Basic Research—Biology
Lipoteichoic Acid of Enterococcus faecalis Inhibits the Differentiation of Macrophages into Osteoclasts Jihyun Yang, PhD,* Ok-Jin Park, PhD,* Jiseon Kim, MS,* Jung Eun Baik, PhD,* Cheol-Heui Yun, PhD,†‡ and Seung Hyun Han, PhD* Abstract Introduction: Enterococcus faecalis is associated with persistent endodontic infection and refractory apical periodontitis. Recently, we have shown that heatkilled E. faecalis attenuates osteoclast differentiation. Because lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we investigated the effect of LTA from E. faecalis (EfLTA) on osteoclast differentiation. Methods: EfLTA was purified through organic solvent extraction, hydrophobic interaction column chromatography, and ion exchange column chromatography. Bone marrow cells from C57BL/6 or Toll-like receptor 2–deficient mice were incubated with macrophage colony-stimulating factor (M-CSF) for 5 days to generate macrophages (bone marrow–derived macrophages [BMMs]). The cells were differentiated into osteoclasts with M-CSF and receptor activator of NF-kB ligand (RANKL) in the presence or absence of EfLTA. The degree of osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining. The expression of NFATc1 and c-Fos transcription factors was determined by Western blotting. A phagocytosis assay was performed by measuring the uptake of carboxyfluorescein diacetate succinimidyl ester–stained E. faecalis. An enzyme-linked immunosorbent assay was used to determine the amount of cytokines and chemokines. Results: When BMMs were treated with EfLTA, osteoclast differentiation was attenuated. EfLTA inhibited the RANKL-induced expression of NFATc1 and c-Fos. EfLTA inhibition of osteoclast differentiation was not observed in TLR2-deficient BMMs. In addition, EfLTA sustained the phagocytic capacity of BMMs even after the differentiation into osteoclasts, whereas it induced the expression of inflammatory cytokines and chemokines. Conclusions: EfLTA inhibits the differentiation of macrophages into osteoclasts and thereby maintains the phagocytic and inflammatory capacities of macrophages, potentially contributing to refractory apical periodontitis. (J Endod 2016;-:1–5)
Key Words Chemokine, cytokine, Enterococcus faecalis, lipoteichoic acid, osteoclast differentiation, phagocytosis
E
nterococcus faecalis is a commensal gram-positive bacterium found in the oral cavity, gastrointestinal tract, and genital tract of humans (1). E. faecalis is also considered to be an opportunistic pathogen that causes endocarditis, bacteremia, and urinary tract infection (2). Of note, E. faecalis is a predominant bacterium implicated in persistent endodontic infection (3). E. faecalis easily invades dentinal tubules and can be isolated from periapical lesions (4, 5). E. faecalis and its major virulence factor, lipoteichoic acid (LTA), induce inflammatory cytokines and chemokines (6, 7), contributing to inflammation and tissue damage through the accumulation of immune cells including neutrophils and monocytes/macrophages in periapical lesions followed by periapical bone loss (8). LTA is known to be a major virulence factor of gram-positive bacteria because it has an important role in bacterial adhesion and in the initiation of host immune responses (9). LTA is shed from bacteria during the growing phase (9). LTA is an amphiphilic molecule that is composed of a glycolipid anchor together with repeating units of glycerol or ribitol phosphate (10). It has been reported that E. faecalis LTA (EfLTA) has a polyglycerol phosphate backbone substituted at the C2 position by D-alanine and kojibiose (11). EfLTA induces inflammatory mediators such as tumor necrosis factor alpha (TNF-a) and nitric oxide (NO) and chemokines such as monocyte chemoattractant protein-1 (MCP-1) through the Toll-like receptor 2 (TLR2) signaling pathway in macrophages (6, 7). It is notable that the glycolipid portion of EfLTA is responsible for its immunostimulating activity because the production of NO, interferon-gammainducible protein 10 (IP-10), and macrophage inflammatory protein-1 alpha (MIP1a) was not observed in macrophages stimulated with calcium hydroxide–treated EfLTA, which is a deacylated form of LTA (12). Macrophages play a role in phagocytosis against bacteria (13) and are well characterized as an important part of innate immunity. In addition, macrophages lead to local inflammatory responses through the induction of proinflammatory cytokines and chemokines (14, 15). On the other hand, macrophages are also considered to be common osteoclast precursors (16). Bone-resorbing osteoclasts are multinucleated giant cells (MNCs) capable of degrading calcified bone matrix such as alveolar bones in chronic periodontitis and roots of teeth in apical periodontitis (16). Bacteria and their virulence factors affect osteoclast differentiation and activation (17). Bacterial lipoproteins and lipopolysaccharides (LPSs) inhibit osteoclast differentiation from macrophages (18, 19). Recently, we reported that heat-killed E. faecalis (HKEF) inhibits osteoclast differentiation from bone marrow–derived macrophages (BMMs)
From the *Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry and †Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea; and ‡Institute of Green Bio Science Technology, Seoul National University, Pyeongchang, Republic of Korea. Address requests for reprints to Prof Seung Hyun Han, Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2016.01.012
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LTA of E. faecalis and Differentiation of Macrophages
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Basic Research—Biology (20). Because LTA is a major virulence factor of gram-positive bacteria, we hypothesized that EfLTA could inhibit osteoclast differentiation, just as HKEF does. Therefore, we prepared highly pure and structurally intact LTA from E. faecalis and investigated the effect of EfLTA on osteoclast differentiation and inflammatory functions.
