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Originally published In Press as doi:10.1074/jbc.M411875200 on January 4, 2005

J. Biol. Chem., Vol. 280, Issue 10, 9482-9488, March 11, 2005
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Gangliosides Inhibit Flagellin Signaling in the Absence of an Effect on Flagellin Binding to Toll-like Receptor 5*

A. Phillip West, Brooke A. Dancho, and Steven B. Mizel{ddagger}

From the Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Received for publication, October 19, 2004 , and in revised form, December 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A recent study (Ogushi, K., Wada, A., Niidome, T., Okuda, T., Llanes, R., Nakayama, M., Nishi, Y., Kurazono, H., Smith, K. D., Aderem, A., Moss, J., and Hirayama, T. (2004) J. Biol. Chem. 279, 12213–12219) concluded that gangliosides serve as co-receptors for flagellin signaling via toll-like receptor 5 (TLR5). In view of several findings in this study that were inconsistent with a role for gangliosides as co-receptors, we re-examined this important issue. Using TLR5-negative RAW 264.7 cells and a TLR5-enhanced yellow fluorescent protein chimera, we established an assay for specific binding of flagellin to cells. Inhibition of clatherin-mediated internalization of flagellin·TLR5-enhanced yellow fluorescent protein complexes did not impair flagellin activation of IRAK-1. Thus flagellin signal occurs at the cell surface and not intracellularly. Exogenous addition of mixed gangliosides (GM1, GD1a, and GT1b) as well as GD1a itself inhibited flagellin-induced interleukin-1 receptor-associated kinase activation as well as tumor necrosis factor {alpha} production in HeNC2, THP-1, and RAW 264.7 cells. Gangliosides inhibited flagellin signaling in the absence of an effect on flagellin binding to TLR5. Depletion of gangliosides in RAW 264.7 cells did not alter the concentration dependence or magnitude of flagellin signaling as measured by interleukin-1 receptor-associated kinase activation or tumor necrosis factor {alpha} production. Our findings are consistent with the conclusions that gangliosides are not essential co-receptors for flagellin and that the inhibitory effect of gangliosides is mediated by at least one mechanism that is distinct from any effect on the binding of flagellin to TLR5.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Flagellin, the major structural protein of flagella from Gramnegative bacteria, is an extraordinarily potent activator of inflammatory cells such as monocytes, macrophages, epithelial cells, osteoblasts, and fibroblasts (1–14). In response to flagellin stimulation, these cell types produce a spectrum of proinflammatory cytokines such as interleukin-1 (IL-1),1 IL-6, IL-8, IL-10, tumor necrosis factor {alpha} (TNF{alpha}), a number of chemokines, nitric oxide, {beta}-defensins, and metalloproteinases. Although flagellin signaling is mediated via toll-like receptor 5 (TLR5) (4, 15, 16), the expression of nitric-oxide synthase and nitric oxide production require signaling via TLR5·TLR4 heteromeric complexes (17). This difference in signaling between TLR5 homomeric complexes and TLR5·TLR4 heteromeric complexes is related to the ability of TLR5·TLR4 but not TLR5·TLR5 complexes to induce the expression of type I interferons, a key step in the induction of nitric-oxide synthase expression in macrophages. The interaction of flagellin with TLR5 involves the recognition of a specific domain of TLR5 encompassing amino acid residues 386–425 within the extracellular domain of the protein (18) and specific regions within the conserved amino and carboxyl domains of flagellin (19–22). Following the interaction of flagellin with TLR5, a signaling cascade is initiated that involves MyD88 (15), the IL-1 receptor-associated kinase (IRAK-1) (16, 23), as well as downstream components such as the extracellular signal-regulated kinase 1/2 and p38 kinases and the transcription factor NF-{kappa}B (4–6, 9, 10, 15, 24).

