690389

research-article2017
JDRXXX10.1177/0022034517690389Journal of Dental ResearchNAMPT Is an Essential Regulator of RA-Mediated Periodontal Inflammation

Research Reports: Biological

Journal of Dental Research
1­–9
NAMPT Is an Essential Regulator of © International & American Associations
for Dental Research 2017

RA-Mediated Periodontal Inflammation Reprints and permissions:

sagepub.com/journalsPermissions.nav
DOI: 10.1177/0022034517690389
https://doi.org/10.1177/0022034517690389
journals.sagepub.com/home/jdr

D. Kim1,2, G. Lee3, Y.H. Huh3, S.Y. Lee1,2, K.H. Park1,2, S. Kim2, J. Kim4,
J. Koh1,2, and J. Ryu1,2

Abstract
Recent studies have indicated a potential correlation between rheumatoid arthritis (RA) and periodontal inflammation. We undertook
this study to verify whether RA mediates periodontitis-like phenotypes in experimental mouse models of RA and to explore the role of
nicotinamide phosphoribosyltransferase (NAMPT) in periodontal inflammation during RA pathogenesis. Periodontal inflammation and
alveolar bone loss have been reported in mice with collagen-induced arthritis (CIA) and in genetically modified tumor necrosis factor–α
(TNF-α) transgenic (TG) mouse models. Among the adipokines examined in our study, NAMPT expression was markedly upregulated
in the periodontal ligament (PDL) tissues in RA mouse models and in human PDL cells stimulated by the proinflammatory cytokines,
interleukin (IL) 1β and TNF-α. When NAMPT was overexpressed with the Nampt-synthesizing adenovirus vector (Ad-Nampt), the PDL
cells exhibited an increased expression of cytokines (IL6), chemokines (IL8 and chemokine [C-C motif] ligand 5 [CCL5]), inflammatory
mediators (cyclooxygenase 2 [COX-2]), and matrix-degrading enzymes (matrix metalloproteinase [MMP] 1 and MMP3). Inhibition of
NAMPT by the intracellular NAMPT (iNAMPT) inhibitor, FK866, or by the sirtuin inhibitor, nicotinamide, in PDL cells led to inhibition
of the IL1β or Ad-Nampt–induced upregulation of catabolic factors, whereas treatment with recombinant NAMPT protein or blockade
of extracellular NAMPT (eNAMPT) with blocking antibody did not. Moreover, NAMPT inhibition by the intraperitoneal or intragingival
injection of FK866 in CIA mice inhibited periodontal tissue damage, under conditions of RA. Thus, our results verified the co-occurrence
of RA and periodontal inflammation using experimental mouse models of RA, suggesting that iNAMPT in PDL cells plays a pivotal role in
the pathogenesis of RA-mediated periodontal inflammation by regulating the expression levels of catabolic genes, such as IL6, IL8, CCL5,
COX-2, MMP1, and MMP3.

