P. aeruginosa LPS stimulates calcium signaling and chloride secretion via CFTR in human bronchial epithelial cells

Journal of Cystic Fibrosis 12 (2013) 60–67 www.elsevier.com/locate/jcf

Original Article

P. aeruginosa LPS stimulates calcium signaling and chloride secretion via CFTR in human bronchial epithelial cells J.M. Buyck a,⁎, V. Verriere b , R. Benmahdi c , G. Higgins d , B. Guery e , R. Matran f , B.J. Harvey b , K. Faure e , V. Urbach d a Laboratoire de Physiologie, EA2689, IMPRT IFR 114, Université de Lille 2, 59945 Lille cedex, France Molecular Medicine Department, RCSI Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland c Hôpital Arnaud de Villeneuve, INSERM U454, Montpellier, France d National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland Service de gestion du risque infectieux et des vigilances, Hôpital Calmette, CHRU de Lille, Université de Lille 2, 59045 Lille cedex, France f Faculty of Medicine, University of Lille, UDSL, EA 4483, F-59 000 Lille, France b

e

Received 6 December 2011; received in revised form 6 June 2012; accepted 14 June 2012 Available online 17 July 2012

Abstract Background: Pseudomonas aeruginosa airway infection is associated with a high mortality rate in cystic fibrosis. Lipopolysaccharide (LPS), a main constituent of the outer membrane of P. aeruginosa, is responsible for activation of innate immune response but its role on airway epithelium ion transport, is not well known. The aim of this study was to determine the role for P. aeruginosa LPS in modulating chloride secretion and intracellular calcium in the human bronchial epithelial cell line, 16HBE14o −. Methods: We used intracellular calcium imaging and short-circuit current measurement upon exposure of cells to P. aeruginosa LPS. Results: Apical LPS stimulated intracellular calcium release and calcium entry and enhanced chloride secretion. This latter effect was significantly inhibited by CFTR(inh)-172 and BAPTA-AM (intracellular Ca 2 + chelator). Conclusions: Our data provides evidence for a new role of P. aeruginosa LPS in stimulating calcium entry and release and a subsequent chloride secretion via CFTR in human bronchial epithelium. © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Human bronchial epithelium; Calcium; Chloride; LPS; Pseudomonas aeruginosa

1. Introduction Abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; AM, acetoxy-methyl ester; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid; CaCC, calcium-activated chloride channel; CFTR, cystic fibrosis transmembrane conductance regulator; DMSO, dimethyl sulfoxide; DPC, diphenylamine-2-carboxylic acid; EGTA, ethylene glycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetra-acetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; [Ca²+]i, intracellular calcium concentration; GdCl3, gadolinium chloride; LaCl3, lanthanum chloride; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; SKF96365, 1-[‐[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride; TRP, transient receptor potential ⁎ Corresponding author at: Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain, LDRI, Avenue E. Mounier 73 bte B1.73.05, B-1200 Bruxelles, Belgium. Tel.: + 32 2 764 72 37; fax: + 32 2 764 73 73. E-mail address: [email protected] (JM. Buyck).

Bronchial infection by Pseudomonas aeruginosa is associated with high mortality rate in cystic fibrosis. Lipopolysaccharide (LPS), a major constituent of its outer membrane, is of great importance in the pathogenic potential of P. aeruginosa by inducing strong response from the immune system. Epithelial ion transport determines the quality of the mucociliary clearance thus protecting the lung from infection and inflammation. Several studies have reported that P. aeruginosa virulence factors regulate ion transport. Rhamnolipids were found to inhibit epithelial Na + absorption [1]. Flagellin has been shown to inhibit Na + absorption in murin tracheal epithelium [2] and to stimulate chloride secretion in human airway epithelial Calu-3 cells [3].