In a recent study, Ogushi et al. (25) concluded that gangliosides, in particular, GD1a, GD1b, and GT1b, act as co-receptors for flagellin. This conclusion was based in part on two key observations. The first was the finding that gangliosides inhibited flagellin signaling in Caco-2 cells, presumably by competing with membrane-associated gangliosides that were part of a TLR5 receptor signaling complex. The second finding was that GD1a protected Salmonella enteritidis flagellin from proteolysis by trypsin, a finding that was consistent with a direct interaction between flagellin and the ganglioside. However, several findings in this study are inconsistent with a role for gangliosides as co-receptors. GD1a did not protect Pseudomonas aeruginosa flagellin from proteolysis by trypsin. If GD1a were a co-receptor for flagellin signaling via TLR5, then the ganglioside should interact with P. aeruginosa flagellin, a flagellin whose TLR5 signaling activity has been well documented (8, 12, 13, 26). CHO-K1 cells, a cell line that does not synthesize gangliosides other than GM3 (27) (a ganglioside that does not inhibit flagellin signaling (25)), exhibited the same flagellin concentration dependence and magnitude of response as ganglioside-producing Chinese hamster ovary cells. If gangliosides were crucial co-receptors for flagellin, then the absence of gangliosides in the membranes of cells should have dramatically reduced flagellin responsiveness. In view of these concerns, we re-examined the issue of gangliosides as co-receptors for flagellin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Reagents—The C3H/HeN-derived macrophage cell line HeNC2 and the human monocytic cell line THP-1 were maintained in RPMI 1640 containing 10% fetal bovine serum. The murine macrophage cell line RAW 264.7 (which is TLR5-negative (17)) was maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. To create a stable RAW 264.7 cell line expressing a TLR5-enhanced yellow fluorescent protein (TLR5-EYFP), the cells were transfected with linearized TLR5-EYFP (see below), rested 48 h, and then incubated in medium containing 200 µg/ml G418. Drug-resistant cells were cloned and evaluated for TLR5-EYFP expression and flagellin responsiveness. A single clone was selected and used in the experiments described in this report. Purified, endotoxin-depleted recombinant His-tagged S. enteritidis, P. aeruginosa (PAO1), and Escherichia coli (EPEC) flagellins were prepared as described previously (2). None of these flagellins exhibit stimulatory activity on TLR5-negative RAW 264.7 cells, indicating that the level of potential contaminants such as lipopolysaccharide are well below the range necessary for bioactivity. The truncated inactive form of flagellin (amino acid residues 297–471) termed 229 (2) was prepared in an identical manner. This protein does not bind to TLR5 (18), nor does it possess any stimulatory activity in flagellin-responsive cells (2). Polyclonal anti-IRAK-1 antibody was obtained from Upstate Biotechnology Inc. Polyclonal anti-flagellin antibody was prepared by immunizing rabbits with purified recombinant S. enteritidis flagellin. Monodansylcadaverine (MDC) was obtained from Sigma. The ganglioside mixture, containing GM1, GD1a, and GT1b, was purchased from EMD Biosciences. Purified GD1a and asialo-GM1 were purchases from Wako Chemical. Asialo GM1 was dissolved in CHCl3:EtOH and GD1a in Me2SO. The glucosylceramide synthase inhibitor, D,L-threo-l-phenyl-2-hexadecanoylamino-3-morpholinopropanol·HCl (PPMP), was purchased from Matreya Inc. (Pleasant Gap, PA) and was dissolved in ethanol and stored at -20 °C. Alexa fluor 488 cholera toxin subunit B conjugate was obtained from Molecular Probes.

mTLR5-EYFP Plasmid—The mTLR5-EYFP chimera contains the complete coding sequence of murine TLR5 with the enhanced yellow fluorescent protein (EYFP) C-terminal to the TLR5. The mTLR5-EYFP fusion was prepared by amplifying a murine TLR5 cDNA via PCR using the forward primer 5'-aaaattatagagctcgccgccaccatggcatgtcaacttgacttg-3' and the reverse primer 5'-atgcgtgcaggtaccccggaaatggttgctatggt-3'. The resultant product was then directionally cloned into the pEYFP-N1 vector (Clontech) at the SacI and KpnI sites. The final product was sequenced to ensure the fidelity of the PCR.

Staining and Confocal Microscopy—To assess flagellin binding, RAW 264.7 cells (2 x 106) were transfected with 2 µg of mTLR5-EYFP plasmid using the Amaxa Biosystems Nucleoefector technology and then plated in chambered covered glass wells (Nalge Nunc). After resting for 24 h, the cells were incubated for 30 min in fresh medium in the absence or presence of 10-9 M flagellin and then washed with ice-cold Tris-buffered saline (TBS) several times to remove unbound flagellin. The samples were then stained with a 1:500 dilution of anti-flagellin antibody at 4 °C for 1 h. After primary staining, the cells were fixed with 4% paraformaldehyde and stained with 5 µg/ml Rhodamine Red-X goat anti-rabbit IgG antibody (Molecular Probes). Confocal images were collected on a Zeiss Axiovert 100-M microscope equipped with a LSM 510 scanning unit and a 1.2 WC 63x plan apochromat objective.

