Archives of Oral Biology 135 (2022) 105370

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

Virulence of Filifactor alocis lipoteichoic acid on human gingival fibroblast

Hyun-Jun Yoo a, Sung-Hoon Lee b, *
a
Department of Preventive Dentistry, College of Dentistry, Dankook University, Cheonan, Republic of Korea
b
Department of Oral Microbiology and Immunology, College of Dentistry, Dankook University, Cheonan, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Objectives: The purpose of this study was to investigate virulence of lipoteichoic acid extracted from Filifactor
Filifactor alocis alocis (F. alocis) through comparison of previously known bacterial virulence factors.
Lipoteichoic acid Design: F. alocis was cultured in columbia media including L-arginine and L-cysteine, and lipoteichoic acid (LTA)
Virulence
from F. alocis was purified using organic solvent and bead extraction. Human gingival fibroblasts (HGFs) were
Signaling pathway
treated with the extracted LTA and other Gram-positive LTA or lipopolysaccharide of other periodontopathogens.
The induction of cytokine expression was examined by real-time RT-PCR and ELISA, and the stimulated signaling
pathway by the LTA was investigated by immunoblotting and various inhibitors.
Results: LTA induced expression of pro-inflammatory cytokines and Matrix-metalloprotein 2. Also, F. alocis LTA
induced expression pro-inflammatory cytokines similar to Porphyromonas gingivalis lipopolysaccharide. The LTA
activated NF-κB and MAP kinase pathway. Furthermore, the induction of TNF-α, IL-6, IL-8, and MMP-2
expression by F. alocis LTA was reduced by the inhibitor of NF-κB, ERK, JNK, and p38 pathway.
Conclusions: LTA of F. alocis, a bacterium recently detected in periodontal patients, may play an important role in
inducing periodontitis by induction of expression of pro-inflammatory cytokines

1. Introduction toll-like receptor 2 (TLR2) (Ellingsen et al., 2002; Hummell, Swift,

Filifactor alocis (F. alocis) is frequently isolated and classified in pa­
tients with chronic periodontitis according to epidemiological study
using 16 S rRNA analysis (Jalava & Eerola, 1999). Furthermore, this
bacterium is emerging as an important bacterium related with peri­
odontitis because this bacterium is detected in endodontic regions,
peri-implantitis, and aggressive periodontitis (Aruni, Chioma, &
Fletcher, 2014; Montagner, Jacinto, Signoretti, Sanches, & Gomes,
2012; Shaddox et al., 2012). F. alocis is gram-positive anaerobe and
co-exists with other gram-negative anaerobe related with periodontitis.
Lipoteichoic acid (LTA) is a major component of cell wall of gram-
positive bacteria and related with bacterial survival, growth, and path­
ogenicity (Seo, Cartee, Pritchard, & Nahm, 2008). Furthermore, LTA has
characteristics of an amphiphilic polymer consisting of poly­
glycerophosphate and a glycolipid anchor with dihexosyldiacylglycerol
and fatty acids inserted into the cell membrane (Shiraishi, Yokota,
Fukiya, & Yokota, 2016). LTA is a pathogen-associated molecular pat­
terns (PAMPs) of gram-positive bacteria like lipopolysaccharide of
gram-negative bacteria and stimulates innate immune response though
Tomasz, & Winkelstein, 1985). LTA has different chemical structure
depending on bacterial species, and these differences of chemical
structure have different biological activity to host cells (Kim et al., 2008;
Lu et al., 2009; Wang et al., 2015).