Keywords: adipokines, arthritis, periodontitis, bone, cytokines, chemokines

Introduction these 2 inflammatory diseases has been clinically confirmed,

Rheumatoid arthritis (RA) and periodontitis are 2 of the most
common chronic inflammatory diseases in the world.
Periodontitis is an inflammatory disease characterized by irre-
versible destruction of the periodontium, which is composed of
the gingiva, cementum, alveolar bone, and periodontal liga-
ment (PDL). Periodontitis is mainly caused by microorgan-
isms, such as Porphyromonas gingivalis and Fusobacterium
nucleatum (Hajishengallis 2015). Several risk factors for peri-
odontal disease include obesity, diabetes, and smoking (Chi
et al. 2010). Recently, increasing attention has been given to
the association between oral health and autoimmune and
inflammatory diseases. RA is a chronic autoimmune disorder
characterized by joint inflammation, leading to severe erosive
arthritis. Recently, a number of clinical and epidemiologic
studies have suggested a link between RA and periodontitis
(Payne et al. 2015; Fuggle et al. 2016). P. gingivalis oral infec-
tion exacerbates the development and severity of collagen-
induced arthritis (CIA) by increasing the immune system
activation favoring Th17 cell responses (Marchesan et al.
2013). Moreover, compared with the general population, sub-
jects with RA are at an increased risk of developing periodon-
titis (Pischon et al. 2008). Although a close association between
the molecular mechanisms involved in preferential targeting of
RA-induced periodontal inflammation remain unclear.
RA and periodontal inflammation have been reported to
share numerous common pathogenic characteristics, such as
immune cell infiltration and bone erosion, and demonstrate a
similar involvement of catabolic factors, including cyclooxy-
genase (COX) and matrix metalloproteinases (MMPs) (Araújo
1
Department of Pharmacology and Dental Therapeutics, School of
Dentistry, Chonnam National University, Gwangju, Republic of Korea
2
Research Center for Biomineralization Disorders, School of Dentistry,
Chonnam National University, Gwangju, Republic of Korea
3
Bioimaging and Cell Logistics Research Center, School of Life Sciences,
Gwangju Institute of Science and Technology, Gwangju, Republic of
Korea
4
Department of Pediatric Dentistry, School of Dentistry, Chonnam
National University, Gwangju, Republic of Korea
A supplemental appendix to this article is available online.
Corresponding Author:
J. Ryu, Department of Pharmacology and Dental Therapeutics, School of
Dentistry, Chonnam National University, Gwangju 61186, Republic of
Korea.
Email: jesryu@jnu.ac.kr
2 Journal of Dental Research 
et al. 2015). They also display similar cytokine profiles, includ-
ing interleukin (IL) 1β, IL6, and tumor necrosis factor–α
(TNF-α), leading to the stimulation of local inflammation and
degradation of extracellular matrix (ECM) (Brennan and
McInnes 2008). Recently, an involvement of adipokines in the
regulation of RA pathogenesis has been reported (Neumann
et al. 2016). The adipose tissue secretes bioactive molecules,
such as nicotinamide phosphoribosyltransferase (NAMPT)
(also called visfatin), leptin, resistin, apelin, and adiponectin,
which are collectively known as adipokines. Adipokines were
originally secreted by adipocytes but now can also be locally
produced by other cell types under pathophysiological condi-
tions (Deschner et al. 2014). Previous studies have demon-
strated an upregulated NAMPT expression in the inflamed
synovium of RA mice and in the plasma and synovial fluid
from RA patients (Otero et al. 2006). NAMPT functions as
both an extracellular form (eNAMPT) acting as an adipokine
and an intracellular form (iNAMPT) exhibiting enzymatic
activity responsible for the synthesis of nicotinamide adenine
dinucleotide (NAD+), which is essential for cell metabolism
(Imai 2009; Yang et al. 2015). Recent studies have suggested
the role of adipokines in periodontal infection, particularly
focusing on the function of eNAMPT as an adipokine
(Deschner et al. 2014). The NAMPT levels in gingival crevicu-
lar fluid have been shown to increase at inflamed sites (Pradeep
et al. 2011). Furthermore, NAMPT is synthesized in PDL cells
under conditions of bacterial infection, and treatment with
