Pathogens and Disease ISSN 2049-632X
SHORT COMMUNICATION
Rothia dentocariosa induces TNF-alpha production in a TLR2-dependent manner Hideo Kataoka, Makoto Taniguchi, Haruka Fukamachi, Takafumi Arimoto, Hirobumi Morisaki & Hirotaka Kuwata Departments of Oral Microbiology and Immunology, Showa University School of Dentistry, Tokyo, Japan
This report addresses a possible mechanism of the little studied Rothia dentocariosa (Rd) to contribute to the inflammation associate with periodontal disease. Notably, the authors demonstrate that two different strains of Rd are able to elicit TNF-alpha production in macrophage in a TLR-2-dependent fashion. This work is some of the first to identify specific mechanisms by which this bacterium contributes to diseases to which it is linked, that is, periodontitis.
Keywords Rothia dentocariosa; inflammation; TNF-a; Toll-like receptor 2. Correspondence Hideo Kataoka, Department of Oral Microbiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Tel.: +81 3 3784 8166 fax: +81 3 3784-4105 e-mail: [email protected] Received 14 June 2013; revised 10 September 2013; accepted 6 November 2013. Final version published online 16 December 2013. doi:10.1111/2049-632X.12115 Editor: Richard T. Marconi
Abstract Previous work suggested that Rothia dentocariosa is associated with periodontal inflammatory disease. However, little is known about the pathogenicity of this bacterium. To characterize host response to this bacterium, we measured (via ELISA) the amount of TNF-a in the culture supernatant following the stimulation of THP-1 cells (a human acute monocytic leukemia cell line) with R. dentocariosa cells (ATCC14189 and ATCC14190). Exposure to bacterial cells induced the production of TNF-a in a dose-dependent manner. The bacterial induction of TNF-a in THP-1 cells was mediated by the Toll-like receptor 2 (TLR2), as demonstrated by gene-specific knockdown via siRNA, which successfully suppressed TLR2 expression and significantly inhibited the production of TNF-a in the culture supernatant. To confirm the role of TLR2, we examined TLR2-dependent NF-jB activation by R. dentocariosa cells in a distinct cell line. Specifically, HEK293 cells were transiently cotransfected with the human TLR2 gene and an NF-jB-dependent luciferase-encoding reporter gene. The bacterial cells induced NF-jB activation in the transfected HEK293 cells in a dose-dependent manner. In contrast, bacterial cells failed to induce NF-jB activation in cells transfected with pEF6 control vector. Taken together, these results suggest that R. dentocariosa induces host TNF-a production by a TLR2-dependent mechanism.
Rothia dentocariosa is a gram-positive pleomorphic bacterium. The bacterium was previously known as Actinomyces dentocariosus (Onishi, 1949), Nocardia dentocariosus (Roth, 1957), and Nocardia salivae (Davis & Freer, 1960). However, because the cell wall constituents of this bacterium differ significantly from both Actinomyces and Nocardia, the species was assigned to a new genus (Rothia) by Georg & Brown (1967). The bacterium was first isolated from carious dentin (Onishi, 1949). Since then, the species has been isolated not only from carious dentin but also from a variety of human sources, such as blood, eyes, tonsils, and respiratory organs (Schiff & Kaplan, 1987; Ohashi et al., 2005; Morley & Tuft, 2006; Yang et al., 2007). Among those sources, the bacterium most frequently has been isolated from the oral cavity, especially from dental plaque taken from periodontal patients (Lesher et al., 1977). This correlation strongly suggests that R. dentocariosa might be
associated with periodontal inflammatory disease. The possible causative nature of R. dentocariosa remains unclear, given previous reports that crude cell wall and cytoplasmic antigens derived from R. dentocariosa strain 477 are not appreciably active in stimulating lymphocyte blastogenesis (Fotos et al., 1982). However, the ability of R. dentocariosa cells to stimulate human macrophages remains unknown, as does the mechanism whereby human macrophages might recognize cells of this species. It is well-known that host immune cells produce Toll-like receptors (TLRs) to detect pathogenic microorganisms and to initiate inflammatory responses. To detect microorganisms, TLRs recognize various microbial components, which are collectively defined as pathogen-associated molecular patterns (PAMPs) (Takeda et al., 2003). Upon recognition of microorganisms through TLRs, host cells release cytokines that initiate and amplify the host’s inflammatory responses.
