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Journal of Dental Research
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Biological

Synergism between TLRs and NOD1/2 in Oral Epithelial Cells

A. Uehara* and H. Takada

Department of Microbiology and Immunology, Tohoku University Graduate School of Dentistry, Sendai, Japan

Correspondence: * corresponding author, kyoro{at}mail.tains.tohoku.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oral epithelium is the first barrier against oral bacteria in periodontal tissue. Oral epithelial cells constitutively express Toll-like receptors (TLRs) and NOD1/2, functional receptors which induce the production of antibacterial factors such as peptidoglycan recognition proteins (PGRPs) and β-defensin 2, but not pro-inflammatory cytokines such as interleukin (IL)-8. In this study, we hypothesized that innate immune responses in the oral epithelium are enhanced in inflamed tissue. We found that NOD1 and NOD2 agonists, in combination with TLR agonists, synergistically induced production of PGRPs and of β-defensin 2 in human oral epithelial cells via NF-{kappa}B. In contrast, co-stimulation with NOD1/2 and TLR ligands had no effect on the production of pro-inflammatory cytokines (IL-6, IL-8, and monocyte chemoattractant protein-1). These findings indicate that, in innate immune responses to invading microbes, a combination of signaling through TLRs and NODs leads to the synergistic activation of antibacterial responses in the oral epithelium.

Key Words: peptidoglycan recognition proteins (PGRPs) • Toll-like receptors (TLRs) • NOD1/2 • oral epithelial cells • β-defensin 2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The immune system provides protection against a wide variety of pathogens. Immunity can be divided into two major categories: innate and adaptive. In the innate immune system, defenses against invasive pathogens are initiated when pathogen-associated molecular patterns are recognized by the pattern recognition molecules of hosts. In mammals, these pathogen-associated molecular patterns are recognized specifically by their respective Toll-like receptors (TLRs): peptidoglycans and lipopeptides mainly by TLR2, double-stranded RNA by TLR3, lipopolysaccharide (LPS) by TLR4, single-stranded RNA by TLR7, and bacterial CpG DNA by TLR9 (Akira et al., 2006). Furthermore, it has been reported that 2 active entities of peptidoglycans, desmuramyl-peptides containing diaminopimelic acid and muramylpeptides, were recognized by intracellular receptors NOD1 and NOD2, respectively (Chamaillard et al., 2003; Girardin et al., 2003a,b; Inohara et al., 2003; Uehara et al., 2006).

Previously, we reported that oral epithelial cells constitutively expressed TLR2, TLR4, NOD1, and NOD2, and that stimulation with ligands for these receptors induced production of antimicrobial peptides such as peptidoglycan recognition proteins (PGRPs) (Uehara et al., 2005b) and β-defensin 2 (Sugawara et al., 2006), but not pro-inflammatory cytokines (Uehara et al., 2005b; Sugawara et al., 2006). In addition, we found the clear expression of TLR2, TLR3, TLR4, TLR7, NOD1, and NOD2 in tongue, salivary gland, pharyngeal, esophageal, intestinal, cervical, breast, lung, and kidney epithelial cells, as well as in oral epithelial cells, and these cells secrete β-defensin 2, but not pro-inflammatory cytokines in response to TLR and NOD agonists (Uehara et al., 2007). Concerning innate immune responses in human monocytic cells, the NOD2-agonistic muramyldipeptide and NOD1-agonistic diaminopimelic acid-containing peptides, in combination with the TLR2-agonistic lipopeptide, TLR4-agonistic lipid A, and TLR9-agonistic CpG DNA, synergistically induced production of IL-8 (Uehara et al., 2005a). In this study, we examined the possible synergistic effects of NOD1/2 and TLR agonists in terms of the production of antimicrobial peptides in oral epithelial cells. We used only chemically synthesized components, because natural microbial preparations are inevitably contaminated with minor bioactive components that might confuse the results.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Synthetic muramyldipeptide (MurNAc-L-Ala-D-isoGln) and an Escherichia coli-type lipid A (LA-15-PP) were purchased from the Protein Research Foundation Peptide Institute (Osaka, Japan). Double-stranded Poly I:C was obtained from Sigma-Aldrich (St. Louis, MO, USA). Single-stranded Poly U was provided by Invivogen (San Diego, CA, USA). A conventional CpG DNA, CpG DNA 1826 (TCCATGACGTTCCTG ACGTT), was purchased from SIGMA Genosys (Tokyo, Japan). A synthetic Mycoplasma-type diacyl lipopeptide FSL-1 (S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-GDPKHPKSF) was obtained from EMC Micro-collections (Tübingen, Germany). A synthetic desmuramylpeptide, a peptidoglycan fragment containing diaminopimelic acid, FK156 (D-lactoyl-L-Ala-{gamma}-D-Glu-meso-DAP-Gly), was supplied by Astellas Pharmaceutical Co. (Tokyo, Japan). Non-enzymatic cell dissociation solution was obtained from Sigma-Aldrich. All other reagents were from Sigma-Aldrich, unless otherwise indicated.

