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Toll-like Receptors, NOD1, and NOD2 in Oral Epithelial Cells

¹ Division of Oral Diagnosis, Department of Oral Medicine and Surgery, and
² Department of Microbiology and Immunology, Tohoku University Graduate School of Dentistry, Sendai, Japan;
³ Department of Chemistry, Graduate School of Science, Osaka University, Japan; and
⁴ Division of Control of Oral Infection, Hokkaido University Graduate School of Dentistry, Sapporo, Japan

^* corresponding authors, dent-ht{at}mail.tains.tohoku.ac.jp, kyoro{at}mail.tains.tohoku.ac.jp

	ABSTRACT

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Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of ß-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

KEY WORDS: TLR • NOD • epithelium • ß-defensin 2 • inflammation

	INTRODUCTION

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All animals and plants possess a means of innate defense against microorganisms. In the innate immune system, pattern recognition of microorganisms should initiate host defense against invasive pathogens, where pathogen-associated molecular patterns are recognized by the pattern recognition molecules of hosts. In bacteria, representative pathogen-associated molecular patterns are distributed mainly on cell surfaces such as peptidoglycans, lipoproteins, and lipopolysaccharides (LPS). Recent studies have demonstrated that, in mammals, these pathogen-associated molecular patterns are recognized specifically by their respective Toll-like receptors (TLRs) on the cell surfaces of hosts; peptidoglycans and lipopeptides are mainly recognized by TLR2, and LPS is recognized by TLR4 (Akira and Takeda, 2004). In this decade, the importance of TLRs in the process of recognition by myeloid cells, such as macrophages or dendritic cells, has been exhaustively studied. Concerning TLR expression on oral epithelial cells, we have recently reported that primary oral epithelial cells, oral squamous cell carcinoma HSC-2, HO-1-u-1, and KB constitutively expressed TLR2 and TLR4 (Uehara et al., 2001). In contrast, gingival epithelial cells transfected with HPV-16 constitutively expressed TLR2, but not TLR4 (Asai et al., 2001). As mentioned above, intact peptidoglycans carrying a polymeric structure linked by glycan chains were recognized by TLR2, whereas TLR2 could not recognize the monomeric structure of the peptidoglycan subunit (Yoshimura et al., 2000; Yang et al., 2001; Vidal et al., 2001). It has been reported that intracellular receptors for two active entities of peptidoglycans—desmuramylpeptides containing diaminopimelic acid and muramyldipeptide—were recognized by NOD1 and NOD2, respectively (Chamaillard et al., 2003; Girardin et al., 2003a,b; Inohara et al., 2003). NOD proteins in human oral epithelium have not been reported so far, except for our report on their function (Uehara et al., 2005).

Previously, we demonstrated that human oral epithelial cells did not show an enhanced production of pro-inflammatory cytokines in response to various bacterial cell-surface components (Uehara et al., 2001). This lack of responsiveness might prevent the tissue destruction caused by excessive inflammatory reactions derived from innate immune responses to bacteria in normal oral flora. In contrast, we have found that oral epithelial cells produce peptidoglycan recognition proteins upon stimulation with bacterial components, including TLR and NOD ligands (Uehara et al., 2005). Peptidoglycan recognition proteins are a novel family of pattern-recognition molecules, involved in innate immunity, which recognize bacterial cell wall peptidoglycans and are suggested to act as antibacterial factors. Some human intestinal epithelial cells also did not produce pro-inflammatory cytokines in response to various bacterial cell-surface components (Eckmann et al., 1993; Schürer-Maly et al., 1994), although these cells produce antibacterial factors upon stimulation with bacterial components, including NOD ligands (Kobayashi et al., 2005).

To elucidate the possible expression of TLR2, TLR4, NOD1, and NOD2 on human oral epithelial cells, we examined the mRNA and protein expression of these molecules by RT-PCR and flow cytometry, and by immunostaining in vitro and in vivo. Additionally, to determine whether these pattern-recognition molecules are functional, we examined whether human oral epithelial cells secreted ß-defensin 2 upon stimulation with TLR and NOD ligands. We used only chemically synthesized bacterial components, because natural bacterial cell-surface preparations are inevitably contaminated with minor bioactive components that might produce confusing results.

	MATERIALS & METHODS

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Tissues
Gingival tissue samples from five patients with chronic periodontitis, characterized by advanced loss of periodontal support, and clinically healthy gingival tissue samples from three patients were used in this study under informed consent. The healthy samples were obtained from teeth extracted for orthodontic reasons. The samples of periodontally involved tissues were obtained from patients undergoing periodontal surgery. We determined their periodontal disease status by measuring probing depth and gingival index and examining radiographs. The Ethical Review Board of Tohoku University Graduate School of Dentistry (Sendai, Japan) approved the experimental procedures.

