Gangliosides Act as Co-receptors for Salmonella enteritidis FliC and Promote FliC Induction of Human β-Defensin-2 Expression in Caco-2 Cells*

Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces. We reported previously that Salmonella enteritidis flagellin (FliC) induced human β-defensin-2 (hBD-2) mRNA expression in Caco-2 cells via NF-κB activation (Ogushi, K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi, A., Asakura, H., Makino, S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) J. Biol. Chem. 276, 30521–30526). In this study, we examined the role of ganglioside as co-receptors with Toll-like receptor 5 (TLR5) on FliC induction of hBD-2 expression in Caco-2 cells. Exogenous gangliosides suppressed FliC induction of hBD-2 promoter activity and binding of FliC to Caco-2 cells. Incorporation of exogenous ganglioside GD1a into Caco-2 cell membranes increased the effect of FliC on hBD-2 promoter activity. In support of a role for endogenous gangliosides, incubation of Caco-2 cells with dl-threo-2-hexadecanoylamino-3-morpholino-1-phenylpropanol, a glucosylceramide synthase inhibitor, reduced FliC induction of hBD-2 promoter activity. GD1a-loaded CHO-K1-expressing TLR5 cells had a higher potential for hBD-2 induction following FliC stimulation than GD1a-loaded CHO-K1 cells not expressing TLR5. FliC increased phosphorylation of mitogen-activated protein kinase, p38, and ERK1/2. Exogenous gangliosides GD1a, GD1b, and GT1b each suppressed FliC induction of p38 and ERK1/2 phosphorylation. Furthermore, FliC did not enhance luciferase activity in Caco-2 cells transfected with a plasmid containing a mutated activator protein 1-binding site. These results suggest that gangliosides act as co-receptors with TLR5 for FliC and promote hBD-2 expression via mitogen-activated protein kinase.

In addition to their antimicrobial activity, hBD-2 and murine ␤-defensins can potentially function as chemokines for immature dendritic cells and memory T cells through interaction with chemokine receptor CCR6 (16,17). Recently, it was reported that murine ␤-defensin 2 is a ligand for Toll-like receptor (TLR) 4 on immature dendritic cells (18). TLRs function as pattern recognition molecules for various microbial components and endogenous ligands and mediate activation of the innate immune response (19,20). Thus, induction of hBD-2 may also affect inflammatory reactions.
Salmonella is a Gram-negative bacterium that causes gastroenteritis and enteric fever in humans. Salmonella infection of cultured intestinal epithelial cells resulted in hBD-2 induction (15). However, the signaling pathways involved in hBD-2 induction in Caco-2 cells, a human colon carcinoma line, by Salmonella are still unclear. From our previous work to evaluate the role of hBD-2 in Salmonella enteritidis infection, we concluded that S. enteritidis flagellin (FliC) increased hBD-2 promoter activity and mRNA levels in Caco-2 cells via NF-B activation (21). Other studies demonstrated that the flagellin of Salmonella dublin (22) or Salmonella typhimurium (23) induced nitric-oxide synthase and IL-8 secretion, respectively.
It has been demonstrated that TLR5 is a receptor for flagellin of Gram-negative, as well as Gram-positive, bacteria (24). After binding of FliC to TLR5 (24,25), myeloid differentiation factor 88 was involved in NF-B activation, resulting in IL-6 expression (24). We reported recently that S. enteritidis FliC increased intracellular Ca 2ϩ concentration via activation of phospholipase C, leading to translocation of NF-B to the nucleus and increased hBD-2 promoter activity (26). However, the signal transduction pathways involved in hBD-2 induction by FliC are still unclear.
Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitous components of eukaryotic cell membranes that have been identified as receptors for bacterial toxins and viruses (27)(28)(29)(30). Although TLR5 was determined to be a FliC receptor, gangliosides such as GM1, GD1a, and asialo-GM1 also bound Pseudomonas aeruginosa flagellin (31). In particular, asialo-GM1, serving as a P. aeruginosa flagellin receptor, was critical for flagellin-dependent signaling in epithelial cells (32). Recently, Yu et al. (33) reported that induction of p38 MAP kinase phosphorylation by S. typhimurium flagellin was mediated by activation of TLR5 rather than through asialo-GM1.
