with a mutant strain lacking flagellin (fliC) (Fig. 1A). Quantification of the Western blots indicates that fliC mutant did not increase Cx43 expression above control levels (Fig. 1B). Next, we exposed polarized Calu-3 cells to HK-PAO1 to determine whether membrane components of the dead bacteria could activate airway epithelial cells. Indeed, HK-PAO1 induced Cx43 expression within hours as detected by both Western blot (Fig. 1C) and mRNA transcription (Fig. 1D). These results suggest that PAO1 induced Cx43 expression in airway epithelial cells via activation of extracellular membrane receptors. Consistent with these observations, we did not observe obvious internalization by Calu-3 cells of PAO1 expressing the GFP. Confocal microscopy revealed that PAO1-GFP accumulates at the surface of Calu-3 cells, close to areas of cell–cell contacts (Supplemental Fig. 1A). At later times of infection (>8 h), we observed disruption of junctional complexes and paracellular migration of the fluorescent bacteria toward the basal side of the airway epithelial cell monolayer (Supplemental Fig. 1A), but again no clear internalization was observed. Finally, expression on the surface of Calu-3 cells of TLR5, the receptor for flagellin, was verified by immunostaining and confocal imaging (Supplemental Fig. 1B). Although Cx43 was not overexpressed with the fliC mutant, infection of Calu-3 cells with this strain in the presence of purified flagellin restored Cx43 induction (Supplemental Fig. 1C, 1D).

FIGURE 1.

Cx43 expression induced by PAO1 is mediated by p38 and JNK MAPKs. Western blots (A, C, and E) for Cx43 (top panels) and GAPDH (bottom panels) were performed from Calu-3 cells exposed to PAO1 (MOI 5) for 6 h. (A) Cx43 expression is increased in Calu-3 cells infected for 6 h with wild-type (wt) and pilin-deficient (pilA) PAO1, but not with a flagellin-deficient (fliC) mutant. CTRL indicates the basal level of Cx43 expression in the absence of bacteria. (B) Quantitative analysis from three Western blots comparing control Calu-3 cells with Calu-3 cells exposed to PAO1 wt, pilA, and fliC, respectively. To these, we added values from two Western blots comparing control Calu-3 cells with Calu-3 cells exposed to PAO1 wt and fliC. Data are expressed as fold increase of the CTRL conditions. *p < 0.05 compared with CTRL. (C) Cx43 expression is also increased in Calu-3 cells exposed to HK-PAO1 (104 CFU/ml and 105 CFU/ml) for 6 h. (D) The expression of Cx43 mRNA was evaluated by quantitative RT-PCR as a function of time of exposure to HK-PAO1 (105 CFU/ml). Data are reported as fold increase of uninfected conditions at time 0 and represent the average of two quantitative RT-PCR experiments performed in triplicate. *p < 0.05 compared with CTRL. (E and F) The expression of Cx43 induced by a 6-h infection with PAO1 (MOI 5) is modulated by inhibitors of protein kinases. The PKC Chel (5 μM), ERK UO126 (UO, 5 μM), and PI3K LY294002 (LY, 10 μM) inhibitors had no effect on Cx43 expression. In contrast, Cx43 expression was decreased by the p38 SB203580 (SB, 10 μM) inhibitor and increased by the JNK SP600125 (SP, 10 μM) inhibitor. n = 5–6, *p < 0.05, compared with DMSO used as vehicle (Veh). Molecular mass (in kDa) markers are indicated on the right side of the blots.

FIGURE 1.

