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Pseudomonas aeruginosa Flagella Activate Airway Epithelial Cells through asialoGM1 and Toll-Like Receptor 2 as well as Toll-Like Receptor 5

Departments of Pediatrics and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York

Address correspondence to: Alice Prince, Departments of Pediatrics and Pharmacology, College of Physicians and Surgeons, Columbia University, BB 416–650 W. 168th Street, New York, NY 10032. E-mail: asp7{at}columbia.edu

	Abstract

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The distribution of specific toll-like receptors and components of the signaling pathways activated by Pseudomonas aeruginosa flagella were studied in airway epithelial cells. Initially flagella bound to the apical surface of polarized epithelial cells, where they prominently colocalized with asialoGM1. By 4 h of exposure to flagella, toll-like receptor (TLR)5 expression was induced, mobilized to the apical surface of the cells, and colocalized with superficial flagella. Interleukin-8 expression in airway cells was activated by flagella through induction of Ca²⁺ fluxes, Src, Ras, and extracellular signal–regulated kinase 1/2 mitogen-activated protein kinase and nuclear factor-B activation, a pathway previously associated with asialoGM1-mediated stimuli. There was evidence for participation of asialoGM1 and TLR2 as well as TLR5 in the response to flagella, and increased asialoGM1 correlated directly with increased signaling. TLR2 DN or TLR5 DN mutations inhibited interleukin-8 induction by 78% and 35%, respectively (P < 0.001 for each). The participation of TLR2 as well as TLR5 was confirmed in Chinese hamster ovary cells transfected with either human TLR2 or TLR5 in which flagella activated a nuclear factor-B–luciferase reporter to the same extent. Flagella signaling in airway cells can be initiated by interactions with asialoGM1 and TLR2 as well as by activation of TLR5. The availability of exposed receptors on the apical surface of polarized airway epithelial cells is a major factor in the activation of signaling pathways by flagella.

Abbreviations: gangliotetraosylceramide (Galß1,2GalNAcß1,4Galß1,4Glcß1,1Cer), asialoGM1 • 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, BAPTA • Chinese hamster ovary, CHO • Dulbecco's modified Eagle's media, DMEM • enzyme-linked immunosorbent assay, ELISA • extracellular signal–regulated kinases(s), ERK(s) • fetal calf serum, FCS • interleukin, IL • interleukin-1 receptor–activated kinase, IRAK • lipopolysaccharide, LPS • mitogen-activated protein kinases, MAPKs • mass spectroscopy, MS • normal human nasal polyp, NHNP • phosphate-buffered saline, PBS • toll-like receptor, TLR

	Introduction

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Mucosal epithelial cells have their own repertoire of innate immune functions, expressing chemokines and cytokines to recruit and activate phagocytic cells in response to pathogens. Epithelial cells respond selectively to specific bacterial components (1, 2), and mucosal cells in different tissues have distinct thresholds for activation in response to commensal or pathogenic bacteria (3). Airway epithelial cells are readily activated by superficial exposure to bacterial ligands (4, 5), as opposed to the mucosal cells of the gastrointestinal tract, which initiate signaling primarily in response to invasive organisms (6). The toll-like receptors (TLRs) mediate such responses (7). Mucosal responses to bacteria depend upon the expression of ligand-specific TLRs and essential coreceptors at the site of infection (8, 9). Unlike nonpolarized cells of hematopoetic origin, the distribution of TLRs in polarized mucosal cells may be important in facilitating their surveillance function. Major differences in proinflammatory signaling events have been observed in polarized epithelial cells as compared with nonpolarized cells. Airway epithelial cells express TLRs as well as CD14 (10). However, to initiate responses to inhaled bacteria, before invasive infection occurs, the appropriate receptors must be present or mobilized to the exposed surfaces of the airway.

Pseudomonas aeruginosa, Staphylococcus aureus, and other pulmonary pathogens bind to the GalNAcß1-4Gal moiety exposed on asialoGM1 (11) on the surface of airway cells and activate epithelial proinflammatory responses though a Ca²⁺-dependent, Src, Ras, extracellular signal–regulated kinase (ERK)1/2, nuclear factor (NF)-B pathway (5). Flagella also bind to gangliotetraosylceramide (Galß1,2GalNAcß1,4Galß1,4Glcß1,1Cer) (asialoGM1) and potently induce airway inflammation (12). P. aeruginosa activate Muc-2 expression through a similar Ca²⁺-dependent signaling pathway (4). The expression of flagella is particularly important for bacterial colonization of the lung, and Fla^– mutants are less virulent in a mouse model of pneumonia (12). P. aeruginosa flagella also induce the expression of matrilysin, a metalloproteinase that functions in host defense and epithelial repair (13). Conserved domains of the major structural component of flagella, FliC, are shared by many different species of bacteria and function as immunostimulatory ligands (14). Salmonella flagella initiate IB degradation, NF-B activation, interleukin (IL)-8 and NO production (15), as well as induce expression of CCL20 in intestinal epithelial cells (16), a chemokine that serves to recruit dendritic cells (17). Salmonella flagella have been recently defined as the dominant bacteria stimulus in the gut mucosa (18).