recombinant NAMPT stimulates an increased expression of
MMP1 and chemokine (C-C motif) ligand (CCL) 2 in these
cells (Nokhbehsaim et al. 2013). Although these observations
suggest a possible role of eNAMPT in general periodontal
destruction, the contribution of iNAMPT remains to be estab-
lished. In this study, we demonstrated that NAMPT expression
is significantly upregulated in the periodontal tissue in RA
mice and that treatment of PDL cells with IL1β and TNF-α, the
major cytokines involved in both RA and periodontal inflam-
mation, further stimulates NAMPT expression. Here we inves-
tigated the role and underlying molecular mechanisms of
NAMPT in RA-mediated periodontal inflammation.
Materials and Methods
Experimental Mouse Model of RA
Male DBA/1J, TNF-α transgenic (TG) mice were employed
for experimental RA studies. CIA was induced by intradermal
injection of bovine type II collagen (100 µg, deliquesced in
acetic acid) emulsified in complete Freund’s adjuvant (CFA) in
8-wk-old DBA/1J mice. After 3 wk, CIA mice were boosted
with bovine type II collagen (100 µg) emulsified in incomplete
Freund’s adjuvant (IFA). To prepare the nonarthritic mouse
model (NI; nonimmunized), mice were injected with IFA and
acetic acid as a vehicle (Brand et al. 2007; Ryu et al. 2014).
Eight-month-old TNF-α TG mice were used as a model for
spontaneous inflammatory arthritis, with non-TG wild-type
(WT) littermates used as controls. CIA mice were injected with
100 µL FK866 (10 mg/kg) (Cayman) in the intraperitonium or
10 µL FK866 (10 mg/kg) under the alveolar mucosa of the
maxilla between the first and second molars using a Hamilton
syringe (32 gauge, 9.25 mm, 30°) or phosphate-buffered saline
as a vehicle, followed by a booster injection once every 3 d
over a 2-wk period. The mice were housed in a specific pathogen-
free barrier facility. All animal experiments were conducted in
accordance with protocols approved by the Animal Care and
Ethics Committees of Chonnam National University, Gwangju,
Republic of Korea.
Micro–Computed Tomography and
Histomorphometric Analysis
The maxilla and ankle obtained from RA mice were fixed in 10%
neutral buffered formalin for 24 h and scanned using the SkyScan
1172 micro–computed tomography (CT) scanner (SkyScan). The
acquisition state was set at 49 kV and 200 µA with a 0.5-mm
aluminum filter and 8.03-µm resolution. Micro-CT (µCT) data
were analyzed to measure alveolar bone volume after setting the
region of interest around the molar of the maxilla with or without
teeth (Park et al. 2007) by using CTAn (SkyScan) and Mimics
14.0 (Materialise) (Choi et al. 2015). Following µCT analysis, the
maxilla and ankle were decalcified in 0.5 M EDTA for 2 wk,
embedded in paraffin, and sectioned at 5-µm thickness. Sections
were subjected to hematoxylin and eosin (H&E) staining.
Synovial inflammation, pannus formation, and cartilage destruc-
tion scored as described previously (Ryu et al. 2014). To evaluate
the extent of alveolar bone loss, the most central sections of 2
molars were used, selected based on the width of the root canal,
and the distance between cement enamel junction (CEJ) and
alveolar bone crest (ABC) was measured using standard param-
eters (Park et al. 2011).
Other Materials and Methods
Additional methods are described in online Appendix Materials
and Methods. These include immunohistochemistry and
immunofluorescence microscopy, cell culture and stimulation,
enzyme-linked immunosorbent assay (ELISA), reverse tran-
scription–polymerase chain reaction (RT-PCR), quantitative
real time-PCR (qPCR), and Western blot analysis.
Results
NAMPT Is Upregulated in PDL Cells of
Experimental RA Mouse
To elucidate the possible mechanism of association between RA
and periodontal inflammation, we initially examined whether
periodontal inflammation co-occurred in experimental RA
mouse models. In this study, CIA and TNF-α TG mice were
employed as inflammatory arthritis models. Severe synovial
inflammation and bone erosion in the ankle joints of CIA (Fig.
1A) and TNF-α TG (Fig. 1B) mice were detected by µCT analy-
sis and the level of synovitis was scored. H&E staining revealed
that experimental RA mice displayed the hallmarks of RA, such as
synovitis, pannus formation, and cartilage destruction (Appendix
NAMPT Is an Essential Regulator of RA-Mediated Periodontal Inflammation 3

