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Lipoproteins of Actinomyces viscosus induce inflammatory responses through TLR2 in human gingival epithelial cells and macrophages
Microbes and Infection 14 (2012) 916e921 www.elsevier.com/locate/micinf
Short communication
Lipoproteins of Actinomyces viscosus induce inflammatory responses through TLR2 in human gingival epithelial cells and macrophages Eri Shimada a,b, Hideo Kataoka a,*, Yasushi Miyazawa b, Matsuo Yamamoto b, Takeshi Igarashi a a
Department of Oral Microbiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan b Department of Periodontology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Received 6 March 2012; accepted 25 April 2012 Available online 4 May 2012
Abstract Actinomyces viscosus has been suggested to be associated with periodontal disease. However, the pathogenicity of this bacterium is not known. In this study, we examined inflammation-inducing activity by A. viscosus. Whole cells and a lipophilic fraction of A. viscosus ATCC19246 induced production of interleukin-8 and tumor necrosis factor alpha from both human oral epithelial cells and human monocytoid cells. This cytokine production was blocked by lipoprotein lipase treatment of the lipophilic fraction. In addition, anti-Toll-like receptor 2 antibody blocked the cytokine production. These results suggest that lipoprotein of A. viscosus triggers inflammatory responses in periodontitis by activation of Toll-like receptor 2. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Actinomyces viscosus; Inflammation; Lipoprotein; Toll-like receptor 2
1. Introduction Actinomyces viscosus is a gram-positive oral bacterium that is frequently isolated from supragingival plaque and root surface caries lesions. Therefore, this bacterium has been suggested to be associated with periodontal inflammation. Although this bacterium has been implicated in the development of gingivitis [1e3], the pathogenicity of A. viscosus is poorly understood. It is generally accepted that periodontal inflammation, including gingivitis, is caused by accumulation of dental plaque bacteria. Therefore, to better understand the mechanisms of this inflammation, it is important to identify the bacterial ligands and host cell receptors that contribute to the triggering of this response. Previous studies indicate that the proportion of A. viscosus increases significantly in the dental plaque at the site of gingivitis [2]. Additionally, disrupted water-soluble cell wall components of A. viscosus induce polyclonal B-cell * Corresponding author. Tel.: þ81 3 3784 8166; fax: þ81 3 3784 4105. E-mail address: [email protected] (H. Kataoka).
activation in murine lymphocytes and activate murine macrophages to produce inflammatory cytokines [3e5]. These reports suggest that one or more A. viscosus cell wall molecules exhibit mitogenic, adjuvant, and inflammation-inducing activities. However, the cell wall components of A. viscosus that induce inflammation have not been identified, nor have the target molecules of human immune cells that serve as receptors for these bacterial components. The host immune and epithelial cells produce Toll-like receptors (TLRs), type-1 transmembrane proteins that detect invasive microbes by recognition of various microbial components, collectively defined as pathogen-associated molecular patterns (PAMPs) [6]. Upon recognition of microbes through TLRs, these host cells release inflammatory cytokines. These cytokines are involved in initiation and amplification of the inflammatory responses in the host organisms. 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 [7e11]. Interestingly, TLR2 also has been implicated
1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2012.04.015
E. Shimada et al. / Microbes and Infection 14 (2012) 916e921
in the inflammatory response triggered by several periodontopathic gram-negative bacteria [12,13], suggesting that TLR2 plays a key role in initiating periodontal inflammation. The molecular mechanisms of TLR2 binding have been demonstrated by X-ray crystallographic studies of TLR2 in the presence of synthetic lipopeptides [14e16]. However, the components of specific gram-positive bacteria that induce inflammatory responses through TLR2 are not fully understood. Recent reports have demonstrated that several wellknown pathogenic gram-positive bacteria (specifically Staphylococcus aureus, Listeria monocytogenes, and Streptococcus agalactiae) activate host immune cells, and that TLR2 plays a key role in this response [17e20]. Additionally, these reports showed that bacterial lipoproteins from these species act as ligands of TLR2 to induce this response. In contrast to what is known in these bacteria, the inflammation-inducing mechanism(s) of gram-positive oral bacteria remain poorly understood. Furthermore, the predominant “sensors” that host cells use to detect gram-positive oral bacteria have not been identified. In the present study, we demonstrate that A. viscosus bacterial lipoproteins can induce periodontal inflammation, and are sensed by TLR2 present on periodontal cells of the host organism. 2. Materials and methods 2.1. Bacterial strain and culturing A. viscosus ATCC19246 was obtained from the American Type Culture Collection (Manassas, VA). All bacterial culturing was performed at 37 C under anaerobic conditions in brain heart infusion (BHI; BactoÔ, NJ) agar or broth. Specifically, the strain was grown on plates for 3 days, then inoculated into broth and incubated overnight. The bacterial cells then were harvested by centrifugation (15,000 g, 5 min, 4 C), washed in phosphate-buffered saline (PBS), and resuspended at an optical density of 0.5 at 600 nm, corresponding to a cell density of approximately 1.1 108 colonyforming units (CFU)/ml.