Compelling data from several experimental systems indicate that TLR5 is responsible for mediating proinflammatory responses to flagella (19). TLR5 has been localized to basolateral compartments of gut epithelial cells (9), where it interacts with flagella that have been internalized, but not those at the apical surface (20). In monocytes lacking polarity, the activation of TLR5 signaling by flagella has been documented by demonstration of IL-1 receptor–activated kinase (IRAK) phosphorylation (21). Similarly, flagella activate NF-B reporters in a heterologous system consisting of Chinese hamster ovary (CHO) cells transfected with human TLR5, suggesting that TLR5-mediated signaling is sufficient to provide downstream gene activation (19). The flagellin-binding domain on TLR5 was recently defined using COS-1 cells, documenting a direct TLR5–flagellin interaction (22). However, in airway cells and gut epithelial cell lines flagella induce signaling through the induction of Ca²⁺ fluxes (4), a response not previously associated with TLR5, but typical of ligands that activate asialoGM1 signaling (5). Thus, flagella could activate airway cells through direct binding to TLR5, if available at the exposed surfaces of the cells, or through ligation of asialoGM1 and the activation of mitogen-activated protein kinases (MAPKs).

To clarify how airway cells signal in response to bacterial flagella, we studied the distribution of asialoGM1, TLR2, and TLR5 in airway epithelial cells, and characterized the signaling pathway activated. We postulated that the apical presentation of asialoGM1 could contribute to epithelial signaling in response to flagella stimulation.

	Materials and Methods

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Isolation of P. aeruginosa Flagella
PAO/NP(pilA), a pilin mutant previously constructed by gene replacement (23), and PAO/NP/fliA, a flagella mutant derived from PAO/NP (used here as a control for possible contaminants in the preparation of flagella) (12), were used to inoculate 1-liter cultures in M9 media, which were grown with aeration for 48 h, harvested by centrifugation, and resuspended in 100 ml of SSC buffer. Flagella were sheared off by blending for 2 min at low speed (whip) in a Waring blender. Bacteria were removed by centrifugation at 10,000 x g for 15 min, and the resulting supernatant was collected and adjusted to 0.5 M with NaCl. Polyethylene glycol (m.w. 15,000–20,000) was added to a final concentration of 1.0%. After 18 h at 4°C, the supernatant was centrifuged at 7,000 x g for 20 min. The pellet was resuspended in 3 ml of 10% (NH₄)₂SO₄ and left at 4°C for 2 h. This solution was centrifuged (20 min at 7,000 x g), the pellet collected and resuspended in 1 ml of dH₂O and dialyzed against phosphate-buffered saline (PBS) for 24 h. Aliquots were assayed for protein content and purity checked by electrophoresis and demonstration of a single 50-kD protein band on a sodium dodecyl sulfate–polyacrylamide gel stained with Coomassie blue, and identified with anti-flagellin antisera by Western hybridization.

The flagella preparations from PAO/NP and the PAO/NP/fliA strains were analyzed by mass spectrometry (MS) to assess purity. The intact preparations were subjected to matrix-assisted laser desorption ionization MS, using a PerSeptive Voyager DE-RP mass spectrometer (PerSeptive Biosystems, Framingham, MA), showing only one protein at 49.5 kD in the PAO/NP preparation and none in the preparation from the flagellin null mutant. The purity was further probed by running a Nano-lc/MS/MS on the trypsin-digested samples. Briefly, 6 µg of the digestion mixtures were separated by an LC Packings (Sunnyvale, CA) nano-lc (200 nL/min flow rate) and injected directly into the nanoelectrospray source of a Micromass Q-Tof hybrid quadrupole/time-of-flight mass spectrometer (Micromass Inc., Beverly, MA). Processed files were then submitted to a MASCOT search at www.matrixscience.com, and sequences checked against the NCBInr database. The sample was identified conclusively as flagellin from P. aeruginosa with 24 peptide matches. There were two minor contaminants found: flagellar capping protein FliD, with three peptide matches, and elongation factor Tu, with two peptide matches. Again, there were no proteins found in an equal volume of the sample derived from the flagellin null mutant.