1569-1993/$ -see front matter © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2012.06.007

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LPS has also been reported to regulate airway epithelial ion transport. LPS from P. aeruginosa inhibits sodium absorption via ENaC in mammalian alveolar epithelial cells [4]. In contrast, LPS from Salmonella enterica stimulated sodium absorption by increasing Na/K ATPase expression in guinea-pig tracheal epithelium [5]. Whereas, Escherichia coli LPS increased expression of a calcium-activated chloride channel, in human airway epithelial cells [6]. LPS was shown to regulate intracellular calcium signaling cytokine and mucin release and ion transport of rat acinar pancreatic cells [7]. However, until the study reported here, there has been no description of the effects of P. aeruginosa LPS on airway epithelial chloride transport and on intracellular calcium homeostasis in airway epithelial cells. 2. Material and methods 2.1. Cell culture The human bronchial epithelial cell line, 16HBE14o− was grown in Eagle's minimal essential medium (EMEM) supplemented by 10% fetal calf serum (FCS), 1% penicillin, 1% streptomycin and 1% L-glutamine, in culture flasks coated with a collagen/ fibronectin solution in a 5% CO2 atmosphere at 37 °C. After reaching confluence, cells were washed twice with a phosphatebuffered saline solution and isolated, using a trypsin solution (1% polyvinylpyrrolidone, 0.2% EGTA, and 0.25% trypsin). 2.2. Measurement of intracellular calcium concentration Cells were grown on glass coverslips as previously described [8]. The cells were loaded with 5 μM of Fura-2 acetoxy-methyl ester (Fura-2 AM) in EMEM for 45 min at 37 °C. Cells were then washed twice in HEPES-buffered Krebs-Henseleit solution (Krebs). The coverslip was placed on fluorescence microscope (Diaphot 200, Nikon, The Netherlands) equipped with a CCD video camera (Darkstar, Photonics Sciences, UK) to acquire images. The fluorescence, depending upon the level of calcium binding to Fura-2 AM, was obtained at each excitation wavelength (F340 and F380) and emission fluorescence was measured at 510 nm. To study calcium entry in cells, we used a calcium-free Krebs (Ca-free) containing 0 mM CaCl2 and 5 mM EGTA. 2.3. Ussing chamber For transepithelial transport measurements, cells were cultured on coated permeable membrane (Snapwell, Corning International, Avon, France). After 8 days of culture, the cells reached a transepithelial electrical resistance of N 800 Ω/cm². The Snapwell membrane was placed in a horizontal Ussing chamber (NaviCyte Scientific, Lexington, MA) bathed in Krebs for 20 min. Then, the apical bath was removed and replaced by low chloride Krebs (Na-gluconate 125 mM; NaCl 15 mM; KCl 5 mM; CaCl2 2 mM; MgSO4 1.2 mM; KH2PO4 1.2 mM; HEPES sodium salt 6 mM, 300 mOsm, pH = 7.4) to create a chloride gradient. The transmembrane potential was measured using a voltage-clamp model amplifier (EVC 4000, World Precision Instruments, Aston, UK) and clamped to 0 mV by

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application of a short-circuit current (scc) corresponding to electrogenic ion transfer. The measures from the amplifier were digitized using a PowerLab system (Chart for Windows v4.0, ADInstruments, Oxfordshire, UK). 2.4. Chemicals Reagents were obtained from Sigma (St-quentin Fallavier, France). LPS (phenol extract, less than 1% of protein contamination) from P. aeruginosa was reconstituted in Krebs at 3 mg/ml. The chemicals were solubilized as a molar stock solution in DMSO (amiloride, DPC, NPPB, glibenclamide, thapsigargin, forskolin, econazole, SKF96365 and CFTR(inh)-172), in 95% ethanol (2-APB) or in Krebs (suramin, verapamil, ruthenium red, LaCl3 and GdCl3). All chemicals were diluted at the final concentration in Krebs before experiments. Fura-2 AM (5 mM stock in DMSO) was provided by FluoProbes (Interchim, Montluçon, France). 2.5. Data analysis The [Ca² +]i levels were evaluated from the F340/F380 ratio. The maximum ratio increase is the variation between the mean ratio measured during the 2 min prior to treatment with LPS and the maximum ratio (peak value) measured after LPS treatment. The area under the curve (AUC) was measured between the LPS-induced peak until 5 min after the peak. The scc variations correspond to the variations between the mean scc measured during the 2 min prior to treatment with LPS and the maximum scc measured after LPS treatment. Data are shown as the means ± S.E.M. of n experiments. Measures of statistical significance were obtained with GraphPad Prism (GraphPad Software INC., San Diego, USA) using either Student's t tests for paired data, or one-way ANOVA for multiple testing. p b 0.05 was deemed to be significant. 3. Results 3.1. LPS effects on [Ca² +]i We determined the effect of P. aeruginosa LPS on [Ca² +]i of 16HBE14o − cells. As shown on Fig. 1 A (solid line), LPS (30 μg/ml) induced a rapid [Ca² +]i increase, with a maximum value reached within 10 s, followed by a rapid decrease to a plateau phase and a slow decline toward baseline. To determine the mechanism involved in LPS-induced calcium signaling, we used an external Ca-free solution. Under these conditions, the LPS-induced [Ca² +]i increase was significantly reduced (Fig. 1 A, dotted line). The peak F340/F380 response was still significant but decreased to 0.02 ± 0.01 (Fig. 1 C). Likewise, the AUC, used to estimate the total amount of calcium mobilized by LPS exposure, was significantly lower in a Ca-free solution (4.04 ± 1.42 AU) compared to normal Krebs (20.50 ± 2.12 AU) (Fig. 1 D). These results indicate that both calcium entry and calcium release from intracellular stores are required to generate the calcium response to LPS. Then, we investigated the contribution of intracellular calcium release in response to LPS. Thapsigargin (15 min, 1 μM), used to