Cx43 expression induced by PAO1 is mediated by p38 and JNK MAPKs. Western blots (A, C, and E) for Cx43 (top panels) and GAPDH (bottom panels) were performed from Calu-3 cells exposed to PAO1 (MOI 5) for 6 h. (A) Cx43 expression is increased in Calu-3 cells infected for 6 h with wild-type (wt) and pilin-deficient (pilA) PAO1, but not with a flagellin-deficient (fliC) mutant. CTRL indicates the basal level of Cx43 expression in the absence of bacteria. (B) Quantitative analysis from three Western blots comparing control Calu-3 cells with Calu-3 cells exposed to PAO1 wt, pilA, and fliC, respectively. To these, we added values from two Western blots comparing control Calu-3 cells with Calu-3 cells exposed to PAO1 wt and fliC. Data are expressed as fold increase of the CTRL conditions. *p < 0.05 compared with CTRL. (C) Cx43 expression is also increased in Calu-3 cells exposed to HK-PAO1 (104 CFU/ml and 105 CFU/ml) for 6 h. (D) The expression of Cx43 mRNA was evaluated by quantitative RT-PCR as a function of time of exposure to HK-PAO1 (105 CFU/ml). Data are reported as fold increase of uninfected conditions at time 0 and represent the average of two quantitative RT-PCR experiments performed in triplicate. *p < 0.05 compared with CTRL. (E and F) The expression of Cx43 induced by a 6-h infection with PAO1 (MOI 5) is modulated by inhibitors of protein kinases. The PKC Chel (5 μM), ERK UO126 (UO, 5 μM), and PI3K LY294002 (LY, 10 μM) inhibitors had no effect on Cx43 expression. In contrast, Cx43 expression was decreased by the p38 SB203580 (SB, 10 μM) inhibitor and increased by the JNK SP600125 (SP, 10 μM) inhibitor. n = 5–6, *p < 0.05, compared with DMSO used as vehicle (Veh). Molecular mass (in kDa) markers are indicated on the right side of the blots.

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To decipher the signaling cascade linked to PAO1-induced activation of Calu-3 cells, we tested the effects of several protein kinase inhibitors on Cx43 expression. In the absence of PAO1, we found that an inhibitor of PI3K (LY) strongly decreased basal level of Cx43 expression (Supplemental Fig. 1E, 1F). In the presence of PAO1, Cx43 induction was not affected by inhibitors of PI3K, PKC (Chel), or ERK (UO) but was inhibited by an inhibitor of p38 MAPK (SB). Surprisingly, a JNK inhibitor (SP) enhanced Cx43 expression elicited by PAO1 (Fig. 1E, 1F).

The consequence of p38 and JNK inhibitors on the function of Cx43 was evaluated by dye coupling. Intracellular microinjection of the Cx43-permeant tracer Lucifer Yellow in well-polarized Calu-3 cells revealed that PAO1 increased gap junctional communication (Fig. 2A). As expected, the fliC mutant had no effect on the resting level of gap junctional communication; in contrast, the gap junction inhibitor αGA fully blocked PAO1-induced increase of dye coupling (Fig. 2A). Increased dye coupling induced by PAO1 was also prevented by p38 inhibition and enhanced by JNK inhibition (Fig. 2B). These results suggest that expression of Cx43 is tightly regulated; basal expression of Cx43 is under the control of PI3K, whereas the PAO1-stimulated expression of Cx43 is modulated by p38 and JNK.

FIGURE 2.

PAO1 modulates the function of Cx43 induced via p38 and JNK MAPKs. (A) Gap junctional communication was evaluated by dye (Lucifer Yellow) coupling. The wild-type (wt, n = 19 dye injections) PAO1 but not the flagellin-deficient (fliC, n = 17) mutant increased the number of coupled cells. PAO1-induced dye coupling was prevented in the presence of a gap junction inhibitor (αGA, 100 μM, n = 13). *p < 0.05 compared with unstimulated control (CTRL, n = 9). #p < 0.05. (B) The extent of dye coupling induced by PAO1 was decreased by the p38 SB203580 (SB, 10 μM, n = 14) inhibitor and increased by the JNK SP600125 (SP, 10 μM, n = 27) inhibitor. Vehicle (Veh):DMSO was used alone as controls for each treatment. *p < 0.05 compared with Veh (n = 7 for SB; n = 28 for SP).

FIGURE 2.

PAO1 modulates the function of Cx43 induced via p38 and JNK MAPKs. (A) Gap junctional communication was evaluated by dye (Lucifer Yellow) coupling. The wild-type (wt, n = 19 dye injections) PAO1 but not the flagellin-deficient (fliC, n = 17) mutant increased the number of coupled cells. PAO1-induced dye coupling was prevented in the presence of a gap junction inhibitor (αGA, 100 μM, n = 13). *p < 0.05 compared with unstimulated control (CTRL, n = 9). #p < 0.05. (B) The extent of dye coupling induced by PAO1 was decreased by the p38 SB203580 (SB, 10 μM, n = 14) inhibitor and increased by the JNK SP600125 (SP, 10 μM, n = 27) inhibitor. Vehicle (Veh):DMSO was used alone as controls for each treatment. *p < 0.05 compared with Veh (n = 7 for SB; n = 28 for SP).