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2.3. Antibodies and reagents Mouse monoclonal antibody (mAb) to human TLR2 (TL2.1) and mouse IgG2a isotype control antibody were purchased from eBioscience (San Diego, CA). Phorbol 12-myristate 13-acetate, lipoprotein lipase from Pseudomonas sp., and Triton X-114 (TX-114) were purchased from SigmaeAldrich. 2.4. Gene cloning The cDNA of human TLR2 was prepared by reverse transcriptionepolymerase chain reaction of RNA isolated from THP-1 cells and cloned into pEF6/V5-His-Topo vector (Invitrogen Co., Carlsbad, CA) using standard molecular biology techniques [21]. The resulting plasmid was designated pEF6-TLR2. 2.5. Extraction of lipophilic fraction of A. viscosus A. viscosus cells were treated with TX-114 to extract the lipophilic fraction. Specifically, A. viscosus was cultured in BHI broth and harvested as described above. The cell pellet was resuspended in buffer solution (150 mM NaCl and 10 mM TriseHCl, pH 8.0). The cell suspensions were mixed with 1/10 volume of 20% (vol/vol) aqueous TX-114 working stock solution. The mixture was rotated at 4 C for 2 h and cell debris was removed by centrifugation (15,000 g, 5 min, 4 C). The supernatant was incubated for 5 min at 37 C and re-centrifuged to separate the lower (lipophilic) phase from the upper (aqueous) phase. Excess methanol was added to the lower phase to precipitate the lipophilic fraction; the mixture was incubated overnight at 80 C prior to centrifugation (15,000 g, 30 min, 4 C) and decanting/discarding of the supernatant. The precipitated lipophilic fraction was resuspended in PBS, and is hereafter referred to as Lipo-fract. The protein concentration of Lipo-fract was measured by BCA assay using BIO-RAD PROTEIN ASSAY (Bio-Rad Laboratories, Hercules, CA). To determine the stimulatory activity of lipoproteins contained in Lipo-fract, aliquots of Lipo-fract were incubated (37 C, 6 h) in the presence or absence of lipoprotein lipase (98,100 U/ml) prior to use in stimulation.
2.2. Cell culture 2.6. ELISA Human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection (CRL-1573). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; SigmaeAldrich, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum, penicillin G (100 U/ml), and streptomycin (100 mg/ml). A human acute monocytic leukemia cell line, THP-1 (RCB1189), and a human cell line derived from oral squamous cell carcinoma, HSC-2 (RCB1145), were purchased from the Cell Engineering Division of RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). Both lines were cultured in RPMI 1640 medium (Wako, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum. All cell culture was performed at 37 C in a humidified 5% CO2 atmosphere.
HSC-2 cells were plated at 1.0 104 cells per well in 24-well plates and cultured for 4 days (to achieve confluency). THP-1 cells were plated at 4.0 105 cells per well in 24-well plates in the presence of 10 nM phorbol 12-myristate 13-acetate and cultured for 24 h (to differentiate into macrophages). These cells were washed three times with serum-free RPMI 1640 and then were stimulated for 6 h with A. viscosus cells (1.1 105, 1.1 106, and 1.1 107 CFU/ml) or Lipo-fract (at protein concentration of 0.01, 0.1, and 1 mg/ml) in serum-free medium. After stimulation, the culture supernatants were collected by centrifugation (7000 g, 10 min, 4 C). The amounts of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-a) in the culture supernatants were determined using enzyme-linked
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immunosorbent assay (ELISA Development Kit Human IL-8 and TNF-a; PeproTech, Rocky Hill, NJ).
assay system (Promega) according to the manufacturer’s instructions. The activity of firefly luciferase was normalized to that of Renilla luciferase.
2.7. Application of TLR2-neutralizing antibody 2.9. Statistical analysis To examine the involvement of TLR2, THP-1 and HSC-2 were cultured in 24-well plates under the same conditions as described above for ELISA. However, following washing with serum-free RPMI 1640, the cells were pretreated with 10 mg/ml of TLR2.1 or isotype control antibody for 1 h, and then stimulated with 1.1 106 CFU/ml of A. viscosus cells or 1 mg/ml of Lipo-fract for 6 h. The culture supernatants were harvested and assayed for IL-8 and TNF-a content by ELISA performed as above.
Statistical analysis was performed using JMP 9.0.2 (SAS Institute Inc., Cary, NC). ELISAs were performed in triplicate (N ¼ 3); individual figures provide data as mean standard deviation (SD) from a single experiment. The statistical differences were assessed by Student’s t-test. For Fig. 1A and B, 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.
2.8. Luciferase reporter assay 3. Results HEK293 cells were plated at 1.0 105 cells per well in 24-well plates. One day later, the cells were transfected transiently with 30 ng of an NF-kB-dependent firefly luciferaseencoding reporter plasmid (pNF-kB-Luc; Stratagene, San Diego, CA), 3.5 ng of a Renilla luciferase-encoding reporter plasmid (pRL-TK; Promega, Madison, WI), and 150 ng of pEF6-TLR2; transfection was performed for 24 h using METAFECTENE Transfection Reagent (Biontex Laboratories, GmbH, Mu¨nchen, Germany). On the following day, the cells were washed with serum-free medium and stimulated for 6 h with A. viscosus cells (1.1 105, 1.1 106 and 1.1 107 CFU/ml) or Lipo-fract (at a protein concentration of 0.01, 0.1, and 1 mg/ml) in serum-free medium. Luciferase activity was determined using a Dual-Luciferase reporter
3.1. A. viscosus cells induce TLR2-mediated inflammatory responses in host cells We examined whether A. viscosus cells induce the production of inflammatory cytokines in host cells. We stimulated lines of oral epithelial cells (HSC-2) and macrophages (THP-1) with A. viscosus. Exposure to bacterial cells induced the production of inflammatory cytokines IL-8 and TNF-a in both HSC-2 and THP-1 cells in a dose-dependent manner (Fig. 1A and B). This result suggested that A. viscosus cells are capable of inducing inflammation in host periodontal tissue. We next examined whether the A. viscosus-induced inflammatory responses were mediated by TLR2. HSC-2 and
Fig. 1. Actinomyces viscosus cells induce production of inflammatory cytokines. The production of IL-8 and TNF-a in the culture supernatants of HSC-2 (A) and THP-1 (B) stimulated with A. viscosus cells were measured by ELISA. A. viscosus cells induce production of inflammatory cytokines in a TLR2-dependent manner. Cells of HSC-2 (C) and THP-1 (D) were pretreated with anti-TLR2 antibody or with isotype control antibody and then stimulated with A. viscosus cells. The production of IL-8 and TNF-a in the culture supernatants were measured by ELISA. (E) A. viscosus cells activate NF-kB in a TLR2-dependent manner. HEK293 was transfected with TLR2-encoding plasmid or control plasmid, along with an NF-kB reporter plasmid and a control reporter plasmid. Transfected cells then were stimulated with A. viscosus cells, and NF-kB-regulated gene expression was measured as relative luciferase activity (*P < 0.05).