Pathogens and Disease (2014), 71, 65–68, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
65
TLR-2-dependent induction of TNF-alpha by Rothia
To date, more than 10 classes of TLRs have been identified. Among them, TLR2 has been reported to recognize the broadest range of PAMPs, including lipoprotein/lipopeptide, lipoteichoic acid, lipoarabinomannan, and yeast zymosan (Aliprantis et al., 1999; Takeuchi et al., 2000; Wang et al., 2000; Schroder et al., 2003; Levitz, 2004). Interestingly, TLR2 also has been implicated in the inflammatory response triggered by several periodontopathic bacteria (Kikkert et al., 2007; Sun et al., 2010), suggesting that TLR2 plays a key role in initiating periodontal inflammation. However, the relationship between TLR2 and specific oral bacteria is poorly understood. Recently, we demonstrated that A. viscosus, an oral gram-positive bacterium frequently isolated from dental plaque, activates human macrophages and oral epithelial cells and that TLR2 plays a key role in this response (Shimada et al., 2012). Based on these observations, we hypothesized that TLR2 also may play a key role in initiating R. dentocariosa-triggered inflammatory responses. It is generally accepted that host macrophages produce inflammatory cytokines to initiate periodontal inflammation when the macrophages detect periodontopathic bacteria such as Porphyromonas gingivalis. The inflammatory cytokines attract neutrophils and macrophages to eliminate the
H. Kataoka et al.
periodontopathic bacteria, but also contribute to the destruction of periodontal tissue. Of those inflammatory cytokines, tumor necrosis factor (TNF)-a is reported to be associated with the development of periodontal inflammation and with the destruction of periodontal tissue (Abu-Amer et al., 1997; Taubman et al., 2005). Thus, we examined whether R. dentocariosa induces TNF-a production in human macrophages and whether TLR2 mediates this response. As a first test, we exposed a human macrophage cell line (THP-1) to R. dentocariosa cells (strains ATCC14189 or ATCC14190) at 1.2 9 105, 1.2 9 106, and 1.2 9 107 colony-forming units (CFU) mL 1, and determined the amounts of TNF-a in the culture supernatants using ELISA (ELISA development kit human TNF-a PeproTech, Rocky Hill, NJ). Exposure of THP-1 cells to the bacteria induced the production of TNF-a in a dose-dependent manner (Fig. 1a). This result shows that R. dentocariosa cells induce TNF-a production in human macrophages. R. dentocariosa has been isolated from dental plaque at the site of periodontal inflammation (Lesher et al., 1977). Using a panel of oral commensal bacteria, Thomas et al. (2012) recently observed that quantities of R. dentocariosa were significantly increased in mature dental plaque compared
(a)
(b)
(c)
(d)
Fig. 1 Rothia dentocariosa cells induce TNF-a production in a TLR2-dependent manner. The production of TNF-a in the culture supernatants of THP-1 stimulated with R. dentocariosa cells (strain ATCC14189 or ATCC14190) was measured by ELISA (a). THP-1 was transfected with TLR2-specific siRNA (siTLR2) or control siRNA and then stimulated by R. dentocariosa cells. The production of TNF-a in the culture supernatants of the transfected THP-1 cells then was measured by ELISA (b). R. dentocariosa cells activate NF-jB in a TLR2-dependent manner. HEK293 was transfected with a TLR2-encoding plasmid (or a control plasmid), along with an NF-jB reporter plasmid. Transfected cells then were stimulated with R. dentocariosa cells, and NF-jB-regulated gene expression was measured as relative luciferase activity (c). An NF-jB inhibitor, BAY11-7082, attenuates TNF-a production induced by R. dentocariosa cells, Pam3CSK4, and lipopolysaccharide in a dose-dependent manner (d). All experiments were performed in triplicate, and data are presented as mean SD from a single experiment. The statistical differences were assessed by Student’s t-test. For (a) and (d), multiple groups were compared using a two-tailed one-way analysis of variance (ANOVA) with Dunnett’s test where significance was indicated. P values of < 0.05 were considered statistically significant (*P < 0.05).