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PAO1 activation of Calu-3 cells is critical to trigger NF-κB activation and efficient IL-8 production (16). In this context, Cx43 has been reported to contribute to the propagation of proinflammatory signals in several epithelial cell models (911). As shown in Fig. 3A, 6-h infection of Calu-3 cells with wild-type PAO1 or pilA mutants triggered strong IL-8 release. As expected, no IL-8 was produced in response to the fliC PAO1. We next evaluated the effects of αGA on the IL-8 response evoked by increasing MOI of PAO1. Gap junction inhibition did not affect IL-8 release (Fig. 3B). Furthermore, no effects of αGA on HK-PAO1–stimulated IL-8 secretion were observed (Fig. 3C), suggesting that gap junctional communication does not extensively contribute to the proinflammatory response of Calu-3 cells evoked by live or dead PAO1.

FIGURE 3.

Cx43 function does not contribute to IL-8 release. (A) The inflammatory response was evaluated by the release of IL-8 6 h postinfection with wild-type (wt) PAO1 (MOI 5). IL-8 release was similarly induced by a PAO1 mutant lacking pilin (pilA) but not flagellin (fliC). n = 3, *p < 0.05 compared with nonstimulated control (CTRL). (B) Focal stimulation of Calu-3 cells was evoked by infection for 6 h with increasing MOI of PAO1, in the presence or absence of 100 μm αGA, a gap junction inhibitor. Again, no difference in IL-8 release was observed after inhibition of gap junctional communication. n = 3. (C) Calu-3 cells were globally stimulated with HK-PAO1 (105 CFU/ml) for 6 h in the presence or absence of 100 μm αGA. Again, gap junction blockade had no effect on IL-8 release. DMSO was used as control of the αGA treatment (vehicle [Veh]). n = 5.

FIGURE 3.

Cx43 function does not contribute to IL-8 release. (A) The inflammatory response was evaluated by the release of IL-8 6 h postinfection with wild-type (wt) PAO1 (MOI 5). IL-8 release was similarly induced by a PAO1 mutant lacking pilin (pilA) but not flagellin (fliC). n = 3, *p < 0.05 compared with nonstimulated control (CTRL). (B) Focal stimulation of Calu-3 cells was evoked by infection for 6 h with increasing MOI of PAO1, in the presence or absence of 100 μm αGA, a gap junction inhibitor. Again, no difference in IL-8 release was observed after inhibition of gap junctional communication. n = 3. (C) Calu-3 cells were globally stimulated with HK-PAO1 (105 CFU/ml) for 6 h in the presence or absence of 100 μm αGA. Again, gap junction blockade had no effect on IL-8 release. DMSO was used as control of the αGA treatment (vehicle [Veh]). n = 5.

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In parallel with proinflammatory responses, P. aeruginosa causes apoptosis of airway epithelial cells, a mechanism that involved pathogen recognition receptors and activation of intracellular signaling pathways, including p38 and JNK (1720). We thus studied the ability of PAO1 to induce apoptosis in Calu-3 cells by detection of active caspase-3 (p17 subunit). As shown in Fig. 4A, infection of Calu-3 cells with PAO1 activated caspase-3 within 6–8 h; this activation was markedly enhanced 8 h postinfection in the presence of the JNK inhibitor SP. PAO1-induced caspase-3 activation was unaffected in the presence of the p38 inhibitor SB (Supplemental Fig. 2A). We also screened for active caspase-3 expression in Calu-3 cells exposed to TNF-α, HK-PAO1, and live PAO1. Caspase-3 activation, however, was only detected with live PAO1 (Supplemental Fig. 2B), indicating that PAO1 infection is essential to trigger apoptosis. As expected, the ability of the fliC mutant to trigger apoptosis was strongly attenuated as compared with wild-type PAO1 (Fig. 4B). Interestingly, we observed that the increase of caspase-3 expression by PAO1 and SP was prevented in the presence of αGA (Fig. 4C), suggesting for a link between gap junctional communication and apoptosis in Calu-3 cells. Strong apoptosis was detected in primary HAECs infected for 16 h with PAO1 (Supplemental Fig. 2C). Activated caspase-3 induced by PAO1 in combination with the JNK inhibitor was also markedly reduced in HAECs pretreated with αGA (Fig. 4D, 4E), confirming the relationship between gap junctional communication and apoptosis in primary HAECs. Finally, the modulation of PAO1-induced apoptosis by JNK inhibition was verified in Calu-3 cells by confocal detection of exogenous annexin V, which binds to the membrane of apoptotic cells. Thus, annexin V binding, which was induced by PAO1, was further increased in the presence of SP (Fig. 4F, 4G). The latter effect was prevented in the presence of αGA (Fig. 4F, 4G).

