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Chlorhexidine Gluconate Attenuates the Ability of Lipoteichoic Acid from Enterococcus faecalis to Stimulate Toll-like Receptor 2
Basic Research—Biology
Chlorhexidine Gluconate Attenuates the Ability of Lipoteichoic Acid from Enterococcus faecalis to Stimulate Toll-like Receptor 2 Jin-Kyung Lee, DDS, MSD,* ,** Jung Eun Baik, BS†,** Cheol-Heui Yun, PhD,‡,§ Kangseok Lee, PhD,储 Seung Hyun Han, PhD,†,¶ WooCheol Lee, DDS, PhD,* Kwang-Shik Bae, DDS, PhD,* Seung-Ho Baek, DDS, PhD,* Yoon Lee, DDS, MSD,# Won-Jun Son, DDS, PhD,* and Kee-Yeon Kum, DDS, PhD* Abstract Chlorhexidine gluconate (CHX) has been widely used as a canal irrigant or an intracanal medicament on account of its antibacterial substantivity. This in vitro study aimed to determine if CHX attenuates the inflammatory activity of Enterococcus faecalis and its major virulence factor, lipoteichoic acid (LTA). An enzymelinked immunosorbent assay showed that CHX-killed E. faecalis was less potent than heat-killed E. faecalis in the production of tumor necrosis factor ␣ (TNF-␣) by a murine macrophage cell line, RAW 264.7 (p ⬍ 0.05). Interestingly, pretreatment of LTA with 2% CHX for 6 hours or with 0.2% CHX for 24 hours almost eliminated the TNF-␣ inducibility (p ⬍ 0.05). Furthermore, CHX abrogated the ability of LTA to stimulate Toll-like receptor 2, resulting in the attenuated induction of TNF-␣ expression. Collectively, our results suggest that CHX can inactivate LTA of E. faecalis leading to the alleviation of inflammatory responses induced by E. faecalis and its LTA. (J Endod 2009;35:212–215)
Key Words Chlorhexidine gluconate, Enterococcus faecalis, intracanal medicament, lipoteichoic acid, Toll-like receptor 2, tumor necrosis factor-␣
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nterococci are human commensals adapted to complex environments of the oral cavity, gastrointestinal tract, and genital tract (1). Although they occupy only a small proportion of the flora in untreated canals (2), Enterococcus faecalis has been frequently recovered from root-filled teeth with refractory apical periodontitis (3, 4). E. faecalis can survive in harsh environmental conditions such as high alkalinity (5) and can invade the dentinal tubules in variable depths (6, 7) followed by colonization of the root canal (8, 9). These abilities of E. faecalis may explain its survival after a root canal treatment and its ability to induce persistent apical periodontitis. Lipoteichoic acid (LTA) is a cell wall component of gram-positive bacteria and appears to be one of the major etiologic factors at various stages of inflammation. LTA isolated from E. faecalis as well as other gram-positive bacteria have been reported to stimulate leukocytes to release the key inflammatory mediators (10, 11). LTA was also shown to exhibit high affinity to hydroxyapatite (12), a feature related to the penetration of dentinal tubules. Chlorhexidine gluconate (CHX) is a cationic bisbiguanide that adsorbs onto the cell wall of microorganisms causing leakage of the intracellular components. CHX has been used as an important constituent of irrigating solutions and intracanal medicaments during root canal therapy on account of its substantive antimicrobial activity (13–15). In various in vivo and in vitro studies, CHX has proven to be effective in reducing or eliminating E. faecalis from the root canal space and dentinal tubules (15–19). Therefore, its use in root canal treatment is believed to be advantageous, particularly in retreatment cases in which high proportions of E. faecalis are frequently found in the root canal system (20). Although the capacity of CHX to kill bacteria has been extensively studied, there are few reports on the mechanisms for its effectiveness against E. faecalis, especially LTA. Therefore, this in vitro study examined the effects of CHX on LTA to determine if CHX can inactivate LTA from E. faecalis and prevent the inflammatory responses.