Cells and Cell Culture
Human gingival epithelial cells were prepared from the normal gingival tissues of a six-year-old patient under informed consent as described previously (Uehara et al., 2001). Human oral epithelial cell lines HSC-2, HO-1-u-1, and KB were obtained from the Cancer Cell Repository, Institute of Development, Aging and Cancer, Tohoku University. HSC-2 and HO-1-u-1 were grown in RPMI 1640 with 10% heat-inactivated FCS. KB was grown in -MEM with 10% FCS. It must be noted here that cell line KB has been known to be a subline of the HeLa, not oral carcinoma.

Reagents
Synthetic muramyldipeptide and Escherichia coli-type lipid A (LA-15-PP) were purchased from the Protein Research Foundation Peptide Institute (Osaka, Japan). E. coli-type lipopeptide Pam₃CSSNA and desmuramylpeptides (iE-diaminopimelic acid; -D-glutamyl-meso-diaminopimelic acid) were chemically synthesized as described previously (Nakamura et al., 2002; Chamaillard et al., 2003). Anti-TLR4 monoclonal antibody HTA125 (mouse IgG2a) and anti-TLR2 monoclonal antibody TL2.1 (mouse IgG2a) were purchased from eBioscience (San Diego, CA, USA). Goat anti-NOD1 polyclonal antibody L-17 was obtained from Cayman Chemical (Ann Arbor, MI, USA). Rabbit anti-NOD2 polyclonal antibody and goat anti-ß-defensin 2 polyclonal antibody C-17 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

RNA Extraction, Reverse Transcription, and Quantitative Polymerase Chain-reaction (PCR)
Total cellular RNA was extracted from cells with the use of Isogen (Nippon Gene, Toyko, Japan) according to the manufacturer’s instructions. Random hexamer-primed reverse transcription was performed on 2.5 µ L of total RNA in a 50-µL reaction, and all PCR procedures were performed in a 20-µL vol. The primers used for PCR were as follows: forward 5'-GCCAAAGTCTTGATTGATTGG-3' and reverse 5'-TTGAAGTTCTCCAGCTCCTG-3'; TLR4, forward 5'-TGGATACGTTTCCTTATAAG-3' and reverse 5'-GAAATGGAGGCACCCCTTC-3'; NOD1, forward 5'-TAGTGCTGTTTCTGCCTCTC-3' and reverse 5'-AATTTGACCCCTGCGTCTAG-3'; NOD2, forward 5'-AGCCATTGTCAGGAGGCTC-3' and reverse 5'-CGTCTCTGCTCCATCATAGG-3'; '-defensin 2, forward 5'-GACTGAGTCTTGCTCTGTCGG-3' and reverse 5'-GGCATGATGGCTTACGCCTATA-3'; and human glyceraldehydes-3-phosphate dehydrogenase (GAPDH), forward 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and reverse 5'-CATGTGGGCCATGAGGTCCACCAC-3'. Amplified samples were visualized on 2.0% agarose gels stained with ethidium bromide and photographed under UV light.

Flow Cytometry
Flow cytometric analyses were performed with a FACSCalibur cytometer (BD Biosciences, Mountain View, CA, USA). The cells were collected and washed in PBS. Cells were stained with anti-TLR2 antibody, anti-TLR4 antibody, or control IgG at 4°C for 30 min, followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (BioSource International, Camarillo, CA, USA) at 4°C for an additional 30 min. For NOD1, NOD2, and ß-defensin 2 stainings, intracellular staining protocols were performed. Briefly, the cells were washed with staining buffer, fixed, and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences) for 15 min at 4°C, and then incubated with anti-NOD1 antibody, anti-NOD2 antibody, anti-ß-defensin 2 antibody, or control IgG for 30 min, followed by FITC-conjugated secondary antibody at 4°C for anther 30 min.

Immunohistochemistry
Immunohistochemistry was conducted as follows. Tissues were fixed in periodate-lysine-4% paraformaldehyde for 4 hrs at 4°C. After being washed in PBS containing sucrose, fixed tissues were embedded in OCT compound (Sakura, Tokyo, Japan) and immediately frozen. Six-micrometer-thick frozen tissue sections were incubated with anti-NOD1 antibody, anti-NOD2 antibody, anti-TLR2 antibody, and anti-TLR4 antibody overnight at 4°C. Subsequently, sections were treated with secondary antibodies, including rabbit anti-goat Simple stain MAX PO (Nichirei, Tokyo, Japan), goat anti-rabbit Envision +/HRP kit, or goat anti-mouse Envision +/HRP kit (DakoCytomation) overnight at 4°C. The chromogen used was 3',3-diaminobenzidine tetrahydrochloride (DakoCytomation). The sections were counterstained with hematoxylin. As negative controls, mouse IgG2a and normal rabbit serum (DakoCytomation) and normal goat serum (ZYMED, San Francisco, CA, USA) were used.