FIGURE 4.

JNK reduces apoptosis induced by PAO1. (A) Apoptosis was evaluated by immunoblotting-activated caspase-3 (p17-Casp3) in Calu-3 cells. PAO1 (MOI 5) increased active caspase-3 in a time-dependent manner. Inhibition of JNK with the JNK SP600125 (SP, 10 μM) inhibitor enhanced the detection of p17-Casp3 (top panels). (B) In contrast to wild-type PAO1 (wt), the flagellin mutant fliC moderately affected p17-Casp3 detection in Calu-3 cells. Molecular mass (in kDa) markers are indicated on the right side of the blots. (C) The increase in p17-Casp3 induced by PAO1 with the JNK inhibitor (PAO1 + SP) in Calu-3 cells was reduced in the presence of 100 μM αGA. (D) The increase in p17-Casp3 induced by PAO1 with the JNK inhibitor (PAO1 + SP) in primary HAECs was reduced in the presence of 100 μM αGA. (E) Quantitative analysis of HAECs infected with PAO1 for 16 h in the presence of the JNK inhibitor alone (SP) or in combination with αGA. n = 6, *p < 0.05. (F) Detection of apoptosis after 8 h of infection with PAO1 by confocal microscopy of annexin V fluorescent staining (green). Annexin V staining was enhanced in the presence of 10 μM JNK inhibitor (PAO1 + SP), an effect that was prevented in the presence of 100 μM of the gap junction inhibitor αGA (PAO1 + SP + αGA). Nuclei are stained in blue with DAPI. Scale bar, 10 μm. (G) Quantitative analysis of the annexin V staining experiments. DMSO was used as vehicle (Veh). n = 6 images, *p < 0.05 compared with unstimulated control (CTRL), #p < 0.05.

FIGURE 4.

JNK reduces apoptosis induced by PAO1. (A) Apoptosis was evaluated by immunoblotting-activated caspase-3 (p17-Casp3) in Calu-3 cells. PAO1 (MOI 5) increased active caspase-3 in a time-dependent manner. Inhibition of JNK with the JNK SP600125 (SP, 10 μM) inhibitor enhanced the detection of p17-Casp3 (top panels). (B) In contrast to wild-type PAO1 (wt), the flagellin mutant fliC moderately affected p17-Casp3 detection in Calu-3 cells. Molecular mass (in kDa) markers are indicated on the right side of the blots. (C) The increase in p17-Casp3 induced by PAO1 with the JNK inhibitor (PAO1 + SP) in Calu-3 cells was reduced in the presence of 100 μM αGA. (D) The increase in p17-Casp3 induced by PAO1 with the JNK inhibitor (PAO1 + SP) in primary HAECs was reduced in the presence of 100 μM αGA. (E) Quantitative analysis of HAECs infected with PAO1 for 16 h in the presence of the JNK inhibitor alone (SP) or in combination with αGA. n = 6, *p < 0.05. (F) Detection of apoptosis after 8 h of infection with PAO1 by confocal microscopy of annexin V fluorescent staining (green). Annexin V staining was enhanced in the presence of 10 μM JNK inhibitor (PAO1 + SP), an effect that was prevented in the presence of 100 μM of the gap junction inhibitor αGA (PAO1 + SP + αGA). Nuclei are stained in blue with DAPI. Scale bar, 10 μm. (G) Quantitative analysis of the annexin V staining experiments. DMSO was used as vehicle (Veh). n = 6 images, *p < 0.05 compared with unstimulated control (CTRL), #p < 0.05.