Cell Culture and Reagents
1HAEo^– cells, SV40 immortalized human airway epithelial cells whose properties have been well characterized (23) and 16HBE cells, SV40 transformed human bronchial epithelial cells, obtained from D. Gruenert (University of Vermont, Burlington VT), were grown in Minimum Essential Medium with Earle's salts supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA) (24). Epithelial cells were isolated from normal human nasal polyp (NHNP) tissue using the protease method and grown in primary culture in a polarized fashion at an air–liquid interface in M3 with antibiotics as previously detailed (23). Unless specified, reagents were purchased from Sigma (St. Louis, MO). CHO-K1 cells obtained from ATCC were grown in Kaighn's modification of Ham's F12 medium (Invitrogen) with 2 mM L-glutamine supplemented with 1.5 g/liter sodium bicarbonate and 10% fetal bovine serum. All media used were supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, and 4 µg/ml amphotericin B. Two cell lines as well as airway cells in primary culture were used because no single airway epithelial cell line has all the necessary properties (tight junctions, apical-baso lateral polarization, low endogenous NF-B activity, and transfectability).

Cells at 70–80% confluence were transfected in 96-well plates using FuGENE 6 reagent (Roche, Indianapolis, IN) with 0.5 µg/ml of Src-RF, RasN17 (Jian Dong Li, House Ear Institute, Los Angeles, CA), PyK2-KM (C. Basbaum, University of California, San Francisco, CA), HMEK (K97R) (A. Saltiel, Parke-Davis Pharmaceutical Research Division, Ann Arbor, MI), JNKK (K116R) (M. Karin, University of California, San Francisco, CA), TLR5DN, and TLR2DN (Genentech, South San Francisco, CA), human constructs which have been previously characterized (20, 25).

Confocal Microscopy and Immunofluorescence Studies
16HBE and NHNP (normal human nasal polyp) cells were grown to confluence on Transwell-Clear filters (Corning-Costar, Corning, NY) with an air–liquid interface to form polarized monolayers. Monolayers were exposed apically to P. aeruginosa flagella (10 µg/ml) conjugated with Alexa Fluor 594 for 1 or 4 h. After four PBS rinses, the cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton-X100 in 4% paraformaldehyde, followed by incubation with 5% normal serum blocking solution for 20 min. Anti-asialoGM1 (Wako Chemical, Richmond, VA), anti-TLR2, anti-TLR5 or anti–caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA) was added for 1 h, followed by Alexa Fluor 488–conjugated secondary antibodies (Molecular Probes, Eugene, OR). Cells were mounted with Vectashield (Vector Laboratories, Burlingame, CA).

Flow Cytometry
16HBE cells grown in a polarized fashion on Transwell-Col were incubated with 10 µg/ml flagella, added apically, for 1 or 4 h, washed three times in PBS, and incubated with anti-asialoGM1 (Wako) anti-TLR2 or anti-TLR5 (Santa Cruz Biotech) and Alexa Fluor 488–conjugated (Molecular Probes) secondary antibodies. Stained cells were detached from the membrane, fixed in 1% paraformaldehyde, and analyzed on a FACSCalibur using CELLQuest software (BD Biosciences, San Diego, CA).

IL-8 and IL-6 Assays
IL-8 and IL-6 were measured by enzyme-linked immunosorbant assay (R&D Systems, Minneapolis, MN and Pierce Endogen, Rockford, IL) following a 60 min exposure of confluent monolayers of 1HAEo- cells, weaned from serum for 24 h in 96-well plates, flagella, or an equal volume of the PAO/NP fliA control prep. For experiments utilizing dominant-negative constructs, cells were stimulated with flagella following an overnight transfection. The effects of various inhibitors were tested by pretreating the cells for 2 h with: 5 µM BAPTA/AM (Molecular Probes), 13 µM PP1 (New England Biolabs, Beverly, MA), 10 µM PP2, 2–20 µM PD89059, and 0.3–30 µM SB202190 (Calbiochem, San Diego, CA). After stimulation the cells were washed three times in PBS Ca²⁺/Mg²⁺ and fresh media and/or inhibitor replaced for 3 h. Supernatants were harvested and duplicate wells were treated with trypan blue to assess epithelial cell viability during the assay, which was > 75%, and normalized to protein content using the Micro BCA Protein Assay Kit (Pierce). Each IL-8 and IL-6 data point was determined in quintuplicate and a mean and standard deviation were calculated. Statistical significance was determined using a one-way ANOVA with Bonferoni's post test (Graph-Pad Instat version 3.0; GraphPad software, San Diego, CA) to test the null hypothesis that there was no difference in the amount of the outcome variable (IL-8 or IL-6 production) under each test condition as compared with the untreated control.