Immunostaining of Cells
The cells were cultured on eight-chamber glass slides until confluent, with or without synthetic components, and were washed with PBS. After fixation with 4% paraformaldehyde for 15 min, the cells were further treated with 0.5% Triton X-100 for 15 min for intracellular staining. Cells were then incubated with anti-TLR2 antibody, anti-TLR4 antibody, anti-NOD1 antibody, anti-NOD2 antibody, or anti-ß-defensin 2 antibody for 3 hrs at room temperature. Samples were then washed and incubated with Alexa Fluor 488 goat anti-mouse IgG2a, Alexa Fluor 488 rabbit anti-goat IgG, and Alexa 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA), respectively. Nuclei were visualized by being stained with 4',6-diamino-2-phenylindole in blue (Molecular Probes) or propidium iodide (Sigma-Aldrich). Samples were photographed with a Leica DC 200 cooled charged-coupled-device camera mounted on a Leica DMR microscope with the Leica Qfluoro system application (Leica Microsystems, Solms, Germany).

	RESULTS

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Histological Analysis of TLR2, TLR4, NOD1, and NOD2 Expression in Human Gingival Epithelial Tissues
First, using immunohistochemistry, we examined whether human oral tissues express TLR2, TLR4, NOD1, and NOD2 molecules. NOD1 and NOD2 molecules were markedly expressed in the epithelial layer of healthy gingival tissue (Figs. 1a, 1c). Expression of NOD1 and NOD2 was also detected in gingival tissue from adult periodontitis patients, similar to that found in healthy gingival tissue (Figs. 1b, 1d). TLR2 and TLR4 molecules were also detected in healthy and inflamed gingival tissue (Figs. 1e–1h), although the expression of TLR2 and TLR4 was weaker than that of NODs. It should be noted that cell-surface localizations of TLR2 and TLR4 were more clearly observed in the inflamed gingival tissue than in healthy gingival tissue (Figs. 2b, 2d).

View larger version (95K): [in this window] [in a new window]   Figure 1. Expression of TLR2, TLR4, NOD1, and NOD2 in human gingival epithelial tissues. Cryosections of healthy gingival tissues (a,c,e,g) and inflamed gingival tissues (b,d,f,h) were stained brown with anti-NOD1 antibody (a,b), anti-NOD2 antibody (c,d), anti-TLR2 antibody (e,f), and anti-TLR4 antibody (g,h). The sections were counterstained blue with hematoxylin (a–h). Scale bars: 100 µm.

View larger version (162K): [in this window] [in a new window]   Figure 2. Up-regulated expression of TLR2 and TLR4 in inflamed gingival epithelial tissues. Cryosections of healthy gingival tissues (a,c) and inflamed gingival tissues (b,d) were stained brown with anti-TLR2 antibody (a,b) and anti-TLR4 antibody (c,d). The sections were counterstained blue with hematoxylin (a–d). Scale bars: 20 µm.

View larger version (45K): [in this window] [in a new window]   Figure 3. Expression of TLR2, TLR4, NOD1, and NOD2 on human oral epithelial cells. (a) Human oral epithelial HSC-2, HO-1-u-1, KB cells, and primary gingival epithelial cells were cultured until confluent at 37°C. After incubation, the total RNA was extracted, and the mRNA expression of TLR2, TLR4, NOD1, and NOD2 was analyzed by PCR. (b) Human oral epithelial cells were cultured until confluent at 37°C. The expression of TLR2, TLR4, NOD1, and NOD2 was assessed by flow cytometry. Thin lines represent the isotype Ab control. The results presented are representative of 4 different experiments demonstrating similar results. (c) Human oral epithelial HSC-2 cells were cultured until confluent at 37°C. After fixation, cells were treated with anti-TLR2 antibody or anti-TLR4 antibody and then visualized with Alexa Fluor 488 (green). Nuclei were visualized by being stained with propidium iodide (red). For NOD staining, cells were treated with anti-NOD1 antibody (brown) or anti-NOD2 antibody (brown). The results are representative of 3 different experiments with similar results. Scale bars: 20 µm.