In this study, to evaluate signal transduction pathways used by FliC, we examined the effect of gangliosides and TLR5 on FliC induction of hBD-2 expression in Caco-2 cells, focusing on the potential role of complex gangliosides as TLR co-receptors.
Transfection and Luciferase Assay-Transfection and luciferase assays were performed as described previously (21) with the following modifications. To assess hBD-2 promoter activity, Caco-2 cells were seeded in 24-well culture plates (0.5 or 1.0 ϫ 10 5 cells in 1 ml of DMEM per well) and incubated at 37°C for 24 h. 2.5 g of the hBD-2 promoter linked to a luciferase reporter gene (pGL3-2110 or pGL3-938) or a mutated hBD-2 promoter construct (pGL3-938/NF-Bmt or pGL3-938/ AP1mt) were incubated with 0.5 g of an internal control Renilla luciferase expression vector (pRL-TK) and 10 l of 3.2 mM dendritic poly-(L-lysine) (KG6) (36) in 250 l of FCS-free DMEM at room temperature for 15 min before addition to Caco-2 cells. After incubation at 37°C for 3 h, 1 ml of DMEM was added, followed by incubation at 37°C for 24 h.
To initiate experiments, the medium was replaced with 200 l of fresh FCS-free DMEM, and 50 l of the sample to be assayed were added. After incubation at 37°C for 3 or 6 h, cells were washed with 1 ml of PBS and lysed by adding 300 l of lysis buffer (Toyo Ink Co.). After 15 min at room temperature, the lysate was centrifuged (18,000 ϫ g, 5 min, 4°C). Luciferase activity of the supernatant, measured using a luminometer (Berthold), was expressed relative to the activity of an internal control Renilla luciferase.
To assess the effects of TLR5 expression and GD1a loading on FliC induction of hBD-2 promoter activity, CHO-K1 cells were seeded in 24-well culture plates (1.0 ϫ 10 5 cells per well) and incubated at 37°C for 24 h. 1.5 g of pGL3-2110 were incubated with 0.5 g of pRL-TK, 1 g of TLR5 expression vector (pEF6V5/HIS TOPO::TLR5), and 5 l of DAC-30 (2.5 g) in 250 l of FCS-free medium at room temperature for 20 min before addition to cells. After incubation at 37°C for 4 h, CHO-K1 cells were incubated without or with GD1a (50 g/ml) in FCS-free medium at 37°C for 24 h. At the end of incubation, cells were washed with medium and then incubated with the indicated concentrations of FliC at 37°C for 6 h. hBD-2 promoter activity was determined by the luciferase reporter gene assay.
Isolation of Flagellin from Bacterial Cells-S. enteritidis FliC and P. aeruginosa flagellin were prepared as previously described (21) with the following modifications. S. enteritidis, and P. aeruginosa grown in 2 liters of tryptic soy broth at 37°C for 16 h were pelleted by centrifugation (5,000 ϫ g, 4°C, 30 min) and dispersed in 40 ml of PBS, which was adjusted to pH 2 with 1 M HCl and maintained at that pH with constant stirring at room temperature for 30 min. After centrifugation (100,000 ϫ g, 4°C, 1 h), the pH of the supernatant containing soluble monomeric flagellin was adjusted to 7.2 with 1 M NaOH, and solid (NH 4 ) 2 SO 4 was added to 65% saturation. After incubation overnight at 4°C, the mixture was centrifuged (15,000 ϫ g, 4°C, 15 min). The precipitate was dissolved in distilled water, dialyzed against distilled water, then heated at 65°C for 15 min, placed on ice, and centrifuged (100,000 ϫ g, 4°C, 1 h). To the supernatant, which contained depolymerized FliC, solid (NH 4 ) 2 SO 4 was added to 65% saturation. After incubation overnight at 4°C, the mixture was centrifuged at 15,000 ϫ g at 4°C for 15 min. The precipitate was dissolved in distilled water and dialyzed against PBS.