Close modal

To demonstrate a causal relationship between gap junctions and caspase-3 activation during JNK inhibition, we developed a Cx43 gene-silencing strategy via lentiviral expression of a specific shRNA (shCx43). Fig. 5A and 5B show the efficiency of the shCx43 in reducing the expression and function of Cx43 as compared with Calu-3 cells infected with a lentivirus expressing a control shRNA (shCTRL). As shown in Fig. 5C and 5D, the activation of caspase-3 in response to PAO1 and JNK inhibition was prevented in cells with reduced Cx43 expression. These results indicate that Cx43-mediated gap junctional communication exerts a proapoptotic role in PAO1-infected airway epithelial cells, whereas JNK signaling acts as a negative regulator of Cx43 function.

FIGURE 5.

Cx43 channels are required for PAO1-induced apoptosis. A silencing strategy by lentiviral expression of a specific Cx43 shRNA (shCx43) was used to knockdown Cx43, as revealed at the protein level by Western blot (A) and at the functional level by dye coupling (B). A scramble shRNA (shCTRL) was used in parallel for control experiments. n = 16 dye injections, *p < 0.05. (C) Knockdown of Cx43 by shCx43 reduced caspase-3 activation induced by PAO1 (MOI 5) and the JNK SP600125 (SP, 10 μM) inhibitor at 8 h, as revealed by Western blot for p17-Casp3 (top panel), GAPDH (middle panel), and Cx43 (bottom panel). (D) Quantitative analysis from a total of three to four experiments, *p < 0.05.

FIGURE 5.

Cx43 channels are required for PAO1-induced apoptosis. A silencing strategy by lentiviral expression of a specific Cx43 shRNA (shCx43) was used to knockdown Cx43, as revealed at the protein level by Western blot (A) and at the functional level by dye coupling (B). A scramble shRNA (shCTRL) was used in parallel for control experiments. n = 16 dye injections, *p < 0.05. (C) Knockdown of Cx43 by shCx43 reduced caspase-3 activation induced by PAO1 (MOI 5) and the JNK SP600125 (SP, 10 μM) inhibitor at 8 h, as revealed by Western blot for p17-Casp3 (top panel), GAPDH (middle panel), and Cx43 (bottom panel). (D) Quantitative analysis from a total of three to four experiments, *p < 0.05.

Close modal

JNK signaling appears as a key regulator of Cx43 expression and apoptosis in PAO1-infected Calu-3 cells. We thus monitored JNK activation at various times following infection via a specific Ab, recognizing the two main isoforms (pp46 and pp54) of phosphorylated JNK (p-JNK). We found that PAO1 transiently increased p-JNK, whereas total JNK expression was unchanged (Fig. 6A). Due to unavoidable variability in host–pathogen interaction experiments, p-JNK could be detected from 30 min up to 6 h following infection with the live bacteria; the peak of JNK phosphorylation was usually observed 2–4 h postinfection. As expected, the fliC mutant had no effect on p-JNK (Fig. 6A).

FIGURE 6.

CFTR activity regulates PAO1-induced JNK signaling in Calu-3 cells. (A) Total JNK expression (bottom panel) and p-JNK (top panel) in Calu-3 cells infected (MOI 5) by wild-type PAO1 (wt) or the fliC flagellin mutant was evaluated as a function of time by Western blots. These blots are representative of three experiments. Molecular mass (in kDa) markers are indicated on the right side of the blots. (B) Western blots showing that CFTR inhibition with 10 μM GlyH-101 (GlyH) consequently enhanced p-JNK detection with time. (C) Quantitative analysis of the effects of GlyH-101 (10 μM) on the two main isoforms of p-JNK (pp46 and pp54). Quantification was made for the whole and peak of p-JNK signals. DMSO was used as vehicle (Veh). n = 4, *p < 0.05. (D) Western blots showing that correction of CF15 cells by adenoviral-mediated expression of wild-type CFTR decreased p-JNK detection following PAO1 (MOI 5) infection. (E) Quantitative analysis of the pp46 and pp54 signals (whole and peak) in PAO1-infected CF15 cells before and after CFTR expression (CF15-CFTR). n = 4, *p < 0.05.

FIGURE 6.

