The FASEB Journal express article 10.1096/fj.05-4454fje. Published online January 12, 2006.
Flagellin of Pseudomonas aeruginosa inhibits Na+ transport in airway epithelia Karl Kunzelmann,* Kerstin Scheidt,* Birgit Scharf,† Jiraporn Ousingsawat,* Rainer Schreiber,* Brandon Wainwright,‡ and Brendan McMorran‡ *Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg Germany; †Institut für Genetik, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany; 3Institute for Molecular Bioscience, The University of Queensland, Australia Corresponding author: Karl Kunzelmann, Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany. E-mail: [email protected]
ABSTRACT Pseudomonas aeruginosa causes severe life-threatening airway infections that are a frequent cause for hospitalization of cystic fibrosis (CF) patients. These Gram-negative pathogens possess flagella that contain the protein flagellin as a major structural component. Flagellin binds to the host cell glycolipid asialoGM1 (ASGM1), which appears enriched in luminal membranes of respiratory epithelial cells. We demonstrate that in mouse airways, luminal exposure to flagellin leads to inhibition of Na+ absorption by the epithelial Na+ channel ENaC, but does not directly induce a secretory response. Inhibition of ENaC was observed in tracheas of wild-type mice and was attenuated in mice homozygous for the frequent cystic fibrosis conductance regulator (CFTR) mutation G551D. Similar to flagellin, anti-ASGM1 antibody also inhibited ENaC. The inhibitory effects of flagellin on ENaC were attenuated by blockers of the purinergic signaling pathway, although an increase in the intracellular Ca2+ concentration by recombinant or purified flagellin or whole flagella was not observed. Because an inhibitor of the mitogen-activated protein kinase (MAPK) pathway also attenuated the effects of flagellin on Na+ absorption, we conclude that flagellin exclusively inhibits ENaC, probably due to release of ATP and activation of purinergic receptors of the P2Y subtype. Stimulation of these receptors activates the MAPK pathway, thereby leading to inhibition of ENaC. Thus, P. aeruginosa reduces Na+ absorption, which could enhance local mucociliary clearance, a mechanism that seem to be attenuated in CF. Key words: EnaC • flagella • cystic fibrosis • infection
seudomonas aeruginosa is a common opportunistic pathogen that causes chronic airway infections in immunocompromised hosts, such as patients with cystic fibrosis (CF). Successful colonization of airways by P. aeruginosa is due mainly to the bacterium’s ability to bind to respiratory cells and mucins, as well as the production of various P. aeruginosa toxins, and cellular invasion (1). Binding of P. aeruginosa to the cell membrane stimulates airway epithelial cells by activation of intracellular second messenger pathways. One of the most important P. aeruginosa factors involved in initial cell stimulation is the protein Page 1 of 22 (page number not for citation purposes)
flagellin, the major constituent of flagella. Indeed, flagella play a central role in the pathogenesis of P. aeruginosa pulmonary infection (2). The flagellin proteins from many different species of bacteria, including P. aeruginosa, share conserved structural components that act as immunostimulatory ligands (3). P. aeruginosa flagellin binds to several membrane receptors, including the class of pathogen-associated pattern-recognition receptors Toll-like receptors (TLR)-2 and TLR-5, and the GalNAcβ1-4Gal moiety exposed on asialoGM1 (ASGM1) on the surface of airway epithelial cells, but attachment to the ASGM1 receptor appears to be the initial event (4, 5). Binding of flagellin to ASGM1 induces translocation of toll-like receptors (TLRs) from the basolateral membrane or subapical compartments to the luminal cell membrane (4, 6, 7). Formation of these receptor-containing signaling complexes in apical caveolin-associated lipid rafts largely potentiates the signaling capabilities of the participating receptors (7). Additionally, binding of this pathogen leads to activation of sphingomyelinase and a release of ceramide into membrane rafts, causing formation of large ceramide-enriched membrane platforms. Ultimately, this may lead to internalization of the pathogen and apoptotic cell death (8). P. aeruginosa activates the CD95/CD95 (Fas/Fas-ligand) system and thus P. aeruginosainfected cells undergo apoptosis, thereby limiting the spread of infection and the development of sepsis (9). Many cellular events are triggered by the binding of P. aeruginosa to epithelial cells, including activation of the transcription factor NF-κB and expression of the inflammatory cytokine IL-8 and the major mucin MUC-2 (10) Flagellin -to ASGM1 interaction is central in these events (10). It has been demonstrated that binding of flagellin from P. aeruginosa to ASGM1 induces a whole cascade of intracellular second messengers, including Ca2+, Src, Ras, ERK1/2, mitogenactivated protein kinase (MAPK), and NF-κB (3, 4, 11, 12). Since it has been demonstrated that several of these second messengers affect ion transport in epithelial cells (13), we examined in the present report whether transport properties of airway epithelial cells are changed during exposure to whole P. aeruginosa and purified flagellin. Previous studies have suggested that heat stable glycolipids secreted from P. aeruginosa decrease active Na+ absorption and reduce Cl– unidirectional flux (14). Another study demonstrated that purified rhamnolipids, tensoactive glycolipids secreted by P. aeruginosa, reduce epithelial Na+ absorption without affecting secretory properties in sheep tracheal epithelium (15). In contrast, P. aeruginosa exotoxin A was suggested to increase fluid absorption in rat respiratory tract (16). Finally, using P. aeruginosa mutants lacking exotoxin A, rhamnolipids, or lipopolysaccharides, an inhibition of fluid absorption was observed in bovine trachea and inhibition of basolateral K+ channels was proposed as the underlying mechanism (17). In addition, in human conjunctiva epithelial (Chang) cells, P. aeruginosa was shown recently to activate an outwardly rectifying Cl– channel (18). Finally, infection with P. aeruginosa reduces expression of ENaC in mouse lung (19). In light of these findings and the discovery that binding of flagellin to ASGM1 receptors is a very early event during infection of airways with P. aeruginosa, we examined whether the activity of ion channels is changed during exposure to flagella and concomitant activation of intracellular signaling. We find that flagellin primarily affects epithelial Na+ channels, thereby switching epithelial transport from absorption toward secretion, which may promote mucociliary clearance of the pathogen.