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Pathogens and Disease (2014), 71, 65–68, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
H. Kataoka et al.
with young dental plaque. Mayer et al. (2000) reported the induction of TNF-a production by Streptococcus gordonii, an oral commensal bacterium. In combination with these reports, our result suggests that R. dentocariosa in dental plaque has the ability to induce inflammatory responses in oral tissues. As noted above, TLR2 is a central component in the development of periodontal inflammation. We therefore tested whether R. dentocariosa-induced TNF-a production was mediated by TLR2. Specifically, we suppressed the expression of TLR2 in THP-1 cells using a gene-specific siRNA (siTLR2; Santa Cruz Biotechnology, Santa Cruz, CA). We confirmed that TLR2 transcript levels were reduced in THP-1 cells transfected with the siTLR2. When these transfected cells were stimulated with R. dentocariosa, TNF-a production was reduced significantly compared with cells that were stimulated with bacteria following transfection with a control siRNA (Fig. 1b). A similar TLR2-dependent effect was seen for stimulation with Pam3CSK4 (Fig. 1b), a synthetic triacylated lipopeptide known to serve as a TLR2 agonist (Aliprantis et al., 1999). In contrast, stimulation of TNF-a production by lipopolysaccharide derived from Escherichia coli (lipopolysaccharide; a ligand for TLR4, not TLR2) was seen in cells transfected with siTLR2 and did not significantly differ from that seen in cells transfected with the control siRNA and stimulated with lipopolysaccharide (Fig. 1b). These results suggest that TLR2 is involved in R. dentocariosa-induced TNF-a production. To further confirm the involvement of TLR2, we examined whether R. dentocariosa cells induce the activation of NF-jB, given that TNF-a production is known to reflect activation of the NF-jB pathway. HEK293 cells (derived from human embryonic kidney cells) were transfected with an NF-jB reporter plasmid together with a TLR2-encoding plasmid (or a control plasmid). We measured NF-jB reporter activities of those cells after stimulation with R. dentocariosa cells. As shown in Fig. 1c, R. dentocariosa cells activated NF-jB in TLR2-transfected cells in a dose-dependent manner. In contrast, this activation was not observed in the cells transfected with the control plasmid. As endogenous TLR2 is not expressed in HEK293 cells (Kirschning et al., 1998), these results demonstrate that R. dentocariosa cells activate inflammatory pathway gene expression (detected with the NF-jB reporter) in a second cell type and that this induction again is mediated via TLR2. To confirm the causal relationship between those NF-jB activation and TNF-a production, we pretreated THP-1 cells with an NF-jB inhibitor (BAY11-7082; InvivoGen, San Diego, CA) before stimulating the cell line with either of two strains of R. dentocariosa. We observed that BAY11-7082 decreased bacterial induction of TNF-a in a dose-dependent manner (Fig. 1d). Exposure of the BAY11-7082-treated cells to Pam3CSK4 (a TLR2 ligand) or lipopolysaccharide (a TLR4 ligand) confirmed that the BAY11-7082 inhibition of TNF-a production reflected a TLR2- and NF-jB-mediated activation process. Other researchers recently have reported that gram-negative periodontopathic bacteria, such as P. gingivalis, interact primarily with TLR2 (Yoshimura et al., 2002),
TLR-2-dependent induction of TNF-alpha by Rothia
such that sonicated extracts of P. gingivalis upregulate the expression of TLR2 (but not that of TLR4) in gingival fibroblasts (Wara-Aswapati et al., 2012). In combination with these recent studies, our results reveal that TLR2 is a key molecule for host recognition of periodontopathic bacteria and for the development of inflammation in periodontal tissue. In the present study, we showed that R. dentocariosa induces host TLR2-dependent TNF-a production. This result suggests that this bacterium may be a causative agent of periodontal inflammation. However, the specific component(s) of the bacterium that interacts with TLR2 remains to be elucidated. Recent reports showed that bacterial lipoproteins from several gram-positive bacteria, including Staphylococcus aureus, Listeria monocytogenesis, and S. agalactiae, induce TLR2-mediated inflammatory responses (Hashimoto et al., 2006; Henneke et al., 2008; Machata et al., 2008), whereas other reports showed that lipoteichoic acid of S. aureus is a primary ligand of TLR2 (von Aulock et al., 2003). In the present study, we observed that TLR2 suppression in THP-1 cells significantly attenuated TNF-a production, although production of the cytokine was not completely abolished by TLR2 gene-specific siRNA (Fig. 1b). This distinction could reflect incomplete suppression by siRNA; alternatively, the observation may indicate that R. dentocariosa cells are recognized by an additional (non-TLR2) receptor. It has been reported that the nucleotide-binding oligomerization domain containing 2 (NOD2) protein, an intracellular receptor, senses peptidoglycans from both gram-positive and gram-negative bacteria to activate NF-jB (Girardin et al., 2003). We hypothesize that NOD2 may detect R. dentocariosa peptidoglycans to provide inflammatory responses in the absence of TLR2. In conclusion, we demonstrate a role for TLR2 in inducing TNF-a production by either of two strains of R. dentocariosa; that is, TLR2 is a primary receptor for induction of this cytokine following exposure to this bacterium. Based on this observation, TLR2 might serve as a new target for regulating excess inflammation induced by R. dentocariosa.