Materials and Methods Reagents and Chemicals LTA was prepared from E. faecalis ATCC 29212 (American Type Culture Collection, Manassas, VA) as described previously (6). Pam2CSK4 and ultrapure LPS from Escherichia coli O111:B4 (EcLPS) were purchased from InvivoGen (San Diego, CA). Recombinant murine macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL) were purchased from PeproTech (Rocky Hill, NJ). Alpha-minimum essential medium and certified fetal bovine serum were obtained from Gibco-BRL (Grand Island, NY). Penicillin/ streptomycin was purchased from HyClone (Logan, UT); 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) and a tartrate-resistant acid phosphatase (TRAP) kit were purchased from Sigma-Aldrich (St Louis, MO). Antibodies specific to NFATc1, c-Fos, or b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase–conjugated antirabbit immunoglobulin G and antimouse immunoglobulin G were purchased from Southern Biotech (New Orleans, LA). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was obtained from Molecular Probes (Eugene, OR). Cells Experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University, Seoul, Korea. Six-weekold C57BL/6 mice were obtained from Orient Bio (Seongnam, Korea). TLR2-deficient C57BL/6 were kindly provided by Dr Shizuo Akira (Osaka University, Osaka, Japan). Mice were housed in specific pathogen-free conditions with food and water ad libitum under a 12hour day/night cycle. Stroma-free bone marrow cells and BMMs were prepared as described previously (17). Briefly, stroma-free bone marrow cells were incubated with 20 ng/mL M-CSF for 5 days to generate macrophages; then, BMMs were plated onto 96-well culture plates at 3 104 cells/200 mL/well and differentiated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the presence or absence of 1, 3, 10, or 30 mg/mL EfLTA for 3 days. For the cell viability assay, 20 mL MTT solution (5 mg/mL) was added into each well. Then, the cells were incubated at 37 C for 2 hours. The culture medium was removed, and the cells were dissolved in 100 mL dimethyl sulfoxide (Sigma-Aldrich). The optical density was measured by spectrophotometric analysis at 570 nm with a reference wavelength of 650 nm. Determination of Osteoclast Differentiation The cells were fixed, washed, and then stained using a TRAP assay kit according to the manufacturer’s instructions. TRAP-positive MNCs with 3 or more nuclei were enumerated as mature osteoclasts with an inverted phase-contrast microscope (CKX41; Olympus, Tokyo, Japan). Western Blotting BMMs were plated onto 60-mm culture dishes at 2 106 cells/ dish and differentiated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of 1, 3, 10, or 30 mg/mL EfLTA for 2 days. The cells were lysed with radio-immunoprecipitation assay (RIPA) buffer containing 50 mmol/L Tris, 150 mmol/L NaCl, 1% sodium deoxycholate, 1% Triton X-100 (Merck, Darmstadt, Germany), and 1% sodium dodecyl sulfate (SDS) and centrifuged at 13,000 g for 2
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15 minutes. The lysates were separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane as described previously (21). The membrane was blocked and then incubated with primary antibodies specific to NFATc1, c-Fos, or b-actin. The membrane was washed and incubated with horseradish peroxidase–conjugated antibody. After washing, the immunoreactive bands were detected with SUPEX ECL reagents (Neuronex, Daegu, Korea) using the ChemiDoc MP System (Bio-Rad Laboratories, Inc, Hercules, CA).
Phagocytosis Assay CFSE-stained E. faecalis was prepared as described previously (20). BMMs were plated onto 24-well culture plates at 2 105 cells/ 500 mL/well and differentiated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of 30 mg/mL EfLTA. On days 1 and 2 after incubation, the cells were cultured with 2 106 colony-forming units (CFUs) of CFSE-labeled E. faecalis for 30 minutes at 4 C or 37 C. The cells were washed and fixed with ice-cold phosphate-buffered saline and 1% paraformaldehyde, respectively. The cells were analyzed by flow cytometry and CellQuest Pro software (BD Biosciences, San Jose, CA). Enzyme-linked Immunosorbent Assay BMMs were plated onto 96-well culture plates at 3 104 cells/ 200 mL/well and differentiated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of 1, 3, 10, or 30 mg/mL EfLTA for 3 days. The expression of TNF-a and MCP-1 in the cell culture supernatants was measured using a commercial enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Statistical Analysis Results were expressed as the mean standard deviation from triplicate samples in each experimental group. The statistical significance of differences was evaluated using the Student t test. All experiments were independently performed at least 4 times. Asterisks indicate a statistically significant difference when compared with the nontreatment control group (*P < .05 and **P < .01).