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Fig. 1. Source of calcium involved in [Ca²+]i increase induced by P. aeruginosa LPS. A. Typical effect of LPS (30 μg/ml) on F340/380 ratio in Krebs solution (solid line) or external calcium-free solution (dotted line). B. Typical effect of thapsigargin pretreatment (15 min, 50 μM) on the LPS (30 μg/ml) induced F340/380 ratio changes measured in Krebs solution (solid line) or calcium-free solution (dotted line). C. Means of maximal F340/380 ratio changes (peak) induced by LPS (30 μg/mL) in Krebs solution (CTL), calcium-free solution (Ca-free), upon exposure to 50 μM thapsigargin (Tg) in Krebs solution and upon exposure to 50 μM thapsigargin in calcium-free solution (Tg +Ca-free). Data are means± SEM (n =5); **: p b 0.01; ***: p b 0.001 statistically different from CTL. D. Means AUC obtained upon LPS (30 μg/ ml) exposure in Krebs solution (CTL), in calcium-free solution (Ca-free), in Krebs solution with 50 μM of thapsigargin (Tg) and in calcium-free solution with 50 μM of thapsigargin (Tg +Ca-free). Data are means ± SEM (n =5); *: p b 0.05; ***: p b 0.001 statistically different from CTL.

inhibit uptake of calcium into intracellular stores via the SERCA pump, induced a sustained [Ca² +]i increase (Fig. 1B, solid line). After thapsigargin-induced calcium mobilization, subsequent challenge with LPS produced a reduced but still significant transient [Ca² +]i increase most likely due to calcium entry. The peak [Ca² +]i and AUC were significantly reduced (Fig. 1 C and D). In external Ca-free solution, thapsigargin induced a reduced calcium increase. This indicates that thapsigargin treatment activates capacitative calcium entry (CCE), in addition to emptying the intracellular calcium stores. Therefore, this also suggests that the LPS-induced calcium signal involves a calcium mobilization from thapsigargin-sensitive intracellular stores and CCE (Fig. 1 B, dotted line). After thapsigargin treatment in external Ca-free solution, LPS failed to produce any calcium increase (Fig. 1 C and D) indicating that the remaining transient calcium response to LPS after thapsigargin (in normal external calcium) was due to a calcium entry distinct from a capacitive calcium entry.

(p b 0.05; n = 5). The non-selective cation channel blockers SKF96365 (3 μM) and econazole (30 μM) both significantly reduced the AUC by 48.8% and 35.2% (p b 0.05; n = 5), respectively; without affecting the peak. Ruthenium red (10 μM), a broad spectrum TRP (transient receptor potential) antagonist, was without effect on the peak while significantly reducing the AUC. The inhibitor of L-type voltage sensitive calcium channel, verapamil (0.1 μM), produced a 47.4% decreased AUC (p b 0.05; n = 5). Taken together these results suggested a simultaneous involvement of TRP and L-type channels in the LPS-induced calcium response. Intracellular calcium increase can be triggered by external ATP or UTP [9]. Suramin (100 μM), an inhibitor of purinoreceptor, did not affect either the peak or the AUC of LPS-induced calcium response (Fig. 2 C and D) indicating that LPS-induced [Ca² + ]i response did not involve nucleotide and purinoreceptor stimulation.