Ca²⁺ Imaging
Subconfluent monolayers of 1HAEo- airway epithelial cells grown on glass coverslips were loaded with 5 µM Fura-2/AM (Molecular Probes) and 0.02% pluronic acid at room temperature for 45 min, then washed twice with PBS. Ratiometric imaging with excitation at wavelengths of 340 and 380 nm was done using a Zeiss Axiovert microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY) and analyzed using VPROBE software. Frames were collected at 6-s intervals following the application of flagella, lipopolysaccharide (LPS), antibody to asialoGM1 or monoclonal anti-human epithelial antigen (Dako, Glostrup, Denmark). Recordings were monitored from 10 cells/field, and at least five fields were examined for each condition.

Western Hybridization
Epithelial cells grown in 6-well plates to 80% confluence and weaned from serum overnight were stimulated with flagella for various time intervals, washed, and lysed by the addition of 0.5% Triton X-100 in PBS for 45 min on ice. Proteins were separated on 4–12% NUPAGE gels (Invitrogen), transferred to Immobilon-P (Millipore, Bedford, MA), and blocked in 5% skim milk overnight. Immunodetection was done with mouse monoclonal antibody to total or antiphosphorylated p44/42 ERK1/2 MAPK (Thr-202/Tyr-204), c-Src (Santa Cruz Biotech), antiphosphorylated p38 MAPK (Tyr-180/Tyr-182) (New England Biolabs), rabbit polyclonal antiphosphorylated Pyk2 (Tyr-402), rabbit polyclonal antiphosphorylated Src (Tyr 418) (Biosource). Anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotech) was used as the secondary antibody and detected with Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA).

Real-Time Polymerase Chain Reaction
16HBE epithelial cells were grown polarized in 10 cm Transwell clear dishes and were weaned from serum overnight. The cells were stimulated with flagella (10 µg/ml), and at different time points cells were lysed and the RNA was isolated using the Qiagen RNeasy Mini Kit (Valencia, CA). cDNA was synthesized from 1 µg of total RNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA). cDNA amplification was done in a Light Cycler using the DNA Master SYBR Green I kit (Roche, Indianapolis, IN). Thirty-five cycles were run with denaturation at 98°C for 8 s, annealing at 57°C for 15 s, and extension at 72°C for 20 s. Primers used were (5'-GCCAAAGTCTTGATTGATTGG-3') and (5'- TTGAAGTTCTCCAGCTCCTG-3') for TLR2, and (5'- CTAGCTCCTAATCCTGATG-3') and (5'- CCATGTGAAGTCTTTGCTGC-3') for TLR5. Each experiment was performed in triplicate and on at least three separate sets of cells.

Activation of NF-B Detected by Luciferase Reporter Constructions
1HAEo- and CHO-K1 cells grown in 12-well plates to 70–80% confluence were washed with PBS and transiently transfected using FuGENE 6 (Roche), 0.5 µg/ml of a TLR2 WT, TLR2 DN, TLR5 WT, TLR5 DN, or control plasmid, 0.5 µg/ml of pNF-B–luciferase (Stratagene, La Jolla, CA), which contains five NF-B binding sites upstream of a luciferase reporter gene, and 0.3 µg/ml of constitutively active pRL-TK (Promega, Madison, WI), to control for transfection efficiency, and incubated at 37°C in 5% CO₂ for 18 h. Cells were washed twice and subsequently stimulated with 10 µg/ml flagella for 4 h. Cells were washed, lysed, and harvested using Passive Lysis Buffer (Promega). Luciferase assays were performed using the reagents and protocol for the Dual-Luciferase Reporter Assay System (Promega) and analyzed with a luminometer. After standardization for transfection efficiency, data were plotted as the mean of quadruplicate samples and are representative of at least two independent experiments.