Results EfLTA Inhibits Osteoclast Differentiation from BMMs through Down-regulation of c-Fos and NFATc1 To examine the effect of EfLTA on osteoclast differentiation, BMMs were differentiated into osteoclasts with 20 ng/mL M-CSF and 40 ng/mL RANKL in the presence or absence of EfLTA for 3 days. EfLTA inhibited the number of TRAP-positive MNCs in a dose-dependent manner, indicating an inhibitory effect of EfLTA on osteoclast differentiation from BMM (Fig. 1A and B). The inhibitory effect of EfLTA on osteoclast differentiation was not caused by cytotoxicity (Fig. 1C). c-Fos and NFATc1 are known as essential transcription factors for osteoclast differentiation (22). Thus, we examined whether the inhibitory effect of EfLTA on osteoclast differentiation from BMMs is caused by downregulation of c-Fos and NFATc1. Western blotting showed that EfLTA inhibited RANKL-induced c-Fos and NFATc1 in a dose-dependent manner (Fig. 1D and E). These results suggest that EfLTA inhibits osteoclast differentiation from BMMs through down-regulation of c-Fos and NFATc1. TLR2 Is Essential for the EfLTA-mediated Inhibition of Osteoclast Differentiation from BMMs We previously reported that EfLTA is recognized by TLR2 (7). To examine whether TLR2 is essential for EfLTA-mediated inhibition of JOE — Volume -, Number -, - 2016
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Figure 1. EfLTA inhibits RANKL-induced osteoclast differentiation from BMMs through down-regulation of c-Fos and NFATc1. BMMs were differentiated into osteoclasts with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of EfLTA (1, 3, 10, or 30 mg/mL) for 3 days. (A) The cells were observed under a microscope with magnification at 100. (B) The cells were subjected to TRAP staining to determine osteoclast differentiation. TRAP-positive MNCs with 3 or more nuclei were enumerated using microscopic analysis. (C) The cells were subjected to MTT assay to determine cell viability. (D) BMMs were differentiated into osteoclasts in the presence of 0, 1, 3, 10, or 30 mg/mL of EfLTA for 2 days. The cells were lysed and subjected to Western blotting using antibodies specific to NFATc1, c-Fos, or b-actin. (E) The band intensity was determined by densitometry to obtain the expression ratio of NFATc1 or c-Fos to b-actin. The values indicate the relative expression ratio to that of the day 0. *P < .05 and **P < .01.
osteoclast differentiation, wild-type and TLR2-deficient BMMs were differentiated into osteoclasts with RANKL in the presence or absence of EfLTA for 3 days. As shown in Figure 2, EfLTA failed to suppress osteoclast differentiation from TLR2-deficient BMMs. It was noted that 250
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Figure 2. TLR2 is essential for the attenuation of osteoclast differentiation by EfLTA. Wild-type or TLR2-deficient BMMs were differentiated into osteoclasts with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of EfLTA (1, 3, 10, or 30 mg/mL), Pam2CSK4 (1 mg/mL), or EcLPS (0.5 mg/ mL) for 3 days. The cells were subjected to TRAP staining to determine osteoclast differentiation. *P < .05 and **P < .01 compared with the nontreatment control group.
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EcLPS (a TLR4 ligand), not Pam2CSK4 (a TLR2 ligand), inhibited osteoclast differentiation of TLR2-deficient BMMs. These results suggest that TLR2 signaling is critical for EfLTA-mediated inhibition of osteoclast differentiation from BMMs.
EfLTA Increases the Phagocytic Capacity and the Expression of Inflammatory Cytokines and Chemokines in Osteoclast Precursors Macrophages phagocytose engulfed bacteria and produce inflammatory cytokines and chemokines that recruit and activate immune cells at the site of infection (23). Interestingly, however, the phagocytic capacity has been shown to decrease after differentiation into osteoclasts (24). To examine whether the EfLTA-mediated inhibition of osteoclast differentiation from BMMs is to maintain the major function of macrophages, phagocytic activity and production of cytokines and chemokines were examined. As shown in Figure 3A, the phagocytic activity decreased in a time-dependent manner during osteoclast differentiation from BMMs. However, when the cells were differentiated in the presence of EfLTA, the phagocytic capacity was maintained. In addition, EfLTA significantly induced the expression of TNF-a (Fig. 3B) and MCP-1 (Fig. 3C) in a dose-dependent manner. These results indicate that EfLTA allows BMMs to retain the abilities of macrophages with regard to their phagocytic capacity and inflammatory functions.
Discussion In the present study, we showed that EfLTA inhibited osteoclast differentiation from BMMs by decreasing NFATc1 and c-Fos expression. In
LTA of E. faecalis and Differentiation of Macrophages
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Figure 3. EfLTA maintains the phagocytic activity of osteoclast precursors and induces proinflammatory cytokines and chemokines in osteoclasts. (A) BMMs were stimulated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of 30 mg/mL EfLTA. On days 1 and 2 after incubation, the cells were incubated with CFSE-labeled E. faecalis (1 106 CFU/mL) for 30 minutes at 4 C or 37 C. The phagocytic capacity was analyzed by flow cytometry. Mean fluorescence intensities at 37 C (upper) and 4 C (lower) are shown in the upper right corner of each histogram. (B and C) BMMs were differentiated into osteoclasts in the absence or presence of 1, 3, 10, or 30 mg/mL EfLTA for 3 days. The culture supernatants were collected to measure the expression of TNF-a and MCP-1 by enzyme-linked immunosorbent assay. **P < .01 compared with nontreated cells. ND, not detected.