CFTR activity regulates PAO1-induced JNK signaling in Calu-3 cells. (A) Total JNK expression (bottom panel) and p-JNK (top panel) in Calu-3 cells infected (MOI 5) by wild-type PAO1 (wt) or the fliC flagellin mutant was evaluated as a function of time by Western blots. These blots are representative of three experiments. Molecular mass (in kDa) markers are indicated on the right side of the blots. (B) Western blots showing that CFTR inhibition with 10 μM GlyH-101 (GlyH) consequently enhanced p-JNK detection with time. (C) Quantitative analysis of the effects of GlyH-101 (10 μM) on the two main isoforms of p-JNK (pp46 and pp54). Quantification was made for the whole and peak of p-JNK signals. DMSO was used as vehicle (Veh). n = 4, *p < 0.05. (D) Western blots showing that correction of CF15 cells by adenoviral-mediated expression of wild-type CFTR decreased p-JNK detection following PAO1 (MOI 5) infection. (E) Quantitative analysis of the pp46 and pp54 signals (whole and peak) in PAO1-infected CF15 cells before and after CFTR expression (CF15-CFTR). n = 4, *p < 0.05.

Close modal

CFTR is endogenously expressed in Calu-3 cells, but its level of expression was not affected by PAO1 infection (Supplemental Fig. 3A). CFTR short-circuit current monitored in Ussing chamber was efficiently blocked by the CFTR inhibitor GlyH-101 (Supplemental Fig. 3B). Interestingly, we observed that CFTR inhibition by GlyH-101 markedly increased PAO1-induced JNK phosphorylation (Fig. 6B). Although time of detection of the peak of phosphorylation was variable between experiments (Supplemental Fig. 3C, 3D), both whole and peak p-JNK signals were increased in Calu-3 cells exposed to the CFTR inhibitor (Fig. 6C). To demonstrate the contribution of CFTR in the regulation of JNK signaling, we measured p-JNK signals in the CF airway CF15 cell line before and after correction of the CF phenotype by adenoviral-mediated expression of wild-type CFTR (Supplemental Fig. 3E). Thus, we found that exogenous expression of CFTR decreased JNK phosphorylation evoked by PAO1 in this CF cell line, also grown on Transwell inserts for accurate comparison with our Calu-3 cell cultures (Fig. 6D, 6E, Supplemental Fig. 3F, 3G). Finally, we observed in Calu-3 cells that expression of Cx43 (Fig. 7A, 7B), activation of caspase-3 (Fig. 7C), and annexin V staining (Fig. 7D, 7E) were markedly decreased in response to PAO1 infection in the presence of Gly-H101. These results suggest that CFTR activity contributes to the response evoked by P. aeruginosa by exerting a negative feedback on PAO1-induced JNK signaling responses.

FIGURE 7.

CFTR activity contributes to PAO1-induced apoptosis in Calu-3 cells. Western blot (A) and quantitative analysis (B) of CFTR inhibition with 10 μM GlyH-101 (GlyH) on Cx43 expression in Calu-3 cells infected with PAO1 (MOI 5) for 6 h. n = 4, *p < 0.05. (C) Western blot showing that CFTR inhibition with GlyH-101 (10 μM) decreased p17-Casp3 detection in Calu-3 cells infected with PAO1 (MOI 5). (D) Detection of apoptosis after 6 h of infection with PAO1 (MOI 5) by confocal microscopy of annexin V fluorescent staining (green). Annexin V staining was reduced in the presence of 10 μM GlyH-101. Nuclei are stained in blue with DAPI. Scale bar, 10 μm. (E) Quantitative analysis of the annexin V staining experiments. n = 6 images, *p < 0.05. In all panels, DMSO was used as vehicle (Veh).

FIGURE 7.

CFTR activity contributes to PAO1-induced apoptosis in Calu-3 cells. Western blot (A) and quantitative analysis (B) of CFTR inhibition with 10 μM GlyH-101 (GlyH) on Cx43 expression in Calu-3 cells infected with PAO1 (MOI 5) for 6 h. n = 4, *p < 0.05. (C) Western blot showing that CFTR inhibition with GlyH-101 (10 μM) decreased p17-Casp3 detection in Calu-3 cells infected with PAO1 (MOI 5). (D) Detection of apoptosis after 6 h of infection with PAO1 (MOI 5) by confocal microscopy of annexin V fluorescent staining (green). Annexin V staining was reduced in the presence of 10 μM GlyH-101. Nuclei are stained in blue with DAPI. Scale bar, 10 μm. (E) Quantitative analysis of the annexin V staining experiments. n = 6 images, *p < 0.05. In all panels, DMSO was used as vehicle (Veh).