The effect of PPMP on membrane ganglioside expression was evaluated by measuring the level of Alexa fluor 488 cholera toxin subunit B conjugate binding to RAW 264.7 cells incubated in the presence or absence of PPMP. RAW 264.7 cells (4 x 104) stably transfected with mTLR5-EYFP were incubated for 4 days in chambered cover glass wells with or without 5 x 10-6 M PPMP. The cells were then washed three times with ice-cold phosphate-buffered saline. The cells were resuspended in 1 ml of TBS containing 5 mg/ml bovine serum albumin, 1 mg/ml glycine, 0.05% Tween 20, 10% goat serum (TBS-BGT), and 10 µg/ml Alexa fluor 488-conjugated cholera toxin B subunit and then incubated on a rocker for 1 h at 4 °C. The cells were washed five times with TBS-BGT, fixed with 4% paraformaldehyde in phosphate-buffered saline, and then washed three times with TBS-BGT prior to examination by confocal microscopy.

IRAK-1 Kinase Assay—IRAK-1 kinase activity in HeNC2 or RAW 264.7 cell lysates was measured as described previously (16, 23). The cells were incubated in serum-free medium to prevent nonspecific absorption of gangliosides by serum proteins. The reaction products were subject to SDS-PAGE, and the phosphorylated form of the substrate, myelin basic protein (MBP), was visualized by autoradiography. The band intensities were quantitated using a Kodak Image Station 2000 RT.

Induction and Measurement of TNF{alpha} Production—HeNC2 and THP-1 cells (2 x 106 cells/ml) were seeded in 24-well tissue culture plates in serum-free medium containing the indicated concentrations of flagellin and gangliosides. After 4 h, the culture supernatants were harvested, and TNF{alpha} production was measured using enzyme-linked immunosorbent assay kits from BD Pharmingen. To assess the effect of gangliosides on TNF{alpha} production in TLR5-EYFP-expressing RAW 264.7 cells, the cells (8 x 104 cells/well) were cultured overnight in 12-well plates in serum-free medium with or without flagellin and 10 µg/ml gangliosides. The culture medium from each well was then harvested and analyzed for TNF{alpha} content by enzyme-linked immunosorbent assay.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Flagellin Binds to Surface-associated TLR5 in RAW 264.7 Cells—As a first step in the evaluation of the action of gangliosides on flagellin signaling, we developed an assay for TLR5-dependent flagellin binding to cells. We took advantage of our earlier observation that although RAW 264.7 cells do not express TLR5, they become flagellin-responsive when transfected with a TLR5 expression plasmid (17). To mark TLR5-expressing cells, we used a chimeric cDNA encoding murine TLR5 and EYFP. In preliminary experiments, we established that the expressed TLR5-EYFP is fully capable of signaling in a flagellin-dependent manner. Therefore, RAW264.7 cells were transiently transfected with TLR5-EYFP, plated in chambered cover glass wells and rested for 24 h prior to incubation in the presence or absence of 10-9 M flagellin or inactive 229 flagellin for 30 min at 37 °C. The cultures were then washed with ice-cold medium and stained with anti-flagellin antibody at 4 °C. After fixation and staining with a secondary rhodamine-labeled anti-rabbit IgG, confocal microscopy was employed to assess TLR5-EYFP expression and the degree to which flagellin bound to TLR5. In the absence of flagellin (Fig. 1A)orinthe presence of the inactive mutant 229 (Fig. 1B), there was no detectable rhodamine staining. However, when cells were incubated with flagellin, a pattern of staining was observed that was consistent with the binding of flagellin to surface TLR5 (Fig. 1C). The specificity of the staining is demonstrated by the presence of staining in only those cells that express TLR5-EYFP, i.e. cells lacking EYFP fluorescence did not bind flagellin. Furthermore, the level of flagellin staining was directly proportional to the level of TLR5-EYFP expression as evidenced by the observation that the rhodamine/EYFP ratio was constant for individual cells expressing TLR5-EYFP (~0.4).



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  		FIG. 1.
Specific binding of flagellin to RAW 264.7 cells expressing TLR5-EYFP. RAW 264.7 cells were transiently transfected with a TLR5-EYFP expression plasmid, rested for 24 h, and then incubated in the absence (A) or presence of 229 mutant flagellin (B) or wild-type flagellin (C) for 30 min. The cells were washed and stained with anti-flagellin antibody followed by rhodamine red-X goat anti-rabbit IgG antibody. DIC, differential interference contrast.