E. Shimada et al. / Microbes and Infection 14 (2012) 916e921
THP-1 were pretreated with anti-TLR2 antibody to block TLR2 at the surface of these cells; the treated cells then were stimulated with A. viscosus cells, and the production of IL-8 and TNF-a was measured by ELISA. Pretreatment with antiTLR2 antibody significantly inhibited production of these cytokines; this effect was not seen with an isotype control antibody (Fig. 1C and D). Furthermore, we used HEK293, a human embryonic kidney cell line that does not produce TLR2 under standard culture conditions [22]. For this experiment, HEK293 cells were transiently transfected with the human TLR2 gene together with an NF-kB-dependent (i.e., TLR2 activated) luciferase-encoding reporter gene; the transfected cells then were exposed to A. viscosus. The bacterial cells induced NF-kB activation in HEK293 transfected with the TLR2 gene in a dose-dependent manner (Fig. 1E). In contrast, these bacterial cells failed to induce NF-kB activation in cells transfected with pEF6 control vector. These results showed that TLR2 recognizes A. viscosus cells and induces inflammatory responses in host cells. 3.2. Lipoproteins of A. viscosus are responsible for inducing inflammatory responses We next investigated whether A. viscosus lipoproteins were responsible for inducing inflammatory cytokine production in host cells. HSC-2 and THP-1 cells were stimulated by Lipofract, and IL-8 and TNF-a production in the culture supernatant was measured by ELISA. Lipo-fract induced production of these cytokines in a dose-dependent manner (Fig. 2). Pretreatment of Lipo-fract with lipoprotein lipase eliminated the stimulatory activity of Lipo-fract in this assay (Fig. 2).
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These results show that A. viscosus lipoproteins induce the host inflammatory response, although this experiment does not rule out a possible role for other A. viscosus components. 3.3. Lipoproteins of A. viscosus induce inflammatory responses in TLR2-dependent manner We next examined whether Lipo-fract is recognized as a ligand by TLR2. As described above, we stimulated HSC-2 and THP-1, pretreated with anti-TLR2 antibody, with Lipo-fract, and we then measured inflammatory cytokine production by ELISA. As seen with A. viscosus whole cells, Lipo-fract’s ability to induced cytokine production was significantly inhibited by pretreatment with anti-TLR2 antibody (Fig. 3A and B). This result revealed that recognition of Lipo-fract by TLR2 contributes to inflammatory cytokine production. To further confirm this TLR2 dependency, we examined TLR2-dependent NF-kB activation by Lipo-fract as described above. As shown in Fig. 3C and D, Lipo-fract activated NF-kB in TLR2-transfected cells in a dosedependent manner; this activity was diminished by lipoprotein lipase pretreatment of Lipo-fract. Together, these results strongly suggest that the inflammatory response induced by A. viscosus lipoproteins is mediated via TLR2. 4. Discussion Periodontal diseases, including gingivitis and periodontitis, are chronic inflammatory diseases of periodontal tissue. Accumulation of supragingival plaque, which mainly includes gram-positive oral bacteria, is thought to be strongly involved
Fig. 2. Lipoproteins extracted from A. viscosus induce production of inflammatory cytokines. The production of IL-8 and TNF-a in the culture supernatants of HSC-2 (A) and THP-1 (B) stimulated by the lipophilic fraction (Lipo-fract) or Lipo-fract treated with lipoprotein lipase were measured by ELISA (*P < 0.05).
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Fig. 3. Lipo-fract induces production of inflammatory cytokines in a TLR2-dependent manner. Cells of HSC-2 (A) and THP-1 (B) were pretreated with anti-TLR2 antibody or with isotype control antibody and then stimulated by Lipo-fract. The production of IL-8 and TNF-a in the culture supernatants were measured by ELISA. (C) Lipo-fract activates NF-kB in a TLR2-dependent manner. HEK293 was transfected with TLR2-encoding plasmid or control plasmid, along with an NF-kB reporter plasmid and a control reporter plasmid. Transfected cells then were stimulated by Lipo-fract and NF-kB-regulated gene expression was measured as relative luciferase activity. (D) Lipoproteins extracted from A. viscosus activate NF-kB in a TLR2-dependent manner. HEK293 was transfected with TLR2-encoding plasmid, along with an NF-kB reporter plasmid and a control reporter plasmid. Transfected cells then were stimulated by Lipo-fract or Lipo-fract treated with lipoprotein lipase, and NF-kB-regulated gene expression was measured as relative luciferase activity (*P < 0.05).
in the initiation and development of gingivitis. However, little is known about the inflammation-inducing activity of grampositive oral bacteria. A. viscosus is one of the gram-positive oral bacteria inhabiting dental plaque; the bacterium is a suspected etiologic agent in the development of periodontal inflammation [1,2]. However, the pathogenicity of A. viscosus, including inflammation-inducing activity, is not fully understood. In this study, we provide the first direct evidence that lipoproteins derived from A. viscosus cells are a major inflammation-inducing bacterial factor, and further demonstrate that TLR2 produced in host cells plays a crucial role in recognition of these lipoproteins. Recently, Mayer et al. have investigated inflammation-inducing components of Streptococcus gordonii. Similar to A. viscosus, S. gordonii is a grampositive oral bacterium that lives commensally on the tooth surface. Mayer et al. reported that lipoproteins of S. gordonii are able to induce inflammatory cytokine production, and further demonstrated that TLR2 plays a crucial role in the recognition of S. gordonii lipoproteins [23]. Those results are consistent with the findings of our study, in which we showed the TLR2-mediated activation of inflammatory responses by A. viscosus-derived bacterial lipoproteins. Our results suggest that the accumulation of A. viscosus in dental plaque might be a causative agent in periodontal inflammation. While we propose a role for TLR2 in this process, Yamaguchi et al.