Acknowledgements This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 21791796), by a Grant-in-Aid for Scientific Research (C) (No. 22592048), and by the Private University High Technology Research Center Project (No. S1001010). References Abu-Amer Y, Ross FP, Edwards J & Teitelbaum SL (1997) Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its p55 receptor. J Clin Invest 100: 1557–1565. Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, Godowski P & Zychlinsky A (1999) Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285: 736–739. Davis GHG & Freer JH (1960) Studies upon an oral aerobic actinomycete. J Gen Microbiol 23: 163–178.
Pathogens and Disease (2014), 71, 65–68, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Fotos PG, Gerencser VF & Gerencser MA (1982) Blastogenic response of human lymphocytes to antigens of Rothia dentocariosa. J Dent Res 61: 640–644. Georg LK & Brown JM (1967) Rothia, gen. nov., and aerobic genus of the family Actinomycetaceae. Int J Syst Bacteriol 17: 79–88. Girardin SE, Boneca IG & Viala J (2003) Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278: 8869–8872. Hashimoto M, Tawaratsumida K, Kariya H, Aoyama K, Tamura T & Suda Y (2006) Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall component. Int Immunol 18: 355–362. Henneke P, Dramsi S, Mancuso G, Chraibi K, Pellegrini E, €bner J & Sierra SS (2008) Lipoproteins are Theilacker C, Hu critical TLR2 activating toxins in Group B Streptococcal sepsis. J Immunol 180: 6149–6158. Kikkert R, Laine ML, Aarden LA & van Winkelhoff AJ (2007) Activation of Toll-like receptors 2 and 4 by gram-negative periodontal bacteria. Oral Microbiol Immunol 22: 145–151. Kirschning CJ, Wesche H, Ayres TM & Rothe M (1998) Human Toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide. J Exp Med 188: 2091–2097. Lesher RJ, Gerencser VF & Morrison DJ (1977) Presence of Rothia dentocariosa strain 477 serotype 2 in gingiva of patients with inflammatory periodontal disease. J Dent Res 56: 189. Levitz SM (2004) Interaction of Toll-like receptors with fungi. Microbes Infect 6: 1351–1355. Machata S, Tchatalbachev S, Mohamad W, Jansch L, Hain LT & Chakraborty T (2008) Lipoproteins of Listeria monocytogenes are critical for virulence and TLR2-mediated immune activation. J Immunol 181: 2028–2035. Mayer ML, Phillips CM, Townsend RA, Halperin SA & Lee SF (2000) Differential activation of dendritic cells by Toll-like receptor agonists isolated from the gram-positive vaccine vector Streptococcus gordonii. Scand J Immunol 69: 351–356. Morley AM & Tuft SJ (2006) Rothia dentocariosa isolated from a corneal ulcer. Cornea 25: 1128–1129. Ohashi M, Yoshikawa T & Akimoto S (2005) Severe acute tonsillitis caused by Rothia dentocariosa in a healthy child. Pediatr Infect Dis J 24: 466–467. Onishi M (1949) Study on Actinomyces isolated from the deeper layers of carious dentin. Shikagaku Zasshi 6: 273–318. Roth GD (1957) Proteolytic organisms of the carious lesion. Oral Surg Oral Med Oral Pathol 10: 1105–1117. Schiff MJ & Kaplan MH (1987) Rothia dentocariosa pneumonia in an immunocompromised patient. Lung 165: 279–282.