 
Clathrin-dependent Endocytosis of Flagellin Is Not Required for IRAK-1 Activation in HeNC2 Cells—Although TLR5, like most other TLRs, is expressed on the cell surface (Fig. 1 and Refs. 4, 16, and 18) and thus is able to bind flagellin (18), it was not known whether the subsequent signaling by TLR5 occurs at the cell surface or is dependent on migration to an intracellular compartment. In this regard, recent studies have revealed that, depending on the cell type, lipopolysaccharide signaling via TLR4 may occur in different cellular compartments. Hornef et al. (28) demonstrated that lipopolysaccharide signaling via TLR4 occurs at the cell surface in RAW 264.7 cells and in the Golgi apparatus in intestinal epithelial cells. A similar conclusion was reached by Guillot et al. (29). In related studies, investigators obtained evidence supporting the notion that TLR9 (30, 31) signaling also occurs in an intracellular compartment in macrophages and dendritic cells. Similar results have also been obtained with TLR2 (32). In view of these observations and the possible involvement of membrane-associated gangliosides in flagellin signaling, we evaluated whether flagellin signaling via TLR5 occurs at the cell surface or in an intracellular compartment. RAW 264.7 cells were transiently transfected with TLR5-EYFP and then incubated with or without 100 µM MDC, an inhibitor of membrane transglutaminase that prevents endocytosis via the clathrin-dependent pathway (33), for 30 min at 37 °C, and then incubated with 10-9 M flagellin for an additional 30 min at 37 °C. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with anti-flagellin antibody. The pattern of flagellin staining was evaluated using confocal microscopy. Representative sections are shown in Fig. 2A. Treatment with MDC decreased intracellular flagellin staining in RAW 264.7 cells as compared with the control sample. This effect was evident in each of the sections through the cells (data not shown). The accumulation of membrane-associated flagellin/TLR5 complexes in the MDC-treated sample most likely reflect enriched invaginations that are blocked from entering the endosomal pathway. Having established the effect of MDC on flagellin internalization in RAW 264.7 cells, we determined whether inhibition of flagellin endocytosis would abrogate TLR5 signaling. TLR5-EYFP-expressing RAW cells (107/sample) were cultured in the presence or absence of 100 µM MDC for 30 min at 37 °C and then stimulated in the presence or absence of 10-9 M flagellin for 5 min at 37 °C. Detergent lysates were prepared and immunoprecipitated with anti-IRAK-1 antibody, and the level of IRAK activity was measured using myelin basic protein as a substrate (16, 23). Although MDC blocked the internalization of flagellin/TLR5 complexes (Fig. 2A), it did not affect the flagellin-dependent activation of IRAK-1 (Fig. 2B). Flagellin induced a 3.3-fold increase in IRAK-1 kinase activity in the absence of MDC and a 2.9-fold increase in the presence of MDC. This difference is well within the experimental error of this type of assay. Thus these results support the notion that internalization is not required for TLR5 signaling in macrophage-like cells.



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  		FIG. 2.
Cell surface signaling by flagellin·TLR5 complexes. A, RAW 264.7 cells were transiently transfected with a TLR5-EYFP expression plasmid, rested for 24 h, and then incubated with or without 100 µM MDC for 30 min at 37 °C prior to incubation of the cells for an additional 30 min with flagellin in the presence or absence of the drug. The cells were then washed, fixed, and analyzed for flagellin binding. B, TLR5-EYFP expressing RAW 264.7 cells were incubated with or without 100 µM MDC for 30 min and then stimulated with or without flagellin for 5 min. The level of IRAK-1 activity was then measured using MBP as a substrate.

 
Gangliosides Inhibit Flagellin-induced TNF{alpha} Production in HeNC2, THP-1, and RAW 264.7 Cells—After establishing an assay for the cell surface binding of flagellin to TLR5 (Fig. 1) and that TLR5 signaling occurs at the cell surface, we focused our analysis on the question of gangliosides as co-receptors for TLR5. We began by determining the effect of gangliosides on the induction of TNF{alpha} production in HeNC2 and THP-1 cells. Each cell type was incubated with 10-9 M flagellin in the presence or absence of 30 µg/ml mixed gangliosides for 4 h at 37 °C. Supernatants were analyzed for TNF{alpha} by enzyme-linked immunosorbent assay. As shown in Fig. 3, exogenous gangliosides inhibited TNF{alpha} production in HeNC2 cells (Fig. 3A) by ~75% and completely abrogated TNF{alpha} production in THP-1 cells (Fig. 3B). These results are consistent with the observed inhibitory effect of gangliosides on flagellin-induced human {beta}-defensin-2 promoter activity in Caco-2 cells (25). The effect of the mixed gangliosides was concentration-dependent. Maximal inhibition was obtained with ~10 µg/ml mixed gangliosides and was absent at concentrations less than ~0.3 µg/ml (data not shown). In addition to HeNC2 and THP-1 cells, we also evaluated the effect of mixed gangliosides on TNF{alpha} production in RAW 264.7 cells stably transfected with TLR5-EYFP. These cells were equally sensitive to the inhibitory action of gangliosides (Fig. 3C).