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MATERIALS AND METHODS Isolation of flagella and mouse infection Flagella were isolated according to Feldman et al. (2). In brief, P. aeruginosa PAO/NP (PAO1pilA), PAO1flicC, and PAO1fliC-minus lacking flagella, were grown for 48 h and then centrifuged at 5000 rpm for 10 min. The pelleted cells were resuspended in 100 ml of 1× SSC buffer and flagella were sheared by blending for 2 min. Afterward, bacteria were removed by centrifugation at 10,000 g for 15 min. The resulting supernatant was collected, and NaCl was added to 0.5 M and stored for 18 h at 4°C in the presence of 1% w/v polyethylene glycol (m.w. 15,000–20,000). After centrifugation at 7000 g for 20 min, the resulting pellet was resuspended in 3 ml of 10% (NH4)2SO4 and incubated at 4°C for 2 h. The solution was centrifuged again for 20 min at 7000 g, and the pellet was collected and resuspended in 1 ml of ddH2O and dialyzed against phosphate-buffered saline (PBS) for 24 h. The purity of the dialysis content was checked by demonstration of a single 53 kDa protein band on a SDS-polyacrylamide gel stained with Coomassie blue, and MALDI-TOF mass spectrometry. Mice, homozygous for the Cftr-G551D mutation (CF), or wild-type strain C57BL/6 (Charles River Laboratories, Wilmington, MA) were infected with P. aeruginosa using the agar bead protocol described in detail in McMorran et al. (20). In brief, mice were anesthetized, and a 50 µl inoculum containing 5 × 104 CFU of viable P. aeruginosa entrapped in agar beads was introduced into the lungs via the trachea. Infected mice were killed 3 days after infection, and their tracheas were subjected to Ussing chamber measurements. Cell culture The human bronchial epithelial cells 9HTEo– and 16HBE14o– were kindly provided by D. C. Gruenert (California Pacific Medical Center Research Institute, San Francisco, CA). T84– and HT29– colonic carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured on cell culture plastic ware (Greiner, Frickenhausen, Germany) coated with collagen and fibronectin (Vitrogen, Palo Alto, CA). 16HBE14o–, 9HTEo–, T84–, and HT29– cells were grown in modified Eagle culture media containing: 5 mmol/l D-glucose, 20 mmol/l D-galactose, 2 mmol/l L-glutamine, 100 g/l fetal calf serum, 100 mg/l penicillin/streptomycin. Cells were grown at 37°C in an atmosphere of 5% CO2 and 95% O2. Immunocytochemistry of ASGM1 Tracheas were dissected, fixed in PBS-buffered 4% paraformaldehyde, and embedded frozen in optimum cutting temperature compound (Sakura Funetek, Torrance, CA). Transverse sections (20 µm) were immunostained with polyclonal rabbit anti-ASGM1 (Wako Chemicals, Osaka, Japan), diluted 1/100 in PBS/2% horse serum/0.2% Triton X100, followed by anti-rabbit IgGAlexa Fluor 488 conjugate (Molecular Probes, Eugene, OR) antibody diluted 1/600. Control staining was performed using nonimmunized rabbit serum instead of anti-ASGM1.