Multi-species bacteria exist in periodontal pocket exist and have
various virulence factors, by which periodontitis including inflamma­
tion and bone resorption is induced (Kim & Lee, 2014; Socransky &
Haffajee, 2005). Periodontitis-related bacteria are mostly known as
gram-negative anaerobes, and they have lipopolysaccharide and various
enzyme to disrupt periodontal tissue. Therefore, there have been many
studies on the response of gingival fibroblasts to PAMPs of these bacteria
(Baek & Lee, 2021; Kim & Lee, 2014; Lee & Baek, 2013; Simpson, Olc­
zak, & Genco, 2004). Periodontitis is initiated to protect tissue upon
microbial infection that leads to release PAMPs and to induce expression
of various proinflammatory cytokines and enzymes by binding to
respective receptors in the host cells (Snyderman, 1971; Socransky &
Haffajee, 2005). PAMPs of periodontitis-related bacteria binds different
host receptors and have different virulence for each bacterium (Baek &
Lee, 2021; Kim & Lee, 2014; Takasaki, Fujise, Miura, Hamachi, &

Abbreviations: LTA, lipoteichoic acid; PAMP, pathogen-associated molecular patterns.

* Correspondence to: 119 Dandae-Ro, Dangnam-gu, Cheonan, Republic of Korea.
E-mail address: dennisyi@dankook.ac.kr (S.-H. Lee).

https://doi.org/10.1016/j.archoralbio.2022.105370
Received 14 November 2021; Received in revised form 14 January 2022; Accepted 3 February 2022
Available online 4 February 2022
0003-9969/© 2022 Elsevier Ltd. All rights reserved.
H.-J. Yoo and S.-H. Lee
Maeda, 2013). Therefore, this study compared virulence of F. alocis LTA
and other PAMP of oral bacteria and investigated effect of F. alocis LTA
on induction of inflammation and tissue destruction of human gingival
fibroblast.
2. Materials and methods
2.1. Bacteria species and cultivation
F. alocis ATCC 35896 was purchased from American type culture
collection and used in this study. The medium for cultivation of this
bacteria obligatorily requires L-cysteine and L-arginine. Therefore, on
the basis of these components, the culture medium was formulated as
follows: 35 g/L of Columbia media (BD bioscience, Sparks, MD), 2 g/L of
L-cysteine⋅HCl, 4 g/L of L-arginine⋅HCl, and 5 g/L of yeast extract. After
autoclaving and cooling, the prepared solution was stored to remove
oxygen in an anaerobic chamber for 48 h and added hemin (1 μg/ml)
and vitamin K (0.2 μg/ml) before inoculating the bacteria. F. alocis was
cultivated under 85% N2, 10% CO2, and 5% H2 atmosphere at 37 ℃.
Enterococcus faecalis (E. faecalis) ATCC 29212 was cultured in brain heart
infusion broth (BD bioscience) at 37 ℃ in an anaerobic condition, and
Staphylococcus aureus (S. aureus) ATCC 29213 was cultivated in nutrient
broth (BD bioscience) at 37 ℃ in a shaking incubator under aerobic
condition. Porphyromonas gingivalis (P. gingivalis) ATCC 33277 and
Tannerella forsythia (T. forsythia) ATCC 43037 were brain heart infusion
broth supplemented with vitamin K (0.2 μg/ml) and hemin (10 μg/ml),
and modified NOS medium (Kim & Lee, 2014) at 37 ℃ in an anaerobic
chamber, respectively.