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  		FIG. 3.
Mixed gangliosides inhibit flagellin induction of TNF{alpha} production in HeNC2 and THP-1 cells. A, HeNC2 cells were incubated in the absence (O/O) or presence of flagellin (O/F), 30 µg/ml gangliosides (G/O), or flagellin and 30 µg/ml mixed gangliosides (G/F) for 4 h in serum-free medium prior to analysis of TNF{alpha} levels in the culture medium. B, THP-1 cells were incubated in the absence (O/O) or presence of flagellin (O/F) or flagellin and 30 µg/ml mixed gangliosides (G/F) for 4 h in serum-free medium prior to analysis of TNF{alpha} levels in the culture medium. C, RAW 264.7 cells stably transfected with TLR5-EYFP were incubated overnight in the absence (O) or presence of flagellin (F) or flagellin and 10 µg/ml gangliosides (F + G) prior to analysis of TNF{alpha} levels.

 
Gangliosides Inhibit Flagellin-induced IRAK-1 Activation— Having established the inhibitory effect of gangliosides on TNF{alpha} production in HeNC2 and THP-1 cells, we next determined whether gangliosides inhibit IRAK-1 activation in flagellin-responsive cells. HeNC2 cells (107) were incubated in the presence or absence of 10-9 M flagellin with or without 10 µg/ml mixed gangliosides for 5 min at 37 °C and then prepared for analysis of IRAK-1 kinase activity. As shown in Fig. 4A, mixed gangliosides inhibited flagellin-induced IRAK-1 activity (by ~75%). This level of inhibition is consistent with the 75% decrease in TNF{alpha} production (Fig. 3). It is important to note that gangliosides had no direct effect on IRAK-1 activity. Incubation of active IRAK-1 from flagellin-stimulated cells with 10 µg/ml mixed gangliosides did not result in any detectable decrease in kinase activity (data not shown). Ogushi et al. (25) reported that GD1a, but not asialo-GM1, inhibited flagellin signaling. Therefore, we examined the effect of these two individual gangliosides on flagellin-induced IRAK-1 activation. RAW 264.7 cells stably expressing TLR5-EYFP were incubated in the presence or absence of flagellin for 1 h at 4 °C to allow for the binding of flagellin, washed, rapidly shifted to 37 °C in the presence or absence of 10 µg/ml GD1a or asialo-GM1, and incubated for 5 min prior to analysis of IRAK-1 kinase activity. As presented in Fig. 4B, GD1a, but not asialo-GM1, inhibited IRAK-1 activation to the same extent as the mixed gangliosides.



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  		FIG. 4.
Mixed gangliosides inhibit flagellin-induced IRAK-1 activation. A, HenC2 cells were incubated in the absence or presence of flagellin and 10 µg/ml mixed gangliosides for 5 min at 37 °C and then analyzed for IRAK-1 activity using MBP as a substrate. B, RAW 264.7 cells stably transfected with TLR5-EYFP were incubated with or without flagellin for 1 h at 4 °C, washed, and rapidly shifted to 37 °C in the presence or absence of 10 µg/ml GD1a or asialo-GM1 for 5 min prior to measurement of IRAK-1 activity. DMSO, dimethyl sulfoxide. C, HeNC2 cells were incubated for4hinthe presence or absence of 10-9 M S. enteriditis (F), P. aeruginosa (P), or E. coli (EPEC) flagellin with or without 10 µg/ml mixed gangliosides (G) prior to the measurement of TNF{alpha} production. D, HeNC2 cells were incubated for 5 min at 37 °C in the absence or presence of S. enteriditis, P. aeruginosa (PAO1), or E. coli (EPEC) flagellin with our without 10 µg/ml mixed gangliosides. IRAK-1 activity was then determined using MBP as a substrate.