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Schroder NW, Morath S, Alexander C, Hamann L, Hartung T, € hringer U, Go € bel UB, Weber JR & Schumann RR. (2003) Za Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved. J Biol Chem 278: 15587–15594. Shimada E, Kataoka H, Miyazawa Y, Yamamoto M & Igarashi T (2012) Lipoproteins of Actinomyces viscosus induce inflammatory responses through TLR2 in human gingival epithelial cells and macrophages. Microbes Infect 14: 916–921. Sun Y, Shu R, Li CL & Zhang MZ (2010) Gram-negative periodontal bacteria induce the activation of Toll-like receptor 2,4 and cytokine production in human periodontal ligament cells. J Periodontol 81: 1488–1496. Takeda K, Kaisho T & Akira S (2003) Toll-like receptors. Annu Rev Immunol 21: 335–376. Takeuchi O, Kaufmann A, Grote K, Kawai T, Hoshino K & Morr M (2000) Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway. J Immunol 164: 554–557. Taubman MA, Valverde P, Han X & Kawai T (2005) Immune response: the key to bone resorption in periodontal disease. J Periodontol 76: 2033–2041. Thomas RZ, Zijnge V, Clcßek A, de Soet JJ, Harmsen HJM & Huysmans MCDNJM (2012) Shifts in the microbial population in relation to in situ caries progression. Caries Res 46: 427–431. von Aulock S, Morath S, Hareng L, Knapp S, van Kessel KP, van Strijp JA & Hartung T (2003) Lipoteichoic acid from Staphylococcus aureus is a potent stimulus for neutrophil recruitment. Immunobiology 208: 413–422. Wang T, Lafuse WP & Zwilling BS (2000) Regulation of Toll-like receptor 2 expression by macrophages 11 following Mycobacterium avium infection. J Immunol 165: 6308–6313. Wara-Aswapati N, Chayasadom A, Surarit R, Pitiphat W, Boch JA, Nagasawa T, Ishikawa I & Izumi Y (2013) Induction of Toll-like receptor expression by Porphyromonas gingivalis. J Periodontol 84: 1010–1018. Yang CY, Hsueh PR, Lu CY, Tsai HY, Lee PI, Shao PL, Wang CY, Wu TZ, Chen SW & Huang LM (2007) Rothia dentocariosa bacteremia in children: report of two cases and review of the literature. J Formos Med Assoc 106: S33–S38. Yoshimura A, Kaneko T, Kato Y, Golenbock DT & Hara Y (2002) Lipopolysaccarides from periodontopathic bacteria Porphyromonas gingivalis and Capnocytophaga ochracea are antagonists for human toll-like receptor 4. Infect Immun 70: 218–225.
Pathogens and Disease (2014), 71, 65–68, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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SHORT COMMUNICATION
Rothia dentocariosa induces TNF-alpha production in a TLR2-dependent manner Hideo Kataoka, Makoto Taniguchi, Haruka Fukamachi, Takafumi Arimoto, Hirobumi Morisaki & Hirotaka Kuwata Departments of Oral Microbiology and Immunology, Showa University School of Dentistry, Tokyo, Japan
This report addresses a possible mechanism of the little studied Rothia dentocariosa (Rd) to contribute to the inflammation associate with periodontal disease. Notably, the authors demonstrate that two different strains of Rd are able to elicit TNF-alpha production in macrophage in a TLR-2-dependent fashion. This work is some of the first to identify specific mechanisms by which this bacterium contributes to diseases to which it is linked, that is, periodontitis.