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Ussing chamber recordings Tracheas were removed from mice after sacrificing the animal by cervical dislocation. After removing connective tissues, we longitudinally cut the tracheas and divided them into two pieces. Tissues were put immediately into an ice-cold buffer solution of the following composition (in mmol/l): NaCl 145, KCI 3.8, D-glucose 5, MgCI2 1, HEPES 5, Ca gluconate 1.3. The tissues were mounted into a perfused micro-Ussing chamber with a circular aperture of 0.95 mm2 as described previously (21). The apical and basolateral surfaces of the epithelium were perfused continuously at a rate of up to 10 ml/min (chamber volume 2 ml). The bath solution contained (in mmol/l): NaCl 145, KH2PO4 0.4, K2HPO4 1.6, D-glucose 5, MgCl2 1, HEPES 5, Ca gluconate 1.3. The pH was adjusted to 7.4, and all experiments were carried out at 37°C. Experiments were performed under open-circuit conditions. Transepithelial resistance (Rte) was determined by applying short (1 s) current pulses (ΔI=0.5 µA), and the corresponding changes in Vte (ΔVte) and basal Vte were recorded continuously. Values for the transepithelial voltage (Vte) were referred to the serosal side of the epithelium. The equivalent short-circuit current (Isc) was calculated according to Ohm’s law from Vte and Rte (Isc=Vte/Rte). After the tissues were mounted in Ussing chambers, an equilibration time of 30 min was allowed for stabilization of basal Vte and Rte. Patch-clamp experiments Cell culture dishes were mounted on the stage of an inverted microscope (IM35, Zeiss, Oberkochen, Germany) and kept at 37°C. The bath was continuously perfused with Ringer solution at a rate of ~10 ml/min. Patch-clamp experiments were performed in the fast whole-cell configuration. The patch pipettes had an input resistance of 2–4 MΩ when filled with a solution containing (in mmol/l) KCl 30, K gluconate 95, NaH2PO4 1.2, Na2HPO4 4.8, EGTA 1, CaCl2 0.726, MgCl2 1.034, D-glucose 5, ATP 1 (32 Cl). The pH was adjusted to 7.2, and the Ca2+ activity was 0.1 µmol/l. The access conductance was measured continuously and was between 30 and 120 ns. Currents (voltage clamp) and voltages (current clamp) were recorded using a patchclamp amplifier (EPC 7, List Medical Electronic, Darmstadt, Germany), and data were stored continuously on a computer hard disc. At regular intervals, membrane voltages (Vc) were clamped in steps of 10 mV from –100 mV to +40 mV. Measurement of the intracellular calcium concentration For fluorescence measurements, cells were perfused with Ringer solution (in mmol/l: NaCl 145, KH2PO4 0.4, K2HPO4 1.6, glucose 5, MgCl2 1, Ca2+ gluconate 1.3) at 37°C. Cell fluorescence was measured continuously using an inverted microscope IMT-2 (Olympus, Hamburg, Germany) and a high-speed polychromator system (VisiChrome, Visitron Systems, Puchheim, Germany). Cells were loaded with 5 µM Fura-2 AM (Molecular Probes) in OptiMEM (Invitrogen, Carlsbad, CA) with 0.02% pluronic (Molecular Probes) for 1 h at room temperature. Fura-2 was excited at 340/380 nm, and emission was recorded between 470 and 550 nm using a CCD camera (CoolSnap HQ, Visitron Systems). Experiments were controlled and analyzed using the software package Meta-Fluor (Universal Imaging, Molecular Devices, Sunnyvale, CA). All optical filters and dichroic mirrors were from AHF (Tübingen, Germany).
