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Basic Research—Biology Sodium Hypochlorite Inactivates Lipoteichoic Acid of Enterococcus faecalis by Deacylation Sun Woong Hong, MS,* Jung Eun Baik, PhD,* Seok-Seong Kang, PhD,* Kee-Yeon Kum, DDS, PhD,† Cheol-Heui Yun, PhD,‡ and Seung Hyun Han, PhD* Abstract Introduction: Enterococcus faecalis is a pathogenic gram-positive bacterium closely associated with apical periodontitis. Although sodium hypochlorite (NaOCl) has been used as a common endodontic irrigant to eradicate bacteria in the root canal, it has not been elucidated whether NaOCl attenuates the inflammatory response induced by the E. faecalis virulence factor lipoteichoic acid (EfLTA). Methods: Structurally intact EfLTA purified from E. faecalis was treated with NaOCl at various concentrations and time periods. Murine macrophage cell line RAW 264.7 was treated with interferon gamma followed by treatment with intact or NaOCl-treated EfLTA to determine the inducibility of inflammatory mediators such as nitric oxide, interferon gamma–inducible protein 10, and macrophage inflammatory protein-1a. Reporter gene assays assessed by flow cytometry were used to examine the ability of intact or NaOCl-treated EfLTA to activate Toll-like receptor 2 (TLR2), which is known to recognize EfLTA on host cells. Structural damage of EfLTA by NaOCl was examined using silver staining and thin-layer chromatography. Results: NaOCl-treated EfLTA showed markedly less induction of nitric oxide, interferon gamma–inducible protein 10, and macrophage inflammatory protein-1a in RAW 264.7 cells compared with intact EfLTA. In contrast to intact EfLTA that potently stimulated TLR2 activation, NaOCl-treated EfLTA did not activate TLR2. Structural analysis showed that NaOCl damaged EfLTA structure by deacylation. Conclusions: NaOCl deacylates the glycolipid moiety of EfLTA, which fails to activate TLR2, leading to the reduced production of inflammatory mediators. (J Endod 2016;42:1503–1508) Key Words Apical periodontitis, Enterococcus faecalis, lipoteichoic acid, sodium hypochlorite, Toll-like receptor 2 E nterococci are gramSignificance positive bacteria freSodium hypochlorite, which is a common endquently found in mucosal odontic irrigant to eradicate bacteria in the root tissues of the oral cavity, canal, could effectively detoxify a major virulence gastrointestinal tract, and factor of Enterococcus faecalis, lipoteichoic acid, genital tract (1). In addileading to the reduced production of inflammatory tion to their role as a mediators in macrophages. common nosocomial pathogen, the enterococci are a major etiologic agent of persistent apical periodontitis (1, 2). Of the enterococci, Enterococcus faecalis is a prevalent species in periradicular lesions of teeth after endodontic treatment and is also involved in persistent apical periodontitis (3, 4). E. faecalis is commonly resistant to disinfectants and antiseptics because it is known to survive in severe environmental conditions such as high alkalinity (5). Virulence factors such as lipoteichoic acid (LTA), lytic enzymes, cytolysin, adhesins, aggregation substance, and pheromones of E. faecalis are known to be involved in pathogenicity (6). Among these virulence factors, LTA is considered a major etiologic agent based on the induction of the inflammatory response and tissue damage (7, 8). For example, it has been shown that E. faecalis LTA (EfLTA) induced several inflammatory mediators such as nitric oxide (NO), macrophage inflammatory protein-1a (MIP-1a), and monocyte chemotactic protein-1 (7, 9). In addition, EfLTA is closely associated with bacterial adhesion and biofilm formation, which are responsible for resistance to disinfectants, antibiotics, and antimicrobial peptides (10). LTA is an amphiphilic molecule consisting of a glycolipid anchor linked with polyglycerolphosphate or polyribitolphosphate backbones (11). Most gram-positive bacteria including E. faecalis contain polyglycerolphosphate-type LTA, whereas a few gram-positive bacteria such as Streptococcus