From the *Department of Conservative Dentistry and Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, Seoul, Republic of Korea; †Department of Oral Microbiology & Immunology, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, Seoul, Republic of Korea; ‡School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea; §Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Korea; 储Department of Life Science, Chung-Ang University, Seoul, Republic of Korea; ¶International Vaccine Institute, Seoul, Republic of Korea; and #Department of Conservative Dentistry, Wonkwang University Dental Hospital, DaeJeon, Republic of Korea. **Jin-Kyung Lee and Jung Eun Baik contributed equally to this article. Supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2008-314-E00223) and by a grant (03-2007-0090) from the Seoul National University Dental Hospital Research Fund, Republic of Korea. Address requests for reprints to Dr Kee-Yeon Kum, Department of Conservative Dentistry and Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, 25-9 Yongun-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2008 American Association of Endodontists. doi:10.1016/j.joen.2008.10.018
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Basic Research—Biology Materials and Methods Bacteria, Reagents, and Chemicals E. faecalis ATCC 29212 was purchased from the American Type Culture Collection (Manassas, VA). Brain heart infusion was obtained from BD Biosciences (Franklin, NJ) and phosphate-buffered saline was obtained from Mediatech Inc (Herndon, VA). Octyl-Sepharose CL-4B, DEAE-Sepharose, and CHX (20% aqueous solution) were purchased from Sigma-Aldrich (St Louis, MO). Unless indicated otherwise, all other reagents were obtained from Sigma-Aldrich. Preparation of Heat-killed Bacteria and CHX-killed Bacteria E. faecalis was grown in brain heart infusion media at 37°C to the midlog phase in which optical density at 600 nm reached 0.6. The cells were harvested by centrifugation, washed 3 times with phosphate-buffered saline, and divided into 4 groups. Two groups of E. faecalis were killed by heating at 60°C for 1 hour or 24 hours as previously described (10). The other two groups were killed by 2% CHX for 1 hour or 24 hours. To ensure that the bacteria had been completely killed, the bacteria subjected to heat or CHX treatment were plated on the corresponding media containing 1.5% agar and cultured overnight at 37°C. No bacterial colony was observed. Preparation of LTA Highly pure LTA of E. faecalis was prepared as previously described (10). Briefly, approximately 100 g (wet weight) of bacterial cells were resuspended in 1,000 mL of 0.1 mol/L sodium citrate buffer (pH 4.7) and disrupted by ultrasonication. After n-butanol extraction, hydrophobic-interaction chromatography was performed by using octyl-Sepharose column CL-4B (2.5 cm ⫻ 10 cm). The LTA-containing fractions were further separated by DEAE-Sepharose ion-exchange chromatography (1.5 cm ⫻ 10 cm). The quantity of purified LTA was determined by measuring the dry weight of the LTA.
membrane whose expression is regulated by NF-B transcription factor. To determine if CHX inhibits the activation of TLR2 by E. faecalis LTA, CHO/CD14/TLR2 cells were stimulated with CHX-treated or -untreated LTA (0, 3, 10, or 30 g/mL) for 16 hours. The level of TLR2dependent nuclear factor-kappa B (NF-B) activation was determined by analysis of CD25 expression by using flow cytometry (FACS Calibur; BD Biosciences, Franklin, NJ).
Statistical Analysis All experiments were performed in triplicate and the mean value ⫾ standard deviation (SD) was calculated for each treatment group. The treatment groups were compared with the nontreated control group, and statistical significance was determined by a Student 2-tailed t test; p values of ⬍0.05 were considered significant.
Results CHX-treated E. Faecalis Induces Less TNF-␣ Secretion Compared with Heat-treated E. Faecalis When RAW 264.7 cells were stimulated with the heat-treated bacteria, the level of TNF-␣ production increased significantly (p ⬍ 0.05) in proportion to the number of cells (Fig. 1). The CHX-treated E. faecalis showed a similar pattern but induced significantly (p ⬍ 0.05) lower levels of TNF-␣ secretion than the heat-treated E. faecalis. This difference was more remarkable at 24 hours of treatment than at 1 hour of treatment. CHX Efficiently Inactivates LTA from E. Faecalis To examine if CHX alters the TNF-␣ inducibility of E. faecalis LTA, LTA at 30 g/mL was treated with 2% CHX for different time durations
Preparation of E. Faecalis LTA Treated with CHX The 300 g of LTA from E. faecalis were treated with 2% CHX for 24 hours followed by dialysis in a semipermeable dialysis membrane (Spectra/Por 6; Spectrum Laboratories, Inc, Ranch Dominquez, CA). Then, the LTA was subjected to dialysis against endotoxin-free distilled water (Dai Han Pharm Co Ltd, Seoul, Republic of Korea) for 48 hours to remove unreacted CHX. Culture of RAW 264.7 The mouse macrophage cell line, RAW 264.7 (TIB-71), was purchased from the American Type Culture Collection. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 U/mL of penicillin, and 100 g/mL of streptomycin at 37°C in a humidified incubator with 5% CO2. Determination of TNF-␣ Production The level of TNF-␣ in the culture supernatants was determined by using a commercially available TNF-␣ enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Measurement of TLR2 Activation A genetically engineered Chinese Hamster Ovary (CHO) cell line, CHO/CD14/TLR2, which coexpresses human TLR2 and CD14, was kindly provided by Dr Douglas Golenbock (Boston Medical Center, Boston, MA) and used to examine the ability of LTA to stimulate TLR2 (21). The cell line has a reporter gene encoding CD25 on the cell JOE — Volume 35, Number 2, February 2009
Figure 1. TNF-␣ production by a murine macrophage cell line in response to heat-killed E. faecalis or CHX-killed E. faecalis. RAW 264.7 cells (1 ⫻ 106 cells/mL) were stimulated with heat-killed or CHX-killed E. faecalis at 104 to 108 CFU/mL for (A) 1 hour or (B) 24 hours. At the end of the incubation period, the culture media were collected for the analysis of TNF-␣ production using enzyme-linked immunosorbent assay. Error bars indicate SD. An asterisk represents significance at p ⬍ 0.05 compared with the nontreatment control.