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Other materials and statistical analysis All compounds used were of highest available grade of purity. 3-Isobutyl-1-methylxanthine (IBMX), forskolin, ATP, PPADS, suramin, hexokinase, imipramine, neomycin, DIDS, BIM, BAPTA-AM, and amiloride were all from Sigma (Deisenhofen, Germany). U-73122, U-0126, and SB-203580 were from Calbiochem (Nottingham, UK). Azosemide was a generous gift from Aventis (Frankfurt, Germany). The recombinant P. aeruginosa flagellin was from Inotek Laboratories (London, UK) and was used at concentrations of 5–10 µg/ml. Student’s t test values of P < 0.05 were considered to be statistically significant. RESULTS ASGM1 in mouse airways and inhibition of amiloride-sensitive transport by flagellin According to previous studies, membranes of human airway epithelial cells contain ASGM1 (4, 22). Here we examined ASGM1 expression in airway epithelial cells of tracheas from both wildtype mice and mice homozygous for the common CFTR mutation G551D (CF mice). Using an anti-ASGM1 antibody, we detected the presence of ASGM1 in the luminal and basolateral membrane of epithelial cells in both mouse strains (Fig. 1A, 1B). Although previous reports indicate increased ASGM1 expression in human CF epithelial cell lines (23), we observed no significant difference in staining intensity between wild-type and CF trachea. Tracheas were prepared from killed mice and mounted into perfused micro-Ussing chambers. Tracheas of wild-type mice demonstrated a spontaneous transepithelial voltage (Vte) of –10.2 ± 2.1 mV, a calculated equivalent short-circuit current (Isc) of –242.2 ± 26.1 µA/cm2, and a transepithelial resistance of 42.1 ± 5.1 Ωcm2 (n=14). Amiloride (10 µmol/l) reduced the transepithelial voltage reversibly (Fig. 2A). Tracheas were exposed for 1 h to 5 µg/ml of flagellin from P. aeruginosa, which reduced the transepithelial voltage (Vte) from –10.8 ± 1.9 to –7.2 ± 1.6 mV (n=5) by inhibiting the amiloride-sensitive short-circuit current (Fig. 2A, 2D). In contrast, transepithelial voltage and amiloride-sensitive Na+ transport were unaffected following 1 h exposure to control buffer solution (Fig. 2C, 2D). These results indicate inhibition of electrogenic amiloride-sensitive Na+ absorption in mouse trachea by flagellin. As shown in Fig. 2B, the inhibitory effect was concentration-dependent, with a half maximal effect at a concentration of 0.1 µg/ml. Because flagellin was prepared from P. aeruginosa, and could therefore still contain other factors released by P. aeruginosa, we tested recombinant flagellin from a commercial source (Inotek). An inhibitory effect on amiloride-sensitive Na+ absorption of similar magnitude was observed with this preparation (Fig. 2E, 2F). Moreover, whole intact flagella were able to inhibit amiloride-sensitive Na+ absorption (Fig. 2F). Effects of flagellin in CF mouse tracheas and tracheas from P. aeruginosa-infected mice Similar experiments were performed on tracheas from transgenic mice homozygous for the frequent CFTR mutation G551D (24). Tracheas from cftrG551D/G551D mice demonstrated a spontaneous transepithelial voltage of –16.2 ± 1.9 mV, a calculated Isc of –292.2 ± 26.1 µA/cm2, and a transepithelial resistance of 55.2 ± 4.1 Ωcm2 (n=5), which is consistent with previous recordings made with this mouse strain. Luminal incubation with flagellin attenuated the effect of amiloride on the transepithelial voltage and significantly inhibited Isc-Amil. But surprisingly, the
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inhibitory effects on amiloride-sensitive Na+ absorption were reduced when compared with wildtype mice (Fig. 3A, 3B). To see whether chronic in vivo infection with P. aeruginosa also interfered with the transport properties of the tracheal epithelium, we infected C57BL/6 mouse lungs with P. aeruginosa entrapped in agar beads for 3 days. Tracheas from infected mice showed a transepithelial voltage of –17.2 ± 2.7 mV, a calculated Isc of –263.2 ± 26.1 µA/cm2, and a transepithelial resistance of 50.2 ± 5.3 Ωcm2 (n=4). In contrast to what we expected, amiloride-sensitive transport was not reduced when compared with noninfected tracheas, and the inhibitory effect of flagellin on IscAmil was comparable to that in noninfected animals. Thus, ~50% of the initial Isc-Amil was inhibited by flagellin in tracheas of both infected and noninfected animals (Fig. 3C, 3D). Moreover, in another series of experiments, mouse airways were infected with a FliC mutant strain of P. aeruginosa, which does not produce flagellin. The Isc-Amil detected in the tracheas of these animals under control conditions was not different to the control animals and was reduced by 54% (n=4), which was similar to the effects in the tracheas of mice infected with the nonmutant Pseudomonas strain. As reported recently for the chronically infected human CF lung, expression of flagellin by P. aeruginosa may be turned off, and therefore Na+ absorption may remain sensitive toward inhibition by flagellin (25). Effects of flagellin are due to binding to ASGM1 receptors and does not cause any secretory responses To prove that flagellin in fact acts on ASGM1 glycolipids, we first incubated tracheas with an ASGM1 antibody (1:100, α-ASGM1), which significantly inhibited Isc-Amil (Fig. 4A). Subsequent incubation with flagellin did not further reduce Isc-Amil, confirming that flagellin acts through binding to ASGM1 glycolipid receptor. Basolateral application of α-ASGM1 or