Cells and Cell Culture
The human oral epithelial cell line HSC-2 was obtained from the Cancer Cell Repository, Institute of Development, Aging and Cancer, Tohoku University. HSC-2 cells were grown in RPMI 1640 medium with 10% heat-inactivated FCS. Human gingival epithelial cells were prepared from explants of normal human gingival tissue, with informed consent from the donors. The experimental procedure was approved by the Ethical Review Board of Tohoku University Graduate School of Dentistry.

Flow Cytometry
Flow cytometric analyses were performed by means of a FACSCalibur cytometer (BD Biosciences, Mountain View, CA, USA). The cells were collected by non-enzymatic cell dissociation solution and washed in PBS. They were stained with anti-PGRP-I{alpha} (mouse IgG), anti-PGRP-Iβ (mouse IgG), or anti-PGRP-S (mouse IgG) antibodies (Imgenex, San Diego, CA), 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. It must be noted that PGRP-L expression was not examined, because no anti-PGRP-L antibodies are yet available commercially.

Cytokine Measurements
To investigate the production of inflammatory cytokines by oral epithelial cells, we collected the supernatant from each culture. The production of cytokines (IL-6, IL-8, and MCP-1) was measured with OptEIA ELISA kits (PharMingen, San Diego, CA, USA). The level of β-defensin 2 was measured with an ELISA Development Kit (PeproTEch EC, London, UK). The concentrations of the cytokines in the supernatants were determined with the LS-PLATEmanager 2004 data analysis program (Wako Pure Chemical Industries, Osaka, Japan).

RNA Interference
Transfections for targeting endogenous NF-{kappa}B p65 and Lamin A/C were carried out with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and short-interfering (si) RNA (final concentration, 200 nM) for 24 hrs at 37°C, according to the manufacturer’s instructions. The viability of the cells after transfection was more than 95%, as assessed by a 0.2% trypan blue exclusion test, and the morphology was not changed by the transfection. The siRNA for NF-{kappa}B p65 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the siRNA for Lamin A/C, from B-Bridge International (Mountain View, CA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of PGRPs in Human Oral Epithelial Cells Stimulated with NOD1-agonistic FK156 and NOD2-agonistic Muramyldipeptide in Combination with Various TLR Ligands
Chemically synthesized TLR and NOD ligands—FSL-1 (TLR2 agonist), Poly I:C (TLR3 agonist), lipid A (TLR4 agonist), Poly U (TLR7 agonist), CpG DNA (TLR9 agonist), FK156 (NOD1 agonist), and muramyldipeptide (NOD2 agonist)—slightly increased the expression of PGRP-I{alpha}, -Iβ, and -S in human oral epithelial cells (Fig. 1Go and APPENDIX). To elucidate the possible synergistic effects of NODs with various TLR agonists, we examined the production of PGRPs in human oral epithelial cells stimulated with NOD1-agonistic FK156 in combination with various TLR ligands. Clear synergistic effects were observed between FK156 and 5 synthetic TLR ligands. NOD2-agonistic muramyldipeptide in combination with TLR agonists also synergistically up-regulated the production of PGRPs (Fig. 1Go and APPENDIX).