2.2. Lipoteichoic acid and lipopolysaccharide extraction
LTA was extracted from F. alocis, E. faecalis, and S. aureus using the
method reported by Lee and Baek (2012). Briefly, the bacteria were
harvested by centrifugation at 8000g for 10 min at 4 ℃ and washed
three times with 0.1 M sodium citrate buffer (pH 4.7). The bacteria were
resuspended and incubated with 0.1 M sodium citrate buffer including
lysozyme (100 μg/ml) at 37 ℃ for 2 h, with proteinase K (100 μg/ml) at
55 ℃ for 2 h and disrupted by ultra-sonication (Sonics & Materials, Inc.,
Newtown, CT). The suspensions were mixed with an equal volume of
n-butanol by stirring them for 1 h at room temperature. After centrifu­
gation at 13,000 g for 20 min, the lower aqueous phase was collected
and transferred into standard RC dialysis membrane tubing (Spectrum
Laboratories, Inc., Ranch Dominquez, CA). The preparation was solu­
bilized by dialysis in 0.1 M sodium citrate with 15% n-propanol, and
LTA was purified by octyl-sepharose CL-4B Fast flow (GE healthcare Life
Sciences, uppsala, Sweden). The fraction was performed ion-exchange
chromatography with DEAE-Sepharose (GE healthcare Life Sciences,
Waukesha, WI, USA). After the inorganic phosphate assay with ammo­
nium molybdate, the fractions containing LTA were pooled and lyoph­
ilized. Lipopolysaccharide was extracted using the method suggested by
Kim and Lee (2014). Briefly, P. gingivalis and T. forsythia were treated
lysis buffer composed with phenol, DNase, and RNase. After adding
chloroform, the mixture was centrifuged at 12,000g for 10 min, and the
upper phase was transferred into a new tube. The preparation was
treated with endonuclease and proteinase K, and re-treated with lysis
buffer. After centrifugation, the supernatant was incubated with iso­
propyl alcohol for 10 min. The mixture was centrifugated at 12,000 g for
10 min, and the supernatant was removed. The pellet was washed with
70% ethyl alcohol and air-dried. The quantity of purified LTA and
lipopolysaccharide was determined by measuring the dry weight of the
LTA and lipopolysaccharide. LTA and lipopolysaccharide were sus­
pended with endotoxin free water and certified using CHO/CD14/TLR2
and TLR4 cell (Lee & Baek, 2012). The LTA was used in this study after
confirming activation of CHO/CD14/TLR2 cell by its.
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Archives of Oral Biology 135 (2022) 105370
2.3. Cell cultivation and treatment with LTA
Human gingival fibroblasts (HGFs; CRL-2014) were used for the
investigation of LTA virulence and signaling pathway and cultivated in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and antibiotics (100
U/ml penicillin and 100 μg/ml streptomycin sulfate). The cells were
plated on 6- well plate and cultured until 90% confluent and then
changed medium with DMEM including 2% human pooled serum
(Sigma-Aldrich Co., San jose, CA, USA) and antibiotics. In order to
compare bioactivity of LTA, the cells were treated with F. alocis LTA (0.1
μg/ml), S. aureus LTA (0.1 μg/ml), and E. faecalis LTA (0.1 μg/ml). Also,
considering the coexistence of Gram-negative anaerobe in periodontal
pocket, the cells were then treated with F. alocis LTA (0.1 μg/ml),
T. forsythia lipopolysaccharide (0.1 μg/ml) and P. gingivalis lipopoly­
saccharide (0.1 μg/ml) for 12 h. In another experiment, HGFs were
pretreated with a NF-κB inhibitor as BAY 11–7082 (5 μM; Sigma-Aldrich
Co. San Jose, CA, USA), a p38 inhibitor as SB203580 (1 μM; Sigma-
Aldrich Co.), a JNK inhibitor as SP600125 (10 μM; Sigma-Aldrich
Co.), and a ERK inhibitor as PD98059 (5 μM; Sigma-Aldrich Co.) for 1
h before treatment with the LTA and the lipopolysaccharide.
2.4. Real-time RT-PCR
The LTA or lipopolysaccharide treated HGFs were washed twice with
phosphate buffered saline (PBS; pH 7.2). Total RNA from HGF was
isolated with a TRIzol® RNA isolation kit (Invitrogen Life Tech., Carls­
bad, CA, USA) according to the manufacturer’s protocol. cDNA was
synthesized by MaximeTM RT premix (iNtRON, Gyeonggi. Republic of
Korea). cDNA was mixed with TB Green® Premix EX Taq II (Takara Co.,
Kyoto, Japan), each specific primers and ROX II dye and subjected 40
PCR cycles by ABI PRISM 7500 real-time PCR system (Applied Bio­
systems, Foster City, CA) as follows; 95 ℃ for 15 s, 60 ℃ for 10 s and 72
℃ for 33 s. The specific amplification of PCR products was analyzed
using a dissociation curve of the product. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as a reference to levels of
normalize expression and quantify changes of inflammatory cytokine
and tissue destruction factors. Critical threshold cycle (Ct) was defined
as the cycle at which fluorescence became detectable as against the
background and was inversely proportional to the logarithm of the
initial number of the template molecules. The sequences of primers for
real-time RT-PCR were as follows: 5′ -AAC CTG TCC ACT GGG CAC A-3′
and 5′ -TCT GGC TCT GAA ACA AAG GAT-3′ for IL-6 gene; 5′ -GTG AAG
GTG CAG TTT TGC CA-3′ and 5′ -TCT CCA CAA CCC TCT GCA C-3′ for
the IL-8 gene; 5′ -CAG GGA CCT CTC TCT AAT CA-3′ and 5′ -AGC TGG
TTA TCT CTC AGC TC-3′ for the TNF-α gene; 5′ -CCC CAA AAC GGA CAA
AGA GT-3′ and 5′ -TCC TGT ATG TGA TCT GGT TCT T-3′ for the MMP-2;
5′ -GTG GTG GAC CTG ACC TGC-3′ and 5′ TGA GCT TGA CAA AGT GGT
CG-3′ for the GAPDH gene.
