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 patho-
genic gram-positive bacterium closely associated with
apical periodontitis. Although sodium hypochlorite
E nterococci are gram-
(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 inter-
feron 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 inflamma-
tory 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 pro-
tein 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 medi-
ators. (J Endod 2016;42:1503–1508)
Key Words
Apical periodontitis, Enterococcus faecalis, lipotei-
choic acid, sodium hypochlorite, Toll-like receptor 2
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: shhan-mi@snu.ac.kr
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
positive bacteria fre-
quently found in mucosal
Significance
Sodium hypochlorite, which is a common end-
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 addi-
leading to the reduced production of inflammatory
tion to their role as a
mediators in macrophages.
common nosocomial pa-
thogen, 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, adhe-
sins, 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
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 Cul-
ture Collection (ATCC, Manassas, VA). Brain-heart infusion medium
was purchased from BD Biosciences (Franklin, NJ). NaOCl was pur-
chased 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 anti-
human TLR2 antibody was purchased from Biolegend (San Diego, CA).
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 sub-
jected to a PD-10 desalting column (GE Healthcare Life Sciences, Buck-
inghamshire, 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 strepto-
mycin at 37 C in a humidified incubator with 5% CO2 (23).
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 deter-
mined using commercial enzyme-linked immunosorbent assay kits
(R&D Systems, Minneapolis, MN) according to the manufacturer’s
instruction.
Determination of TLR2 Activation
CHO/CD14/TLR2 cells are Chinese hamster ovary cells constitu-
tively 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 acti-
vation, the cells were stained with FITC-conjugated mouse monoclonal
antihuman TLR2, and then the expression was analyzed by flow cytom-
1504 Hong et al.
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 electro-
phoresis 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
When Compared with EfLTA, NaOCl-treated EfLTA Showed
No or Little Immunostimulating Activities
We previously showed that EfLTA induced the production of in-
flammatory mediators such as NO, IP-10, and MIP-1a in murine mac-
rophages (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, suggest-
ing that NaOCl treatment completely abolished EfLTA-induced inflam-
matory responses. In addition, as a positive control, NaOH-treated
EfLTA showed significantly less induction of NO, IP-10, and MIP-1a
compared with EfLTA.
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 inacti-
vate 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).
Compared with EfLTA, NaOCl-treated EfLTA
Fails to Activate TLR2
We previously observed that EfLTA-induced inflammatory res-
ponses were through TLR2 activation (7). To examine whether the
reduction of inflammatory responses was caused by impaired TLR2 acti-
vation, CHO/CD14/TLR2 cells were incubated with NaOCl-treated EfLTA
to examine its activation. EfLTA increased TLR2 activation in a dose-
dependent 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.
JOE — Volume 42, Number 10, October 2016
 Basic Research—Biology
25 15
* *
20
Nitrite (μM)

15 10

Nitrite (μM)
10
5 5 *
0
EfLTA - - - + + +
0
NaOCl - + - - + -
EfLTA (μg/ml) - 10 10 10 10 10 10
NaOH - - + - - +
NaOCl (%) - - 10-1 10-2 10-3 10-4 10-5

30 *
10
*
IP-10 (ng/ml)

20
8

Nitrite (μM)
6
10
4
0
EfLTA - - - + + + 2
NaOCl - + - - + -
0
NaOH - - + - - +
EfLTA (μg/ml) - 10 10 10 10 10
NaOCl treatment (min) - - 0.5 1 2.5 5

300 Figure 2. A low NaOCl concentration effectively reduces EfLTA-induced NO

* production at early time points. (A) EfLTA (10 mg/mL) was pretreated with
MIP-1α (ng/ml)

various concentrations (10 1, 10 2, 10 3, 10 4, or 10 5 %) of NaOCl for

200
100
* was pretreated with 5.25% NaOCl for various time periods (0.5, 1, 2.5, or 5 mi-
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 prein-
cubated 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.
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
1 hour followed by desalting. RAW 264.7 cells (1  106 cells/mL) were pre-
incubated 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)
nutes). 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
We previously observed that EfLTA is responsible for the induction
of inflammatory mediators by E. faecalis (7). Furthermore, calcium hy-
droxide, 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 mac-
rophages.
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

JOE — Volume 42, Number 10, October 2016 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 inflamma-
tory response. Our data also show that NaOCl-treated EfLTA impaired
TLR2 activation and the induction of inflammatory mediators. In addi-
tion, TLC assays showed that NaOCl damages the EfLTA structure, poten-
tially 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,
and 5% NaOCl may reduce flexural strength and the modulus of elastic-
ity (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 inflamma-
tory response (7). Thus, based on the current results, the minimal
concentration of NaOCl, which is far lower than the bactericidal concen-
tration, 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 hy-
droxide affects the immunostimulatory potential of EfLTA (28). Simi-
larly, the current study shows that the complete inactivation of EfLTA

1506 Hong et al. JOE — Volume 42, Number 10, October 2016

Figure 4. NaOCl alters the structural integrity of EfLTA. (A) EfLTA, NaOCl-
treated 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, reten-
tion factor.
is observed by NaOCl treatment at a minimal concentration within less
than 1 minute. NaOCl is a common endodontic irrigant to eradicate mi-
croorganisms 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 (NRF-
2015R1A2A1A15055453 and NRF-2015M2A2A6A01044894) and
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.
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