Figure 1.  Periodontal destruction was induced in experimental mouse models of rheumatoid arthritis (RA). collagen-induced arthritis (CIA) and
tumor necrosis factor–α (TNF-α) transgenic (TG) mice are used as representative experimental RA mice models. (A) Left: Representative micro–
computed tomography (µCT) images of the ankle joints of DBA/1J mice, either immunized with type II collagen (CIA) or not immunized (NI) (n = 6).
Right: Scoring of synovial inflammation. (B) Typical µCT images and synovial inflammation scores of the ankle joints of 8-mo-old TNF-α TG or its wild-
type (WT) mice (n = 5). (C, D) Representative µCT reconstructions and bone surface (BS) to bone volume (BV) ratio of the mice maxilla under CIA
conditions (C) and of TNF-α TG mice (D). (E, F) Hematoxylin and eosin–stained sections of intact periodontal tissues in CIA (E) and TNF-α TG mice
(F) and measurement of bone levels by comparing the distance from cemento-enamel junction (CEJ) to alveolar bone crest (ABC) in µm. The distance
from the CEJ to ABC was measured between the first and second molars of the maxilla. Values are presented as mean ± standard error of the mean
(SEM) (*P < 0.001, **P < 0.0001). Scale bar: 10 µm.

Fig. 1). To examine RA-mediated periodontal destruction in CIA
and TNF-α TG mice, µCT and histomorphometric analyses were
performed in the mouse maxilla. Three-dimensional reconstruc-
tion images and volumetric measurements from µCT analysis
revealed an alveolar bone loss, and the level of exposed root
surface was found to be significantly higher in the maxillae of
CIA and TNF-α TG mice compared with control (NI) and WT
mice, respectively (Fig. 1C, D). Furthermore, histomorphomet-
ric measurements of H&E-stained tissue sections also showed
that the distance from the CEJ to ABC was significantly greater
in RA mice than in control mice (Fig. 1E, F).
To investigate the regulatory mechanisms underlying
RA-mediated periodontal inflammation, we examined the
expression levels of adipokines, which have recently evidenced
their relation to inflammatory diseases, such as RA. The mes-
senger RNA (mRNA) levels of adipokines, such as Adiponectin,
Apelin, Leptin, Resistin, and Nampt, were examined in the gingi-
val tissue. Among the examined adipokines, only Nampt expres-
sion was highly upregulated in the gingival tissue of CIA mice
(Fig. 2A). Upregulation of NAMPT expression was further veri-
fied by immunohistochemistry of the RA tissue sections from
CIA mice (Fig. 2B) and TNF-α TG mice (Fig. 2C). Increased
local NAMPT expression in the gingiva correlates with disease
severity (Appendix Fig. 2A). However, circulating serum
NAMPT level was not altered in these mouse models (Appendix
Fig. 2B). To explore possible functions of local NAMPT in
RA-mediated periodontal inflammation, we examined the
expression pattern of NAMPT by immunostaining the gingival
tissues of RA mice. NAMPT was highly expressed in PDL cells
as determined by double immunostaining (Appendix Fig. 3).
NAMPT-positive cells were also observed in some tartrate-resis-
tant acid phosphatase (TRAP)–positive osteoclasts but not in
monocytes/macrophages (Appendix Fig. 3). Thus, we investi-
gated the pattern of NAMPT expression using a primary culture
of human PDL cells. The proinflammatory cytokines, IL1β and
TNF-α, which are involved in RA pathogenesis, induced an
upregulation of cytokines (IL6), chemokines (IL8 and CCL5),
inflammatory mediators (PTGS2), and matrix-degrading
enzymes (MMP1 and MMP3). In primary cultured pathogenic
PDL cells, treatment with IL1β (Fig. 2D) or TNF-α (Fig. 2E)
dramatically increased NAMPT expression in a dose-dependent
manner but not that of other examined adipokines. NAMPT
functions in 2 different forms: intracellular and extracellular.
Western blot analysis revealed that both iNAMPT and eNAMPT
were produced by IL1β or TNF-α treatment (Fig. 2F). IL1β
treatment of PDL cells triggered the activation of nuclear factor–
κB (NF-κB) and mitogen-activated protein kinase subtypes,
including extracellular signal-regulated kinase (ERK) and p38
kinase (Fig. 2G). Inhibition of p38 kinase and NF-κB with
SB203580 and SC514, respectively, markedly inhibited the
IL1β-induced increase of NAMPT expression, but inhibition of
ERK with PD98059 had no effect on NAMPT expression (Fig.
2H). These data indicate that NAMPT expression is increased in
PDL cells during RA-mediated periodontal inflammation and
that this cytokine-induced upregulation of NAMPT is mediated
via the p38 kinase and NF-κB signaling pathways.
4 Journal of Dental Research 

Figure 2.  Nicotinamide phosphoribosyltransferase (NAMPT) is upregulated in periodontal ligament (PDL) cells during rheumatoid arthritis (RA)–
mediated periodontal inflammation. (A) Total RNA was extracted from whole gingival tissues of the not immunized (NI) or collagen-induced arthritis
(CIA) mouse. Indicated messenger RNA (mRNA) levels were determined by reverse transcription–polymerase chain reaction (RT-PCR) analysis.
(B, C) Immunostaining of gingival tissue for NAMPT in CIA or NI mice (B) and tumor necrosis factor–α (TNF-α) transgenic (TG) or its control (wild-
type [WT]) mice (C). Scale bar: 50 µm. (D, E) Primary cultures of human PDL cells were left untreated or were treated with the indicated amounts
of interleukin (IL) 1β (D) and TNF-α (E). Transcript levels of the indicated genes were determined by RT-PCR and quantified by quantitative real-time
PCR (qPCR). (F) Western blot analysis of the indicated proteins in primary cultured human PDL cells treated with IL1β and TNF-α for 24 h. (G)
Western blot analysis of inhibitor of κB (IκB) and mitogen-activated protein (MAP) kinase subtypes in human PDL cells treated with IL1β (1 ng/mL) for
the indicated time periods. (H) RT-PCR was performed in PDL cells treated with IL1β for 24 h, in the presence of the indicated inhibitors: PD98059,
an ERK inhibitor; SB203580, a p38 MAP kinase inhibitor; and SC514, an inhibitor of nuclear factor κBα (IκBα). Values are presented as mean ± SEM
(*P < 0.05, **P < 0.005).
NAMPT Is an Essential Regulator of RA-Mediated Periodontal Inflammation 5