2.5. ELISA
The conditioned media of the cells treated with F. alocis LTA, other
Gram-positive bacteria LTA and Gram-negative anaerobe lipopolysac­
charide were collected by centrifugation at 4000 × g for 10 min at 4 ℃ to
remove cell and debris. The supernatants were analyzed for IL-6, IL-8,
TNF-α, and MMP-2 level using ELISA kit (R&D systems, Minneapolis,
MN) according to the manufacturer’s protocol.
2.6. Immunoblotting
Cells were treated with F. alocis LTA, P. gingivalis lipopolysaccharide,
and T. forsythia lipopolysaccharide for 1 h. After washing with cold PBS,
the cells were treated with RIPA buffer (20 mM Tris-HCl (pH 7.4), 1 mM
Na2EDTA, 150 mM NaCl, 1% Triton X-100, 1 mM β-glycerophosphate,
2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM
H.-J. Yoo and S.-H. Lee
phenylmethylsulfonyl fluoride, and 1 μg/ml leupeptin). The lysates were
centrifuged at 5000 g for 10 min at 4 ℃ to remove debris. The super­
natants were immediately transferred into new 1.5 ml tube. After
measuring total protein concentration with BCA assay, the lysates (75
μg) were mixed with 5 × sample buffer and heated at 95 ℃ for 5 min to
denature protein. The proteins were separated by 12% SDS-PAGE and
transferred to polyvinylidene difluoride membrane (Millipore). The
membrane was blocked with 5% bovine serum albumin for 1 hr. The
membranes were rinsed with buffered saline (TBS, pH 7.2), and then
treated with primary antibodies (1:1000 dilution with TBS including 5%
bovine serum albumin and 0.1% tween 20) overnight at 4 ℃. After
washing with TBS containing 1% tween 20 (TBST) three times each for
10 min, the membranes were incubated with a horseradish peroxidase
(HRP)-conjugated anti-rabbit IgG Antibody (R&D Systems, Minneapolis,
MN, USA) (1:1000 dilution with TBS) for 2 hr at room temperature.
After washing with TBST three times each for 10 min, the membrane was
incubated with a solution containing the chemiluminescent substrate
(DyneBio, Gyeonggi, Korea) and detected with a ChemiDoc (Micro­
Chemi, DNR Bio-Imaging systems, Neve Yamin, Israel). Band levels were
analyzed using Multi Gauge V3.0 software (Fuji Photo Film Co., Tokyo,
Japan). The primary antibodies were used rabbit anti-IκB-α mAb
(#9242, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-
phospho-SAPK/JNK mAb (#9251, Cell Signaling Technology), rabbit
anti-SAPK/JNK mAb (#9252, Cell Signaling Technology), rabbit anti-
phospho-ERK mAb (#9101, Cell Signaling Technology), rabbit anti-
ERK mAb (#9102, Cell Signaling Technology), rabbit anti-phospho-
p38 MAPK mAb (#9211, Cell Signaling Technology), rabbit anti-p38
MAPK mAb (#9212, Cell Signaling Technology), and rabbit anti-
β-actin mAb (#4970, Cell Signaling Technology).