addition, EfLTA maintained the phagocytic capacity and increased the expression of inflammatory cytokines and chemokines in osteoclast precursors. Therefore, these results suggest that EfLTA inhibits the differentiation of macrophages into osteoclasts, just as HKEF does (20). In the present study, EfLTA inhibited osteoclast differentiation from BMMs. Coincidently, we previously reported that LTA of Staphylococcus aureus (SaLTA) attenuates osteoclast differentiation (24). In addition, peptidoglycan, LPS, and lipoproteins are known to inhibit osteoclast differentiation (25). Thus, the inhibitory effect of osteoclast differentiation from BMMs seems to be a general phenomenon of pathogen-associated molecular patterns. In addition, EfLTA inhibited the expressions of c-Fos and NFATc1 in the present study. It is well-known that c-Fos and NFATc1 are key transcription factors for osteoclast differentiation (22) and the suppression of cFos and NFATc1 expression leads to inhibition of osteoclast differentiation (26, 27). Therefore, it is likely that EfLTA could inhibit osteoclast differentiation from BMMs through down-regulation of c-Fos and NFATc1. The TLR2 signaling pathway appears to be essential for EfLTAinhibited osteoclast differentiation because it was not observed in TLR2-deficient BMMs. These results are concordant with a previous study showing that TLR2 is required for the SaLTA-mediated inhibition of osteoclast differentiation (24). LTA can induce the expression of chemokines and inflammatory mediators such as TNF-a and NO through TLR2 activation of macrophages (7, 28). Furthermore, structural analysis with x-ray crystallography showed that the glycolipid portion of LTA interacts with the hydrophobic pocket of TLR2 (29). In addition, the deacylated form of LTA neither stimulated TLR2 nor induced the ex4
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pressions of NO or TNF-a (28, 30). Collectively, these results imply that EfLTA activates the TLR2 signaling pathway via c-Fos and NFATc1, leading to the inhibition of osteoclast differentiation and the induction of inflammatory responses. It is well-known that the phagocytic capacity gradually decreases as macrophages differentiate into osteoclasts (31). However, in our study, EfLTA increased the phagocytic capacity of osteoclast precursors. Concordantly, SaLTA also elicited an increase in the endocytic capacity and the production of TNF-a in the same experimental model (24). Also, TLR ligands including peptidoglycan, polyinosinic-polycytidylic acid [Poly (I:C)], LPS, and CpG have been shown to maintain the phagocytic capacity of osteoclast precursors (25). Thus, the suppressed osteoclast differentiation by pathogen-associated molecular patterns is inclined to preserve the phagocytic function of macrophages. Together with the retention of phagocytic potential, we also observed that EfLTA induced the expression of inflammatory cytokines and chemokines in osteoclast precursors. Notably, increased levels of proinflammatory cytokines, including TNF-a and interleukin 6, as well as chemokines such as MCP-1 and keratinocyte chemoattractant (KC) are known to be present in periapical lesions (32, 33). Increased levels of proinflammatory cytokines and chemokines caused by EfLTA might contribute to the inflammation in periapical lesions. In conclusion, EfLTA inhibits osteoclast differentiation from BMMs though the TLR2 signaling pathway, as shown in the present study. Also, osteoclast precursors that differentiate in the presence of EfLTA retain their phagocytic capacity and their ability to induce proinflammatory cytokines and chemokines. These results provide insights into the role of EfLTA in the pathogenesis of apical periodontitis. JOE — Volume -, Number -, - 2016
Basic Research—Biology Acknowledgments Jihyun Yang and Ok-Jin Park contributed equally to this work. Supported by grants from the National Research Foundation of Korea funded by the Korean government (MISIP) (NRF2015R1A2A1A15055453 and NRF-2015M2A2A6A01044894) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (HI14C0469), Republic of Korea. The authors deny any conflicts of interest related to this study.
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15. O’Riordan M, Yi CH, Gonzales R, et al. Innate recognition of bacteria by a macrophage cytosolic surveillance pathway. Proc Natl Acad Sci U S A 2002;99: 13861–6. 16. Wu Y, Humphrey MB, Nakamura MC. Osteoclasts - the innate immune cells of the bone. Autoimmunity 2008;41:183–94. 17. Kim J, Yang J, Park OJ, et al. Lipoproteins are an important bacterial component responsible for bone destruction through the induction of osteoclast differentiation and activation. J Bone Miner Res 2013;28:2381–91. 18. Zou W, Bar-Shavit Z. Dual modulation of osteoclast differentiation by lipopolysaccharide. J Bone Miner Res 2002;17:1211–8. 19. Zhang P, Liu J, Xu Q, et al. TLR2-dependent modulation of osteoclastogenesis by Porphyromonas gingivalis through differential induction of NFATc1 and NF-kappaB. J Biol Chem 2011;286:24159–69. 20. Park OJ, Yang J, Kim J, et al. Enterococcus faecalis attenuates the differentiation of macrophages into osteoclasts. J Endod 2015;41:658–62. 21. Kim AY, Kwak JH, Je NK, et al. Epithelial-mesenchymal transition is associated with acquired resistance to 5-fluorocuracil in HT-29 colon cancer cells. Toxicol Res 2015;31:151–6. 22. Takayanagi H. The role of NFAT in osteoclast formation. Ann N Y Acad Sci 2007; 1116:227–37. 23. Soehnlein O, Lindbom L. Phagocyte partnership during the onset and resolution of inflammation. Nat Rev Immunol 2010;10:427–39. 