Effect of Exogenous Ganglioside on FliC Induction of hBD-2 Promoter Activity-Caco-2 cells, transfected with luciferase reporter gene, were incubated with FliC (100 ng/ml) and the indicated concentration of ganglioside at 37°C for 3 or 6 h. hBD-2 promoter activity was determined using the luciferase reporter gene assay.
Effect of Anti-ganglioside Antibodies on FliC Induction of hBD-2 Promoter Activity-Caco-2 cells were seeded in 24-well culture plates (5 ϫ 10 4 cells per well) and incubated at 37°C for 24 h before transfection with luciferase reporter gene as described above and incubation with the indicated concentration of anti-GD1a, -GD1b, or -GT1b antibodies at 37°C for 1 h followed by incubation with FliC (100 ng/ml) at 37°C for 6 h. hBD-2 promoter activity was determined using the luciferase reporter gene assay.
Proteolytic Digestion of FliC-Protection against tryptic digestion of FGF-2 by gangliosides has been reported (37). S. enteritidis FliC (3 g), VacA (3 g), or P. aeruginosa flagellin (3 g) was incubated in 50 mM Tris-HCl, pH 7.5, with or without the indicated amount of ganglioside at 37°C for 5 min, followed by addition of trypsin (Sigma), 60 ng in a final volume of 100 l, and incubation at 37°C for 3 h, before addition of an equal volume of SDS sample buffer (0.1 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.1% (w/v) bromphenol blue, 50 mM dithiothreitol), and heating at 95°C for 5 min. Samples were subjected to SDS-PAGE in 10% gels, which were stained with Coomassie Brilliant Blue.
Western Blotting Analysis-Caco-2 cells were seeded in 6-well culture plates (3 ϫ 10 5 cells per well). After 24 h, the medium was replaced with FCS-free DMEM, and the cells were incubated at 37°C for 24 h. FliC (10 g/ml) was added, and after 30 min at 37°C, cells were washed with PBS and lysed in 300 l of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% glycerol, 0.1% (w/v) bromphenol blue, 50 mM dithiothreitol, 1 mM Na 3 VO 4 ). Proteins were separated by SDS-PAGE in 10% gels and transferred to polyvinylidine difluoride membranes, which were washed with TBS (20 mM Tris-HCl, 133 mM NaCl, 1 mM Na 3 VO 4 ) at room temperature for 5 min, and then incubated with 5% (w/v) milk powder in TBS-T (20 mM Tris-HCl, 133 mM NaCl, 0.1% Tween 20, 1 mM Na 3 VO 4 ) at room temperature for 1 h. Membranes were washed with TBS-T, followed by incubation in a 1:1,000 dilution of anti-phospho-p38 or -phospho-ERK1/2 antibodies overnight at 4°C. After washing in TBS-T, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:4,000 dilution) at room temperature for 1 h and then washed in TBS-T, followed by incubation for 5 min in TBS before detection by ECL system (Amersham Biosciences).
To assess TLR5 expression, CHO or CHO-K1 cells transfected with TLR5 expression vector were washed with PBS and lysed in 200 l of SDS sample buffer. Proteins were resolved by SDS-PAGE in 10% gels and transferred to polyvinylidine difluoride membranes, which were washed with TBS at room temperature for 5 min, and then incubated with 5% (w/v) milk powder in TBS-T at room temperature for 1 h. Membranes were washed with TBS-T, followed by incubation in a 1:2,000 dilution of horseradish peroxidase-conjugated anti-V5 antibody at room temperature for 1 h, then washed in TBS-T, followed by incubation for 5 min in TBS before detection by ECL system.