2.7. Statistical analysis
IBM SPSS statistics ver. 23 (IBM, Armonk, NY, USA) was used for
Fig. 1. Induction of cytokine expression by LTA of various gram-positive bacteria. HGFs were treated with LTA for 12 h. The induction of TNF-α (A and D), IL-6 (B
and E), IL-8 (C and F), and MMP-2 (D and H) expression by LTA was examined. The experiments were performed three times in triplicate, and data are represented as
the median (horizontal lines), interquartile range (boxes), and full ranges (whiskers). Asterisk (*) indicates statistically significant differences compared with the
control group (P < 0.05). The dotted line indicates the control level.
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Archives of Oral Biology 135 (2022) 105370
statistical analysis. Data distribution was checked using the
Kolmogorov-Smirnov test, and intergroup data was analyzed by the non-
parametric Kruskal-Wallis test. Differences with P value of less than 0.05
were considered statistically significant. Post-hoc analysis to compare
differences between individual groups was analyzed by the Mann-
Whitney U test, which was corrected for multiple comparisons by the
Bonferroni methods. The values are expressed as the median and
interquartile range.
3. Results
3.1. Comparison of bioactivity among various lipoteichoic acid
To compare the bioactivity of F. alocis LTA and other gram-positive
bacterial LTA, the ability to induce expression of pro-inflammatory cy­
tokines and MMP-2 was compared with LTA of other gram-positive
bacteria. F. alocis LTA induced pro-inflammatory cytokines weaker
than S. aureus LTA but stronger than E. faecalis LTA (Fig. 1A and B).
3.2. Induction of inflammatory factors by F. alocis LTA
Next, when the ability to induce the expression of pro-inflammatory
cytokines and MMP-2 by PAMP of periodontitis-related bacteria was
compared, F. alocis LTA induced expression of the cytokines weaker than
T. forsythia lipopolysaccharide but slightly stronger than P. gingivalis
lipopolysaccharide (Fig. 2). Also, MMP-2 expression was induced by
F. alocis LTA, and showed a similar trend to the induction of cytokines
expression by lipopolysaccharide of other periodontal pathogens
(Fig. 2D and H).
3.3. Activation of NF-κB and MAPK signaling pathway by F. alocis LTA
Next, Immunoblotting was performed to examine whether F. alocis
H.-J. Yoo and S.-H. Lee
Fig. 2. Induction of cytokine expression by PAMP of various periodontopathogens. HGFs were treated with F. alocis LTA or lipopolysaccharide of P. gingivalis and
T. forsythia for 12 h. The induction of TNF-α (A and E), IL-6 (B and F), IL-8 (C and G), and MMP-2 (D and H) expression by PAMP was investigated. Data are expressed
as the median (horizontal lines), interquartile range (boxes), and full ranges (whiskers). Asterisk (*) indicates statistically significant differences compared with the
control group (P < 0.05). The dotted line indicates the control level.
LTA activates which signaling pathway. As shown Fig. 3, P. gingivalis
lipopolysaccharide and T. forsythia lipopolysaccharide, as the positive
controls, activated NF-κB and MAPK (ERK, JNK, and p38) signaling
pathway. F. alocis LTA activated the MAPK signaling pathway consisting
of ERK, JNK, and p38 pathway. Also, the LTA activated the NF-κB
Fig. 3. Activation of signaling pathway by F. alocis LTA. HGFs were treated
with F. alocis LTA or lipopolysaccharide of P. gingivalis and T. forsythia for 1 h.
The cytosolic proteins of the cells were transferred onto PVDF membrane after
separating by SDS-PAGE. Activation of NF-κB and MAPK signaling pathway was
analyzed using specific antibodies.
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Archives of Oral Biology 135 (2022) 105370
signaling pathway (Fig. 3). When the extracted LTA from F. alocis in this
study investigated to certify the purity using CHO/CD14/TLR2 and
TLR4 cells, the LTA activated CHO/CD14/TLR2 cell (data not shown).