3.2. Pharmacological inhibition of calcium channel 3.3. Effects of LPS on short-circuit current To characterize calcium channels involved in the LPS-induced [Ca² +]i increase, we used inhibitors of known calcium channels and of non-selective cation channels (Fig. 2). Both LaCl3 (1 μM) and GdCl3 (1 μM) significantly decreased the AUC and the peak

Since [Ca² +]i plays a fundamental role in the regulation of ion transport, we investigated the consequence of LPS-induced [Ca² +]i increase on the transepithelial short-circuit current (scc).

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Fig. 2. Effect of calcium channel inhibitors and suramin on the calcium response induced by P. aeruginosa LPS. A. Mean of the maximum F340/380 ratio increases (peak) induced by LPS (30 μg/ml) in Krebs solution (CTL), or in presence of calcium channel inhibitors (LaCl3: 1 μM, GdCl3: 1 μM, SKF96365: 3 μM, Econazole: 30 μM, Ruthenium Red: 10 μM, Verapamil: 0.1 μM and 2-APB: 10 μM). Data are means ± SEM (n = 5; *: p b 0.05; **: p b 0.01; statistically different from CTL). B. Means of AUC increase induced by LPS (30 μg/ml) in Krebs solution (CTL), or in presence of calcium channel inhibitors. Data are means ± SEM (n = 5; *: p b 0.05; **: p b 0.01; statistically different from CTL). C. Mean of maximum F340/380 ratio increases (peak) induced by LPS (30 μg/ml) in Krebs solution (CTL), or in presence of suramin (100 μM). Data are means ± SEM (n = 5). D. Mean AUC increase induced by LPS (30 μg/ml) in Krebs solution (CTL), or during treatment with suramin (100 μM). Data are means ± SEM (n = 5).

Fig. 3. Effect of P. aeruginosa LPS on short-circuit current (scc). A. Typical effect of LPS on the scc measured in Ussing chambers (left) and means of scc values obtained before (CTL) and after exposure to LPS (LPS, 30 μg/ml). B. Dose effect of apical (from 10 to 50 μg/mL) and basolateral (BL 30, 30 μg/mL) application of LPS. Data are mean of the scc variations compared to the basal scc ± SEM (n = 3).

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The basal scc measured in Krebs solution was 8.4 ± 5.8 μA/cm² (n = 5). Under these conditions, LPS added on either side did not affect the scc (not shown). In the apical low-chloride solution, the basal scc increased to 22.2 ± 2.1 μA/cm² (n = 5). Apical application of LPS (30 μg/ml) increased the scc to 31.8 ± 2.5 μA/cm² (p b 0.05; n = 5) (Fig. 3 A). This effect was dose-dependent and significant from 10 μg/ml while basolateral application of LPS did not affect scc (Fig. 3 B). 3.4. Role for chloride transport A scc increase may be due to either the stimulation of Na + absorption and/or of Cl − secretion. Amiloride (10 μM),

used to inhibit the epithelial Na + channel (ENaC) did not affect the basal scc nor the LPS-induced scc increase (Fig. 4 A). This result indicated that ENaC activity was not responsible for the LPS-induced scc increase in 16HBE14o − cells. We investigated the role for chloride channels in the LPSinduced scc increase. NPPB (1 μM), used as a non selective inhibitor of CaCC did not have any significant effect on both basal and LPS-induced scc increase (Fig. 4 B). The apical treatment of the epithelium by DPC, glibenclamide, used as non specific inhibitors of CFTR channel, decreased the basal scc and inhibited the LPS-induced scc (Fig. 4 B). The specific inhibitor CFTR(inh)-172 (10 μM) decreased the basal scc by

Fig. 4. Pharmacological inhibition of scc increase induced by P. aeruginosa LPS. A. Typical effect of LPS in presence of amiloride (left) and mean of variation of scc (Δ scc ± SEM, n = 5) (right). B. Typical effects of NPPB (100 μM), DPC (100 μM) and Glibenclamide (100 μM) on scc increase induced by LPS (30 μg/ml) (left) and mean variation of scc (Δ scc ± SEM; n = 5; *: p b 0.05, **: p b 0.01) (right). C. Typical effects of CFTR(inh)-172 and forskolin (10 μM) on the scc increase induced by LPS (30 μg/ml) (left), and mean of variation of scc (Δ scc ± SEM; n = 5; *: p b 0.05) (right).