Effect of Exogenous Gangliosides on FliC Induction of hBD-2
Promoter Activity in Caco-2 Cells-The hBD-2 promoter was coupled to luciferase, and activity was quantified in pGL3-2110-transfected Caco-2 cells incubated with FliC (100 ng/ml) and various amounts of ganglioside mixtures. Gangliosides inhibited hBD-2 promoter activity in a concentration-dependent manner (Fig. 1A). To determine the specificity of ganglioside inhibition, several gangliosides were tested in the luciferase reporter gene assay. Asialo-GM1 and GM3 had no effect, whereas GM1, GM2, and GD3 were weakly inhibitory, and GD1a, GD1b, and GT1b strongly inhibited FliC action (Fig.  1B). To examine further the inhibitory effects of GD1a, GD1b, and GT1b, we used the luciferase reporter gene assay. Gangliosides GD1a, GD1b, and GT1b each inhibited FliC induction of hBD-2 promoter activity in a concentration-dependent manner (Fig. 1C), with more than 90% inhibition at 10 M. These results showed that exogenous gangliosides suppress FliC action when added to Caco-2 cell culture medium.
Effects of Anti-ganglioside Antibodies on FliC Induction of hBD-2 Promoter Activity in Caco-2 Cells-To confirm the role of FliC interaction with gangliosides on signal transduction, gangliosides on the cell surface were masked by reaction with anti-ganglioside antibody, and then FliC induction of hBD-2 promoter activity was evaluated with the luciferase assay. After masking of native GD1a, GD1b, and GT1b on Caco-2 cells by antibodies, FliC induction of hBD-2 promoter activity was inhibited in a concentration-dependent manner (Fig. 2). Antiasialo-GM1 and isotype control antibodies had no effects (data not shown). These results suggested that native gangliosides on Caco-2 cells are functionally important for FliC induction of signaling, leading to hBD-2 expression.
Exogenous GD1a Binds to FliC and Prevents Binding of FliC to Caco-2 Cells-Rusnati et al. (37) reported that gangliosides directly bind to fibroblast growth factor 2 (FGF-2) and protect it from tryptic digestion. Furthermore, they recently reported (41) that GM1 acts as functional co-receptor for FGF-2. To determine whether gangliosides directly bind to FliC, we determined whether ganglioside interaction with FliC can prevent its digestion by trypsin. GD1a protected FliC from tryptic digestion in a concentration-dependent manner, but asialo-GM1 had no effect (Fig. 3A), and GD1a did not protect an unrelated bacterial toxin VacA from tryptic digestion (Fig. 3B). Both GD1a and asialo-GM1 had no effect on tryptic digestion of P. aeruginosa flagellin (Fig. 3C).
We investigated the effect of exogenous GD1a on binding of FliC to Caco-2 cells using fluorescence-activated cell sorting. FliC bound to Caco-2 cells in a concentration-dependent manner, and exogenous GD1a suppressed FliC binding to Caco-2 cells (Fig. 4), consistent with the notion that exogenous GD1a directly interacts with FliC and prevents binding of FliC to Caco-2 cells.
Effects of Ganglioside Depletion and GD1a Loading on FliC Induction of hBD-2 Promoter Activity in Caco-2 Cells-To con- firm that native gangliosides in Caco-2 cells serve as a FliC co-receptor, Caco-2 cells were incubated with 5 M PPMP, an inhibitor of glucosylceramide synthase before stimulation with FliC. FliC induction of hBD-2 promoter activity was reduced in PPMP-treated Caco-2 cells. GD1a-loaded Caco-2 cells exhibited greater hBD-2 expression by following FliC stimulation than did untreated cells. Furthermore, incubation of gangliosidedepleted cells with GD1a restored FliC responsiveness (Fig. 5). These results also supported the hypothesis that ganglioside is functionally important as a FliC co-receptor.
Effects of TLR5 Expression and GD1a Loading on FliC induction of hBD-2 Promoter Activity in CHO-K1 Cells-To confirm further that the gangliosides serve as a FliC co-receptor, we determined the effects of TLR5 expression and GD1a loading on FliC induction of hBD-2 promoter activity in CHO-K1 cells. First TLR5 expression in cells was confirmed using Western blotting analysis (Fig. 6A). Both lines of TLR5-expressing cells respond to FliC in a concentration-dependent manner (Fig. 6, B and C). Induction of hBD-2 was increased by prior loading of TLR5-expressed CHO-K1 cells with GD1a, but CHO-K1 cells not expressing TLR5 were unresponsive to FliC (Fig. 6D). These results supported the hypothesis that gangliosides serve as a co-receptor for FliC.