pneumoniae express polyribitolphosphate-type LTA (12, 13). LTA exclusively activates Toll-like receptor 2 (TLR2), which leads to the production of various proinflammatory chemokines and cytokines (14). It has been well described that the glycolipid moiety of the LTA structure is critical for its immunostimulating potential. Delipidation of LTA completely abolishes the immunostimulating potential (15). On the other hand, LTA with more acyl chains shows higher immunostimulating activity (16). Therefore, the glycolipid moiety of the LTA structure can be a determinant to elicit immune responses including the production of inflammatory mediators. Endodontic irrigants are used to eliminate causative bacteria including E. faecalis in the infected root canal. Sodium hypochlorite (NaOCl) has been widely used as an From the *Department of Oral Microbiology and Immunology, Dental Research Institute, and BK21 Plus Program, School of Dentistry, †Department of Conservative Dentistry and Dental Research Institute, School of Dentistry, and ‡Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea. Address requests for reprints to Dr Seung Hyun Han, Department of Oral Microbiology and Immunology, Dental Research Institute, and BK21 Plus Program, School of Dentistry, Seoul National University, Building 86 (Room 304), 1 Gwanak-ro, Gwanak-gu, Seoul 08826, 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.2016.06.018 JOE — Volume 42, Number 10, October 2016 NaOCl Inactivates LTA of E. faecalis 1503 Basic Research—Biology endodontic irrigant for effective bactericidal and nonspecific proteolytic activity (17) and is strongly alkaline and hypertonic (18). In addition, it is known to dissolve organic tissues containing fatty acids and lipids via a saponification reaction (19). Although the antibacterial effect of NaOCl is well recognized, it is poorly understood if NaOCl detoxifies a major virulence factor of gram-positive bacteria, LTA. Therefore, the aim of the study was to investigate whether NaOCl inactivates EfLTA, leading to the reduction of the inflammatory response. Materials and Methods Bacteria, Reagents, and Chemicals E. faecalis ATCC29212 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Brain-heart infusion medium was purchased from BD Biosciences (Franklin, NJ). NaOCl was purchased from Duksan Pure Chemicals (Ansan, Korea). All other reagents were obtained from Sigma-Aldrich (St Louis, MO) unless otherwise stated. Fluorescein isothiocyanate–conjugated mouse monoclonal antihuman TLR2 antibody was purchased from Biolegend (San Diego, CA). etry using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences). Thin-layer Chromatographic Separation and Silver Staining Thin-layer chromatographic (TLC) separation was performed on a silica gel TLC plate (Silica gel 60; Merck, Whitehouse Station, NJ) (28), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining was performed as described previously (29). Statistical Analysis All experiments were conducted in triplicate, and treatment groups with EfLTA or NaOCl-treated EfLTA were compared with the nontreatment group; statistical significance was determined using the Student t test. An asterisk indicates a significant difference (P < .05) when compared with the nontreatment group. Results EfLTA Preparation and Treatment with NaOCl E. faecalis was cultured in brain-heart infusion medium at 37 C, and the highly pure and structurally intact EfLTA was prepared as described previously (7, 20, 21). Unwanted biological molecules including endotoxin, nucleic acids, and proteins in the preparation were not detectable (data not shown). NaOCl-treated EfLTA was subjected to a PD-10 desalting column (GE Healthcare Life Sciences, Buckinghamshire, UK) to remove NaOCl and debris that may have formed by structural damage. After lyophilization, the concentration of EfLTA was determined using phosphate assay as previously described (22). Culture of RAW 264.7 Cells The murine macrophage cell line RAW 264.7 obtained from the ATCC was maintained in Dulbecco’s Modified Eagle’s Medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 C in a humidified incubator with 5% CO2 (23). When Compared with EfLTA, NaOCl-treated EfLTA Showed No or Little Immunostimulating Activities We previously showed that EfLTA induced the production of inflammatory mediators such as NO, IP-10, and MIP-1a in murine macrophages (9). To investigate whether the NaOCl treatment abolishes the immunostimulating activity of EfLTA, RAW 264.7 cells were treated with EfLTA or NaOCl-treated EfLTA for 24 hours. EfLTA induced a significant amount of NO, IP-10, and MIP-1a (Fig. 1A–C, respectively). However, NaOCl-treated EfLTA failed to induce the respective mediators, suggesting that NaOCl treatment completely abolished EfLTA-induced inflammatory responses. In addition, as a positive control, NaOH-treated EfLTA showed significantly less induction of NO, IP-10, and MIP-1a compared with EfLTA. Measurement of NO and Proinflammatory Cytokines RAW 264.7 cells (1 106 cells/mL) were treated with EfLTA or NaOCl- or sodium hydroxide (NaOH)-treated EfLTA for 24 hours. Then, the culture supernatants were collected, and the production of NO was measured as described previously (24, 25). The secretion of interferon-g-inducible protein-10 (IP-10) and MIP-1a was determined using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instruction. NaOCl Reduced Ability of EfLTA to Induce NO Production at Low Concentrations and at Early Time Points To identify the optimal concentration and duration of NaOCl needed for inactivation, EfLTA was pretreated with 10 1, 10 2, 10 3, 10 4, or 10 5 % NaOCl for 1 hour. Then, RAW 264.7 cells were incubated with NaOCl-treated EfLTA for 24 hours. As shown in Figure 2A, the production of NO was not induced by EfLTA treated with 10 1 to 10 4 % (v/v) NaOCl. However, it was noted that 10 5 % NaOCl–treated EfLTA slightly increased the production of NO. This concentration of NaOCl appears to be insufficient to completely inactivate EfLTA and thus it might induce the expression of inducible nitric oxide synthase resulting in NO production (30). Furthermore, when EfLTA was treated with 5.25% NaOCl for various time points, a complete reduction of NO was observed as early as 0.5 minutes (Fig. 2B). Determination of TLR2 Activation CHO/CD14/TLR2 cells are Chinese hamster ovary cells constitutively expressing human CD14 and TLR2 that are stably transfected with a nuclear factor kappa B–dependent reporter plasmid to express membrane-bound CD25 in proportion to TLR2 activation (26). For the determination of TLR2 activation, the cells (3 105 cells/mL) were stimulated with EfLTA (0, 1, 3, 10, or 30 mg/mL); NaOCl-treated EfLTA (0, 1, 3, 10, or 30 mg/mL); or Pam2CSK4, a synthetic lipopeptide mimicking gram-positive bacterial lipoproteins that is widely used as a TLR2 ligand (27) (0.1 mg/mL), for 24 hours. To determine TLR2 activation, the cells were stained with FITC-conjugated mouse monoclonal antihuman TLR2, and then the expression was analyzed by flow cytom- Compared with EfLTA, NaOCl-treated EfLTA Fails to Activate TLR2 We previously observed that EfLTA-induced inflammatory responses were through TLR2 activation (7). To examine whether the reduction of inflammatory responses was caused by impaired TLR2 activation, CHO/CD14/TLR2 cells were incubated with NaOCl-treated EfLTA to examine its activation. EfLTA increased TLR2 activation in a dosedependent manner, as previously shown (7). However, NaOCl-treated EfLTA did not increase TLR2 activation (Fig. 3A and B), indicating that NaOCl treatment impaired TLR2 activation and that the reduction of inflammatory responses by NaOCl treatment is attributable to impaired TLR2 activation. 