flagellin had no inhibitory effects on Isc-Amil (Fig. 4B), although ASGM1 receptors are present on the basolateral surfaces of respiratory epithelium (see Fig. 1). We further examined if flagellin also affects other membrane conductances, apart from amiloride-sensitive Na+ absorption. To that end, we examined electrolyte transport after inhibition of Na+ absorption by amiloride, which completely abolished Isc in tracheal epithelia (Fig. 4C). This indicates that mouse trachea is dominated by Na+ absorption. Inhibition of the basolateral Na+K+2Cl– cotransporter NKCC1 by basolateral application of azosemide (100 µmol/l) or luminal application of the inhibitor of Ca2+-activated Cl– channels, DIDS (100 µmol/l) did not show any effects either before or after 1 h incubation with flagellin (Fig. 4C). Using a microapplication pipette in patch-clamp experiments, we applied in close proximity to human bronchial epithelial cells (16HBE14o–) flagellin (5 µg/ml) or ATP (1 µmol/l). We found that ATP, but not flagellin, activated a whole-cell conductance due to activation of Ca2+-activated K+ and Cl– channels (26) (Fig. 4D, 4E). Taken together, these results show that ~50% of the amiloride-sensitive electrolyte absorption is inhibited by flagellin, whereas no significant secretory transport is activated by flagellin in mouse trachea or human airway epithelial cells. Mechanism for the inhibition of amiloride-sensitive transport by flagellin We further examined the mechanisms underlying the inhibition of Isc-Amil by flagellin. It has been reported previously that binding of flagellin to ASGM1 glycolipids leads to a cellular release of ATP and subsequent activation of purinergic receptors (10). We speculated that inhibition of Na+
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absorption by flagellin may be due to autocrine stimulation of purinergic receptors located in the luminal side of airway epithelial cells. We therefore exposed mouse airways to flagellin in the presence of two different inhibitors of purinergic receptors, suramin and PPADS (both 100 µmol/l). Both compounds reduced the inhibitory effect of flagellin on Isc-Amil. Moreover, we found that scavenging of ATP, presumably secreted by airway epithelial cells, with hexokinase (5 U/ml) and glucose (15 mmol/l) in the apical perfusate, also attenuated the inhibitory effects of flagellin on Isc-Amil. Tracheas of this batch of mice generally showed a lower Isc-Amil. However, also in these tracheas, luminal application of 5 µg/ml flagellin under control conditions inhibited Isc-Amil from 116 ± 8.2 to 57 ± 8.5 µA/cm2 (n=4). This suggests that flagellin inhibits ENaC through release of ATP and binding of ATP to luminal ATP receptors, similar to what has been demonstrated previously for the inhibition of ENaC by parainfluenza virus (27) (Fig. 5A). Increase of intracellular Ca2+ by basolateral but not luminal application of flagellin We further examined if binding of flagellin to airway epithelial cells causing a putative release of ATP, triggers an increase in the intracellular Ca2+ concentration. Luminal application of 5 µg/ml of recombinant flagellin to normal (16HBE; Fig. 5B) or CF (CFDE; Fig. 5C) epithelial cells did not increase intracellular Ca2+, whereas application of 1 µmol/l ATP showed a clear increase in [Ca2+]i. Similar results were obtained with purified flagellin or whole flagella. In contrast to luminal application, basolateral application of purified flagellin induced a small but reproducible increase in [Ca2+]i, which confirms results of previous studies (28) (Fig. 5D). Although no global changes in cytosolic Ca2+ were observed upon luminal application of flagellin, it is entirely possible that a localized increase in intracellular Ca2+ remains undetected by standard imaging techniques (29) (see Fig. 7C, 7D). Moreover, membrane adherent ecto-5′-nucleotidase activity may cleave a significant portion of the secreted ATP, and the downstream products ADP, AMP, and adenosine may act through binding to P2Y6 and other purinergic receptors, which are inhibited by the compound PPADS (Fig. 5A) (30). Inhibition of ENaC by phosphatidylinositol bisphosphate (PIP2) hydrolysis and activation of MAPKs Stimulation of purinergic receptors activates multiple signaling molecules, such as phospholipase C (PLC) and MAPKs (5). To test the contribution of these signaling pathways to Na+ absorption by flagellin, we incubated mouse tracheas with the inhibitor of phospholipase Cβ, U-73122 (10 µmol/l, 15 min), which significantly attenuated the inhibitory effects of flagellin on Isc-Amil (Fig. 6A). A similar effect was observed when tracheas were incubated for 1 h with 5 mmol/l neomycin, which competes with PLC for binding to PIP2. In contrast, imipramine (100 µmol), an activator of PLC (31), reduced Isc-Amil, and Isc-Amil was only slightly further inhibited by subsequent flagellin (Fig. 6A). Chelation of intracellular Ca2+ by BAPTA-AM (10 µmol/l) also interfered with the inhibitory effects of flagellin on Na+ absorption, whereas inhibition of protein kinase C by bisindolylmaleimide (BIM, 1 µmol/l) had no significant effects (Fig. 6B). Furthermore, the inhibitory effects of flagellin on Na+ absorption were reduced significantly by the P44/42 kinase inhibitor U-0126 (25 µmol/l), but not by the P38 kinase inhibitor SB-203580 (25 µmol/l) (Fig. 6C) The inhibitory effect of flagellin on Isc-Amil was completely abolished by simultaneous inhibition of purinergic receptors and the P44/42 kinase.