Effects of Exogenous Gangliosides on FliC Induction of MAP Kinase Phosphorylation in Caco-2 Cells-
To examine the effect of ganglioside on MAP kinase phosphorylation, Caco-2 cells were incubated with FliC plus gangliosides. p38 and ERK1/2 phosphorylation by FliC were significantly inhibited by the presence of GD1a, GD1b, or GT1b (Fig. 7).
Effect of Mutation in the AP-1 Site of the hBD-2 Promoter on Response of Caco-2 Cells to FliC-To determine the importance of AP-1 for induction of hBD-2, we constructed pGL3-938/AP-1mt, a plasmid having a mutated AP-1 site. FliC increased activity in cells transfected with pGL3-938 but not pGL3-basic (empty vector), pGL3-938/NF-Bmt, and pGL3-938/AP-1mt (Fig. 8). This result suggested that AP-1 is also an important transcriptional factor for hBD-2 induction in Caco-2 cells by FliC.

DISCUSSION
Binding of P. aeruginosa flagellin to gangliosides such as GM1, GD1a, and asialo-GM1 had been reported in 1998 (31). In addition, asialo-GM1 on epithelial cells interacted with P. aeruginosa flagellin, leading to activation of phospholipase C, Ca 2ϩ mobilization, phosphorylation of ERK1/2, and activation of mucin transcription (32). We demonstrate here that higher order gangliosides serve as a S. enteritidis FliC coreceptor and influence FliC induction of hBD-2 promoter activity in Caco-2 cells.
FliC induction of hBD-2 promoter activity in Caco-2 cells was inhibited by exogenous gangliosides, GD1a, GD1b, and GT1b ( Fig. 1). These three gangliosides have a common backbone (GM1) plus additional one or two sialic acids (Fig. 9). Because exogenous GD1a protected FliC from tryptic digestion (Fig. 3A) and suppressed binding of FliC to Caco-2 cells (Fig. 4), it appears that exogenous GD1a directly interacts with FliC. Moreover, these data are consistent with the inhibitory effects of GD1a on FliC induction of hBD-2 promoter activity. In contrast to the S. enteritidis FliC, P. aeruginosa flagellin digestion by trypsin was not inhibited by GD1a and asialo-GM1 (Fig. 3C), suggesting that the sialic acids components of gangliosides have an important role in FliC binding and induction of promoter activity. In agreement, anti-GD1a, GD1b, and GT1b antibodies partially blocked FliC induction of hBD-2 promoter activity (Fig. 2). Increasing the GD1a content of Caco-2 cells enhanced FliC responsiveness; similarly, inhibition of ganglioside synthesis by treatment with PPMP decreased sensitivity to FliC, which was restored by GD1a loading (Fig. 5). In addition, as shown in Fig. 6, GD1a loading of TLR5-expressing CHO-K1 cells increased hBD-2 induction by FliC, whereas GD1a-loaded CHO-K1 cells were not responsible to FliC.
Glycosphingolipid, particularly gangliosides, sphingomyelin, and glycosylphosphatidylinositol-anchored proteins exist in glycosphingolipid-enriched microdomain (lipid rafts) in the plasma membrane. Li et al. (42) provided a perspective on the biological effect of gangliosides on cell proliferation. Depletion of the gangliosides conceivably abolishes the formation of glycosphingolipid clusters in the cell plasma membrane, and consequently, this could be one mechanism leading to blocked growth factor-induced proliferation. Conversely, preincubation of the cells with gangliosides, which causes enrichment of gangliosides (GD1a) in the plasma membrane and thereby potentially enhances the formation of glycosphingolipid cluster, promoted growth factor-induced proliferation (42) through enhancement of growth factor signaling and activation of Src kinase by gangliosides (43). In addition, by coupling to glycosphingolipid, Escherichia coli P fimbriae utilize the TLR4-dependent pathway to trigger cytokine responses, consistent with the conclusion that the glycosphingolipid receptors for P fimbriae recruit TLR4 as a co-receptor (44,45). Thus, these results supported our hypothesis that cell membrane gangliosides such as GD1a, GD1b, and GT1b function as co-receptors for FliC with TLR5.