Inflammatory Responses in tlr2^–/– Mice
C57Bl/6 and tlr2^–/– mice (provided by S. Akira, Osaka University, Osaka, Japan), 7–10 d old, were intranasally inoculated with 50 µg of flagella in 10 µl of PBS or PBS (control) and killed 16 h later using pentobarbital. Cell suspensions were obtained from lung homogenates and red cells were lysed. Remaining cells were suspended in PBS containing 10% normal mouse serum and incubated for 30 min at 37°C. Cells were then double-stained with PE-labeled anti-CD45 and FITC-labeled anti-Ly6G (BD Pharmingen, San Diego, CA). Negative control samples were incubated with irrelevant, isotype-matched antibodies. Cells were gated based on their FSC/SSC profile and analyzed for the double expression of CD45 and Ly6G.

Peritoneal lavage was performed in C57Bl/6 wild-type and tlr2^–/– mice. Cells were washed twice, resuspended in RPMI 1640 media with 10% fetal bovine serum and cultured in the presence of medium alone or 10 µg/ml flagella for 10 h with monensin (Pharmingen). IL-6 production was evaluated at the single cell level by surface staining with anti-F4/80 and intracellular staining by using the Citofix/Cytoperm Plus kit (Pharmingen) according to the manufacturer's instructions. Briefly, cultured cells were washed, incubated with anti-F4/80 (Pharmingen), washed again, and treated with Cytofix/Cytoperm solution. PE-conjugated anti–IL-6 antibody (Pharmingen) was added. The cells were then washed with Perm/wash solution (Pharmingen) and finally with PBS with 2% fetal calf serum. Flow cytometry analysis was performed. Negative control samples were incubated with irrelevant, isotype-matched antibodies in parallel with all experimental samples. The percentage of F4/80 positive cells producing IL-6 was determined. Each mouse acted as his own control, comparing the responses of macrophages with or without exposure to flagella.

	Results

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P. aeruginosa Flagella Activate NF-B and IL-8 Expression in Airway Epithelial Cells through a Ca²⁺-Dependent Pathway
Various bacterial components (peptidoglycan and lipoprotein, LPS, flagella) have been shown to initiate signaling through interactions with specific TLRs (TLR2, TLR4, and TLR5, respectively). We verified the purity of our flagella preparation by mass spectroscopy (as detailed in MATERIALS AND METHODS) and demonstrated that corresponding material isolated from the flagella null mutant lacks the immunostimulatory capabilities of the flagella isolated from the Fla⁺ PAO1/NP strain (Figure 1). Although there could be nanogram amounts of lipopeptides remaining in the prep below the detection of mass spectroscopy, the lack of immunostimulation associated with an identical prep from the Fli^– mutant supports the hypothesis that flagella are the major immunostimulus in these experiments using purified flagella.

View larger version (29K): [in this window] [in a new window]   Figure 1. Immunogenicity of purified flagella. (A) Equal volumes of samples expected to contain flagella isolated from P. aeruginosa strains PAO/NP and an equivalent preparation from PAO/NP/fliA (Fla–) were compared by electrophoresis on a sodium dodecyl sulfate–polyacrylamide gel stained with Coomassie blue. (B) IL-8 was assayed by ELISA following 1 h exposure of 1HAEo- cells or 16HBE cells to flagella from PAO/NP (10 µg/ml, 1 µg/ml) and the mock flagella prep of the PAO/NP/fliA strain (volume equal to the PAO/NP prep at 10 µg/ml). Mean and SD were calculated from sextuplicate wells.

View larger version (22K): [in this window] [in a new window]   Figure 2. Flagella activation of airway epithelial cells. (A) The induction of NF-B activity as assessed by luciferase reporter was measured in 1HAEo exposed to media alone (control) or to increasing concentrations of P. aeruginosa flagella. (B) IL-8 and IL-6 were assayed by ELISA after exposure of 1HAEo- cells to increasing concentrations of flagella, or flagella (10 µg/ml) + BAPTA (5 µM). A mean and SD were calculated from quintuplicate wells. Background IL-8 and IL-6 expression from unstimulated cells in media is indicated as the control. (C) Spectrophotometric monitoring of Ca2+ transients in 1HAEo- cells loaded with Fura-2/AM and exposed to flagella, LPS, or antibody to asialoGM1 as indicated by the arrows. This tracing is representative of > 3 experiments performed with this and 16HBE airway epithelial cell lines.