View larger version (20K): [in this window] [in a new window]   Figure 4. Induction of ß-defensin 2 triggered by TLR2, TLR4, NOD1, or NOD2 ligand in human oral epithelial cells. (a) Human oral epithelial HSC-2 cells were incubated for 8 hrs in the presence or absence of Pam3CSSNA (100 pg/mL), lipid A (100 ng/mL), muramyldipeptide (100 µg/mL), or iE-diaminopimelic acid (100 µg/mL). After incubation, total RNA was extracted, and the mRNA expression of ß-defensin 2 was analyzed by real-time PCR. (b) Human oral epithelial HSC-2 cells were incubated for 24 hrs in the presence or absence of Pam3CSSNA (100 pg/mL), lipid A (100 ng/mL), muramyldipeptide (100 µg/mL), or iE-diaminopimelic acid (100 µg/mL). After fixation, cells were treated with anti-ß-defensin 2 antiboby and then visualized with Alexa Fluor 488 (green). Nuclei were visualized by being stained with 4',6-diamino-2-phenylindole (blue). Scale bars: 20 µ m. (c) Human oral epithelial HSC-2 cells were incubated for 24 hrs in the presence or absence of Pam3CSSNA (100 pg/mL), lipid A (100 ng/mL), muramyldipeptide (100 µg/mL), or iE-diaminopimelic acid (100 µ g/mL). The expression of ß-defensin 2 was assessed by flow cytometry. Thin lines represent the isotype antibody control. The results presented are representative of 4 different experiments demonstrating similar results.

	DISCUSSION

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TLR4 mRNA was clearly expressed in 3 simian-virus-transformed cell lines (OBA-9, SHGE-1, and SHGE-2), but only faintly in primary gingival epithelial cells, whereas TLR2 mRNA was strongly expressed in both cell lines and primary cells by RT-PCR (Kusumoto et al., 2004). In the latter report, the investigators claimed that the prominent expression of TLR2 was observed in both cell lines and primary cells, but neither was stained by anti-TLR4. In a related report, human gingival epithelial cells transfected with human papillomavirus predominantly expressed TLR2 mRNA and protein, but not TLR4 mRNA or protein (Asai et al., 2001). In these studies, papillomavirus- and simian-virus-40-transformed human oral epithelial cell lines were used on the basis of their statement that papillomavirus- and SV40-immortalized cell lines retain the parental cell phenotypes and preserve the contact inhibition, while the transformed cells have a substantially increased growth rate, although the phenotypes of the cell lines tested showed slight divergences in TLR expression (Asai et al., 2001; Kusumoto et al., 2004). These authors also reported that TLR2 was observed in epithelial cells, whereas epithelial cells were only faintly stained with anti-TLR4 Ab.

In the present study, TLR4 as well as TLR2 were also detectable in gingival tissues, and more marked expression of both TLRs on the cell surface was observed in inflamed tissues compared with healthy tissues (Fig. 2). The significantly higher expression of TLR2 and TLR4 in inflamed epithelium may have resulted from stimulation by a variety of bacterial products and inflammatory cytokines. We previously revealed that treatment with pro-inflammatory cytokines up-regulated the expression of TLRs (Uehara et al., 2002). TLRs are expressed at high levels in cells that respond to LPS, such as peripheral blood leukocytes, macrophages, and monocytes. These findings suggest that up-regulated expression of TLRs and NODs on oral epithelium possibly results in the higher innate immune response to bacterial products in periodontal tissues.

In contrast to colonic epithelial cells, however, oral epithelial cells did not secrete cytokines—such as IL-8, monocyte chemoattractant protein-1, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, and vascular endothelial growth factor—upon stimulation with bacterial components (Uehara et al., 2001, 2005). Therefore, we hypothesized that the oral epithelial cells may be partially desensitized to avoid tissue destruction by excessive innate immune responses to bacterial stimuli, because the cells are constitutively interacting with bacteria. Recently, we demonstrated that oral epithelial cells are highly responsive to bacterial components, resulting in the production of high levels of peptidoglycan recognition proteins via TLRs and NODs (Uehara et al., 2005). Consistent with the up-regulation of peptidoglycan recognition proteins by bacterial components, antibacterial ß-defensin 2 mRNA and protein were significantly up-regulated upon stimulation with bacterial components via TLRs and NODs (Fig. 4). Therefore, pattern-recognition molecules on oral epithelial cells are functional, and oral epithelial cells might actively participate in bacterial clearance in the oral mucosa without inflammation, which also prevents excessive innate immune responses to bacteria, which might result in excessive inflammatory reactions followed by tissue destruction.

	ACKNOWLEDGMENTS

 
We thank D. Mrozek (Medical English Service, Kyoto, Japan) for reviewing the paper. This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (14370576, 16390519, 17591959). A.U. was supported by a research fellowship from the Japan Society for the Promotion of Science (PD6961).

Received July 15, 2005; Last revision January 10, 2006; Accepted January 30, 2006

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