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Basic Research—Biology (1, 6, 12, 24, or 48 hours). LTA treated with 2% CHX for 1 hour significantly reduced TNF-␣ production by RAW 264.7 cells (p ⬍ 0.05), but the activity of LTA was not completely inactivated. LTA treated for 6 hours showed similar TNF-␣ level with the nontreatment group (Fig. 2A). This notion was further confirmed by using LTA treated with different concentrations of CHX at 0.02%, 0.2%, or 2%. Pretreatment of LTA with 0.2% CHX for 24 hours attenuated the ability to stimulate TNF-␣ production. However, LTA treated with 0.02% CHX had no significant difference with the positive control (Fig. 2B).
CHX-treated LTA Is Not Able to Stimulate TLR2 TLR2 is known to mediate E. faecalis LTA-induced TNF-␣ expression (9). To further address the mechanism for CHX inactivation of E. faecalis LTA, the LTA was pretreated with CHX before use for the stimulation of CHO/CD14/TLR2 cells, which express CD25 reporter protein proportionally to TLR2 stimulation and NF-B activation. Figure 3 showed that CD25 expression on the CHO/CD14/TLR2 cells increased
Figure 3. The inability to stimulate TLR2 by CHX-treated LTA. CHO/CD14/TLR2 cells were stimulated with CHX-treated or nontreated LTA (0, 3, 10, or 30 g/mL) for 16 hours. After incubation, TLR2-dependent NF-B activation was measured using flow cytometric analysis of CD25 expression on the cell surface as described in the Materials and Methods section. The gray-filled area and thick line in each histogram indicate the isotype control and each experimental group, respectively. A percentage of CD25-positive cells is shown in the upper right in each histogram.
proportionally with treated LTA concentration but not with the CHXtreated LTA. These results indicate that CHX abrogates the ability of LTA to stimulate TLR2, resulting in the attenuated induction of TNF-␣ expression.
Discussion
Figure 2. TNF-␣ production by a murine macrophage cell line in response to LTA or CHX-treated LTA. E. faecalis LTA was treated with (A) 2% CHX for 1, 6, 12, 24, or 48 hours at 37°C or with (B) CHX at 0.02%, 0.2%, or 2% for 24 hours at 37°C. Then, the LTA was dialyzed three times in nonpyrogenic distilled water for 48 hours with a semipermeable membrane to separate unreacted CHX. RAW 264.7 cells (1 ⫻ 106 cells/mL) were stimulated with CHX-treated LTA at 30 g/mL for 18 hours. After the incubation, culture supernatants were obtained and measured for TNF-␣ level using an ELISA assay. Error bars indicate SD. An asterisk represents significance at p ⬍ 0.05 compared with the LTA-treated positive control.
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Together with calcium hydroxide, CHX is widely used as an intracanal disinfectant during root canal treatment. Recently, we have shown that calcium hydroxide inactivates LTA from E. faecalis (22). However, those two compounds seem to use different mechanisms for their antimicrobial activities. Unlike calcium hydroxide, which dissociates lipids from the virulence factors such as LPS (23), CHX binds and neutralizes LPS (24). Moreover, CHX has been reported to have a higher antimicrobial activity against gram-positive bacteria than gram-negative bacteria (25). It could be because of the different cell wall structure between two bacteria because gram-negative bacteria, which have a more complex cell wall, are less permeable and susceptible to CHX (26). In the present study, we found that CHX treatment is able to inactivate the LTA of E. faecalis. These results will help us understand the action mechanisms of CHX and provide a theoretic basis of its clinical application. The level of TNF-␣ production by heat-treated E. faecalis was increased in proportion on the road to the number of cells. This implies that the remnants of dead E. faecalis can still sustain an inflammatory potential. However, CHX-treated E. faecalis showed significantly lower TNF-␣ secretion than heat-treated E. faecalis (p ⬍ 0.05). This inhibitory effect of CHX increased with the treatment time. Furthermore, treatment of the purified LTA with CHX abrogated the capacity of LTA to stimulate TNF-␣ expression. These results suggest that CHX is able to detoxify virulence factors of E. faecalis, and one potential target might be LTA. However, we could not exclude the possibility that CHX inactivates not only LTA but also other virulence factors such as peptidoglycan and/or lipoproteins. TLR2 appears to be the primary mediator of the innate immune response to LTAs from various gram-positive bacteria (27). It was reported that LTA from E. faecalis preferentially stimulates TLR2 rather than TLR4 (10). Thus, we would suggest a series of sequential responses in which CHX may alter LTA structure, thereby disabling LTA from stimulating TLR2, and therefore NF-B that is essential for the transcriptional activation of TNF-␣ is not activated. The action mechaJOE — Volume 35, Number 2, February 2009
Basic Research—Biology nism of CHX was previously proposed that the initial interaction of CHX with organic molecules is generally thought to occur via electrostatic attraction, whereby the positively charged CHX interacts with negatively charged hydrophilic groups of bacterial cell wall membrane and then penetrates into the cell and interacts with the cytoplasmic constituents (26). More recently, direct binding of CHX to LTA as well as LPS was proven by fluorescen displacement assay and isothermal titration (24). Interestingly, CHX bindings to LTA and LPS are different in that electrostatic interaction seems to be important in CHX-LTA binding while both hydrophobic and electrostatic interactions in CHX-LPS binding (24). In the present study, we found that, for complete neutralization of the inflammatory potential of LTA, CHX of more than 0.2% concentration was needed at 24 hours of treatment and treatment time longer than 6 hours was required when 2% CHX was used. Abdullah et al. (28) reported that 100% bacterial killing was achieved after exposure to 0.2% CHX for 15 minutes when E. faecalis was grown in planktonic state. However, in the biofilm state, 0.2% CHX could not achieve 100% killing after 60 minutes of treatment. Thus, CHX does not exhibit its full antibacterial action when applied for a short exposure time in root canal during irrigation (25). As a result, a large number of bacteria may persist within the dentinal tubules and remain viable, and, thus, the inhibitory effect of CHX on LTA is limited. Therefore, prolonged application of CHX as an intracanal medicament is needed between appointments rather than being used only as an irrigant. Several researchers recommended CHX medication for at least 7 days (15–17, 29, 30). In conclusion, CHX is able to inactivate LTA of E. faecalis leading to alleviation of inflammatory responses induced by E. faecalis and its LTA.