 
Gangliosides Inhibit PAO1- and EPEC-induced TNF{alpha} Production and IRAK-1 Activation in HeNC2 Cells—Because Ogushi et al. (25) reported that gangliosides protected S. enteriditis flagellin but not P. aeruginosa flagellin from proteolysis by trypsin, we evaluated the effect of gangliosides on signaling by P. aeruginosa and E. coli flagellins. HeNC2 cells were incubated in the presence or absence of 10-9 M S. enteriditis flagellin, PAO1 flagellin, or EPEC flagellin with or without 10 µg/ml mixed gangliosides and analyzed for TNF{alpha} production (Fig. 4C) and IRAK-1 kinase activity (Fig. 4D). As with S. enteriditis flagellin, gangliosides inhibited PAO1- and EPEC-induced TNF{alpha} production in HeNC2 cells by ~80% (Fig. 4C). Additionally, exogenous gangliosides had a comparable inhibitory effect on PAO1- and EPEC-induced IRAK-1 activation (Fig. 4D). These findings demonstrate that gangliosides inhibit signaling induced by flagellins from different sources and that trypsin protection does not correlate with the inhibitory effect of gangliosides on flagellin signaling.

Gangliosides Inhibit Flagellin Signaling in the Absence of an Effect on Flagellin Binding to TLR5—Ogushi et al. proposed that exogenous gangliosides bind flagellin and thus prevent flagellin from interacting with TLR5 receptor complexes containing ganglioside. If this were the case, then gangliosides should not inhibit signaling if added after flagellin was bound to TLR5. To test this hypothesis, TLR5-EYFP-expressing cells were incubated with 10-9 M flagellin at 4 °C for 1 h, washed to remove unbound flagellin, and then assessed for IRAK-1 kinase activity following a shift to 37 °C for 5 min in the presence or absence of 10 µg/ml gangliosides (Fig. 5B). Under these conditions, gangliosides inhibited IRAK-1 activity in RAW 264.7 cells even when flagellin was previously bound to TLR5. Because it was possible that gangliosides might promote the dissociation of flagellin from the TLR5 receptor complex, we assessed this possibility in cultures of TLR5-EYFP-expressing RAW 264.7 cells that were incubated with flagellin at 4 °C prior to exposure to gangliosides (Fig. 5A). RAW 264.7 cells transfected with TLR5-EYFP were incubated with 10-9 M flagellin for 2 h at 4 °C, washed, and then incubated with or without 10 µg/ml gangliosides for 5 min at 37 °C. The extent of residual flagellin binding was assessed using confocal microscopy. As is evident in the image presented in Fig. 5A, gangliosides had no detectable effect on flagellin binding compared with untreated cells. By quantitating the mean pixelation for the rhodamine and EYFP channels for each of 22 TLR5-EYFP-positive cells incubated in the presence or absence of gangliosides, we were able to calculate the ratio of rhodamine/EYFP intensities. Because the binding of flagellin is proportional to the level of TLR5-EYFP expression (Fig. 1), a decrease in this ratio would indicate a loss of flagellin binding. However, we obtained identical values in control and ganglioside-treated cells (rhodamine/EYFP ratios of ~0.42 ± 0.07 for 22 control cells and 0.43 ± 0.05 for 22 ganglioside-treated cells). The results in Fig. 5 clearly establish that gangliosides inhibit flagellin signaling in the absence of any detectable effect on flagellin binding to TLR5.



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  		FIG. 5.
Mixed gangliosides inhibit flagellin-induced IRAK-1 activation in the absence of an effect on flagellin binding. A, RAW 264.7 cells were transiently transfected with a TLR5-EYFP expression plasmid, rested for 24 h, and then incubated with flagellin for 2 h at 4 °C. The cells were washed and then shifted to 37 °C in the presence or absence of 10 µg/ml mixed gangliosides for 5 min. The cells were subsequently evaluated for bound flagellin. In addition, the ratio of rhodamine to EYFP fluorescence was determined for 22 representative cells from the control and ganglioside-treated groups. B, TLR5-EYFP-expressing RAW 264.7 cells were incubated in the presence or absence of flagellin for 1 h at 4 °C. The cells were then shifted to 37 °C in the presence or absence of 10 µg/ml mixed gangliosides and incubated for 5 min. IRAK-1 activity was then measured using MBP as a substrate.