Figure 3.  Overexpression of nicotinamide phosphoribosyltransferase (NAMPT) regulates the expression of interleukin (IL) 6, IL8, chemokine [C-C
motif] ligand 5 (CCL5), cyclooxygenase 2 (COX-2), matrix metalloproteinase (MMP) 1, and MMP3 in periodontal ligament (PDL) cells. Human PDL
cells were infected with empty virus (Ad-C) or Nampt adenovirus (Ad-Nampt). (A) Expression levels of catabolic factors determined by reverse
transcription–polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) in PDL cells infected with Ad-C (800 multiplicity of infection
[MOI]) or Ad-Nampt for 24 h. (B) Western blots of cellular and secreted NAMPT, IL6, COX-2, and MMP3 in PDL cells infected with the indicated
MOI of Ad-Nampt. (C) Immunofluorescence microscopy results of NAMPT, IL6, CCL5, and MMP3 in PDL cells infected at an MOI of 800 with Ad-C
or Ad-Nampt. DAPI was used for nuclear staining. Values are presented as mean ± SEM (*P < 0.05, **P < 0.005). Scale bar: 50 µm.

NAMPT Upregulates Catabolic Factor
Expression in PDL Cells
We next investigated the possible role of NAMPT in the
expression of catabolic factors (cytokines, chemokines, inflam-
matory mediators, and matrix-degrading enzymes) involved in
periodontal inflammation. Overexpression of NAMPT by
Ad-Nampt infection led to a marked increase in the mRNA lev-
els of IL6, IL8, CCL5, PTGS2, MMP1, and MMP3 (Fig. 3A).
In addition, increased protein levels of eNAMPT, iNAMPT,
IL6, COX-2, and MMP3 were detected by immunoblotting
(Fig. 3B). Double-staining for iNAMPT and its target genes in
the PDL cells infected with Ad-Nampt indicated that cells with
a high expression of iNAMPT were strongly positive for the
tested genes (IL6, CCL5, and MMP3) (Fig. 3C). These obser-
vations suggest that NAMPT expression plays an important
role in RA-mediated periodontal destruction.
To further investigate the regulatory mechanism of NAMPT
during RA-mediated periodontal inflammation, the inflamma-
tory actions of iNAMPT and eNAMPT were determined. The
significance of NAMPT enzymatic activity was determined
using FK866, a highly specific noncompetitive inhibitor of
6 Journal of Dental Research 

upregulation of target genes was sig-

nificantly reduced by inhibition of
SIRT with nicotinamide (Fig. 4F).
Taken together, these results indicated
that iNAMPT/SIRT activity, rather than
eNAMPT, promotes the upregulation
of catabolic genes in PDL by NAMPT
overexpression.