Keywords Rothia dentocariosa; inflammation; TNF-a; Toll-like receptor 2. Correspondence Hideo Kataoka, Department of Oral Microbiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Tel.: +81 3 3784 8166 fax: +81 3 3784-4105 e-mail: [email protected] Received 14 June 2013; revised 10 September 2013; accepted 6 November 2013. Final version published online 16 December 2013. doi:10.1111/2049-632X.12115 Editor: Richard T. Marconi
Abstract Previous work suggested that Rothia dentocariosa is associated with periodontal inflammatory disease. However, little is known about the pathogenicity of this bacterium. To characterize host response to this bacterium, we measured (via ELISA) the amount of TNF-a in the culture supernatant following the stimulation of THP-1 cells (a human acute monocytic leukemia cell line) with R. dentocariosa cells (ATCC14189 and ATCC14190). Exposure to bacterial cells induced the production of TNF-a in a dose-dependent manner. The bacterial induction of TNF-a in THP-1 cells was mediated by the Toll-like receptor 2 (TLR2), as demonstrated by gene-specific knockdown via siRNA, which successfully suppressed TLR2 expression and significantly inhibited the production of TNF-a in the culture supernatant. To confirm the role of TLR2, we examined TLR2-dependent NF-jB activation by R. dentocariosa cells in a distinct cell line. Specifically, HEK293 cells were transiently cotransfected with the human TLR2 gene and an NF-jB-dependent luciferase-encoding reporter gene. The bacterial cells induced NF-jB activation in the transfected HEK293 cells in a dose-dependent manner. In contrast, bacterial cells failed to induce NF-jB activation in cells transfected with pEF6 control vector. Taken together, these results suggest that R. dentocariosa induces host TNF-a production by a TLR2-dependent mechanism.
Rothia dentocariosa is a gram-positive pleomorphic bacterium. The bacterium was previously known as Actinomyces dentocariosus (Onishi, 1949), Nocardia dentocariosus (Roth, 1957), and Nocardia salivae (Davis & Freer, 1960). However, because the cell wall constituents of this bacterium differ significantly from both Actinomyces and Nocardia, the species was assigned to a new genus (Rothia) by Georg & Brown (1967). The bacterium was first isolated from carious dentin (Onishi, 1949). Since then, the species has been isolated not only from carious dentin but also from a variety of human sources, such as blood, eyes, tonsils, and respiratory organs (Schiff & Kaplan, 1987; Ohashi et al., 2005; Morley & Tuft, 2006; Yang et al., 2007). Among those sources, the bacterium most frequently has been isolated from the oral cavity, especially from dental plaque taken from periodontal patients (Lesher et al., 1977). This correlation strongly suggests that R. dentocariosa might be
associated with periodontal inflammatory disease. The possible causative nature of R. dentocariosa remains unclear, given previous reports that crude cell wall and cytoplasmic antigens derived from R. dentocariosa strain 477 are not appreciably active in stimulating lymphocyte blastogenesis (Fotos et al., 1982). However, the ability of R. dentocariosa cells to stimulate human macrophages remains unknown, as does the mechanism whereby human macrophages might recognize cells of this species. It is well-known that host immune cells produce Toll-like receptors (TLRs) to detect pathogenic microorganisms and to initiate inflammatory responses. To detect microorganisms, TLRs recognize various microbial components, which are collectively defined as pathogen-associated molecular patterns (PAMPs) (Takeda et al., 2003). Upon recognition of microorganisms through TLRs, host cells release cytokines that initiate and amplify the host’s inflammatory responses.
Pathogens and Disease (2014), 71, 65–68, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
65
TLR-2-dependent induction of TNF-alpha by Rothia
To date, more than 10 classes of TLRs have been identified. Among them, TLR2 has been reported to recognize the broadest range of PAMPs, including lipoprotein/lipopeptide, lipoteichoic acid, lipoarabinomannan, and yeast zymosan (Aliprantis et al., 1999; Takeuchi et al., 2000; Wang et al., 2000; Schroder et al., 2003; Levitz, 2004). Interestingly, TLR2 also has been implicated in the inflammatory response triggered by several periodontopathic bacteria (Kikkert et al., 2007; Sun et al., 2010), suggesting that TLR2 plays a key role in initiating periodontal inflammation. However, the relationship between TLR2 and specific oral bacteria is poorly understood. Recently, we demonstrated that A. viscosus, an oral gram-positive bacterium frequently isolated from dental plaque, activates human macrophages and oral epithelial cells and that TLR2 plays a key role in this response (Shimada et al., 2012). Based on these observations, we hypothesized that TLR2 also may play a key role in initiating R. dentocariosa-triggered inflammatory responses. It is generally accepted that host macrophages produce inflammatory cytokines to initiate periodontal inflammation when the macrophages detect periodontopathic bacteria such as Porphyromonas gingivalis. The inflammatory cytokines attract neutrophils and macrophages to eliminate the