reported that the induction (in human peripheral blood mononuclear cells) of cytokine production by supragingival plaque was inhibited by anti-TLR4 mAb, and not by antiTLR2 mAb [24]. However, Yamaguchi et al. did not distinguish which component(s) of supragingival plaque were responsible for inducing inflammation via TLR4. Thus, the proposed roles for TLR2 and TLR4 may not be contradictory or mutually exclusive. Lipo-fract is a crude fraction, so we could not characterize the specific lipoprotein(s) that provided the inflammation-inducing activity. However, it has been shown that TX-114 extraction is an efficacious method for extracting bacterial lipoproteins, and that lipoprotein lipase treatment of this fraction impedes the induction of inflammation [17,25], providing an independent confirmation that lipoproteins included in the TX-114 fraction are responsible for inducing inflammation. We cannot rule out the possibility that unfractionated components also may induce inflammatory responses. However, both whole cells and lipoproteins of A. viscosus induced similar magnitudes of cytokine production; with both, induction exhibited TLR2 dependency. These results suggest that lipoproteins may be a major inflammationinducing factor of A. viscosus. We note that pretreatment with anti-TLR2 antibody significantly reduced, although it did not completely inhibit, the stimulation of inflammatory cytokine production by A. viscosus cells. This observation suggests that
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another receptor (or receptors) produced in host cells also may play a role in recognizing A. viscosus cells. In summary, we demonstrated a role for A. viscosus lipoproteins in TLR2-mediated inflammatory responses by host cells. Targeting of bacterial lipoprotein synthesis and/or host inflammatory pathways may provide new routes for treatment of periodontal disease. Acknowledgments 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 [1] P.R. Ellen, Establishment and distribution of Actinomyces viscosus and Actinomyces naeslundii in the human oral cavity, Infect. Immun. 14 (1976) 1119e1124. [2] W.J. Loesche, S.A. Syed, Bacteriology of human experimental gingivitis: effect of plaque and gingivitis score, Infect. Immun. 21 (1978) 830e839. [3] P. Chen, J.J. Farrar, R.J. Genco, Immunological properties of Actinomyces viscosus: comparison of blastogenic and adjuvant activities, Infect. Immun. 28 (1980) 212e219. [4] J. Clagett, D. Engel, E. Chi, In vitro expression of immunoglobulin M and G subclasses by murine B lymphocytes in response to a polyclonal activator from Actinomyces, Infect. Immun. 29 (1980) 234e243. [5] H. Takada, S. Kimura, S. Hamada, Induction of inflammatory cytokines by soluble moiety prepared from an enzyme lysate of Actinomyces viscosus cell walls, J. Med. Microbiol. 38 (1993) 395e400. [6] K. Takeda, T. Kaisho, S. Akira, Toll-like receptors, Annu. Rev. Immunol. 21 (2003) 335e376. [7] A.O. Alliprantis, R.B. Yang, M.R. Mark, S. Suggett, B. Devaux, J.D. Radolf, G.R. Klimpel, P. Godowski, A. Zychlinsky, Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2, Science 285 (1999) 736e739. [8] O. Takeuchi, A. Kaufmann, K. Grote, T. Kawai, K. Hoshino, M. Morr, 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 (2000) 554e557. [9] N.W. Schroder, S. Morath, C. Alexander, L. Hamann, T. Hartung, U. Za¨hringer, U.B. Go¨bel, J.R. Weber, R.R. Schumann, Lipoteicoic 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 (2003) 15587e15594.