View larger version (33K): [in this window] [in a new window]   Figure 3. Kinases involved in flagella signaling. (A) The effects of the Src inhibitors PP1 and PP2, ERK inhibitor PD98059, and the p38 inhibitor SB202190 on flagella-induced IL-8 expression in 1HAEo- cells, as measured by ELISA, are shown as compared with an unstimulated control and with flagella stimulation considered 100%. A mean and SD were calculated from quintuplicate wells and the experiment was repeated three times. A representative experiment is shown. (B) The effects of the dominant-negative mutants Src-RF, JNKK (K116R), Ras (N17), and HMEK (K97R) on flagella-induced IL-8 expression were examined as described above. (C) The phosphorylation of Src, ERK1/2 (p42/44), and p38 following exposure of 1HAEo- cells to flagella was detected by Western hybridization using phospho-specific antibodies. Anti-asialoGM1 added to the monolayers for 60 min was used as a positive control.

The role of the ERK MAPKs in flagella-induced IL-8 expression was further substantiated. Although there was similar inhibition of IL-8 expression by the ERK inhibitor PD98059 (P < 0.01) and the p38 inhibitor SB202190 (P < 0.05) (Figure 3A), the kinetics of ERK phosphorylation within 15 min of exposure to flagella and absence of detectable p38 phosphorylation in these cells over the same time period are consistent with a predominant role of ERK 1/2 in this pathway (Figure 3C). Thus, flagella-induced IL-8 expression in airway cells is mediated by Ca²⁺ fluxes and the activation of Src, Ras, and downstream MAPKs.

Colocalization of Flagella and Receptors on the Apical Surface of Polarized Airway Cells
The generation of 100 nM Ca²⁺ fluxes is characteristic of bacterial ligands that recognize the GalNAcß1–4Gal moiety exposed on asialoGM1, and is sufficient to induce NF-B translocation and IL-8 transcription (5). We postulated that flagella, by inducing similar Ca²⁺ fluxes, must be able to activate epithelial cells through this common asialoGM1-dependent pathway. The involvement of Ca²⁺ fluxes in toll-mediated signaling pathways has not been fully characterized. Polarized 16HBE cells, with tight junctions and apical–basolateral polarization when grown at an air–liquid interface on semipermeable supports, were examined by confocal microscopy to determine the distribution of potential receptors for flagella: asialoGM1 and TLR5 (Figure 4A). As shown in z-sections of permeabilized cells, asialoGM1 was most prominent on the apical surface of the 16HBE cells, whereas TLR5 was distributed more basolaterally. Alexa Fluor 594–labeled flagella added to the monolayers for 1 h colocalized along the apical surface with asialoGM1 in discrete clusters but only minimally with TLR5. This distribution of TLR5 was confirmed in human airway cells in primary culture, which are polarized and retain tight junctions (Figure 4B). Following a more prolonged exposure to flagella (4 h), the pattern of TLR5 expression was different, and substantial amounts of the receptor were now present apically and colocalized with the flagella (Figure 4C). The distribution of flagella was clearly apical, as shown in comparison with caveolin-1, which is present throughout the cells.

View larger version (37K): [in this window] [in a new window]   Figure 4. Distribution and colocalization of asialoGM1, TLR2, TLR5, and flagella in permeabilized human airway epithelial cells. (A) 16HBE cells or (B) human airway cells in primary culture (NHNP) grown at an air–liquid interface were examined by confocal microscopy (z-sections) 1 h following the addition of Alex Fluor 594–tagged flagella (red) and treated with either Alexa Fluor 488 (green)–labeled asialoGM1, TLR2, or TLR5 (magnification: x100). Areas of colocalization are yellow, indicated by arrows. (C) 16HBE cells after 4 h of exposure to flagella, now have apical TLR5 colocalized with the flagella, which remain on the apical surface, as compared with a caveolin-1 control, which is present throughout the cell.

View larger version (22K): [in this window] [in a new window]   Figure 5. Effect of flagella stimulation on TLR2 and TLR5 mRNA expression and mobilization. (A) 16HBE cells were analyzed by real-time PCR under unstimulated conditions or following stimulation with flagella (10 µg/ml) for the times indicated. mRNA expression was normalized to ß-actin and is shown as the fold change relative to the endogenous level of TLR2 in unstimulated cells. Each bar represents the mean of triplicate samples. *P < 0.05, Student t test. Open bars, TLR2; filled bars, TLR5. (B) Surface expression of TLR5 and asialoGM1 was assessed by flow cytometry in unstimulated 16HBE cells, and after 1 h (open bars) and 4 h (filled bars) of exposure to flagella.