NAMPT Is Required for

RA-Mediated Periodontal
Inflammation
Because iNAMPT enzymatic activity is
necessary for cytokine and MMP expres-
sion, we examined whether iNAMPT
regulates RA-mediated periodontal
inflammation in experimental RA mice
models. Intraperitoneal injection of
FK866 significantly inhibited CIA-
induced inflammation and bone
destruction in teeth (Fig. 5A), with con-
comitant reduction of NAMPT targets,
Figure 4.  Nicotinamide phosphoribosyltransferase (NAMPT) enzymatic activity is required for such as IL6 and MMP3 (Fig. 5B). In
the upregulation of catabolic genes in periodontal ligament (PDL) cells. (A, B) Expression levels addition, CD3-positive T-cell infiltra-
of catabolic factors as determined by reverse transcription–polymerase chain reaction (RT-PCR) tion was reduced in the gingiva of CIA
in empty virus-infected PDL cells (Ad-C) or PDL cells infected with Ad-Nampt (800 multiplicity of
infection [MOI]) (A) or interleukin (IL) 1β (1 ng/mL) (B), in the absence or presence of indicated
mice, supporting RA-mediated peri-
amounts of FK866 for 24 h. (C) RT-PCR results of the indicated catabolic factors in PDL cells odontal inflammation (Fig. 5B). In
treated with recombinant NAMPT (rNAMPT). (D, E) PDL cells infected with Ad-Nampt (D) or addition, the role of NAMPT in
treated with IL1β (E), in the absence or presence of the indicated concentrations of the neutralizing RA-mediated alveolar bone destruction
antibody against human NAMPT. (F) Messenger RNA levels of the NAMPT target genes that were
detected in PDL cells infected with Ad-C or Ad-Nampt (800 MOI) for 24 h, in the absence or followed by periodontal inflammation
presence of nicotinamide (NIC). Representative RT-PCR results are shown (n = 5). in mice was examined in vivo by intra-
gingival injection of FK866, which
iNAMPT. FK866 pretreatment significantly blocked the resulted in the inhibition of NAMPT function in PDL cells
Ad-Nampt–induced (Fig. 4A) or IL1β-induced (Fig. 4B) (Fig. 5C). As shown in Figure 5A, consistent with intraperito-
upregulation of IL6, IL8, PTGS2, MMP1, and MMP3. neal injection, intragingival injection of FK866 showed a sig-
eNAMPT action was assessed by treating the PDL cells with nificant blockade of periodontal inflammation and alveolar
recombinant NAMPT (rNAMPT). In contrast to the effects of bone destruction in CIA mice, with concomitant reduction of
FK866, rNAMPT only slightly increased PTGS2 and MMP1 IL6, MMP3, and CD3 in the PDL region (Fig. 5D). These
expression among the examined tested genes (Fig. 4C). In results collectively indicated that iNAMPT enzymatic activity
addition, treatment with a neutralizing antibody against is essential for RA-mediated periodontal inflammation.
NAMPT did not alter the Ad-Nampt–induced upregulation of
catabolic factors (Fig. 4D). IL1β-induced upregulation of cata-
Discussion
bolic factors remained unchanged by treatment with NAMPT-
neutralizing antibody (Fig. 4E). In addition, the expression of In this study, our results demonstrated that NAMPT levels are
the NAMPT receptor, Igf1r, was not altered in the gingival tis- highly increased in PDL cells during periodontal inflamma-
sue of CIA mice (Appendix Fig. 2C), supporting the effects of tion, using experimental mouse models of RA. NAMPT stimu-
iNAMPT, but not eNAMPT, including circulating NAMPT, on lates IL6, IL8, CCL5, COX-2, MMP1, and MMP3 expression
RA-mediated periodontal inflammation. NAMPT enzymatic by regulating their enzymatic activities. Inhibition of iNAMPT
activity is an essential cofactor of sirtuin (SIRT) protein function by FK866 ameliorates RA-induced periodontal
deacetylases. SIRT regulates gene expression by modulating inflammation in mice, suggesting the NAMPT activation is
chromatin function via direct deacetylation of histones and required for periodontal inflammation and alveolar bone loss
transcription factors (Imai 2009; Zhang et al. 2011). To exam- under conditions of RA.
ine whether the effects of iNAMPT in PDL cells were due to In our current study, we used 2 different experimental mod-
the NAD+-SIRT axis, SIRT activity was inhibited by nicotin- els of RA for in vivo analysis: CIA mice for the induced arthri-
amide in the presence of Ad-Nampt. Ad-Nampt–induced tis model and TNF-α TG mice for the genetically modified
NAMPT Is an Essential Regulator of RA-Mediated Periodontal Inflammation 7