24. Yang J, Ryu YH, Yun CH, Han SH. Impaired osteoclastogenesis by staphylococcal lipoteichoic acid through Toll-like receptor 2 with partial involvement of MyD88. J Leukoc Biol 2009;86:823–31. 25. Takami M, Kim N, Rho J, Choi Y. Stimulation by toll-like receptors inhibits osteoclast differentiation. J Immunol 2002;169:1516–23. 26. Choi SW, Son YJ, Yun JM, Kim SH. Fisetin Inhibits Osteoclast Differentiation via Downregulation of p38 and c-Fos-NFATc1 Signaling Pathways. Evid Based Complement Alternat Med 2012;2012:810563. 27. Kim H, Hyeon S, Kim H, et al. Dynein light chain LC8 inhibits osteoclast differentiation and prevents bone loss in mice. J Immunol 2013;190:1312–8. 28. Hong SW, Baik JE, Kang SS, et al. Lipoteichoic acid of Streptococcus mutans interacts with Toll-like receptor 2 through the lipid moiety for induction of inflammatory mediators in murine macrophages. Mol Immunol 2014;57: 284–91. 29. Kang JY, Nan X, Jin MS, et al. Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. Immunity 2009;31:873–84. 30. Ryu YH, Baik JE, Yang JS, et al. Differential immunostimulatory effects of Grampositive bacteria due to their lipoteichoic acids. Int Immunopharmacol 2009;9: 127–33. 31. Chang EJ, Ha J, Huang H, et al. The JNK-dependent CaMK pathway restrains the reversion of committed cells during osteoclast differentiation. J Cell Sci 2008; 121:2555–64. 32. Silva TA, Garlet GP, Fukada SY, et al. Chemokines in oral inflammatory diseases: apical periodontitis and periodontal disease. J Dent Res 2007;86: 306–19. 33. Prso IB, Kocjan W, Simic H, et al. Tumor necrosis factor-alpha and interleukin 6 in human periapical lesions. Mediators Inflamm 2007;2007:38210.
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Lipoteichoic Acid of Enterococcus faecalis Inhibits the Differentiation of Macrophages into Osteoclasts Jihyun Yang, PhD,* Ok-Jin Park, PhD,* Jiseon Kim, MS,* Jung Eun Baik, PhD,* Cheol-Heui Yun, PhD,†‡ and Seung Hyun Han, PhD* Abstract Introduction: Enterococcus faecalis is associated with persistent endodontic infection and refractory apical periodontitis. Recently, we have shown that heatkilled E. faecalis attenuates osteoclast differentiation. Because lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we investigated the effect of LTA from E. faecalis (EfLTA) on osteoclast differentiation. Methods: EfLTA was purified through organic solvent extraction, hydrophobic interaction column chromatography, and ion exchange column chromatography. Bone marrow cells from C57BL/6 or Toll-like receptor 2–deficient mice were incubated with macrophage colony-stimulating factor (M-CSF) for 5 days to generate macrophages (bone marrow–derived macrophages [BMMs]). The cells were differentiated into osteoclasts with M-CSF and receptor activator of NF-kB ligand (RANKL) in the presence or absence of EfLTA. The degree of osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining. The expression of NFATc1 and c-Fos transcription factors was determined by Western blotting. A phagocytosis assay was performed by measuring the uptake of carboxyfluorescein diacetate succinimidyl ester–stained E. faecalis. An enzyme-linked immunosorbent assay was used to determine the amount of cytokines and chemokines. Results: When BMMs were treated with EfLTA, osteoclast differentiation was attenuated. EfLTA inhibited the RANKL-induced expression of NFATc1 and c-Fos. EfLTA inhibition of osteoclast differentiation was not observed in TLR2-deficient BMMs. In addition, EfLTA sustained the phagocytic capacity of BMMs even after the differentiation into osteoclasts, whereas it induced the expression of inflammatory cytokines and chemokines. Conclusions: EfLTA inhibits the differentiation of macrophages into osteoclasts and thereby maintains the phagocytic and inflammatory capacities of macrophages, potentially contributing to refractory apical periodontitis. (J Endod 2016;-:1–5)
Key Words Chemokine, cytokine, Enterococcus faecalis, lipoteichoic acid, osteoclast differentiation, phagocytosis
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nterococcus faecalis is a commensal gram-positive bacterium found in the oral cavity, gastrointestinal tract, and genital tract of humans (1). E. faecalis is also considered to be an opportunistic pathogen that causes endocarditis, bacteremia, and urinary tract infection (2). Of note, E. faecalis is a predominant bacterium implicated in persistent endodontic infection (3). E. faecalis easily invades dentinal tubules and can be isolated from periapical lesions (4, 5). E. faecalis and its major virulence factor, lipoteichoic acid (LTA), induce inflammatory cytokines and chemokines (6, 7), contributing to inflammation and tissue damage through the accumulation of immune cells including neutrophils and monocytes/macrophages in periapical lesions followed by periapical bone loss (8). LTA is known to be a major virulence factor of gram-positive bacteria because it has an important role in bacterial adhesion and in the initiation of host immune responses (9). LTA is shed from bacteria during the growing phase (9). LTA is an amphiphilic molecule that is composed of a glycolipid anchor together with repeating units of glycerol or ribitol phosphate (10). It has been reported that E. faecalis LTA (EfLTA) has a polyglycerol phosphate backbone substituted at the C2 position by D-alanine and kojibiose (11). EfLTA induces inflammatory mediators such as tumor necrosis factor alpha (TNF-a) and nitric oxide (NO) and chemokines such as monocyte chemoattractant protein-1 (MCP-1) through the Toll-like