View larger version (23K): [in this window] [in a new window]   Figure 6. TLR2 and TLR5 can mediate flagella signaling. (A) The effects of dominant-negative mutants of TLR2 and TLR5 on flagella induction of IL-8 expression were examined. 1HAEo- cells were transfected with plasmid DNA expressing one of the dominant-negative mutants indicated or an empty vector control. After exposure to flagella, culture supernatants, standardized by protein content, were assayed by ELISA for IL-8. Each condition was tested in quintuplicate, a mean and SD calculated, and each experiment was repeated at least three times. The decrease in IL-8 production by either the TLR5 or TLR2 dominant-negative mutants was statistically significant (P < 0.001) as compared with cells transfected with a control vector. A representative experiment is shown. 100% IL-8 is equivalent to 3 ng/µg protein. (B) The activity of an NF-B luciferase reporter construct was monitored in CHO-K1 cells, which were transiently transfected with wild-type TLR2, wild-type TLR5, or the empty vector control. The cells were stimulated with flagella (open bars) or media as the unstimulated control (filled bars), and results expressed as fold activity. The difference in NF-B activity between either of the TLR5 or TLR2 transfected cells and the control vector is statistically significant (P < 0.001), as were the differences between the unstimulated cells and those exposed to flagella (P < 0.01 for TLR5, P < 0.05 for TLR2).

TLR2 Contributes to Flagella-Induced IL-6 Expression in Macrophages
Further evidence for the involvement of TLR2 in flagella signaling was obtained from studies comparing the response of peritoneal macrophages from tlr2^–/– and wild-type mice (Figure 7A). Following a 10-h incubation with flagella, a significantly greater number of the macrophages from wild-type mice expressed IL-6 than the TLR2 null mice (37% versus 15%, P < 0.05), consistent with a role for TLR2 in signaling. These findings confirm that the ability of TLR2 to signal flagella is not limited to airway cells. However, the participation of TLR2 in the pulmonary response to flagella is not essential. We compared the in vivo inflammatory responses of wild-type and tlr2^–/– mice (Figure 7B). There was no significant defect in polymorphonuclear leukocyte (PMN) recruitment into the lung in response to flagella in the tlr2^–/– mice, indicating that TLR5 is sufficient to mediate a proinflammatory response in the intact animal.

View larger version (17K): [in this window] [in a new window]   Figure 7. IL-6 production and in vivo inflammatory responses induced by flagella in wild-type and tlr2–/– mice. (A) Peritoneal macrophages from C57Bl/6 wild-type (filled circles) and tlr2–/– (open circles) mice were incubated in the presence of medium alone or 10 µg/ml flagella for 10 h and stained for intracellular expression of IL-6 as detected by flow cytometry. Values obtained from individual mice are connected. Horizontal short lines indicate the median values obtained for each group. P values were determined by using the Wilcoxon signed-rank test for paired samples to compare the percentage of cells producing IL-6 after stimulation with flagella versus medium alone (*P < 0.05). (B) Lung cell suspensions from wild-type and tlr2–/– mice that had been inoculated with flagella (filled circles) or PBS (control; open circles) were stained for CD45 and Ly-6G. Leukocytes were gated on the basis of CD45 expression and the percentage of Ly-6G+ cells (PMN) was determined. Individual mice values are shown, and the short horizontal lines indicate the median values of each group (*P < 0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Epithelial proinflammatory responses to bacterial ligands clearly differ from those activated in nonpolarized cells. Epithelial responses to bacterial flagella have been best characterized in the gastrointestinal tract, where flagella have been shown to interact with TLR5 to activate host proinflammatory gene expression (15). The basolateral location of TLR5 in the gut mucosa is consistent with observations that only invasive bacteria, or flagella that access the basolateral aspects of the cell, activate proinflammatory responses (6). Other polarized epithelial cells appear to have a similar distribution of TLR5. The IL-8 signaling pathway in MDCK cells that is activated by flagella also involves interactions with basolateral TLR5, but is mediated by activation of p38 and not by NF-B (31). Flagella from enteropathogenic Escherichia coli activate both p38 and ERK MAPKs in polarized T84 cells, which express TLR5 basolaterally (30). However, in these studies IL-8 was expressed in response to both apically and basolaterally applied flagella, prompting the authors to suggest that there may be an additional non-TLR5 receptor available at the apical surface of T84 cells. The situation in the airway epithelium appears to be similar, in that TLR5 is abundant, but not immediately accessible to flagella when they are first presented at the apical surface of the mucosal cells. However, unlike the gut mucosa, despite an initial lack of apical TLR5 the airway epithelium is nonetheless stimulated by flagella, and responds immediately with Ca²⁺ fluxes followed by the activation of Src, Ras, ERK, NF-B, to stimulate IL-8 expression. Thus, an alternative receptor must be available to mediate this response.