H. Kataoka et al.
periodontopathic bacteria, but also contribute to the destruction of periodontal tissue. Of those inflammatory cytokines, tumor necrosis factor (TNF)-a is reported to be associated with the development of periodontal inflammation and with the destruction of periodontal tissue (Abu-Amer et al., 1997; Taubman et al., 2005). Thus, we examined whether R. dentocariosa induces TNF-a production in human macrophages and whether TLR2 mediates this response. As a first test, we exposed a human macrophage cell line (THP-1) to R. dentocariosa cells (strains ATCC14189 or ATCC14190) at 1.2 9 105, 1.2 9 106, and 1.2 9 107 colony-forming units (CFU) mL 1, and determined the amounts of TNF-a in the culture supernatants using ELISA (ELISA development kit human TNF-a PeproTech, Rocky Hill, NJ). Exposure of THP-1 cells to the bacteria induced the production of TNF-a in a dose-dependent manner (Fig. 1a). This result shows that R. dentocariosa cells induce TNF-a production in human macrophages. R. dentocariosa has been isolated from dental plaque at the site of periodontal inflammation (Lesher et al., 1977). Using a panel of oral commensal bacteria, Thomas et al. (2012) recently observed that quantities of R. dentocariosa were significantly increased in mature dental plaque compared
(a)
(b)
(c)
(d)
Fig. 1 Rothia dentocariosa cells induce TNF-a production in a TLR2-dependent manner. The production of TNF-a in the culture supernatants of THP-1 stimulated with R. dentocariosa cells (strain ATCC14189 or ATCC14190) was measured by ELISA (a). THP-1 was transfected with TLR2-specific siRNA (siTLR2) or control siRNA and then stimulated by R. dentocariosa cells. The production of TNF-a in the culture supernatants of the transfected THP-1 cells then was measured by ELISA (b). R. dentocariosa cells activate NF-jB in a TLR2-dependent manner. HEK293 was transfected with a TLR2-encoding plasmid (or a control plasmid), along with an NF-jB reporter plasmid. Transfected cells then were stimulated with R. dentocariosa cells, and NF-jB-regulated gene expression was measured as relative luciferase activity (c). An NF-jB inhibitor, BAY11-7082, attenuates TNF-a production induced by R. dentocariosa cells, Pam3CSK4, and lipopolysaccharide in a dose-dependent manner (d). All experiments were performed in triplicate, and data are presented as mean SD from a single experiment. The statistical differences were assessed by Student’s t-test. For (a) and (d), multiple groups were compared using a two-tailed one-way analysis of variance (ANOVA) with Dunnett’s test where significance was indicated. P values of < 0.05 were considered statistically significant (*P < 0.05).
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Pathogens and Disease (2014), 71, 65–68, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
H. Kataoka et al.
with young dental plaque. Mayer et al. (2000) reported the induction of TNF-a production by Streptococcus gordonii, an oral commensal bacterium. In combination with these reports, our result suggests that R. dentocariosa in dental plaque has the ability to induce inflammatory responses in oral tissues. As noted above, TLR2 is a central component in the development of periodontal inflammation. We therefore tested whether R. dentocariosa-induced TNF-a production was mediated by TLR2. Specifically, we suppressed the expression of TLR2 in THP-1 cells using a gene-specific siRNA (siTLR2; Santa Cruz Biotechnology, Santa Cruz, CA). We confirmed that TLR2 transcript levels were reduced in THP-1 cells transfected with the siTLR2. When these transfected cells were stimulated with R. dentocariosa, TNF-a production was reduced significantly compared with cells that were stimulated with bacteria following transfection with a control siRNA (Fig. 1b). A similar TLR2-dependent effect was seen for stimulation with Pam3CSK4 (Fig. 1b), a synthetic triacylated lipopeptide known to serve as a TLR2 agonist (Aliprantis et al., 1999). In contrast, stimulation of TNF-a production by lipopolysaccharide derived from Escherichia coli (lipopolysaccharide; a ligand for TLR4, not TLR2) was seen in cells transfected with siTLR2 and did not significantly differ from that seen in cells transfected with the control siRNA and stimulated with lipopolysaccharide (Fig. 1b). These results suggest that TLR2 is involved in R. dentocariosa-induced TNF-a production. To further confirm the involvement of TLR2, we examined whether R. dentocariosa cells induce the activation of NF-jB, given that TNF-a production is known to reflect