receptor 2 (TLR2) signaling pathway in macrophages (6, 7). It is notable that the glycolipid portion of EfLTA is responsible for its immunostimulating activity because the production of NO, interferon-gammainducible protein 10 (IP-10), and macrophage inflammatory protein-1 alpha (MIP1a) was not observed in macrophages stimulated with calcium hydroxide–treated EfLTA, which is a deacylated form of LTA (12). Macrophages play a role in phagocytosis against bacteria (13) and are well characterized as an important part of innate immunity. In addition, macrophages lead to local inflammatory responses through the induction of proinflammatory cytokines and chemokines (14, 15). On the other hand, macrophages are also considered to be common osteoclast precursors (16). Bone-resorbing osteoclasts are multinucleated giant cells (MNCs) capable of degrading calcified bone matrix such as alveolar bones in chronic periodontitis and roots of teeth in apical periodontitis (16). Bacteria and their virulence factors affect osteoclast differentiation and activation (17). Bacterial lipoproteins and lipopolysaccharides (LPSs) inhibit osteoclast differentiation from macrophages (18, 19). Recently, we reported that heat-killed E. faecalis (HKEF) inhibits osteoclast differentiation from bone marrow–derived macrophages (BMMs)
From the *Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry and †Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea; and ‡Institute of Green Bio Science Technology, Seoul National University, Pyeongchang, Republic of Korea. Address requests for reprints to Prof Seung Hyun Han, Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2016.01.012
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Basic Research—Biology (20). Because LTA is a major virulence factor of gram-positive bacteria, we hypothesized that EfLTA could inhibit osteoclast differentiation, just as HKEF does. Therefore, we prepared highly pure and structurally intact LTA from E. faecalis and investigated the effect of EfLTA on osteoclast differentiation and inflammatory functions.
Materials and Methods Reagents and Chemicals LTA was prepared from E. faecalis ATCC 29212 (American Type Culture Collection, Manassas, VA) as described previously (6). Pam2CSK4 and ultrapure LPS from Escherichia coli O111:B4 (EcLPS) were purchased from InvivoGen (San Diego, CA). Recombinant murine macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL) were purchased from PeproTech (Rocky Hill, NJ). Alpha-minimum essential medium and certified fetal bovine serum were obtained from Gibco-BRL (Grand Island, NY). Penicillin/ streptomycin was purchased from HyClone (Logan, UT); 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) and a tartrate-resistant acid phosphatase (TRAP) kit were purchased from Sigma-Aldrich (St Louis, MO). Antibodies specific to NFATc1, c-Fos, or b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase–conjugated antirabbit immunoglobulin G and antimouse immunoglobulin G were purchased from Southern Biotech (New Orleans, LA). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was obtained from Molecular Probes (Eugene, OR). Cells Experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University, Seoul, Korea. Six-weekold C57BL/6 mice were obtained from Orient Bio (Seongnam, Korea). TLR2-deficient C57BL/6 were kindly provided by Dr Shizuo Akira (Osaka University, Osaka, Japan). Mice were housed in specific pathogen-free conditions with food and water ad libitum under a 12hour day/night cycle. Stroma-free bone marrow cells and BMMs were prepared as described previously (17). Briefly, stroma-free bone marrow cells were incubated with 20 ng/mL M-CSF for 5 days to generate macrophages; then, BMMs were plated onto 96-well culture plates at 3 104 cells/200 mL/well and differentiated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the presence or absence of 1, 3, 10, or 30 mg/mL EfLTA for 3 days. For the cell viability assay, 20 mL MTT solution (5 mg/mL) was added into each well. Then, the cells were incubated at 37 C for 2 hours. The culture medium was removed, and the cells were dissolved in 100 mL dimethyl sulfoxide (Sigma-Aldrich). The optical density was measured by spectrophotometric analysis at 570 nm with a reference wavelength of 650 nm. Determination of Osteoclast Differentiation The cells were fixed, washed, and then stained using a TRAP assay kit according to the manufacturer’s instructions. TRAP-positive MNCs with 3 or more nuclei were enumerated as mature osteoclasts with an inverted phase-contrast microscope (CKX41; Olympus, Tokyo, Japan). Western Blotting BMMs were plated onto 60-mm culture dishes at 2 106 cells/ dish and differentiated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of 1, 3, 10, or 30 mg/mL EfLTA for 2 days. The cells were lysed with radio-immunoprecipitation assay (RIPA) buffer containing 50 mmol/L Tris, 150 mmol/L NaCl, 1% sodium deoxycholate, 1% Triton X-100 (Merck, Darmstadt, Germany), and 1% sodium dodecyl sulfate (SDS) and centrifuged at 13,000 g for 2
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15 minutes. The lysates were separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane as described previously (21). The membrane was blocked and then incubated with primary antibodies specific to NFATc1, c-Fos, or b-actin. The membrane was washed and incubated with horseradish peroxidase–conjugated antibody. After washing, the immunoreactive bands were detected with SUPEX ECL reagents (Neuronex, Daegu, Korea) using the ChemiDoc MP System (Bio-Rad Laboratories, Inc, Hercules, CA).