References 1. Jett BD, Huycke MM, Gilmore MS. Virulence of enterococci. Clin Microbiol Rev 1994;7:462–78. 2. Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbial Immunol 1992;257– 62. 3. Gomes BP, Pinheiro ET, Jacinto RC, Zaia AA, Ferraz CC, Souza-Filho FJ. Microbial analysis of canals of root-filled teeth with periapical lesions using polymerase chain reaction. J Endod 2008;34:537– 40. 4. Rôças IN, Hülsmann M, Siqueira JF Jr. Microorganisms in root canal-treated teeth from a German population. J Endod 2008;34:926 –31. 5. Flahaut S, Hartke A, Giard JC, Auffray Y. Alkaline stress response in Enterococcus faecalis: adaptation, cross-protection, and changes in protein synthesis. Appl Environ Microbiol 1997;63:812– 4. 6. Love RM. Enterococcus faecalis: a mechanism for its role in endodontic failure. Int Endod J 2001;34:399 – 405. 7. Ørstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endod Dent Traumatol 1990;6:142–9. 8. Chavez de Paz LE. Redefining the persistent infection in root canals: possible role of biofilm communities. J Endod 2007;33:652– 62. 9. Duggan JM, Sedgley CM. Biofilm formation of oral and endodontic Enterococcus faecalis. J Endod 2007;33:815– 8. 10. Baik JE, Ryu YH, Han JY, et al. Lipoteichoic acid partially contributes to the inflammatory responses to Enterococcus faecalis. J Endod 2008;34:975– 82.
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11. Han SH, Kim JH, Martin M, Michalek SM, Nahm MH. Pneumococcal lipoteichoic acid (LTA) is not as potent as staphylococcal LTA in stimulating Toll-like receptor 2. Infect Immun 2003;71:5541– 8. 12. Ciardi JE, Rolla G, Bowen WH, Reilly JA. Adsorption of Streptococcus mutans lipoteichoic acid to hydroxyapatite. Scand J Dent Res 1977;85:387–91. 13. Fardal O, Turnbull RS. A review of the literature on use of chlorhexidine in dentistry. J Am Dent Assoc 1986;112:863–9. 14. Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of chlorhexidine-treated bovine root dentin. J Endod 2000;26:315–7. 15. Siqueira JF Jr, Pavia SSM, Rôças IN. Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol. J Endod 2007;33:541–7. 16. Basrani B, Santos JM, Tjaderhane L, et al. Substantive antimicrobial activity in chlorhexidine-treated human root dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:240 –5. 17. Gomes BP, Souza SF, Ferraz CC, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267–75. 18. Krithikadatta J, Indira R, Dorothykalyani AL. Disinfection of dentinal tubules with 2% chlorhexidine, 2% metronidazole, bioactive glass when compared with calcium hydroxide as intracanal medicaments. J Endod 2007;33:1473– 6. 19. Portenier I, Haapasalo H, Ørstavik D, Yamauchi M, Haapasalo M. Inactivation of the antibacterial activity of iodine potassium iodide and chlorhexidine digluconate against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heatkilled microbial whole cells. J Endod 2002;28:634 –7. 20. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93– 8. 21. Medvedev AE, Henneke P, Schromm A, et al. Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4. J Immunol 2001;167: 2257– 67. 22. Baik JE, Kum KY, Yun CH, et al. Calcium hydroxide inactivates lipoteichoic acid from Enterococcus faecalis. J Endod 2008;11:1355–9. 23. Barthel CR, Levin LG, Reisner HM, Trope M. TNF-alpha release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS. Int Endod J 1997;30:155–9. 24. Zorko M, Jerala R. Alexidine and chlorhexidine bind to lipopolysaccharide and lipoteichoic acid and prevent cell activation by antibiotics. J Antimicrob Chemother 2008;62:730 –7. 25. Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007; 52:S64 – 82. 26. Sheldon AT Jr. Antiseptic ”resistance”: real or perceived threat? Clin Infect Dis 2005;40:1650 – 6. 27. Schroder NW, Morath S, Alexander C, et al. Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved. J Biol Chem 2003;278:15587–94. 28. Abdullah M, Ng YL, Gulabivala K, Moles DR, Spratt DA. Susceptibilties of two Enterococcus faecalis phenotypes to root canal medications. J Endod 2005;31:30 – 6. 29. Lin S, Zuckerman O, Weiss EI, Mazor Y, Fuss Z. Antibacterial efficacy of a new chlorhexidine slow release device to disinfect dentinal tubules. J Endod 2003;29:416 – 8. 30. Lee Y, Han SH, Hong SH, Lee JK, Ji H, Kum KY. Antimicrobial efficacy of a polymeric chlorhexidine release device using in vitro model of Enterococcus faecalis dentinal tubule infection. J Endod 2008;34:855– 8.