activation of the NF-jB pathway. HEK293 cells (derived from human embryonic kidney cells) were transfected with an NF-jB reporter plasmid together with a TLR2-encoding plasmid (or a control plasmid). We measured NF-jB reporter activities of those cells after stimulation with R. dentocariosa cells. As shown in Fig. 1c, R. dentocariosa cells activated NF-jB in TLR2-transfected cells in a dose-dependent manner. In contrast, this activation was not observed in the cells transfected with the control plasmid. As endogenous TLR2 is not expressed in HEK293 cells (Kirschning et al., 1998), these results demonstrate that R. dentocariosa cells activate inflammatory pathway gene expression (detected with the NF-jB reporter) in a second cell type and that this induction again is mediated via TLR2. To confirm the causal relationship between those NF-jB activation and TNF-a production, we pretreated THP-1 cells with an NF-jB inhibitor (BAY11-7082; InvivoGen, San Diego, CA) before stimulating the cell line with either of two strains of R. dentocariosa. We observed that BAY11-7082 decreased bacterial induction of TNF-a in a dose-dependent manner (Fig. 1d). Exposure of the BAY11-7082-treated cells to Pam3CSK4 (a TLR2 ligand) or lipopolysaccharide (a TLR4 ligand) confirmed that the BAY11-7082 inhibition of TNF-a production reflected a TLR2- and NF-jB-mediated activation process. Other researchers recently have reported that gram-negative periodontopathic bacteria, such as P. gingivalis, interact primarily with TLR2 (Yoshimura et al., 2002),
TLR-2-dependent induction of TNF-alpha by Rothia
such that sonicated extracts of P. gingivalis upregulate the expression of TLR2 (but not that of TLR4) in gingival fibroblasts (Wara-Aswapati et al., 2012). In combination with these recent studies, our results reveal that TLR2 is a key molecule for host recognition of periodontopathic bacteria and for the development of inflammation in periodontal tissue. In the present study, we showed that R. dentocariosa induces host TLR2-dependent TNF-a production. This result suggests that this bacterium may be a causative agent of periodontal inflammation. However, the specific component(s) of the bacterium that interacts with TLR2 remains to be elucidated. Recent reports showed that bacterial lipoproteins from several gram-positive bacteria, including Staphylococcus aureus, Listeria monocytogenesis, and S. agalactiae, induce TLR2-mediated inflammatory responses (Hashimoto et al., 2006; Henneke et al., 2008; Machata et al., 2008), whereas other reports showed that lipoteichoic acid of S. aureus is a primary ligand of TLR2 (von Aulock et al., 2003). In the present study, we observed that TLR2 suppression in THP-1 cells significantly attenuated TNF-a production, although production of the cytokine was not completely abolished by TLR2 gene-specific siRNA (Fig. 1b). This distinction could reflect incomplete suppression by siRNA; alternatively, the observation may indicate that R. dentocariosa cells are recognized by an additional (non-TLR2) receptor. It has been reported that the nucleotide-binding oligomerization domain containing 2 (NOD2) protein, an intracellular receptor, senses peptidoglycans from both gram-positive and gram-negative bacteria to activate NF-jB (Girardin et al., 2003). We hypothesize that NOD2 may detect R. dentocariosa peptidoglycans to provide inflammatory responses in the absence of TLR2. In conclusion, we demonstrate a role for TLR2 in inducing TNF-a production by either of two strains of R. dentocariosa; that is, TLR2 is a primary receptor for induction of this cytokine following exposure to this bacterium. Based on this observation, TLR2 might serve as a new target for regulating excess inflammation induced by R. dentocariosa.
Acknowledgements This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 21791796), by a Grant-in-Aid for Scientific Research (C) (No. 22592048), and by the Private University High Technology Research Center Project (No. S1001010). References Abu-Amer Y, Ross FP, Edwards J & Teitelbaum SL (1997) Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its p55 receptor. J Clin Invest 100: 1557–1565. Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, Godowski P & Zychlinsky A (1999) Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285: 736–739. Davis GHG & Freer JH (1960) Studies upon an oral aerobic actinomycete. J Gen Microbiol 23: 163–178.
Pathogens and Disease (2014), 71, 65–68, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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