These results indicate that F. alocis LTA activated MAPK and NF-κB
signaling pathway through TLR2 receptor.
3.4. Effect of signaling inhibitors on induction of inflammatory cytokines
by F. alocis LTA
Finally, specific inhibitors of signaling pathway were used to inves­
tigate which signaling pathway was associated with the induction of
pro-inflammatory cytokine expression by F. alocis LTA. Treatment with
Bay 11-7082 inhibited induction of TNF-α, IL-6 and IL-8 expression as
well as MMP-2 expression. activation of NF-κB signaling pathway. In
case of MAPK signaling pathway, treatment of SB203580, SP 600125,
and PD98059 significantly reduced the production of pro-inflammatory
cytokines and MMP-2 (Fig. 4).
4. Discussion
F. alocis is the first gram-positive bacterium to be detected in chronic
periodontitis patients and associated with periodontitis on basis of
epidemiological studies (Aruni et al., 2015; Kumar et al., 2006). This
bacterium has the characteristics of asaccharolytic and obligate anaer­
obic rod, and is a marker organism of periodontitis because of identifi­
cation in the pathogenetic structure of biofilms related with periodontal
inflammation (Aruni, Roy, & Fletcher, 2011). However, due to the lack
of studies on the PAMPs of F. alocis, the understanding of the relation­
ship between this bacterium and the induction of periodontitis is poor.
Therefore, the virulence of F. alocis has been investigated in vitro. This
study investigated the virulence of LTA first among various virulence
factors of F. alocis.
F. alocis LTA was certified purity with activated CHO/CD14/TLR2
H.-J. Yoo and S.-H. Lee Archives of Oral Biology 135 (2022) 105370

Fig. 4. Effect of inhibitors on expression of pro-inflammatory cytokines and MMP-2 induced by F. alocis LTA. HGFs were pretreated with NF-κB (BAY11-7082), ERK
(PD98059), JNK(SP600125), and p38 (SB203580) inhibitor and then treated with F. alocis LTA. The expression of TNF-α (A and E), IL-6 (B and F), IL-8 (C and G), and
MMP-2 (D and H) was investigated. Asterisk (*) indicates statistically significant differences compared with the control group (P < 0.05). Sharp (#) indicates
statistically significant differences compared with F. alocis LTA treated cell (P < 0.001). Data are represented as the median (horizontal lines), interquartile range
(boxes), and full ranges (whiskers). The dotted line indicates the control level.

cells and, proteinase K was treated to remove lipoprotein in extracting
F. alocis LTA. Also, LTA from E. faecalis and S. aureus was extracted in the
same method as extraction of F. alocis LTA. When the induction of
proinflammatory cytokine expression by LTA of the three bacteria was
compared, S. aureus LTA induced the most expression of inflammatory
cytokines, followed by F. alocis LTA did. Also, compared with oral
bacteria, F. alocis LTA induced more expression of inflammatory cyto­
kines than E. faecalis LTA. High concentration of E. faecalis LTA induced
expression of inflammatory proteins on mouse macrophage (Park et al.,
2013) and weakly induced expression of pro-inflammatory cytokines on
human periodontal ligament cell (Im et al., 2015). Furthermore,
E. faecalis LTA reduced expression of the cytokines induced by Aggre­
gatibacter actinomycetemcomitans lipopolysaccharide (Im et al., 2015).
On the basis of E. faecalis LTA, F. alocis LTA may have sufficient viru­
lence to induce periodontitis. The virulence of LTA differs according to
the structure of LTA, which is depending on the bacterial species. is due
to the different chemical structure (Lu et al., 2009; Wang et al., 2015).