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[10] S.M. Levitz, Interaction of Toll-like receptors with fungi, Microbes Infect. 6 (2004) 1351e1355. [11] T. Wang, W.P. Lafuse, B.S. Zwilling, Regulation of Toll-like receptor 2 expression by macrophages following Mycobacterium avium infection, J. Immunol. 165 (2000) 6308e6313. [12] Y. Sun, R. Shu, C.L. Li, M.Z. Zhang, Gram-negative periodontal bacteria induce the activation of Toll-like receptor 2,4 and cytokine production in human periodontal ligament cells, J. Periodontol. 81 (2010) 1488e1496. [13] R. Kikkert, M.L. Laine, L.A. Aarden, A.J. van Winkelhoff, Activation of toll-like receptors 2 and 4 by gram-negative periodontal bacteria, Oral Microbiol. Immunol. 22 (2007) 145e151. [14] M.S. Jin, S.E. Kim, J.Y. Heo, M.E. Lee, H.M. Kim, S.G. Paik, H. Lee, J.O. Lee, Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide, Cell 130 (2007) 1071e1082. [15] M.S. Jin, J.O. Lee, Structures of the toll-like receptor family and its ligand complexes, Immunity 29 (2008) 182e191. [16] J.Y. Kang, X. Nan, M.S. Jin, S.J. Youn, Y.H. Ryu, S. Mah, S.H. Han, H. Lee, S.G. Paik, J.O. Lee, Recognition of lipopeptide patterns by Tolllike receptor 2-Toll-like receptor 6 heterodimer, Immunity 31 (2009) 873e884. [17] M. Hashimoto, K. Tawaratsumida, H. Kariya, K. Aoyama, T. Tamura, Y. Suda, Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall component, Int. Immunol. 18 (2006) 355e362. [18] M. Hashimoto, K. Tawaratsumida, H. Kariya, A. Kiyohara, Y. Suda, F. Krikae, T. Kirikae, F. Go¨tz, Not lipoteicoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococus aureus, J. Immunol. 177 (2006) 3162e3169. [19] S. Machata, S. Tchatalbachev, W. Mohamad, L. Jansch, T. Hain, T. Chakraborty, Lipoproteins of Listeria monocytogenes are critical for virulence and TLR2-mediated immune activation, J. Immunol. 181 (2008) 2028e2035. [20] P. Henneke, S. Dramsi, G. Mancuso, K. Chraibi, E. Pellegrini, C. Theilacker, J. Hu¨bner, S.S. Sierra, Lipoproteins are critical TLR2 activating toxins in Group B Streptococcal sepsis, J. Immunol. 180 (2008) 6149e6158. [21] J. Sambrook, E.F. Fritsch, T. Manuatis, Molecular Cloning: a Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. [22] C.J. Kirschning, H. Wesche, T.M. Ayres, M. Rothe, Human Toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide, J. Exp. Med. 188 (1998) 2091e2097. [23] M.L. Mayer, C.M. Phillips, R.A. Townsend, S.A. Halperin, S.F. Lee, Differential activation of dendritic cells by Toll-like receptor agonists isolated from the gram-positive vaccine vector Streptococcus gordonii, Scand. J. Immunol. 69 (2000) 351e356. [24] R. Yamaguchi, A. Yoshimura, H. Yoshioka, T. Kaneko, Y. Hara, Ability of supragingival plaque to induce Toll-like receptor 4-mediated stimulation is associated with cytokine production by peripheral blood mononuclear cells, J. Periodontol. 80 (2009) 512e520. [25] Q. Li, A. Kumar, J.F. Gui, F.S.X. Yu, Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2, Microb. Pathog. 44 (2008) 426e434.
Short communication
Lipoproteins of Actinomyces viscosus induce inflammatory responses through TLR2 in human gingival epithelial cells and macrophages Eri Shimada a,b, Hideo Kataoka a,*, Yasushi Miyazawa b, Matsuo Yamamoto b, Takeshi Igarashi a a
Department of Oral Microbiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan b Department of Periodontology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Received 6 March 2012; accepted 25 April 2012 Available online 4 May 2012
Abstract Actinomyces viscosus has been suggested to be associated with periodontal disease. However, the pathogenicity of this bacterium is not known. In this study, we examined inflammation-inducing activity by A. viscosus. Whole cells and a lipophilic fraction of A. viscosus ATCC19246 induced production of interleukin-8 and tumor necrosis factor alpha from both human oral epithelial cells and human monocytoid cells. This cytokine production was blocked by lipoprotein lipase treatment of the lipophilic fraction. In addition, anti-Toll-like receptor 2 antibody blocked the cytokine production. These results suggest that lipoprotein of A. viscosus triggers inflammatory responses in periodontitis by activation of Toll-like receptor 2. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Actinomyces viscosus; Inflammation; Lipoprotein; Toll-like receptor 2
1. Introduction Actinomyces viscosus is a gram-positive oral bacterium that is frequently isolated from supragingival plaque and root surface caries lesions. Therefore, this bacterium has been suggested to be associated with periodontal inflammation. Although this bacterium has been implicated in the development of gingivitis [1e3], the pathogenicity of A. viscosus is poorly understood. It is generally accepted that periodontal inflammation, including gingivitis, is caused by accumulation of dental plaque bacteria. Therefore, to better understand the mechanisms of this inflammation, it is important to identify the bacterial ligands and host cell receptors that contribute to the triggering of this response. Previous studies indicate that the proportion of A. viscosus increases significantly in the dental plaque at the site of gingivitis [2]. Additionally, disrupted water-soluble cell wall components of A. viscosus induce polyclonal B-cell * Corresponding author. Tel.: þ81 3 3784 8166; fax: þ81 3 3784 4105. E-mail address: [email protected] (H. Kataoka).
activation in murine lymphocytes and activate murine macrophages to produce inflammatory cytokines [3e5]. These reports suggest that one or more A. viscosus cell wall molecules exhibit mitogenic, adjuvant, and inflammation-inducing activities. However, the cell wall components of A. viscosus that induce inflammation have not been identified, nor have the target molecules of human immune cells that serve as receptors for these bacterial components. The host immune and epithelial cells produce Toll-like receptors (TLRs), type-1 transmembrane proteins that detect invasive microbes by recognition of various microbial components, collectively defined as pathogen-associated molecular patterns (PAMPs) [6]. Upon recognition of microbes through TLRs, these host cells release inflammatory cytokines. These cytokines are involved in initiation and amplification of the inflammatory responses in the host organisms. 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 [7e11]. Interestingly, TLR2 also has been implicated
1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2012.04.015
E. Shimada et al. / Microbes and Infection 14 (2012) 916e921
in the inflammatory response triggered by several periodontopathic gram-negative bacteria [12,13], suggesting that TLR2 plays a key role in initiating periodontal inflammation. The molecular mechanisms of TLR2 binding have been demonstrated by X-ray crystallographic studies of TLR2 in the presence of synthetic lipopeptides [14e16]. However, the components of specific gram-positive bacteria that induce inflammatory responses through TLR2 are not fully understood. Recent reports have demonstrated that several wellknown pathogenic gram-positive bacteria (specifically Staphylococcus aureus, Listeria monocytogenes, and Streptococcus agalactiae) activate host immune cells, and that TLR2 plays a key role in this response [17e20]. Additionally, these reports showed that bacterial lipoproteins from these species act as ligands of TLR2 to induce this response. In contrast to what is known in these bacteria, the inflammation-inducing mechanism(s) of gram-positive oral bacteria remain poorly understood. Furthermore, the predominant “sensors” that host cells use to detect gram-positive oral bacteria have not been identified. In the present study, we demonstrate that A. viscosus bacterial lipoproteins can induce periodontal inflammation, and are sensed by TLR2 present on periodontal cells of the host organism. 2. Materials and methods 2.1. Bacterial strain and culturing A. viscosus ATCC19246 was obtained from the American Type Culture Collection (Manassas, VA). All bacterial culturing was performed at 37 C under anaerobic conditions in brain heart infusion (BHI; BactoÔ, NJ) agar or broth. Specifically, the strain was grown on plates for 3 days, then inoculated into broth and incubated overnight. The bacterial cells then were harvested by centrifugation (15,000 g, 5 min, 4 C), washed in phosphate-buffered saline (PBS), and resuspended at an optical density of 0.5 at 600 nm, corresponding to a cell density of approximately 1.1 108 colonyforming units (CFU)/ml.