Phagocytosis Assay CFSE-stained E. faecalis was prepared as described previously (20). BMMs were plated onto 24-well culture plates at 2 105 cells/ 500 mL/well and differentiated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of 30 mg/mL EfLTA. On days 1 and 2 after incubation, the cells were cultured with 2 106 colony-forming units (CFUs) of CFSE-labeled E. faecalis for 30 minutes at 4 C or 37 C. The cells were washed and fixed with ice-cold phosphate-buffered saline and 1% paraformaldehyde, respectively. The cells were analyzed by flow cytometry and CellQuest Pro software (BD Biosciences, San Jose, CA). Enzyme-linked Immunosorbent Assay BMMs were plated onto 96-well culture plates at 3 104 cells/ 200 mL/well and differentiated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of 1, 3, 10, or 30 mg/mL EfLTA for 3 days. The expression of TNF-a and MCP-1 in the cell culture supernatants was measured using a commercial enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Statistical Analysis Results were expressed as the mean standard deviation from triplicate samples in each experimental group. The statistical significance of differences was evaluated using the Student t test. All experiments were independently performed at least 4 times. Asterisks indicate a statistically significant difference when compared with the nontreatment control group (*P < .05 and **P < .01).
Results EfLTA Inhibits Osteoclast Differentiation from BMMs through Down-regulation of c-Fos and NFATc1 To examine the effect of EfLTA on osteoclast differentiation, BMMs were differentiated into osteoclasts with 20 ng/mL M-CSF and 40 ng/mL RANKL in the presence or absence of EfLTA for 3 days. EfLTA inhibited the number of TRAP-positive MNCs in a dose-dependent manner, indicating an inhibitory effect of EfLTA on osteoclast differentiation from BMM (Fig. 1A and B). The inhibitory effect of EfLTA on osteoclast differentiation was not caused by cytotoxicity (Fig. 1C). c-Fos and NFATc1 are known as essential transcription factors for osteoclast differentiation (22). Thus, we examined whether the inhibitory effect of EfLTA on osteoclast differentiation from BMMs is caused by downregulation of c-Fos and NFATc1. Western blotting showed that EfLTA inhibited RANKL-induced c-Fos and NFATc1 in a dose-dependent manner (Fig. 1D and E). These results suggest that EfLTA inhibits osteoclast differentiation from BMMs through down-regulation of c-Fos and NFATc1. TLR2 Is Essential for the EfLTA-mediated Inhibition of Osteoclast Differentiation from BMMs We previously reported that EfLTA is recognized by TLR2 (7). To examine whether TLR2 is essential for EfLTA-mediated inhibition of JOE — Volume -, Number -, - 2016
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Figure 1. EfLTA inhibits RANKL-induced osteoclast differentiation from BMMs through down-regulation of c-Fos and NFATc1. BMMs were differentiated into osteoclasts with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of EfLTA (1, 3, 10, or 30 mg/mL) for 3 days. (A) The cells were observed under a microscope with magnification at 100. (B) The cells were subjected to TRAP staining to determine osteoclast differentiation. TRAP-positive MNCs with 3 or more nuclei were enumerated using microscopic analysis. (C) The cells were subjected to MTT assay to determine cell viability. (D) BMMs were differentiated into osteoclasts in the presence of 0, 1, 3, 10, or 30 mg/mL of EfLTA for 2 days. The cells were lysed and subjected to Western blotting using antibodies specific to NFATc1, c-Fos, or b-actin. (E) The band intensity was determined by densitometry to obtain the expression ratio of NFATc1 or c-Fos to b-actin. The values indicate the relative expression ratio to that of the day 0. *P < .05 and **P < .01.
osteoclast differentiation, wild-type and TLR2-deficient BMMs were differentiated into osteoclasts with RANKL in the presence or absence of EfLTA for 3 days. As shown in Figure 2, EfLTA failed to suppress osteoclast differentiation from TLR2-deficient BMMs. It was noted that 250
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Figure 2. TLR2 is essential for the attenuation of osteoclast differentiation by EfLTA. Wild-type or TLR2-deficient BMMs were differentiated into osteoclasts with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of EfLTA (1, 3, 10, or 30 mg/mL), Pam2CSK4 (1 mg/mL), or EcLPS (0.5 mg/ mL) for 3 days. The cells were subjected to TRAP staining to determine osteoclast differentiation. *P < .05 and **P < .01 compared with the nontreatment control group.
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EcLPS (a TLR4 ligand), not Pam2CSK4 (a TLR2 ligand), inhibited osteoclast differentiation of TLR2-deficient BMMs. These results suggest that TLR2 signaling is critical for EfLTA-mediated inhibition of osteoclast differentiation from BMMs.
EfLTA Increases the Phagocytic Capacity and the Expression of Inflammatory Cytokines and Chemokines in Osteoclast Precursors Macrophages phagocytose engulfed bacteria and produce inflammatory cytokines and chemokines that recruit and activate immune cells at the site of infection (23). Interestingly, however, the phagocytic capacity has been shown to decrease after differentiation into osteoclasts (24). To examine whether the EfLTA-mediated inhibition of osteoclast differentiation from BMMs is to maintain the major function of macrophages, phagocytic activity and production of cytokines and chemokines were examined. As shown in Figure 3A, the phagocytic activity decreased in a time-dependent manner during osteoclast differentiation from BMMs. However, when the cells were differentiated in the presence of EfLTA, the phagocytic capacity was maintained. In addition, EfLTA significantly induced the expression of TNF-a (Fig. 3B) and MCP-1 (Fig. 3C) in a dose-dependent manner. These results indicate that EfLTA allows BMMs to retain the abilities of macrophages with regard to their phagocytic capacity and inflammatory functions.