Direct binding studies (12), as well as the confocal microscopy data provided in this article, suggest that flagella bind to apically displayed asialoGM1 on the airway cell surface. These observations are entirely consistent with the IL-8 signaling pathway activated in the airway cells, identical to that previously associated with ligation of asialoGM1 by intact bacteria or anti-asialoGM1 (5). Ligation of asialoGM1 and generation of 100 nM Ca²⁺ fluxes is involved in flagellar activation of both MUC-2 and IL-8 expression. However, in HM3 MUC-2 reporter system, this Ca²⁺ flux was followed by ATP release and ligation of a purinergic receptor (4). In the airway cells studied herein, IL-8 expression was not inhibited by either apyrase or Reactive Blue (data not shown), indicating that the release of ATP is not required in these cells, although the other constituents of the flagella signaling pathway appear to be the same.

In the respiratory tract, TLR5 does mobilize to the apical surface of the airway cells following flagella exposure, and is then fully capable of signaling the presence of superficial, adherent flagella. We postulate that the initial asialoGM1–flagella interaction activates an epithelial response that results in TLR5 transcription and mobilization. By 4 h of flagella stimulation, TLR5 is accessible on the apical surfaces of the cells, where it can mediate further signaling of superficial flagella. We have focused entirely on the bacterial–epithelial interactions at the apical surfaces of the respiratory mucosa, as this is the site of initial infection and likely to be critical in inflammatory airway diseases such as cystic fibrosis in which the epithelial barrier remains intact and organisms are trapped in the airway lumen (32). In addition, flagella appear to be most important in the establishment of lung infection, whereas bacteria isolated from chronic infections in CF often have become Fla^– (33).

The participation of TLR2 in flagella signaling was unexpected. The published data clearly demonstrate that TLR5 both binds and signals bacterial flagella. Although we did not find that TLR2 binds flagella, we do provide several lines of evidence that TLR2 can contribute to signaling in response to flagella. TLR2, as well as TLR5, was sufficient to mediate flagellar signaling when expressed in CHO cells, and TL2 DN mutants inhibited flagellar stimulation of the cells. Previous reports (19) also using CHO cells transfected with TLR2 and stimulated with flagella, albeit from a different organism, did not demonstrate activation. This may be due to differences in available asialoGM1 on CHO cells, which we have found to be highly variable and dependent upon cell culture conditions. Moreover, the contribution of TLR2 to inflammatory signaling was not limited to epithelial cells. Peritoneal macrophages from TLR2 null mice were found to be significantly impaired in their ability to produce IL-6 in response to flagella. Although TLR2 was not necessary to recruit PMN's into the lungs in response to a flagella challenge, it appears to be able to activate epithelial proinflammatory responses in the absence of available TLR5. Because flagella bind to asialoGM1, we postulate that TLR2 may be involved in mediating signals initiated by the flagella–asialoGM1 interaction.

The participation of specific receptors in the response to bacterial ligands at the airway surface is dependent upon their accessibility to bacterial ligands. TLR5 by virtue of its direct binding to flagella is clearly the key receptor for flagella once it is exposed on the airway surface. asialoGM1, which is available to bind either shed flagella or flagella expressed on intact bacteria, appears to initiate responses at the airway mucosal surface. In cystic fibrosis, with increased availability of asialoGM1 (34), this could contribute to the increased proinflammatory state in the lungs, even in patients with minimal infection. Once the proinflammatory circuit is primed, TLR5 is mobilized to increase epithelial responsiveness. Thus, superficial interactions of bacterial flagella, asialoGM1, TLR2, and TLR5 on the apical surface of the airway initiate an immediate and sustained host response to infection.

	Acknowledgments

 
The authors thank Dr. Mary Ann Gawinowicz of the Columbia University Protein Chemistry Core Facility for assistance with mass spectroscopy. Confocal imaging was done at Herbert Irving Optical Microscopy facility at Columbia University. This work was supported by National Institutes of Health Grants HL60293 and DK39693 to A.P, and by the Cystic Fibrosis Foundation.

Received in original form July 10, 2003

Received in final form November 3, 2003

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