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We finally examined the direct effects of luminal application of ATP on Na+ absorption and intracellular Ca2+. Maximal stimulation of luminal purinergic receptors by 100 µM ATP induced a transient Cl– secretion and inhibited amiloride-sensitive Na+ absorption (Fig. 7A, 7B). ATP (100 µM) also induced a large increase in intracellular Ca2+ (Fig. 5, 7C). However, ATP concentrations as low as 1 or 10 nM did not produce measurable Ca2+ increases (Fig. 7C). Despite of the lack of detectable Ca2+ increase, long lasting (1 h) stimulation by 1 nM luminal ATP significantly inhibited amiloride-sensitive Na+ absorption (Fig. 7D). Taken together, these results suggest that binding of flagella from P. aeruginosa to ASGM1 glycolipids inhibits electrogenic Na+ absorption by inhibition of epithelial Na+ channels through ATP release, activation of purinergic receptors, which induces PIP2 hydrolysis, and activation of the MAPK pathway. DISCUSSION P. aeruginosa causes severe lung infections in patients with CF. The pathogen binds to airway mucus and to airway epithelial cells and binding to both is enhanced in patients with CF (4, 5, 32). In contrast to the gastrointestinal mucosa, where defense mechanisms are generally activated only in response to invasive pathogens and through another set of TLRs, the airway epithelium does not tolerate chronic exposure to bacteria (33). The present study examined the effects of flagellin from P. aeruginosa on electrolyte transport in the airways. Flagellin has been demonstrated recently to bind to ASGM1 glycolipid receptors and to induce mucin transcription via activation of PLC, Ca2+ mobilization, and phosphorylation of a MAPK (10). Apart from the glycolipid receptor, flagellin is also recognized by TLR2 and TLR5, but not TLR4 (4). Notably, in preliminary experiments with TLR-4 (−/−) mice, we saw a similar down-regulation of the amiloride-sensitive Na+ transport by luminal application of flagellin as in wild-type mice (data not shown). Apart from the inhibition of ENaC by luminal flagellin, basolateral application did not affect electrogenic Na+ absorption, although intracellular Ca2+ was increased by basolateral flagellin. This is somehow different from the pro-inflammatory effects in the intestine induced by basolateral flagellin from enteropathogenic Salmonella (34). We found reduced inhibition of amiloride-sensitive Na+ absorption by flagellin of P. aeruginosa in the cftrG551D/G551D mice, lacking functional CFTR. This remains currently unexplained since expression of membrane ASGM1 receptors appeared not reduced in cftrG551D/G551D mice when compared with wild-type animals. Moreover, results obtained in the mouse respiratory tract cannot be extrapolated linearly to human airways, since Na+ absorption is not enhanced in tracheas of CF mice and because expression of CFTR in mouse airways is largely reduced when compared with human airways (35). We demonstrated previously that enhanced P. aeruginosa burden in the airways of cftrG551D/G551D mice was corrected by overexpression of wild-type CFTR, which enhanced fluid transport and normalized pathogen clearance (36). Along this line, it has been proposed that CFTR serves as a receptor for P. aeruginosa, thereby helping to clear pathogens from the respiratory tract (37). In the present study, we found a similar inhibition of Na+ absorption in tracheas of P. aeruginosainfected animals (38). Notably, previous studies showed that flagellin expression by P. aeruginosa is shut off during chronic airway infection and when in contact with the airway surface liquid of CF patients. This response was interpreted as an adaptive response, since it allows the pathogen to escape from detection by host pattern recognition receptors (25). A similar phenomenon may take place in the chronically infected animals in the present study.