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Chlorhexidine Gluconate Attenuates the Ability of Lipoteichoic Acid from Enterococcus faecalis to Stimulate Toll-like Receptor 2 Jin-Kyung Lee, DDS, MSD,* ,** Jung Eun Baik, BS†,** Cheol-Heui Yun, PhD,‡,§ Kangseok Lee, PhD,储 Seung Hyun Han, PhD,†,¶ WooCheol Lee, DDS, PhD,* Kwang-Shik Bae, DDS, PhD,* Seung-Ho Baek, DDS, PhD,* Yoon Lee, DDS, MSD,# Won-Jun Son, DDS, PhD,* and Kee-Yeon Kum, DDS, PhD* Abstract Chlorhexidine gluconate (CHX) has been widely used as a canal irrigant or an intracanal medicament on account of its antibacterial substantivity. This in vitro study aimed to determine if CHX attenuates the inflammatory activity of Enterococcus faecalis and its major virulence factor, lipoteichoic acid (LTA). An enzymelinked immunosorbent assay showed that CHX-killed E. faecalis was less potent than heat-killed E. faecalis in the production of tumor necrosis factor ␣ (TNF-␣) by a murine macrophage cell line, RAW 264.7 (p ⬍ 0.05). Interestingly, pretreatment of LTA with 2% CHX for 6 hours or with 0.2% CHX for 24 hours almost eliminated the TNF-␣ inducibility (p ⬍ 0.05). Furthermore, CHX abrogated the ability of LTA to stimulate Toll-like receptor 2, resulting in the attenuated induction of TNF-␣ expression. Collectively, our results suggest that CHX can inactivate LTA of E. faecalis leading to the alleviation of inflammatory responses induced by E. faecalis and its LTA. (J Endod 2009;35:212–215)
Key Words Chlorhexidine gluconate, Enterococcus faecalis, intracanal medicament, lipoteichoic acid, Toll-like receptor 2, tumor necrosis factor-␣
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nterococci are human commensals adapted to complex environments of the oral cavity, gastrointestinal tract, and genital tract (1). Although they occupy only a small proportion of the flora in untreated canals (2), Enterococcus faecalis has been frequently recovered from root-filled teeth with refractory apical periodontitis (3, 4). E. faecalis can survive in harsh environmental conditions such as high alkalinity (5) and can invade the dentinal tubules in variable depths (6, 7) followed by colonization of the root canal (8, 9). These abilities of E. faecalis may explain its survival after a root canal treatment and its ability to induce persistent apical periodontitis. Lipoteichoic acid (LTA) is a cell wall component of gram-positive bacteria and appears to be one of the major etiologic factors at various stages of inflammation. LTA isolated from E. faecalis as well as other gram-positive bacteria have been reported to stimulate leukocytes to release the key inflammatory mediators (10, 11). LTA was also shown to exhibit high affinity to hydroxyapatite (12), a feature related to the penetration of dentinal tubules. Chlorhexidine gluconate (CHX) is a cationic bisbiguanide that adsorbs onto the cell wall of microorganisms causing leakage of the intracellular components. CHX has been used as an important constituent of irrigating solutions and intracanal medicaments during root canal therapy on account of its substantive antimicrobial activity (13–15). In various in vivo and in vitro studies, CHX has proven to be effective in reducing or eliminating E. faecalis from the root canal space and dentinal tubules (15–19). Therefore, its use in root canal treatment is believed to be advantageous, particularly in retreatment cases in which high proportions of E. faecalis are frequently found in the root canal system (20). Although the capacity of CHX to kill bacteria has been extensively studied, there are few reports on the mechanisms for its effectiveness against E. faecalis, especially LTA. Therefore, this in vitro study examined the effects of CHX on LTA to determine if CHX can inactivate LTA from E. faecalis and prevent the inflammatory responses.