TLRs can activate not only NF-B signaling pathways but also MAP kinase-dependent signaling. MAP kinase pathways are thought to play a major role in the activation of gene transcription by proinflammatory cytokines and antimicrobial peptides. Hobbie et al. (46) showed that the inflammatory response induced by S. typhimurium may be because of the specific stimulation of MAP kinase signaling pathways, leading to nuclear responses. In agreement, FliC increased phosphorylation of MAP kinases, p38 and ERK1/2, whereas exogenous GD1a, GD1b, and GT1b blocked FliC induction of p38 and ERK1/2 phosphorylation (Fig. 7). The promoter region of the hBD-2 gene contains an AP-1-binding site as well as NF-Bbinding sites. AP-1 is a homo-or heterodimer of proteins of the Jun and Fos families, transcription factors activated by MAP kinase pathways. Recently, Krisanaprakornkit et al. (47) reported that MAP kinase pathways involving the AP-1 transcription family are important for Fusobacterium nucleatumdependent hBD-2 induction in gingival epithelial cells. We had reported that FliC enhanced hBD-2 promoter activity in Caco-2 cells transfected with pGL3-938, containing the 5Ј-flanking region of the hBD-2 gene (Ϫ938 to Ϫ1) linked to a luciferase reporter but did not enhance activity with pGL3-938/NF-Bmt containing a mutated NF-B-binding site (21). We constructed a reporter gene pGL3-938/AP-1mt with a mutated AP-1 site. FliC increased luciferase activity in Caco-2 cells transfected with pGL3-938 but not with pGL3-basic (empty vector), PGL3-938/NF-Bmt, and pGL3-938/AP-1mt (Fig. 8). These data suggest that both NF-B and AP-1 are important for hBD-2 induction by S. enteritidis FliC in Caco-2 cells.
P. aeruginosa can activate MAP kinase signaling pathways through effects on intracellular Ca 2ϩ concentration (48). Gouy et al. (49) showed that an increase in intracellular Ca 2ϩ concentration can be elicited by human Jurkat T cell line by using cholera toxin B subunits to ligate ganglioside GM1. It would appear that GM1 may act as a cell activation molecule associated with unidentified transmembrane protein(s) (49). We reported previously (26) that FliC increased intracellular Ca 2ϩ concentrations, followed by induction of hBD-2 expression via an NF-B-dependent pathway. In this study, we reported that exogenous gangliosides inhibited not only FliC induction of hBD-2 promoter activity but also MAP kinase phosphorylation. Endogenous gangliosides by serving as co-receptors with TLR5 may increase intracellular Ca 2ϩ concentration and MAP kinase phosphorylation by FliC. Moon et al. (50) showed that an Src-dependent Raf-MEK1/2-ERK signaling pathway is required for IL-1␣-induced ␤-defensin 2 in human middle ear epithelial cells. Hazeki et al. (51) also showed that TLR regulates the function of paxillin through an Src familydependent mechanism. FliC-initiated signals may activate related pathways because TLRs and the IL-1 receptor have homologous cytoplasmic domains and can induce expression of some of the same target genes.
In summary, we have shown that gangliosides such as GD1a, GD1b, and GT1b act as co-receptors for FliC and promote hBD-2 induction in Caco-2 cells. FliC can activate not only the NF-B pathway but also MAP kinase pathways followed by enhancement of hBD-2 induction. Understanding the signaling pathways used by FliC could lead to novel therapeutic strategies for preventing the toxic effects of bacterial products.