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2.3. Antibodies and reagents Mouse monoclonal antibody (mAb) to human TLR2 (TL2.1) and mouse IgG2a isotype control antibody were purchased from eBioscience (San Diego, CA). Phorbol 12-myristate 13-acetate, lipoprotein lipase from Pseudomonas sp., and Triton X-114 (TX-114) were purchased from SigmaeAldrich. 2.4. Gene cloning The cDNA of human TLR2 was prepared by reverse transcriptionepolymerase chain reaction of RNA isolated from THP-1 cells and cloned into pEF6/V5-His-Topo vector (Invitrogen Co., Carlsbad, CA) using standard molecular biology techniques [21]. The resulting plasmid was designated pEF6-TLR2. 2.5. Extraction of lipophilic fraction of A. viscosus A. viscosus cells were treated with TX-114 to extract the lipophilic fraction. Specifically, A. viscosus was cultured in BHI broth and harvested as described above. The cell pellet was resuspended in buffer solution (150 mM NaCl and 10 mM TriseHCl, pH 8.0). The cell suspensions were mixed with 1/10 volume of 20% (vol/vol) aqueous TX-114 working stock solution. The mixture was rotated at 4 C for 2 h and cell debris was removed by centrifugation (15,000 g, 5 min, 4 C). The supernatant was incubated for 5 min at 37 C and re-centrifuged to separate the lower (lipophilic) phase from the upper (aqueous) phase. Excess methanol was added to the lower phase to precipitate the lipophilic fraction; the mixture was incubated overnight at 80 C prior to centrifugation (15,000 g, 30 min, 4 C) and decanting/discarding of the supernatant. The precipitated lipophilic fraction was resuspended in PBS, and is hereafter referred to as Lipo-fract. The protein concentration of Lipo-fract was measured by BCA assay using BIO-RAD PROTEIN ASSAY (Bio-Rad Laboratories, Hercules, CA). To determine the stimulatory activity of lipoproteins contained in Lipo-fract, aliquots of Lipo-fract were incubated (37 C, 6 h) in the presence or absence of lipoprotein lipase (98,100 U/ml) prior to use in stimulation.
2.2. Cell culture 2.6. ELISA Human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection (CRL-1573). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; SigmaeAldrich, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum, penicillin G (100 U/ml), and streptomycin (100 mg/ml). A human acute monocytic leukemia cell line, THP-1 (RCB1189), and a human cell line derived from oral squamous cell carcinoma, HSC-2 (RCB1145), were purchased from the Cell Engineering Division of RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). Both lines were cultured in RPMI 1640 medium (Wako, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum. All cell culture was performed at 37 C in a humidified 5% CO2 atmosphere.
HSC-2 cells were plated at 1.0 104 cells per well in 24-well plates and cultured for 4 days (to achieve confluency). THP-1 cells were plated at 4.0 105 cells per well in 24-well plates in the presence of 10 nM phorbol 12-myristate 13-acetate and cultured for 24 h (to differentiate into macrophages). These cells were washed three times with serum-free RPMI 1640 and then were stimulated for 6 h with A. viscosus cells (1.1 105, 1.1 106, and 1.1 107 CFU/ml) or Lipo-fract (at protein concentration of 0.01, 0.1, and 1 mg/ml) in serum-free medium. After stimulation, the culture supernatants were collected by centrifugation (7000 g, 10 min, 4 C). The amounts of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-a) in the culture supernatants were determined using enzyme-linked
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immunosorbent assay (ELISA Development Kit Human IL-8 and TNF-a; PeproTech, Rocky Hill, NJ).
assay system (Promega) according to the manufacturer’s instructions. The activity of firefly luciferase was normalized to that of Renilla luciferase.
2.7. Application of TLR2-neutralizing antibody 2.9. Statistical analysis To examine the involvement of TLR2, THP-1 and HSC-2 were cultured in 24-well plates under the same conditions as described above for ELISA. However, following washing with serum-free RPMI 1640, the cells were pretreated with 10 mg/ml of TLR2.1 or isotype control antibody for 1 h, and then stimulated with 1.1 106 CFU/ml of A. viscosus cells or 1 mg/ml of Lipo-fract for 6 h. The culture supernatants were harvested and assayed for IL-8 and TNF-a content by ELISA performed as above.
Statistical analysis was performed using JMP 9.0.2 (SAS Institute Inc., Cary, NC). ELISAs were performed in triplicate (N ¼ 3); individual figures provide data as mean standard deviation (SD) from a single experiment. The statistical differences were assessed by Student’s t-test. For Fig. 1A and B, 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.