LTA, which is an amphiphilic glycopolymer, is composed of a hydro­
philic backbone of poly (glycerol-phosphate) units, substituted with
D-alanine and sugar, and two hydrophobic anchor of fatty acid (13
carbon atoms in average) (Deininger et al., 2003; Reichmann & Grün­
dling, 2011). Hydrophilic part of LTA binds to carrier proteins such as
LPS-binding protein and CD14, which transfer LTA to toll-like receptor 2
(Schroder et al., 2003). Also, High degree of D-alanine substitutions in
glycerol-phosphate shows strong cell response and a complete absence
of D-alanine substitution in LTA exhibited weak cell response (Grangette
et al., 2005; Schroder et al., 2003). Also, the stimulation of TLR2 was
different according to the structure of fatty acid as acyl chain in LTA
(Han, Kim, Martin, Michalek, & Nahm, 2003). It was predicted that
F. alocis LTA contained a lower amount of D-alanine susstitution than
S. aureus LTA and a higher amount of D-alanine susstitution than
E. faecalis LTA in relation to TLR2 activity. In addition, the structure of
acyl chain in F. alocis LTA may also be different from LTA of the two
bacteria. Further research is needed for accurate structural analysis of
F. alocis LTA.
Next, the characteristics of F. alocis LTA was investigated. The
virulence of PAMP of periodontitis-related bacteria was compared.
T. forsythia lipopolysaccharide induced the most expression of inflam­
matory cytokines, and P. gingivalis lipopolysaccharide and F. alocis LTA
similarly induced the expression of the cytokines. This study is the first
comparing PAMP of periodontitis-related bacteria, and when the bac­
teria grow in subgingival pocket, the surface molecules of the bacteria
release to gingival crevicular fluid, and the released molecules induces
expression of inflammatory cytokines (Snyderman, 1971; Socransky &
Haffajee, 2005). Investigating the virulence of F. alocis LTA may be the
data needed to understand the induction of periodontitis, and LTA of
F. alocis may also release and stimulate human gingival fibroblast.
F. alocis LTA induced the expression of pro-inflammatory cytokines such
as TNF-α, IL-6, and IL-8. Although P. gingivalis lipopolysaccharide and
F. alocis LTA induced similar levels of pro-inflammatory cytokines and
are TLR2 ligands, the dimerizing TLR for TLR2 in the two PAMPS are
may be different. Therefore, when the signaling pathway activated by
F. alocis LTA was investigated, the LTA activated NF-κB and MAPK
signaling pathway including ERK, JNK, and p38. Furthermore, the
amount of signaling molecules by the LTA is similar to that of P. gingivalis
lipopolysaccharide.

5
H.-J. Yoo and S.-H. Lee
Finally, in order to investigate which signaling pathway induces the
expression of pro-inflammatory cytokines, the induction of the cytokines
was examined using each signaling pathway inhibitor. Before evaluating
the inhibitory effects of each inhibitor, the concentration at which
cytotoxicity did not appear was determined. Each inhibitor reduced the
expression of TNF-α, IL-6, IL-8, and MMP-2 induced by F. alocis LTA.
5. Conclusions
F. alocis LTA induced expression of tissue-destruction factor and pro-
inflammatory cytokines in human gingival fibroblasts with a level of
virulence similar to that of P. gingivalis lipopolysaccharide, and these
cytokines and tissue destruction factor of the LTA was induced through
NF-κB, ERK, JNK, and p38 signaling pathway. Basis on the results of this
study, F. alocis LTA may play an important role in inducing periodontitis
by induction of expression of pro-inflammatory cytokines.
Ethics approval and consent to participate
Since cells used in this study is a cell line, ethical approval was not
required.
Funding
This study was supported by the Basic Science Research Program of
the National Research foundation of Korea (NRF), which is funded by
the Ministry of Education, Science and Technology (NRF-
2019R1F1A1061315).
CRediT authorship contribution statement
Hyun-Jun Yoo and Sung-Hoon Lee conceived the experiments.
Hyun-Jun Yoo and Sung-Hoon Lee designed the experiments. Sung-
Hoon Lee conducted the experiments. The data were collected,
analyzed by Hyun-Jun Yoo and Sung-Hoon Lee. Hyun-Jun Yoo
drafted the manuscript. Sung-Hoon Lee revised and submitted the final
approval of the manuscript.
Conflict interest
The authors declare that no conflict of interest exists.
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