 
Depletion of Gangliosides Does Not Alter the Flagellin Responsiveness of Cells—If gangliosides function as co-receptors for flagellin, then the depletion of these molecules should either prevent flagellin signaling or invoke a requirement for significantly higher concentrations of flagellin to stimulate cells. To evaluate these possibilities, we used PPMP, a glucosylceramide synthase inhibitor (34), to deplete cellular gangliosides and then examined flagellin responsiveness. RAW264.7 cells stably transfected with TLR5-EYFP were incubated for 4 days in the presence or absence of 5 x 10-6 M PPMP and then analyzed for their ability to produce TNF{alpha} in response to increasing concentrations of flagellin. To verify that the drug was indeed reducing the cellular content of gangliosides, control and PPMP-treated cells were analyzed for cell surface GM1 expression using cholera toxin subunit B labeled with Alexa fluor 488. Even though PPMP treatment markedly reduced the cellular levels of gangliosides as evidenced by the substantial decrease in cholera toxin binding (Fig. 6A), the drug did not prevent flagellin induction of TNF{alpha}, nor did it significantly alter the concentrations of flagellin required for this induction (Fig. 6B). It should be noted that PPMP treatment resulted in a decreased rate of cellular growth but not viability (data not shown). Thus it was critical to normalize the results with regard to the number of cells/sample. In addition to evaluating the effect of PPMP treatment on flagellin-induced TNF{alpha} production, we also tested the effect of ganglioside depletion on flagellin-induced IRAK-1 activation. RAW 264.7 cells stably transfected with TLR5-EYFP were incubated with PPMP for 4 days, washed, and then incubated in the presence or absence of flagellin for 5 min prior to analysis of IRAK-1 activity. As shown in Fig. 6C, depletion of gangliosides did not affect the ability of flagellin to activate IRAK-1. In both instances (± PPMP), flagellin induced an ~3-fold increase in IRAK-1 activity. These results provide additional support for the conclusion that although gangliosides inhibit flagellin signaling, they do not function as essential co-receptors with TLR5.



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  		FIG. 6.
Depletion of cellular gangliosides does not impair flagellin signaling. RAW 264.7 cells stably transfected with TLR5-EYFP were incubated in the presence or absence of 5 µM PPMP for 4 days. A, cell surface expression of GM1 was measured using Alexa fluor 488-labeled cholera toxin B subunit. The cells were washed, incubated with 10 µg/ml Alexa fluor 488-conjugated cholera toxin B subunit for 1 h at 4 °C, washed, and then examined by confocal microscopy. B, cultures incubated in the absence (•) or the presence ({circ}) of PPMP were then stimulated with increasing concentrations of flagellin for 4 h prior to determination of TNF{alpha} levels in the culture medium. In addition, the cell numbers in each culture were determined after 4 days of incubation with or without PPMP. C, control and PPMP-treated cells were also analyzed for the ability of flagellin to induce IRAK-1 activation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although several TLRs can signal intracellularly, for example TLR2, TLR4, and TLR9 (28–32), our results are consistent with the notion that TLR5 signaling occurs at the cell surface in macrophage-like cells. This conclusion is based on the observation that inhibition of flagellin·TLR5 receptor signaling complexes by MDC did not impair flagellin-induced activation of IRAK-1 (Fig. 2). The finding that IRAK-1 activation in response to flagellin occurs very rapid, i.e. within 1–2 min (23), is also consistent with cell surface signaling.

The results reported in this study are also consistent with the conclusion that although gangliosides inhibit flagellin signaling, they are not essential co-receptors. This conclusion is based on the following lines of evidence. First, gangliosides exert an inhibitory effect in the presence of TLR5-bound flagellin (Fig. 5). Second, depletion of gangliosides does not alter the concentration dependence or the magnitude of the flagellin-induced response (Fig. 6). Our results with PPMP appear to be in conflict with those of Ogushi et al. (25). These investigators observed that PPMP treatment decreased the flagellin response by 40–50%. However, these investigators did not correct their results for the growth inhibitory effect of PPMP. In our experiments, PPMP reduced RAW 264.7 cell growth in the range of 25–55%. Third, CHO-K1 cells that lack gangliosides other than GM3 (27) are as flagellin-responsive as other cell types that produce a broad array of gangliosides (25). The observation that reduction of gangliosides does not result in an elevated flagellin response supports the notion that gangliosides, under physiological conditions, are present in insufficient amounts to modify TLR5 signaling. This conclusion is also supported by the finding that relatively high concentrations of gangliosides are required to significantly inhibit flagellin signaling (this study and Ref. 25).