2.8. Luciferase reporter assay 3. Results HEK293 cells were plated at 1.0 105 cells per well in 24-well plates. One day later, the cells were transfected transiently with 30 ng of an NF-kB-dependent firefly luciferaseencoding reporter plasmid (pNF-kB-Luc; Stratagene, San Diego, CA), 3.5 ng of a Renilla luciferase-encoding reporter plasmid (pRL-TK; Promega, Madison, WI), and 150 ng of pEF6-TLR2; transfection was performed for 24 h using METAFECTENE Transfection Reagent (Biontex Laboratories, GmbH, Mu¨nchen, Germany). On the following day, the cells were washed with serum-free medium and stimulated for 6 h with A. viscosus cells (1.1 105, 1.1 106 and 1.1 107 CFU/ml) or Lipo-fract (at a protein concentration of 0.01, 0.1, and 1 mg/ml) in serum-free medium. Luciferase activity was determined using a Dual-Luciferase reporter
3.1. A. viscosus cells induce TLR2-mediated inflammatory responses in host cells We examined whether A. viscosus cells induce the production of inflammatory cytokines in host cells. We stimulated lines of oral epithelial cells (HSC-2) and macrophages (THP-1) with A. viscosus. Exposure to bacterial cells induced the production of inflammatory cytokines IL-8 and TNF-a in both HSC-2 and THP-1 cells in a dose-dependent manner (Fig. 1A and B). This result suggested that A. viscosus cells are capable of inducing inflammation in host periodontal tissue. We next examined whether the A. viscosus-induced inflammatory responses were mediated by TLR2. HSC-2 and
Fig. 1. Actinomyces viscosus cells induce production of inflammatory cytokines. The production of IL-8 and TNF-a in the culture supernatants of HSC-2 (A) and THP-1 (B) stimulated with A. viscosus cells were measured by ELISA. A. viscosus cells induce production of inflammatory cytokines in a TLR2-dependent manner. Cells of HSC-2 (C) and THP-1 (D) were pretreated with anti-TLR2 antibody or with isotype control antibody and then stimulated with A. viscosus cells. The production of IL-8 and TNF-a in the culture supernatants were measured by ELISA. (E) A. viscosus cells activate NF-kB in a TLR2-dependent manner. HEK293 was transfected with TLR2-encoding plasmid or control plasmid, along with an NF-kB reporter plasmid and a control reporter plasmid. Transfected cells then were stimulated with A. viscosus cells, and NF-kB-regulated gene expression was measured as relative luciferase activity (*P < 0.05).
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THP-1 were pretreated with anti-TLR2 antibody to block TLR2 at the surface of these cells; the treated cells then were stimulated with A. viscosus cells, and the production of IL-8 and TNF-a was measured by ELISA. Pretreatment with antiTLR2 antibody significantly inhibited production of these cytokines; this effect was not seen with an isotype control antibody (Fig. 1C and D). Furthermore, we used HEK293, a human embryonic kidney cell line that does not produce TLR2 under standard culture conditions [22]. For this experiment, HEK293 cells were transiently transfected with the human TLR2 gene together with an NF-kB-dependent (i.e., TLR2 activated) luciferase-encoding reporter gene; the transfected cells then were exposed to A. viscosus. The bacterial cells induced NF-kB activation in HEK293 transfected with the TLR2 gene in a dose-dependent manner (Fig. 1E). In contrast, these bacterial cells failed to induce NF-kB activation in cells transfected with pEF6 control vector. These results showed that TLR2 recognizes A. viscosus cells and induces inflammatory responses in host cells. 3.2. Lipoproteins of A. viscosus are responsible for inducing inflammatory responses We next investigated whether A. viscosus lipoproteins were responsible for inducing inflammatory cytokine production in host cells. HSC-2 and THP-1 cells were stimulated by Lipofract, and IL-8 and TNF-a production in the culture supernatant was measured by ELISA. Lipo-fract induced production of these cytokines in a dose-dependent manner (Fig. 2). Pretreatment of Lipo-fract with lipoprotein lipase eliminated the stimulatory activity of Lipo-fract in this assay (Fig. 2).
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These results show that A. viscosus lipoproteins induce the host inflammatory response, although this experiment does not rule out a possible role for other A. viscosus components. 3.3. Lipoproteins of A. viscosus induce inflammatory responses in TLR2-dependent manner We next examined whether Lipo-fract is recognized as a ligand by TLR2. As described above, we stimulated HSC-2 and THP-1, pretreated with anti-TLR2 antibody, with Lipo-fract, and we then measured inflammatory cytokine production by ELISA. As seen with A. viscosus whole cells, Lipo-fract’s ability to induced cytokine production was significantly inhibited by pretreatment with anti-TLR2 antibody (Fig. 3A and B). This result revealed that recognition of Lipo-fract by TLR2 contributes to inflammatory cytokine production. To further confirm this TLR2 dependency, we examined TLR2-dependent NF-kB activation by Lipo-fract as described above. As shown in Fig. 3C and D, Lipo-fract activated NF-kB in TLR2-transfected cells in a dosedependent manner; this activity was diminished by lipoprotein lipase pretreatment of Lipo-fract. Together, these results strongly suggest that the inflammatory response induced by A. viscosus lipoproteins is mediated via TLR2. 4. Discussion Periodontal diseases, including gingivitis and periodontitis, are chronic inflammatory diseases of periodontal tissue. Accumulation of supragingival plaque, which mainly includes gram-positive oral bacteria, is thought to be strongly involved
Fig. 2. Lipoproteins extracted from A. viscosus induce production of inflammatory cytokines. The production of IL-8 and TNF-a in the culture supernatants of HSC-2 (A) and THP-1 (B) stimulated by the lipophilic fraction (Lipo-fract) or Lipo-fract treated with lipoprotein lipase were measured by ELISA (*P < 0.05).