From the *Department of Conservative Dentistry and Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, Seoul, Republic of Korea; †Department of Oral Microbiology & Immunology, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, Seoul, Republic of Korea; ‡School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea; §Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Korea; 储Department of Life Science, Chung-Ang University, Seoul, Republic of Korea; ¶International Vaccine Institute, Seoul, Republic of Korea; and #Department of Conservative Dentistry, Wonkwang University Dental Hospital, DaeJeon, Republic of Korea. **Jin-Kyung Lee and Jung Eun Baik contributed equally to this article. Supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2008-314-E00223) and by a grant (03-2007-0090) from the Seoul National University Dental Hospital Research Fund, Republic of Korea. Address requests for reprints to Dr Kee-Yeon Kum, Department of Conservative Dentistry and Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, 25-9 Yongun-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2008 American Association of Endodontists. doi:10.1016/j.joen.2008.10.018
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Basic Research—Biology Materials and Methods Bacteria, Reagents, and Chemicals E. faecalis ATCC 29212 was purchased from the American Type Culture Collection (Manassas, VA). Brain heart infusion was obtained from BD Biosciences (Franklin, NJ) and phosphate-buffered saline was obtained from Mediatech Inc (Herndon, VA). Octyl-Sepharose CL-4B, DEAE-Sepharose, and CHX (20% aqueous solution) were purchased from Sigma-Aldrich (St Louis, MO). Unless indicated otherwise, all other reagents were obtained from Sigma-Aldrich. Preparation of Heat-killed Bacteria and CHX-killed Bacteria E. faecalis was grown in brain heart infusion media at 37°C to the midlog phase in which optical density at 600 nm reached 0.6. The cells were harvested by centrifugation, washed 3 times with phosphate-buffered saline, and divided into 4 groups. Two groups of E. faecalis were killed by heating at 60°C for 1 hour or 24 hours as previously described (10). The other two groups were killed by 2% CHX for 1 hour or 24 hours. To ensure that the bacteria had been completely killed, the bacteria subjected to heat or CHX treatment were plated on the corresponding media containing 1.5% agar and cultured overnight at 37°C. No bacterial colony was observed. Preparation of LTA Highly pure LTA of E. faecalis was prepared as previously described (10). Briefly, approximately 100 g (wet weight) of bacterial cells were resuspended in 1,000 mL of 0.1 mol/L sodium citrate buffer (pH 4.7) and disrupted by ultrasonication. After n-butanol extraction, hydrophobic-interaction chromatography was performed by using octyl-Sepharose column CL-4B (2.5 cm ⫻ 10 cm). The LTA-containing fractions were further separated by DEAE-Sepharose ion-exchange chromatography (1.5 cm ⫻ 10 cm). The quantity of purified LTA was determined by measuring the dry weight of the LTA.
membrane whose expression is regulated by NF-B transcription factor. To determine if CHX inhibits the activation of TLR2 by E. faecalis LTA, CHO/CD14/TLR2 cells were stimulated with CHX-treated or -untreated LTA (0, 3, 10, or 30 g/mL) for 16 hours. The level of TLR2dependent nuclear factor-kappa B (NF-B) activation was determined by analysis of CD25 expression by using flow cytometry (FACS Calibur; BD Biosciences, Franklin, NJ).
Statistical Analysis All experiments were performed in triplicate and the mean value ⫾ standard deviation (SD) was calculated for each treatment group. The treatment groups were compared with the nontreated control group, and statistical significance was determined by a Student 2-tailed t test; p values of ⬍0.05 were considered significant.
Results CHX-treated E. Faecalis Induces Less TNF-␣ Secretion Compared with Heat-treated E. Faecalis When RAW 264.7 cells were stimulated with the heat-treated bacteria, the level of TNF-␣ production increased significantly (p ⬍ 0.05) in proportion to the number of cells (Fig. 1). The CHX-treated E. faecalis showed a similar pattern but induced significantly (p ⬍ 0.05) lower levels of TNF-␣ secretion than the heat-treated E. faecalis. This difference was more remarkable at 24 hours of treatment than at 1 hour of treatment. CHX Efficiently Inactivates LTA from E. Faecalis To examine if CHX alters the TNF-␣ inducibility of E. faecalis LTA, LTA at 30 g/mL was treated with 2% CHX for different time durations
Preparation of E. Faecalis LTA Treated with CHX The 300 g of LTA from E. faecalis were treated with 2% CHX for 24 hours followed by dialysis in a semipermeable dialysis membrane (Spectra/Por 6; Spectrum Laboratories, Inc, Ranch Dominquez, CA). Then, the LTA was subjected to dialysis against endotoxin-free distilled water (Dai Han Pharm Co Ltd, Seoul, Republic of Korea) for 48 hours to remove unreacted CHX. Culture of RAW 264.7 The mouse macrophage cell line, RAW 264.7 (TIB-71), was purchased from the American Type Culture Collection. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 U/mL of penicillin, and 100 g/mL of streptomycin at 37°C in a humidified incubator with 5% CO2. Determination of TNF-␣ Production The level of TNF-␣ in the culture supernatants was determined by using a commercially available TNF-␣ enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Measurement of TLR2 Activation A genetically engineered Chinese Hamster Ovary (CHO) cell line, CHO/CD14/TLR2, which coexpresses human TLR2 and CD14, was kindly provided by Dr Douglas Golenbock (Boston Medical Center, Boston, MA) and used to examine the ability of LTA to stimulate TLR2 (21). The cell line has a reporter gene encoding CD25 on the cell JOE — Volume 35, Number 2, February 2009
Figure 1. TNF-␣ production by a murine macrophage cell line in response to heat-killed E. faecalis or CHX-killed E. faecalis. RAW 264.7 cells (1 ⫻ 106 cells/mL) were stimulated with heat-killed or CHX-killed E. faecalis at 104 to 108 CFU/mL for (A) 1 hour or (B) 24 hours. At the end of the incubation period, the culture media were collected for the analysis of TNF-␣ production using enzyme-linked immunosorbent assay. Error bars indicate SD. An asterisk represents significance at p ⬍ 0.05 compared with the nontreatment control.