Close modal

In the current study, we addressed the connection between cell-to-cell communication and the response of airway epithelial cells infected with the CF-associated pathogen P. aeruginosa. We report that the P. aeruginosa laboratory strain PAO1 induced functional expression of Cx43 in Calu-3 cells. We demonstrate that JNK signaling is critical to the modulation of Cx43 expression, and that Cx43-mediated cell-to-cell communication contributes to apoptosis but not inflammation of the airway epithelium. We further show that CFTR negatively regulates JNK activity. The tightly regulated expression of Cx43 may confer in normal airway epithelia a mechanism to balance the inflammatory and apoptotic responses.

Gap junctions and hemichannels are targets of bacterial pathogens and their toxins (reviewed by Ceelen et al.) (7). S. flexneri, an invasive bacterium that replicates within intestinal epithelial cells, received particular attention. The activity of connexin-made hemichannels was found crucial to the invasion and dissemination of this microorganism in intestinal epithelial cells (21, 22). More recently, it was reported in an intestinal cell line that gap junction channels favor the cell-to-cell spread of an inflammatory response dominated by IL-8 production upon S. flexneri internalization and activation of the intracellular pattern recognition receptor Nod1 in the infected cells (11). Low incidence of P. aeruginosa internalization has been previously reported (23, 24). This phenomenon was, however, not observed in our cultures of polarized Calu-3 cells. Cell polarization was confirmed by the high transepithelial resistance reached. In a close cell culture system, long-term infection led to epithelium destruction due to excessive bacterial toxin and virulence factor production. Even under these conditions, we observed that PAO1 transmigrate between epithelial cells to accumulate on the basal side of the epithelium without being obviously internalized. Thus, activation of innate immune response in this system relies on the interaction of pathogen-associated patterns with epithelial surface receptors, as also suggested by the observation that HK-PAO1 induced Cx43 expression and IL-8 release in Calu-3 cells. We found that a flagellin-deficient PAO1 mutant failed to induce Cx43 expression as well as IL-8 secretion. Infection of Calu-3 cells with fliC strain in the presence of purified flagellin restored the induction of Cx43, indicating that activation of an innate immune response in airway epithelia by PAO1 requires the binding of this bacterial component to pathogen recognition receptors. In vertebrates, at least two major types of flagellin receptors have been identified, as follows: surface TLR5 (25) and intracellular NLRC4 inflammasome (26, 27). Although TLR5 is expressed in our cell system, we cannot rule out that other signaling pathways like the NLRC4 inflammasome are involved in the host response to PAO1 (28). Of note, we did not detect the production of the inflammasome cytokine IL-1β in the medium of infected Calu-3 cells (data not shown).

Using a variety of protein kinase inhibitors, we have identified p38 and JNK as key modulators of PAO1-induced Cx43 expression. Interestingly, p38 stimulated Cx43 expression, whereas JNK exerted a negative regulation. In epithelial cells, the opposite regulation of Cx43 expression by p38 and JNK has been reported in keratinocytes and mammary glands in response to TNF-α and TGF-β, respectively (29, 30). JNK was also pointed out as an important mediator of stress-induced Cx43 downregulation in the failing heart (31). p38 was previously shown to activate CFTR-dependent chloride secretion in response to flagellin stimulation, which is necessary for efficient water transport and mucus hydration (32). Interestingly, gap junctions were also involved in CFTR function and airway surface liquid hydration (12). Thus, Cx43 induction by PAO1 can be considered as a component of the response of airway epithelial cells to innate immune activation, leading to a regulated increase in gap junctional intercellular communication.

We did not detect quantitative differences in the release of the proinflammatory chemokine IL-8 by airway epithelial cells treated with gap junction blockers. These results are of importance regarding the previous demonstration that gap junctions were required to spread signals to activate MAPKs in neighbors of intestinal epithelial cells infected with S. flexneri, a mechanism thought to amplify IL-8 production (11). In contrast to this finding, we report in this study that Cx43 is a target of MAPKs but does not contribute efficiently to the modulation of IL-8 secretion of airway epithelial cells in response to PAO1 infection. It is worth pointing out that a role for gap junctions in the propagation of calcium waves evoked by TLR2 stimulation was shown to transiently increase IL-8 secretion in airway epithelial cells (9). Mechanisms of intercellular communication independent of gap junctions may also coexist, as it was recently reported in intestinal epithelial cells infected with Listeria monocytogenes (33). Collectively, these observations may indicate that involvement of gap junctions in the spreading of proinflammatory signals may be dependent on the type of pattern-recognition receptors that are activated and/or of toxins that are released by pathogens.