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Thus, despite the fact that flagellin inhibits ENaC, this mechanism may be shut off in the Pseudomonas-infected CF lung, thereby allowing for continuous high Na+ absorption. According to the present data and previous studies, flagellin induces a release of ATP from airway epithelial cells. Higher concentrations of ATP and flagellin clearly induce Ca2+ increases (4, 10, 27). In agreement with a previous report, we only detected an increase in intracellular Ca2+ by basolateral application of flagellin (27). Nevertheless, a small and nondetectable increase of [Ca2+]i may occur through both luminal flagellin or low concentrations of ATP (Fig. 5, 7). According to previous reports, ATP release by airway epithelial cells is likely to stay clearly below 100 nM, which may therefore lead to only minimal changes in cytosolic Ca2+, nondetectable in standard fluorescence imaging (4, 10). Even by sensitive patch-clamp techniques, we did not detect a Ca2+-dependent activation of ion channels due to flagellin. However, previous studies demonstrated that IL-8 and mucin responses evoked by flagellin were blocked in the presence of the intracellular Ca2+ chelator BAPTA/AM, indicating that flagellin acts on the intracellular Ca2+-signaling cascade (4, 10). Also in the present study, inhibition of ENaC by flagellin was attenuated in the presence of BAPTA/AM, suggesting a contribution of Ca2+ to the inhibitory effects on ENaC. Stimulation of purinergic receptors leads to hydrolysis of PIP2, which reduces binding of ENaC to the phospholipid and thus decreases the open probability of the channel (22, 39–41). The present data supply evidence that such a mechanism is in charge of the inhibition of ENaC by flagellin. However, we also present data showing that ENaC is inhibited through activation of the MAPK ERK1/2 (42). In fact, stimulation of purinergic P2Y2 receptors, which are most abundant on airway epithelial cells, activates the p42/p44 (ERK1/2) MAPK pathway (42). ERK-mediated phosphorylation facilitates interaction between the ubiquitin ligase Nedd4 and β/γ-subunits of EnaC, which down-regulates Na+ channel activity (43–46). Ubiquitination leads to retrieval of ENaC from the plasma membrane, a process that is antagonized by phosphorylation of Nedd4-2 through serum- and glucocorticoid-dependent kinase (Sgk1) (47). Flagellin acts on Na+ conductance by induction of ATP release from epithelial cells by a still unknown release mechanism. ATP release via CFTR-dependent mechanisms is a controversial issue (29, 48–51). It is, however, well accepted that epithelial cells release ATP under baseline conditions and upon mechanical stress stimuli (29, 52–55). ATP release may even be enhanced during host cell killing by pathogens (56). A substantial portion of the released ATP is rapidly hydrolyzed by ecto-ATPases, and adenosine is formed through further action of ecto-5′nucleotidase. Adenosine binds to apical A2B adenosine receptors, which activates luminal CFTR receptors and basolateral cAMP-activated K+ channels, thereby enhancing the secretory response (29, 57). Conversion to adenosine and subsequent activation of CFTR might be difficult to detect with our continuously perfused setup since products of ecto-enzymatic activity may be washed away. However, the importance of the ecto-5′-nucleotidase in purinergic activation of fluid secretion is demonstrated in ecto-5′-nucleotidase knockout mice (58), which show largely reduced purinergic airway secretion (unpublished data). As inhibition of epithelial Na+ transport by flagellin was also blocked by PPADS, other P2Y receptors such as P2Y6 are involved in the flagellin response (59, 60). What are the clinical implications of the flagellin-sensitive airway Na+ absorption? It has been suggested that decreased fluid absorption induced by P. aeruginosa leads to a buildup of mucus, Page 9 of 22 (page number not for citation purposes)
which could reduce clearance (17). However, our data and those of others (52) are consistent with an alternative scenario, based on a compartmentalized autocrine signaling mechanism. This involves host pathogen receptors, ATP release that activates purinergic receptors, and activity of ecto-ATPase and ecto-5′-nucleotidase as well as ion channels. We propose that pathogens such as P. aeruginosa induce a rapid and localized release of ATP, resulting in localized generation of intracellular messengers that leads to reduced electrolyte absorption and thus enhances clearance of the pathogen from the airways. ACKNOWLEDGMENTS Supported by German Mukoviszidose e.V. and Else Kröner-Fresenius-Stiftung. We gratefully acknowledge the supply of the human airway epithelial cell lines 16HBE140– and CFDE by Prof. Dr. D. Gruenert (California Pacific Medical Center Research Institute, San Francisco, CA). REFERENCES 1.
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Figure 1. Immunocytochemistry of the glycolipid receptor asialoGM1. Transverse sections of trachea from WT and CF (G551D) mice were stained with the anti-ASGM1 or control antibodies and viewed under low (A) and high (B) magnifications.