Figure 1
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  		Figure 1. Synergistic effects of NOD1 and NOD2 agonists in combination with synthetic TLR agonists for induction of PGRP-I{alpha} in oral epithelial cells. Oral epithelial HSC-2 cells were stimulated with FK156 (100 µg/mL) or muramyldipeptide (MDP) (100 µg/mL) plus FSL-1 (1 nM), Poly I:C (10 µg/mL), lipid A (10 ng/mL), Poly U (10 µg/mL), or CpG DNA (10 nM) for 24 hrs in triplicate. The expression of PGRP-I{alpha} was assessed by flow cytometry. The unshaded curve represents medium alone. The results presented are representative of 3 different experiments.

 
NOD1-agonistic FK156 and NOD2-agonistic Muramyldipeptide in Combination with Chemically Synthesized TLR Agonists Synergistically Induced the Production of β-defensin 2 in Human Oral Epithelial Cells
Next, we examined the synergistic effects of NOD and TLR ligands in terms of the production of β-defensin 2 by ELISA. Consistent with the results regarding PGRP production, FK156 and muramyl-dipeptide, in combination with TLR agonists, synergistically induced the production of β-defensin 2 in oral epithelial cells (Fig. 2aGo). Primary gingival epithelial cells also showed synergistic production of β-defensin 2 upon stimulation with NOD and TLR ligands (Fig. 2bGo).


Figure 2
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  		Figure 2. Synergistic effects of NOD1 and NOD2 agonists in combination with synthetic TLR agonists for induction of β-defensin 2 secretion in oral epithelial cells. Oral epithelial HSC-2 cells (a) and primary gingival epithelial cells (b) were stimulated with FK156 (100 µg/mL) or muramyldipeptide (MDP) (100 µg/mL) plus FSL-1 (1 nM), Poly I:C (10 µg/mL), lipid A (10 ng/mL), Poly U (10 µg/mL), or CpG DNA (10 nM) for 24 hrs in triplicate. Concentrations of β-defensin 2 in the culture supernatants were determined by ELISA, and expressed as means ± SD. *,#Marked values differed significantly from those obtained with medium alone or from respective cultures stimulated with the indicated ligands, respectively. The results presented are representative of 3 different experiments.

 
FK156 and Muramyldipeptide in Combination with Chemically Synthesized TLR Agonists Did Not Induce the Production of Pro-inflammatory Cytokines in Human Oral Epithelial Cells
Oral epithelial cells did not secrete pro-inflammatory cytokines upon stimulation with TLR and NOD ligands (Uehara et al., 2005b, 2007). Contrary to PGRPs and β-defensin 2, co-stimulation with NOD1/2 and TLR ligands had no effect on the production of pro-inflammatory cytokines (IL-6, IL-8, and monocyte chemoattractant protein-1) (Fig. 3Go).


Figure 3
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  		Figure 3. Stimulation with NOD1 and NOD2 agonists in combination with synthetic TLR agonists did not induce the secretion of pro-inflammatory cytokines in oral epithelial cells. Oral epithelial cells were stimulated with FK156 (100 µg/mL) or muramyldipeptide (MDP) (100 µg/mL) plus FSL-1 (1 nM), Poly I:C (10 µg/mL), lipid A (10 ng/mL), Poly U (10 µg/mL), or CpG DNA (10 nM) for 24 hrs in triplicate. IL-1{alpha} (10 ng/mL) and TNF-{alpha} (10 ng/mL) were used as positive controls. Concentrations of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by ELISA, and expressed as means ± SD. *P < 0.01 vs. medium alone. The results presented are representative of 4 different experiments.