Discussion In the present study, we showed that EfLTA inhibited osteoclast differentiation from BMMs by decreasing NFATc1 and c-Fos expression. In
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Figure 3. EfLTA maintains the phagocytic activity of osteoclast precursors and induces proinflammatory cytokines and chemokines in osteoclasts. (A) BMMs were stimulated with 20 ng/mL M-CSF and 40 ng/mL RANKL in the absence or presence of 30 mg/mL EfLTA. On days 1 and 2 after incubation, the cells were incubated with CFSE-labeled E. faecalis (1 106 CFU/mL) for 30 minutes at 4 C or 37 C. The phagocytic capacity was analyzed by flow cytometry. Mean fluorescence intensities at 37 C (upper) and 4 C (lower) are shown in the upper right corner of each histogram. (B and C) BMMs were differentiated into osteoclasts in the absence or presence of 1, 3, 10, or 30 mg/mL EfLTA for 3 days. The culture supernatants were collected to measure the expression of TNF-a and MCP-1 by enzyme-linked immunosorbent assay. **P < .01 compared with nontreated cells. ND, not detected.
addition, EfLTA maintained the phagocytic capacity and increased the expression of inflammatory cytokines and chemokines in osteoclast precursors. Therefore, these results suggest that EfLTA inhibits the differentiation of macrophages into osteoclasts, just as HKEF does (20). In the present study, EfLTA inhibited osteoclast differentiation from BMMs. Coincidently, we previously reported that LTA of Staphylococcus aureus (SaLTA) attenuates osteoclast differentiation (24). In addition, peptidoglycan, LPS, and lipoproteins are known to inhibit osteoclast differentiation (25). Thus, the inhibitory effect of osteoclast differentiation from BMMs seems to be a general phenomenon of pathogen-associated molecular patterns. In addition, EfLTA inhibited the expressions of c-Fos and NFATc1 in the present study. It is well-known that c-Fos and NFATc1 are key transcription factors for osteoclast differentiation (22) and the suppression of cFos and NFATc1 expression leads to inhibition of osteoclast differentiation (26, 27). Therefore, it is likely that EfLTA could inhibit osteoclast differentiation from BMMs through down-regulation of c-Fos and NFATc1. The TLR2 signaling pathway appears to be essential for EfLTAinhibited osteoclast differentiation because it was not observed in TLR2-deficient BMMs. These results are concordant with a previous study showing that TLR2 is required for the SaLTA-mediated inhibition of osteoclast differentiation (24). LTA can induce the expression of chemokines and inflammatory mediators such as TNF-a and NO through TLR2 activation of macrophages (7, 28). Furthermore, structural analysis with x-ray crystallography showed that the glycolipid portion of LTA interacts with the hydrophobic pocket of TLR2 (29). In addition, the deacylated form of LTA neither stimulated TLR2 nor induced the ex4
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pressions of NO or TNF-a (28, 30). Collectively, these results imply that EfLTA activates the TLR2 signaling pathway via c-Fos and NFATc1, leading to the inhibition of osteoclast differentiation and the induction of inflammatory responses. It is well-known that the phagocytic capacity gradually decreases as macrophages differentiate into osteoclasts (31). However, in our study, EfLTA increased the phagocytic capacity of osteoclast precursors. Concordantly, SaLTA also elicited an increase in the endocytic capacity and the production of TNF-a in the same experimental model (24). Also, TLR ligands including peptidoglycan, polyinosinic-polycytidylic acid [Poly (I:C)], LPS, and CpG have been shown to maintain the phagocytic capacity of osteoclast precursors (25). Thus, the suppressed osteoclast differentiation by pathogen-associated molecular patterns is inclined to preserve the phagocytic function of macrophages. Together with the retention of phagocytic potential, we also observed that EfLTA induced the expression of inflammatory cytokines and chemokines in osteoclast precursors. Notably, increased levels of proinflammatory cytokines, including TNF-a and interleukin 6, as well as chemokines such as MCP-1 and keratinocyte chemoattractant (KC) are known to be present in periapical lesions (32, 33). Increased levels of proinflammatory cytokines and chemokines caused by EfLTA might contribute to the inflammation in periapical lesions. In conclusion, EfLTA inhibits osteoclast differentiation from BMMs though the TLR2 signaling pathway, as shown in the present study. Also, osteoclast precursors that differentiate in the presence of EfLTA retain their phagocytic capacity and their ability to induce proinflammatory cytokines and chemokines. These results provide insights into the role of EfLTA in the pathogenesis of apical periodontitis. JOE — Volume -, Number -, - 2016
Basic Research—Biology Acknowledgments Jihyun Yang and Ok-Jin Park contributed equally to this work. Supported by grants from the National Research Foundation of Korea funded by the Korean government (MISIP) (NRF2015R1A2A1A15055453 and NRF-2015M2A2A6A01044894) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (HI14C0469), Republic of Korea. The authors deny any conflicts of interest related to this study.
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