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Figure 2. Inhibition of airway epithelial Na+ absorption by flagellin. A) Original Ussing chamber recordings of the transepithelial voltage measured in the trachea of a non-CF mouse. Amiloride (10 µmol/l) inhibits the transepithelial voltage by blocking epithelial Na+ absorption via ENaC. Subsequent incubation of the luminal side of the tissue by flagellin from Pseudomonas aeruginosa (5 µg/ml) attenuates the effects of amiloride on Vte, indicating the inhibition of amiloride-sensitive Na+ absorption by flagellin. B) Concentration response curve for the inhibitory effect of flagellin on IscAmil. C) Ussing chamber recordings of Vte and effects of amiloride before and after 1 h incubation by luminal buffer and in the absence of flagella. D) Summaries of the experiments shown in A and C. E, F) Original recordings and summary of the effects of recombinant flagellin and whole flagella on amiloride-sensitive Na+ absorption in mouse trachea. *Significant effects of amiloride (paired t test); (n) = number of experiments. Page 17 of 22 (page number not for citation purposes)
Figure 3. Inhibition of epithelial Na+ absorption by flagellin in the airways of CF mice. A) Original Ussing chamber recordings of the transepithelial voltage measured in the trachea of a homozygous G551D-mouse. Amiloride (10 µmol/l) inhibited the transepithelial voltage by blocking epithelial Na+ absorption via ENaC. Subsequent incubation of the luminal side of the tissue by flagella from Pseudomonas aeruginosa (5 µg/ml) attenuated the effects of amiloride on Vte; 0 indicates voltage zero after transient removal of the insert carrying the tissue from the chamber. B) Summaries of the experiments shown in A. C) Ussing chamber recording of Vte measured in a trachea of a mouse infected with P. aeruginosa. Amiloride (10 µmol/l) inhibits the transepithelial voltage by blocking ENaC. Subsequent incubation of the luminal side of the tissue by flagellin from P. aeruginosa (5 µg/ml) attenuates the effects of amiloride on Vte; 0 indicates voltage zero after transient removal of the insert carrying the tissue from the chamber. D) Summaries of the experiments shown in C. *Significant effects of amiloride (paired t test); #significant difference when compared with the effect of flagellin under control conditions (unpaired t test); (n) = number of experiments. Page 18 of 22 (page number not for citation purposes)
Figure 4. A) Summary of the effects of luminal incubation with the ASGM1 antibody (α-ASGM1; 1:20 dilution) in the absence and presence of flagellin on Isc-Amil. B) Summary of the effects of basolateral application of α-ASGM1 and flagellin on Isc-Amil. C) Summary of the application of luminal amiloride (10 µmol/l), basolateral azosemide (100 µmol/l), or luminal DIDS (100 µmol/l) in the absence or presence of flagellin (5 µg/ml). D, E) Original whole-cell recording and summary of whole-cell patch-clamp experiments with human bronchial epithelial cells. ATP (1 µmol/l) but not flagellin (5 µg/ml) activates a whole-cell conductance and hyperpolarizes the membrane voltage Vm. *Significant effect of α-ASGM1 or ATP (paired t test); (n) = number of experiments. Page 19 of 22 (page number not for citation purposes)
Figure 5. A) Summary of the effects of flagellin (5 µg/ml) on Isc-Amil in the presence of inhibitors of the purinergic signaling pathway. The inhibitors of P2Y2 and P2Y6 receptors (100 µmol/l suramin and PPADS, respectively) antagonized the inhibitory effects of flagellin on Isc-Amil. The ATP scavengers, hexokinase (5 U/ml) and glucose (15 mmol/l), also attenuated the inhibitory effects of flagellin. B, C) Luminal ATP (1 µmol/l) and recombinant flagellin (5 µg/ml) enhanced [Ca2+]i in non-CF (16HBE140–) (B) and CF (CFDE) (C) airway epithelial cells. D) Basolateral application of recombinant flagellin (5 µg/ml) and 100 µM ATP increased [Ca2+]i in non-CF (16HBE140–) epithelial cells. *Significant inhibition of Isc-Amil by flagellin (paired t test); #Significant difference when compared with the effects of flagellin under control conditions (unpaired t test); (n) = number of experiments. Page 20 of 22 (page number not for citation purposes)
Figure 6. A) Summary of the effects of flagellin (5 µg/ml) on Isc-Amil in the presence of the PLC inhibitor U-73122 (10 µmol/l), the PLC- binding compound neomycin (1 mmol/l), and the PLC activator imipramine (100 µmol/l). B) Summary of the effects of flagellin (5 µg/ml) on Isc-Amil in the presence of the Ca2+ chelator BAPTA-AM (BAPTA, 10 µmol/l) and the inhibitor of protein kinase C bisindolylmaleimide (BIM, 0.1 µmol/l). C) Summary of the effects of flagellin (5 µg/l) on IscAmil in the presence of inhibitors of P38 kinase (SB-203580, 25 µmol/l, 30 min preincubation), P44/42 kinase (U-0126, 25 µmol/l, 30 min preincubation), and coapplication of U-0126/suramin. *Significant inhibition of Isc-Amil by flagellin (paired t test), #Significant difference when compared with the effects of flagellin under control conditions (unpaired t test); (n) = number of experiments. Page 21 of 22 (page number not for citation purposes)
Figure 7. Inhibition of airway epithelial Na+ absorption and increase in intracellular Ca2+ by ATP. A) Original Ussing chamber recording of the transepithelial voltage measured in the trachea of a non-CF mouse. Amiloride (A; 10 µmol/l) inhibits the transepithelial voltage by blocking epithelial Na+ absorption via ENaC. Subsequent stimulation with 100 µM ATP induces a transient Cl– secretion and attenuates the effect of amiloride on Vte, indicating the inhibition of amiloridesensitive Na+ absorption by ATP. B) Summaries of the experiments shown in A. C) Effect of luminal application of various concentrations of ATP on intracellular Ca2+. D) Summaries of the experiments shown in C. *Significant effects of amiloride (paired t test); (n) = number of experiments.
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