1504 Hong et al. JOE — Volume 42, Number 10, October 2016 Basic Research—Biology 15 * 15 10 5 0 EfLTA NaOCl - + - + - + + + - NaOH - - + - - + * 5 0 - NaOCl (%) - 10 10-1 10 - 10 10-3 10 10-2 10 10-4 10 10-5 10 * 20 8 Nitrite (μM) IP-10 (ng/ml) 10 EfLTA (μg/ml) * 30 10 0 EfLTA NaOCl - + - + - + + + - NaOH - - + - - + 300 MIP-1α (ng/ml) * 20 Nitrite (μM) Nitrite (μM) 25 * 200 100 * 0 EfLTA NaOCl - + - + - + + + - NaOH - - + - - + Figure 1. NaOCl abolishes EfLTA-induced proinflammatory responses. EfLTA was pretreated with 5.25% NaOCl or 0.5 N NaOH at 37 C for 1 hour. After the treatment, salts in the EfLTA solution were removed from the preparation using a PD-10 desalting column. RAW 264.7 cells (1 106 cells/mL) were preincubated with interferon gamma (0.1 ng/mL) for 1 hour followed by stimulation with EfLTA, NaOCl-treated EfLTA, or NaOH-treated EfLTA (10 mg/mL) for 24 hours. Then, the culture supernatants were collected, and the production of (A) NO, (B) IP-10, and (C) MIP-1a were determined. Data shown are mean values standard deviations, and an asterisk indicates the treatment group with statistical significance at P < .05 compared with the nontreatment group. One of 3 independent results is shown. 6 4 2 0 EfLTA (μg/ml) - 10 10 10 10 10 NaOCl treatment (min) - - 0.5 1 2.5 5 Figure 2. A low NaOCl concentration effectively reduces EfLTA-induced NO production at early time points. (A) EfLTA (10 mg/mL) was pretreated with various concentrations (10 1, 10 2, 10 3, 10 4, or 10 5 %) of NaOCl for 1 hour followed by desalting. RAW 264.7 cells (1 106 cells/mL) were preincubated with interferon gamma (0.1 ng/mL) for 1 hour, and then the cells were stimulated with NaOCl-treated EfLTA for 24 hours. (B) EfLTA (10 mg/mL) was pretreated with 5.25% NaOCl for various time periods (0.5, 1, 2.5, or 5 minutes). RAW 264.7 cells (1 106 cells/mL) were preincubated with interferon gamma (0.1 ng/mL) for 1 hour followed by stimulation with NaOCl-treated EfLTA for 24 hours. At the end of the incubation, the culture supernatants were collected, and the production of NO was determined. Data are mean values standard deviations, and an asterisk indicates the treatment group with statistical significance at P < .05 compared with the nontreatment group. One of 3 similar results is shown. glycolipids from EfLTA were clearly detected in the TLC because of the distinct length and saturation degree of the fatty acid chains. In contrast, free fatty acids released from the NaOCl-treated EfLTA were detected, and the patterns were similar to those of calcium hydroxide or NaOH-treated EfLTA, suggesting that EfLTA was deacylated by NaOCl treatment. Discussion Structural Integrity of EfLTA Is Altered by NaOCl To assess the structural integrity of EfLTA treated with NaOCl, we performed silver stain analysis. As shown in Figure 4A, EfLTA showed a staining pattern displaying 2 distinct major bands between 7 and 20 kDa. However, NaOCl-treated EfLTA exhibited a smaller-sized band (<17 kDa), and the staining pattern was weak and fragmented. Furthermore, the <7-kDa band of NaOCl-treated EfLTA was similar to that of NaOH-treated EfLTA, suggesting that the structural integrity of EfLTA was altered upon treatment with NaOCl. To further investigate whether NaOCl deacylates the glycolipid moiety of the EfLTA structure, we performed TLC separation to examine the release of fatty acids from the EfLTA glycolipids treated with NaOCl. Figure 4B shows that various JOE — Volume 42, Number 10, October 2016 We previously observed that EfLTA is responsible for the induction of inflammatory mediators by E. faecalis (7). Furthermore, calcium hydroxide, an endodontic medicament, reduced the immunostimulatory function of EfLTA (28). NaOCl has been widely used as an endodontic irrigant that exhibits potent antimicrobial action against pathogens in the root canal. In the present study, we further showed that treatment of EfLTA with NaOCl results in the impairment of immunostimulating activity by the delipidation of the EfLTA glycolipid moiety structure, potentially leading to a reduction of the inflammatory response in macrophages. Accumulating results have pointed out that LTA from gram-positive bacteria including EfLTA directly binds to TLR2 (31) through its lipid moieties (32). Furthermore, LTA induced the production of NaOCl Inactivates LTA of E. faecalis 1505 Basic Research—Biology Figure 3. NaOCl-treated EfLTA fails to activate TLR2. (A) CHO/CD14/TLR2 cells (3 105 