 
Specific Suppression by siRNA Targeting NF-βB against Synergistic β-defensin 2 Production Induced by a Combination of FK156 or Muramyldipeptide in Combination with Synthetic TLR Agonists
To clarify the signaling pathway of the synergistic effects of NOD1 and NOD2 agonists with TLR agonists, we utilized RNA interference assays, targeting NF-{kappa}B p65 mRNA. NF-{kappa}B protein levels determined by flow cytometry were suppressed by ca. 80% with specific siRNA in oral epithelial HSC-2 cells up to 72 hrs (Uehara et al., 2007). The synergistic effects induced by FK156 or muramyldipeptide and TLR ligands were almost completely diminished in NF-{kappa}B-silenced cells, but not in Lamin siRNA-silenced cells (Fig. 4Go).


Figure 4
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  		Figure 4. Specific suppression of synergistic effects of NOD1 and NOD2 agonists in combination with TLR ligands in oral epithelial cells with siRNA for NF-{kappa}B p65. Oral epithelial cells were transfected with NF-{kappa}B- or Lamin-specific siRNA. After 24 hrs, the transfected cells were stimulated with FK156 (100 µg/mL) or muramyldipeptide (MDP) (100 µg/mL) plus FSL-1 (1 nM), Poly I:C (10 µg/mL), lipid A (10 ng/mL), Poly U (1 µg/mL), or CpG DNA (1 µM) for an additional 24 hrs in triplicate. Concentrations of β-defensin 2 in the culture supernatants were determined by ELISA, and expressed as means ± SD. *,#Marked values differed significantly from those obtained with medium alone or from respective cultures stimulated with the indicated ligands, respectively. The results presented are representative of 3 different experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we clearly demonstrated that NOD1 and NOD2 agonists exhibited synergistic effects with TLR2, TLR3, TLR4, TLR7, and TLR9 agonists to induce the production of antimicrobial peptides such as PGRPs and β-defensin 2 in human oral epithelial cells. We also demonstrated that NF-{kappa}B is involved in the synergistic effects of NOD1 and NOD2 agonists in combination with TLR agonists.

It has been shown that mura-myldipeptide can act synergistically with LPS to induce production of inflammatory cytokines (Yang et al., 2001; Takada et al., 2002; Traub et al., 2004, 2006; Tada et al., 2005; Takada and Uehara, 2006). As well as NOD2-agonistic muramyl-dipeptide, NOD1-agonistic iE-DAP was also reported to induce TNF-{alpha} and IL-6 secretion, and, when combined with LPS, generated a greater response than that generated by LPS alone (Chamaillard et al., 2003). Our studies with the human myelomonocytic cell line THP-1 revealed that a NOD1-agonistic FK156 or FK565 induced IL-8 production by itself and in synergy with TLR2, TLR4, and TLR9 ligands (Uehara et al., 2005a). Furthermore, NOD1- and NOD2-agonists are reported to be able to stimulate the release of cytokines and chemokines in synergy with LPS in human monocytes and dendritic cells (Fritz et al., 2005; van Heel et al., 2005). This is the first report that NOD1 and 2 have synergistic effects with TLR2-, TLR3-, TLR7-, and TLR9-agonists, as well as TLR4-agonistic lipid A (LPS), to induce production of an antimicrobial peptide in human epithelial cells.

It is reasonable for oral epithelial cells to produce anti -microbial factors synergistically, without the accompanying inflammatory cytokines, upon stimulation with microbial components. Therefore, pattern recognition molecules on oral epithelial cells are functional, and oral epithelial cells actively participate in bacterial clearance without accompanying inflammatory responses in the oral mucosa.


   ACKNOWLEDGMENTS
 
We thank D. Mrozek (Medical English Service, Kyoto, Japan) for careful review of this paper. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (18390484 to H.T.), and the Ministry of Education, Sports, Science and Culture, Japan (18689901 to A.U.).


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/7/682/DC1.

Received for publication November 18, 2007. Revision received February 4, 2008. Accepted for publication February 16, 2008.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Dental Research, Vol. 87, No. 7, 682-686 (2008)
DOI: 10.1177/154405910808700709
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