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6.2 ± 2.2 μA/cm 2 (n = 5) and the LPS-induced scc was reduced to 3.4 ± 2.1 μA/cm 2 (n = 5). Forskolin (10 μM), used to activate CFTR channels by inducing a cAMP increase, stimulated the basal scc and reduced the subsequent LPS-induced scc. Furthermore, CFTR(inh)-172 completely inhibited the scc increase induced by forskolin and exposure to LPS did not produce any further scc increase. Taken together, these results indicate that CFTR channel activity plays a major role in the LPS-induced chloride secretion in 16HBE14o − cells. 3.5. Effects of intracellular calcium increase on chloride transport We tested the role of calcium in the effect of LPS on chloride secretion. BAPTA-AM (50 μM, 20 min), an intracellular calcium chelator, slightly decreased the basal scc and totally abolished the LPS-induced scc increase (Fig. 5), indicating that the LPS-induced chloride secretion was mediated by the increase in [Ca² +]i . We further showed that 2-APB used as a blocker of the store operated calcium entry and GdCl3 and SKF96365 used as inhibitors of plasma membrane calcium channel significantly decreased the LPS-induced scc increase. 4. Discussion In this paper, we demonstrated that apical P. aeruginosa LPS induced a [Ca² +]i increase leading to the stimulation of chloride secretion in human bronchial epithelial cells. Calcium responses are caused by different virulence factors, excluding LPS [10,11]. Thus, Jacob et al. demonstrated that basolateral but not apical addition of P. aeruginosa elicited a calcium signal in airway epithelial cells [10]. Our data contrasts with that of Ratner et al. who reported that purified P. aeruginosa LPS alone did not affect [Ca² +]i, in 1HAEo − human airway epithelial cells [12]. This difference might be due to the origin of

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the cells. Indeed, Paradiso et al. have described heterogeneous responses of cell calcium depending of the airway epithelial cell types and cell culture conditions [13]. Otherwise, as reported by Ratner et al., LPS used in our study did not produce lethal effect on 16HBE14o − cells. The effect of LPS varies according to its structure. Indeed, P. aeruginosa lipid A of LPS exhibits less toxicity than E. coli lipid A due to its penta-acylated form [14] while A hexa-acylated lipid A is associated with a stronger inflammatory response [15]. Moreover, the length of the O-antigen of LPS is also a determinant for virulence [16]. We used LPS pseudotype 10 from Sigma, a penta-acylated lipid A with O-side chain LPS, while Ratner et al. have isolated LPS directly from the PAO1 strain. These differences of the origin of LPS could also explain the discrepancy. A capacitive calcium entry involving the release of calcium from intracellular stores followed by activation of a plasma membrane calcium channel has been described in 16HBE14o − cells [9]. Our experiments showed that LPS induced a biphasic calcium signal characterized by an initial peak followed by a plateau phase. In Ca-free external solution, the response to LPS was significantly reduced and the initial calcium peak disappeared. Furthermore, after thapsigargin-induced calcium mobilization, LPS still produced a reduced but significant transient calcium increase which was completely abolished with Ca-free external solution. These experiments indicate that both calcium entry and calcium release from intracellular stores are required to generate the calcium response to LPS and that the transient calcium peak was due to a calcium entry distinct from CCE. Similar calcium signaling has been reported as calcium-induced calcium release. However, the sustained plateau phase was completely inhibited by thapsigargin pretreatment. In external Ca-free solution, thapsigargin induced a reduced calcium increase indicating that thapsigargin treatment, in addition to emptying intracellular calcium stores, also activates CCE. Therefore, the inhibition of the LPS-induced plateau phase after thapsigargin indicates that this component of the calcium signal involves

Fig. 5. Role of intracellular calcium modulators on the scc induced by P. aeruginosa LPS. Typical effects of BAPTA (50 μM), 2-APB (10 μM), SKF96365 (3 μM) and GdCl3 (1 μM) on the scc before and after exposure to LPS (30 μg/ml) (left). Mean of variation of scc induced by LPS after BAPTA (n = 5), 2-APB (n = 5), SKF96365 (n = 3) and GdCl3 (n = 4). *: p b 0.05, **: p b 0.001 (right).