cells/mL) were stimulated with EfLTA or NaOCl-treated EfLTA (0–30 mg/ mL) for 24 hours. After the stimulation, CD25 expression by TLR2-dependent nuclear factor kappa B activation was analyzed using flow cytometric analysis. Pam2CSK4 (0.1 mg/mL) was used as a positive control. (B) The percentage of CD25-positive cells is illustrated by a bar graph. Data are mean values standard deviations, and an asterisk indicates the treatment group with statistical significance at P < .05 compared with the nontreatment group. One of 3 similar results is shown. inflammatory mediators such as tumor necrosis factor a and NO in macrophages (7, 20), whereas deacylated LTA failed to activate TLR2 for the production of tumor necrosis factor a and NO (20). Thus, the lipid moiety seems to be crucial for the induction of the inflammatory response. Our data also show that NaOCl-treated EfLTA impaired TLR2 activation and the induction of inflammatory mediators. In addition, TLC assays showed that NaOCl damages the EfLTA structure, potentially through deacylation. Therefore, NaOCl, which is one of the common endodontic irrigants, could effectively detoxify EfLTA by removing the lipid moieties. The use of NaOCl for root canal treatment is mainly because of its low cost and ability to remove residual microorganisms (33). However, effective NaOCl solution concentrations (2.6%–5.25%) are cytotoxic (34); 1% NaOCl results in reduced mechanical strength of dentine, 1506 Hong et al. and 5% NaOCl may reduce flexural strength and the modulus of elasticity (35, 36). In this study, we found that <0.0001% NaOCl could effectively inhibit the EfLTA-induced inflammatory response. Notably, E. faecalis is known as a major etiologic pathogen causing persistent apical periodontitis (2), and EfLTA could contribute to the inflammatory response (7). Thus, based on the current results, the minimal concentration of NaOCl, which is far lower than the bactericidal concentration, could immediately inactivate EfLTA, probably to reduce the inflammatory response in the infected root canal. It is believed that the complete removal of bacteria or bacterial toxins during endodontic treatment is beneficial for the prevention of persistent apical periodontitis. Previously, we showed that calcium hydroxide affects the immunostimulatory potential of EfLTA (28). Similarly, the current study shows that the complete inactivation of EfLTA JOE — Volume 42, Number 10, October 2016 Basic Research—Biology the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, which is funded by the Ministry of Health and Welfare (HI14C0469), Republic of Korea. The authors deny any conflicts of interest related to this study. References Figure 4. NaOCl alters the structural integrity of EfLTA. (A) EfLTA, NaOCltreated EfLTA, and NaOH-treated EfLTA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by silver staining to examine the structural integrity of EfLTA. Distilled water (DW), NaOCl, or NaOH was used as each control, respectively. SM denotes the size marker. Numbers are the size of molecules in kDa. (B) TLC separation was performed on a silica gel TLC plate, and fatty acids were visualized with 5% ethanolic phosphomolybdic acid. One of 3 similar results is shown. Rf, retention factor. is observed by NaOCl treatment at a minimal concentration within less than 1 minute. NaOCl is a common endodontic irrigant to eradicate microorganisms in the root canal and could be applied to rapidly detoxify EfLTA, a major virulence factor of E. faecalis. Acknowledgments Supported by grants from the National Research Foundation of Korea, which is funded by the Korean government (NRF2015R1A2A1A15055453 and NRF-2015M2A2A6A01044894) and JOE — Volume 42, Number 10, October 2016 1. Wisplinghoff H, Seifert H, Tallent SM, et al. Nosocomial bloodstream infections in pediatric patients in United States hospitals: epidemiology, clinical features and susceptibilities. Pediatr Infect Dis J 2003;22:686–91. 2. Hancock HH 3rd, Sigurdsson A, Trope M, Moiseiwitsch J. 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