Infection with P. aeruginosa causes apoptosis of airway epithelial cells, a mechanism that was shown to involve flagellin and JNK (1720). Indeed, the ability of the fliC mutant to induce apoptosis was strongly attenuated as compared with wild-type PAO1, whereas HK-PAO1 or the inflammatory mediator TNF-α did not cause apoptosis. Therefore, PAO1 infection is essential to trigger apoptosis, but the epithelial innate immune response induced by flagellin is important to modulate the strength of the apoptotic signals. Moreover, we found that JNK inhibition enhanced PAO1-induced caspase-3 activation and annexin V binding to the surface Calu-3 cells, suggesting that the stress-activated protein kinase negatively regulates apoptosis. In addition, our data unmasked the contribution of Cx43-mediated gap junctional communication on the extent of apoptosis. In this regard, the lower extent of epithelial apoptosis observed in response to fliC strain might be due to the lack of Cx43 modulation. There is accumulating evidence that gap junctions may facilitate the intercellular diffusion of death signals and/or may initiate apoptosis during disruption of homeostasis (34). However, the nature of the intercellular cell death molecules remains elusive, and the exact contribution of connexin channels still remains controversial (35). Importantly, the relationship between gap junctional communication and apoptosis was confirmed in primary HAECs infected with PAO1.

Lung infection with P. aeruginosa is a hallmark of CF, although the mechanisms underlying enhanced affinity of airway epithelial cells to this pathogen are still poorly understood. To date the links with CFTR are not known. In this study, we show that CFTR contributes to the response evoked by PAO1 by exerting a negative feedback on JNK signaling cascade. Pharmacological inhibition of CFTR activity led to enhanced duration of PAO1-induced JNK activity, whereas correction of the CF phenotype in a homozygous F508del airway epithelial cell line (CF15 cells) reduced p-JNK detection. Our results are consistent with those of Saadane et al. (36), who reported increased p-JNK in CF airway epithelial cell lines and in primary necropsy human tracheal epithelial cells treated with a CFTR inhibitor. Clearly, activation of inflammation, apoptosis, and fluid transport by airway epithelia during P. aeruginosa are central to host-defense mechanisms. Excessive JNK activation, however, may have major consequences, including longer expression of proinflammatory genes and strong inhibition of Cx43 expression, thus decreasing fluid transport and apoptosis. We suggest in this study that these events are related in CF airway epithelial cells by enhanced PAO1-induced JNK activity due to defective CFTR function. Our results also suggest that abnormal regulation of JNK signaling may provide an explanation to the misbalance between inflammation and apoptosis induced by P. aeruginosa in the CF disease. The molecular mechanism of JNK inhibition by CFTR remains, however, to be elucidated.

We thank Dr. Giulio Cabrini (University of Verona) for the gift of the heat-killed PAO1 and for performing preliminary experiments. We thank Joanna Bou Saab, Richard Ruez, and Brenda Kwak for helpful discussion during the progression of this project. We thank Sergei Startchik from the BioImaging Core Facility for the assistance in quantification of annexin V staining.

This work was supported by Swiss National Science Foundation Grants 310030_134907/1 (to M.C.) and 32473B_140929 (to C.v.D.). S.C. and J.B. were supported by Vaincre la Mucoviscidose. D.L. was supported by Italian Cystic Fibrosis Research Foundation Grant 19/2009 (adopted by Fondazione Ricerca Fibrosi Cistica delegation La Bottega delle Donne) and by Fondation pour des Bourses d'Etudes Italo-Suisses.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CF

cystic fibrosis

CFTR

CF transmembrane conductance regulator

Chel

Chelerythrin

EGFP

enhanced GFP

HAEC

human airway epithelial cell

HK

heat-killed

MOI

multiplicity of infection

PKC

protein kinase C

shRNA

short hairpin RNA

siRNA

small interfering RNA.

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The authors have no financial conflicts of interest.

Supplementary data