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calcium mobilization from thapsigargin-sensitive intracellular stores and CCE. Taken together our results suggest that LPS stimulates a calcium signaling cascade involving an initial transient signal due to calcium entry (independent from CCE), a calcium-induced calcium release from internal stores and capacitive calcium entry. We established the pharmacological sensitivity of LPSstimulated calcium entry across the membrane. Recent studies have described the presence of the TRP channel family in human airway epithelial cells [17]. Nevertheless, their role in CCE remains not well understood and none of these channels are selective for calcium. LaCl3 and GdCl3, which inhibit nonselective cation channels and L-type calcium channels at micromolar concentration [18], decreased the LPS-stimulated calcium mobilization. The non-selective cation channel blockers, SKF96365 and econazole, both significantly reduced the total amount of calcium mobilization without affecting the maximum calcium increase induced by LPS, which was consistent with a contributory role of TRPC [19]. However, ruthenium red also induced a significant inhibition which is consistent with a role for TRPV activity [20]. The inhibition of L-type calcium channels using verapamil [21], decreased the LPS stimulation of [Ca² +]i indicating a role for these channels. We have identified a role for the TRP channel in the effect of LPS on [Ca² + ]i, despite the lack of specific inhibitors and the multiple activation signals sensed by this channels. We demonstrated a role for L-type channel in this response and further showed that, the LPS response was not due to an ATP release and purinoreceptor-mediated calcium entry. The role of PLC in generating calcium release has been described in airway epithelial cells and LPS can stimulate PLC in alveolar epithelial cells [4]. It cannot be excluded that such mechanism occurs in 16HBE14o − cells upon LPS stimulation. In our study, 2-APB, used as an inhibitor of the inositol 1,4,5-triphosphate receptor involved in intracellular calcium release mediated by PLC activation [22], had no significant effect on LPS-stimulated [Ca² +]i. However, 2-APB was demonstrated to also block TRPC [23] and activate TRPV1-3 channels [24]. In our study the lack of effect of 2-APB could be explained by the LPS stimulation of a complex mechanism involving calcium release and calcium entry by both TRPV and TRPC channels. Likewise, we have noticed that higher concentrations of 2-APB (100 μM) induced large changes in basal [Ca² + ]i with marked variation in the magnitude of the response between cells and rendered the interpretation impractical. In vivo, airway epithelium has the capacity for both chloride secretion and sodium absorption. P. aeruginosa inhibits sodium absorption via ENaC in alveolar epithelial cells [4]. However, it has been reported that airway epithelial cell lines have a tendency to lose their amiloride-sensitivity in culture and that ENaC activity varies according culture conditions of the 16HBE14o − cells [25]. In our hands, amiloride did not affect basal scc of the 16HBE14o − cells, indicating that ENaC did not contribute to the basal scc. Therefore, the absence of effect of LPS on ENaC activity in our model can be explained by the absence of basal

amiloride-sensitive current and cannot be generalized. The LPS-induced transient scc increase was due to chloride secretion since this effect was significantly inhibited by chloride channel inhibitors DPC, glibenclamide, CFTR(inh)-172 but not NPPB. This inhibitor profile supports the involvement of CFTR channels in the LPS-stimulated chloride secretion. BAPTA-AM, an intracellular calcium chelator, produced the total inhibition of the LPS-induced chloride secretion, indicating that it was completely dependent on the [Ca² + ]i. This response indicates that the CFTR stimulation by LPS is mediated by the [Ca² + ]i increase. In airway epithelium, the common view is that CFTR channel is activated by cAMP and not directly by intracellular calcium. However, a recent report indicated that most of the chloride current elicited by calcium agonists in human bronchial epithelial cells is mediated by CFTR via a mechanism involving calcium activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling and that the AC1-CFTR association is responsible for Ca 2+/cAMP cross-talk [26]. Changes in [Ca² +]i may enhance cAMP production through the various calcium-sensitive isoforms of adenylyl cyclase (AC) [27] and, LPS has been reported to induce intracellular cAMP increase in epithelium [28]. Recent studies described a store-operated cAMP signaling activated by P. aeruginosa in which the content of the calcium stores is connected to cAMP signaling and CFTR activation in human airway epithelia [29]. In our study, the inhibition by 2-APB of the LPS-induced scc could result from the effect of calcium store depletion on cAMP level. Taken together, our results demonstrate a novel role of P. aeruginosa LPS to induce calcium release from thapsigarginsensitive stores and calcium entry by both TRP and L-type calcium channels and a further stimulating chloride secretion by CFTR channels in human bronchial epithelium cells. These effects occur within minutes and could be the first signal sensed by the epithelial cells during bacterial infection. In cystic fibrosis, the lack of functional CFTR would be expected to reduce the pro-secretory effect of LPS and this might contribute to the persistence of infection by reduced mucociliary clearance which is otherwise enhanced by P. aeruginosa as a primary defense in normal lung.

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