An increasing number of studies have revealed that exogenous gangliosides can modulate signaling by a wide range of receptors. For example, gangliosides modify signaling by the epidermal growth factor receptor (35–39), the platelet-derived growth factor receptor (40–42), TLR4 (43–46), CD4 (47), Fc{epsilon}RI (48), and IL-4 (49). In most cases, gangliosides are inhibitory, but in some instances they actually enhance receptor signaling (for example, see Ref. 39). The latter type of effect may occur at lower concentrations of gangliosides, whereas the inhibitory effect is clearly associated with high concentrations of exogenous gangliosides. Work in various systems has revealed three basic mechanisms by which gangliosides inhibit signaling (see Ref. 50 for a review). Gangliosides may interact with the ligand and thus block interaction with a receptor. Alternatively, they may inhibit receptor dimerization, a key event in receptor signaling. Also, gangliosides may modulate the activation state or cellular localization of receptors. This latter mechanism may involve receptor modification by phosphatases or kinases or displacement of receptors from specific membrane microdomains.

In the case of flagellin, it is likely that the inhibitory effect of gangliosides is due to at least two types of mechanisms. The first may involve a direct interaction between flagellin and high concentrations of gangliosides that blocks the binding of flagellin to TLR5. The available evidence indicates that gangliosides containing multiple N-acetylneuraminic acid molecules interact with flagellin in a manner that is relatively nonspecific and low affinity (25, 51). In view of the observation that gangliosides do not reduce flagellin binding after flagellin is bound to TLR5 (Fig. 5), we contend that if exogenous gangliosides impair flagellin binding to TLR5, they do so by interacting with regions of flagellin that are involved in the binding to TLR5 and not to membrane-associated gangliosides. The observations that gangliosides are not required for the high affinity binding of flagellin to TLR5 (18) nor for flagellin signaling (Fig. 6) support this conclusion. Furthermore, the absence of flagellin binding in TLR5-negative RAW 264.7 cells (Fig. 1) provides additional evidence against the notion that membrane-associated gangliosides serve as co-receptors for flagellin.

In preliminary experiments, we found that mixed gangliosides do not inhibit the ability of TLR5 to form homodimers (as measured by the co-immunoprecipitation of TLR5 tagged with FLAG or hemagglutinin epitopes). However, it is possible that TLR5 signaling requires higher order oligomers and that gangliosides inhibit the formation of these oligomers and, in so doing, prevent the association or activation of adapter molecules such as MyD88, IRAK-1, and IRAK-4. This possibility is a subject of current investigation.

Although gangliosides do not inhibit flagellin signaling via an effect on TLR5 dimerization, they may affect the membrane microenvironment of TLR5 and thus reduce its signaling potential. As noted earlier, gangliosides have been shown to inhibit the phosphorylation of a number of receptors including the platelet-derived growth factor (40–42) and epidermal growth factor receptors (36, 37). Although phosphorylation of TLR5 has not been documented, it is interesting to note that the IL-1 receptor, a member of the TLR family of proteins, is phosphorylated following the binding of IL-1 (52) or stimulation with phorbol esters (53). Arbibe et al. (54) have reported that TLR2 is also phosphorylated following stimulation with a TLR2 agonist. Thus it is possible that gangliosides may block phosphorylation of TLR5. Alternatively, gangliosides may inhibit the phosphorylation of IRAK-1 or other TLR5-associated adaptor proteins.

A number of studies (55–58) have suggested that exogenous gangliosides can induce the displacement of receptors from glycolipid-enriched domains in the cell membrane and by this action convert the involved receptors into an inactive state. Thus it is possible that the inhibitory effect of gangliosides on TLR5 signaling may be mediated by a similar mechanism. This is a particularly attractive notion given the observation that lipopolysaccharide promotes the accumulation of TLR4 in glycolipid-enriched membrane microdomains (59, 60).

In summary, our findings support the conclusion that gangliosides are not essential co-receptors for flagellin signaling via TLR5. Furthermore, the inhibitory effect of gangliosides on flagellin signaling is mediated by at least one mechanism that is distinct from any effect on the binding of flagellin to TLR5. The elucidation of this mechanism may provide new and important insights into the regulation of TLR5 signaling and the events that occur following the binding of flagellin.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant AI51319. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. E-mail: smizel{at}wfubmc.edu.

1 The abbreviations used are: IL, interleukin; TNF, tumor necrosis factor; TLR, toll-like receptor; IRAK, interleukin-1 receptor-associated kinase; EYFP, enhanced yellow fluorescent protein; PPMP, D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-propanol HCl; TBS, Tris-buffered saline; MBP, myelin basic protein; MDC, monodansylcadaverine. Back


   ACKNOWLEDGMENTS
 
We acknowledge Aaron Graff for outstanding technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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