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Basic Research—Biology (1, 6, 12, 24, or 48 hours). LTA treated with 2% CHX for 1 hour significantly reduced TNF-␣ production by RAW 264.7 cells (p ⬍ 0.05), but the activity of LTA was not completely inactivated. LTA treated for 6 hours showed similar TNF-␣ level with the nontreatment group (Fig. 2A). This notion was further confirmed by using LTA treated with different concentrations of CHX at 0.02%, 0.2%, or 2%. Pretreatment of LTA with 0.2% CHX for 24 hours attenuated the ability to stimulate TNF-␣ production. However, LTA treated with 0.02% CHX had no significant difference with the positive control (Fig. 2B).
CHX-treated LTA Is Not Able to Stimulate TLR2 TLR2 is known to mediate E. faecalis LTA-induced TNF-␣ expression (9). To further address the mechanism for CHX inactivation of E. faecalis LTA, the LTA was pretreated with CHX before use for the stimulation of CHO/CD14/TLR2 cells, which express CD25 reporter protein proportionally to TLR2 stimulation and NF-B activation. Figure 3 showed that CD25 expression on the CHO/CD14/TLR2 cells increased
Figure 3. The inability to stimulate TLR2 by CHX-treated LTA. CHO/CD14/TLR2 cells were stimulated with CHX-treated or nontreated LTA (0, 3, 10, or 30 g/mL) for 16 hours. After incubation, TLR2-dependent NF-B activation was measured using flow cytometric analysis of CD25 expression on the cell surface as described in the Materials and Methods section. The gray-filled area and thick line in each histogram indicate the isotype control and each experimental group, respectively. A percentage of CD25-positive cells is shown in the upper right in each histogram.
proportionally with treated LTA concentration but not with the CHXtreated LTA. These results indicate that CHX abrogates the ability of LTA to stimulate TLR2, resulting in the attenuated induction of TNF-␣ expression.
Discussion
Figure 2. TNF-␣ production by a murine macrophage cell line in response to LTA or CHX-treated LTA. E. faecalis LTA was treated with (A) 2% CHX for 1, 6, 12, 24, or 48 hours at 37°C or with (B) CHX at 0.02%, 0.2%, or 2% for 24 hours at 37°C. Then, the LTA was dialyzed three times in nonpyrogenic distilled water for 48 hours with a semipermeable membrane to separate unreacted CHX. RAW 264.7 cells (1 ⫻ 106 cells/mL) were stimulated with CHX-treated LTA at 30 g/mL for 18 hours. After the incubation, culture supernatants were obtained and measured for TNF-␣ level using an ELISA assay. Error bars indicate SD. An asterisk represents significance at p ⬍ 0.05 compared with the LTA-treated positive control.
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Together with calcium hydroxide, CHX is widely used as an intracanal disinfectant during root canal treatment. Recently, we have shown that calcium hydroxide inactivates LTA from E. faecalis (22). However, those two compounds seem to use different mechanisms for their antimicrobial activities. Unlike calcium hydroxide, which dissociates lipids from the virulence factors such as LPS (23), CHX binds and neutralizes LPS (24). Moreover, CHX has been reported to have a higher antimicrobial activity against gram-positive bacteria than gram-negative bacteria (25). It could be because of the different cell wall structure between two bacteria because gram-negative bacteria, which have a more complex cell wall, are less permeable and susceptible to CHX (26). In the present study, we found that CHX treatment is able to inactivate the LTA of E. faecalis. These results will help us understand the action mechanisms of CHX and provide a theoretic basis of its clinical application. The level of TNF-␣ production by heat-treated E. faecalis was increased in proportion on the road to the number of cells. This implies that the remnants of dead E. faecalis can still sustain an inflammatory potential. However, CHX-treated E. faecalis showed significantly lower TNF-␣ secretion than heat-treated E. faecalis (p ⬍ 0.05). This inhibitory effect of CHX increased with the treatment time. Furthermore, treatment of the purified LTA with CHX abrogated the capacity of LTA to stimulate TNF-␣ expression. These results suggest that CHX is able to detoxify virulence factors of E. faecalis, and one potential target might be LTA. However, we could not exclude the possibility that CHX inactivates not only LTA but also other virulence factors such as peptidoglycan and/or lipoproteins. TLR2 appears to be the primary mediator of the innate immune response to LTAs from various gram-positive bacteria (27). It was reported that LTA from E. faecalis preferentially stimulates TLR2 rather than TLR4 (10). Thus, we would suggest a series of sequential responses in which CHX may alter LTA structure, thereby disabling LTA from stimulating TLR2, and therefore NF-B that is essential for the transcriptional activation of TNF-␣ is not activated. The action mechaJOE — Volume 35, Number 2, February 2009