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Fig. 3. Lipo-fract induces production of inflammatory cytokines in a TLR2-dependent manner. Cells of HSC-2 (A) and THP-1 (B) were pretreated with anti-TLR2 antibody or with isotype control antibody and then stimulated by Lipo-fract. The production of IL-8 and TNF-a in the culture supernatants were measured by ELISA. (C) Lipo-fract activates NF-kB in a TLR2-dependent manner. HEK293 was transfected with TLR2-encoding plasmid or control plasmid, along with an NF-kB reporter plasmid and a control reporter plasmid. Transfected cells then were stimulated by Lipo-fract and NF-kB-regulated gene expression was measured as relative luciferase activity. (D) Lipoproteins extracted from A. viscosus activate NF-kB in a TLR2-dependent manner. HEK293 was transfected with TLR2-encoding plasmid, along with an NF-kB reporter plasmid and a control reporter plasmid. Transfected cells then were stimulated by Lipo-fract or Lipo-fract treated with lipoprotein lipase, and NF-kB-regulated gene expression was measured as relative luciferase activity (*P < 0.05).
in the initiation and development of gingivitis. However, little is known about the inflammation-inducing activity of grampositive oral bacteria. A. viscosus is one of the gram-positive oral bacteria inhabiting dental plaque; the bacterium is a suspected etiologic agent in the development of periodontal inflammation [1,2]. However, the pathogenicity of A. viscosus, including inflammation-inducing activity, is not fully understood. In this study, we provide the first direct evidence that lipoproteins derived from A. viscosus cells are a major inflammation-inducing bacterial factor, and further demonstrate that TLR2 produced in host cells plays a crucial role in recognition of these lipoproteins. Recently, Mayer et al. have investigated inflammation-inducing components of Streptococcus gordonii. Similar to A. viscosus, S. gordonii is a grampositive oral bacterium that lives commensally on the tooth surface. Mayer et al. reported that lipoproteins of S. gordonii are able to induce inflammatory cytokine production, and further demonstrated that TLR2 plays a crucial role in the recognition of S. gordonii lipoproteins [23]. Those results are consistent with the findings of our study, in which we showed the TLR2-mediated activation of inflammatory responses by A. viscosus-derived bacterial lipoproteins. Our results suggest that the accumulation of A. viscosus in dental plaque might be a causative agent in periodontal inflammation. While we propose a role for TLR2 in this process, Yamaguchi et al.
reported that the induction (in human peripheral blood mononuclear cells) of cytokine production by supragingival plaque was inhibited by anti-TLR4 mAb, and not by antiTLR2 mAb [24]. However, Yamaguchi et al. did not distinguish which component(s) of supragingival plaque were responsible for inducing inflammation via TLR4. Thus, the proposed roles for TLR2 and TLR4 may not be contradictory or mutually exclusive. Lipo-fract is a crude fraction, so we could not characterize the specific lipoprotein(s) that provided the inflammation-inducing activity. However, it has been shown that TX-114 extraction is an efficacious method for extracting bacterial lipoproteins, and that lipoprotein lipase treatment of this fraction impedes the induction of inflammation [17,25], providing an independent confirmation that lipoproteins included in the TX-114 fraction are responsible for inducing inflammation. We cannot rule out the possibility that unfractionated components also may induce inflammatory responses. However, both whole cells and lipoproteins of A. viscosus induced similar magnitudes of cytokine production; with both, induction exhibited TLR2 dependency. These results suggest that lipoproteins may be a major inflammationinducing factor of A. viscosus. We note that pretreatment with anti-TLR2 antibody significantly reduced, although it did not completely inhibit, the stimulation of inflammatory cytokine production by A. viscosus cells. This observation suggests that
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another receptor (or receptors) produced in host cells also may play a role in recognizing A. viscosus cells. In summary, we demonstrated a role for A. viscosus lipoproteins in TLR2-mediated inflammatory responses by host cells. Targeting of bacterial lipoprotein synthesis and/or host inflammatory pathways may provide new routes for treatment of periodontal disease. Acknowledgments 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 [1] P.R. Ellen, Establishment and distribution of Actinomyces viscosus and Actinomyces naeslundii in the human oral cavity, Infect. Immun. 14 (1976) 1119e1124. [2] W.J. Loesche, S.A. Syed, Bacteriology of human experimental gingivitis: effect of plaque and gingivitis score, Infect. Immun. 21 (1978) 830e839. [3] P. Chen, J.J. Farrar, R.J. Genco, Immunological properties of Actinomyces viscosus: comparison of blastogenic and adjuvant activities, Infect. Immun. 28 (1980) 212e219. [4] J. Clagett, D. Engel, E. Chi, In vitro expression of immunoglobulin M and G subclasses by murine B lymphocytes in response to a polyclonal activator from Actinomyces, Infect. Immun. 29 (1980) 234e243. [5] H. Takada, S. Kimura, S. Hamada, Induction of inflammatory cytokines by soluble moiety prepared from an enzyme lysate of Actinomyces viscosus cell walls, J. Med. Microbiol. 38 (1993) 395e400. [6] K. Takeda, T. Kaisho, S. Akira, Toll-like receptors, Annu. Rev. Immunol. 21 (2003) 335e376. [7] A.O. Alliprantis, R.B. Yang, M.R. Mark, S. Suggett, B. Devaux, J.D. Radolf, G.R. Klimpel, P. Godowski, A. Zychlinsky, Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2, Science 285 (1999) 736e739. [8] O. Takeuchi, A. Kaufmann, K. Grote, T. Kawai, K. Hoshino, M. Morr, 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 (2000) 554e557. [9] N.W. Schroder, S. Morath, C. Alexander, L. Hamann, T. Hartung, U. Za¨hringer, U.B. Go¨bel, J.R. Weber, R.R. Schumann, Lipoteicoic 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 (2003) 15587e15594.
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