Basic Research—Biology nism of CHX was previously proposed that the initial interaction of CHX with organic molecules is generally thought to occur via electrostatic attraction, whereby the positively charged CHX interacts with negatively charged hydrophilic groups of bacterial cell wall membrane and then penetrates into the cell and interacts with the cytoplasmic constituents (26). More recently, direct binding of CHX to LTA as well as LPS was proven by fluorescen displacement assay and isothermal titration (24). Interestingly, CHX bindings to LTA and LPS are different in that electrostatic interaction seems to be important in CHX-LTA binding while both hydrophobic and electrostatic interactions in CHX-LPS binding (24). In the present study, we found that, for complete neutralization of the inflammatory potential of LTA, CHX of more than 0.2% concentration was needed at 24 hours of treatment and treatment time longer than 6 hours was required when 2% CHX was used. Abdullah et al. (28) reported that 100% bacterial killing was achieved after exposure to 0.2% CHX for 15 minutes when E. faecalis was grown in planktonic state. However, in the biofilm state, 0.2% CHX could not achieve 100% killing after 60 minutes of treatment. Thus, CHX does not exhibit its full antibacterial action when applied for a short exposure time in root canal during irrigation (25). As a result, a large number of bacteria may persist within the dentinal tubules and remain viable, and, thus, the inhibitory effect of CHX on LTA is limited. Therefore, prolonged application of CHX as an intracanal medicament is needed between appointments rather than being used only as an irrigant. Several researchers recommended CHX medication for at least 7 days (15–17, 29, 30). In conclusion, CHX is able to inactivate LTA of E. faecalis leading to alleviation of inflammatory responses induced by E. faecalis and its LTA.
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11. Han SH, Kim JH, Martin M, Michalek SM, Nahm MH. Pneumococcal lipoteichoic acid (LTA) is not as potent as staphylococcal LTA in stimulating Toll-like receptor 2. Infect Immun 2003;71:5541– 8. 12. Ciardi JE, Rolla G, Bowen WH, Reilly JA. Adsorption of Streptococcus mutans lipoteichoic acid to hydroxyapatite. Scand J Dent Res 1977;85:387–91. 13. Fardal O, Turnbull RS. A review of the literature on use of chlorhexidine in dentistry. J Am Dent Assoc 1986;112:863–9. 14. Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of chlorhexidine-treated bovine root dentin. J Endod 2000;26:315–7. 15. Siqueira JF Jr, Pavia SSM, Rôças IN. Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol. J Endod 2007;33:541–7. 16. Basrani B, Santos JM, Tjaderhane L, et al. Substantive antimicrobial activity in chlorhexidine-treated human root dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:240 –5. 17. Gomes BP, Souza SF, Ferraz CC, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267–75. 18. Krithikadatta J, Indira R, Dorothykalyani AL. Disinfection of dentinal tubules with 2% chlorhexidine, 2% metronidazole, bioactive glass when compared with calcium hydroxide as intracanal medicaments. J Endod 2007;33:1473– 6. 19. Portenier I, Haapasalo H, Ørstavik D, Yamauchi M, Haapasalo M. Inactivation of the antibacterial activity of iodine potassium iodide and chlorhexidine digluconate against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heatkilled microbial whole cells. J Endod 2002;28:634 –7. 20. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93– 8. 21. Medvedev AE, Henneke P, Schromm A, et al. Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4. J Immunol 2001;167: 2257– 67. 22. Baik JE, Kum KY, Yun CH, et al. Calcium hydroxide inactivates lipoteichoic acid from Enterococcus faecalis. J Endod 2008;11:1355–9. 23. Barthel CR, Levin LG, Reisner HM, Trope M. TNF-alpha release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS. Int Endod J 1997;30:155–9. 24. Zorko M, Jerala R. Alexidine and chlorhexidine bind to lipopolysaccharide and lipoteichoic acid and prevent cell activation by antibiotics. J Antimicrob Chemother 2008;62:730 –7. 25. Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007; 52:S64 – 82. 26. Sheldon AT Jr. Antiseptic ”resistance”: real or perceived threat? Clin Infect Dis 2005;40:1650 – 6. 27. Schroder NW, Morath S, Alexander C, et al. Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved. J Biol Chem 2003;278:15587–94. 28. Abdullah M, Ng YL, Gulabivala K, Moles DR, Spratt DA. Susceptibilties of two Enterococcus faecalis phenotypes to root canal medications. J Endod 2005;31:30 – 6. 29. Lin S, Zuckerman O, Weiss EI, Mazor Y, Fuss Z. Antibacterial efficacy of a new chlorhexidine slow release device to disinfect dentinal tubules. J Endod 2003;29:416 – 8. 30. Lee Y, Han SH, Hong SH, Lee JK, Ji H, Kum KY. Antimicrobial efficacy of a polymeric chlorhexidine release device using in vitro model of Enterococcus faecalis dentinal tubule infection. J Endod 2008;34:855– 8.
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