Figure 5.  Blockade of nicotinamide phosphoribosyltransferase (NAMPT) enzymatic activity ameliorates rheumatoid arthritis (RA)–mediated
periodontal inflammation. FK866 was intraperitoneally (A, B) or intragingivally (C, D) injected after type II collagen induction in mice. (A, C)
Periodontal inflammation and alveolar bone erosion were detected by micro–computed tomography (µCT) reconstruction, volumetric measurements,
and histomorphometric analysis in collagen-induced arthritis (CIA) mice injected with FK866, intragingivally (A) or intraperitoneally (C). Scale
bar: 10 µm. (B, D) IL6 and MMP3 protein levels and T-cell infiltrations in CIA gingival tissue with or without injection of FK866 were examined by
immunostaining. Scale bar: 50 µm. Values are presented as mean ± SEM (*P < 0.05).

model. Despite the evolutionary distance between mice and
humans, prominent features of arthritis in various experimental
mouse models are similar to that in human RA. The most com-
monly used model for RA in mice is CIA. Park et al. (2011)
demonstrated that CIA mouse models develop periodontal
destruction with alveolar bone loss, which is characterized by
increased osteoclast activity and adipocyte production, as well
as decreased osteoblastic activity in alveolar bone cells. Our
data are consistent with these observations, demonstrating
severe inflammation of PDL and alveolar bone loss in CIA
mouse models, even though the duration of immunization with
type II collagen was different. Here, we also used another rep-
resentative model of RA, the TNF-α TG mice. Our results from
µCT volumetric measurements and histomorphometric analy-
sis revealed that TNF-α TG mice showed similar signs and dis-
ease progression, reflecting periodontal inflammation. These
data suggest that TNF-α TG mice can serve as a good experi-
mental model for RA-induced periodontal inflammation.
Various proinflammatory cytokines, such as TNF-α, IL1β,
IL6, and IL17, are overexpressed in the joints of RA patients
and mice (Brennan and McInnes 2008), suggesting the impor-
tance of cytokines in RA development. Direct evidence of a
critical role of TNF-α was determined by the spontaneous RA
development in mice overexpressing the human TNF-α trans-
gene (Moudgil et al. 2011). As with TNF-α, several members of
the IL1 superfamily have been implicated in the pathogenesis
of RA. In addition, mice deficient in IL1 receptor antagonist
have been shown to spontaneously develop arthritis (Horai et al.
2000). Like RA, periodontitis also has similar proinflamma-
tory cytokine profiles, including high amounts of TNF-α and
IL1β in chronic inflammatory lesions (Garlet 2010). In patients
with periodontitis, high levels of TNF-α and IL1β are present
in both gingival crevicular fluid and diseased periodontal tis-
sues, where they are positively correlated with MMPs and
receptor activator of nuclear factor kappa-B ligand (RANKL)
expression (Graves and Cochran 2003). Data from experimen-
tal periodontitis in rats using a ligature model have also clearly
demonstrated that TNF-α and IL1β play a central role in
immune response, inflammation, and alveolar bone resorption
(Graves et al. 2011). Thus, in our study, we used both the cyto-
kines, TNF-α and IL1β, as a common pathological cause of RA
and periodontal inflammation for in vitro analysis.
Systemic adipokines are known to be related to inflamma-
tory diseases, such as RA. Alteration of adipokine expression
produced by adipocytes in experimental RA mouse models has
been also reported, suggesting the possible role of serum
NAMPT in our experimental mouse models. To clarify this
issue, we examined secretion level of Nampt in CIA and
TNF-α TG mice. ELISA assay revealed no significant increase
of Nampt levels in serum from mice with CIA and TNF-α TG
mice, consistent with the report by Evans et al. (2011). Here,
we demonstrated that NAMPT in PDL cells plays an essential
8 Journal of Dental Research 
role in RA-mediated periodontal inflammation by upregulating
the matrix-degrading enzymes and inflammatory mediators.
Our gain-of-function studies using Ad-Nampt demonstrated
that NAMPT upregulates various catabolic factors, including
IL6, MMP1, MMP3, and COX-2, in PDL cells. Our loss-of-
function studies by FK866 treatment, in the presence of cyto-
kines or Ad-Nampt, suggested that iNAMPT enzyme activity
plays an important role in the regulation of catabolic factor
expression and is sufficient to induce pathogenesis of
RA-induced periodontal inflammation. Consistent with this,
administration of FK866 inhibited CIA-induced periodontal
destruction. It has recently reported that periodontal inflamma-
tion and oral microbiota are risk factors of RA (Leech and
Bartold 2015). This suggests that hallmarks of RA, including
paw swelling, can be regulated by the inhibition of NAMPT
enzymatic activity via IG injection of FK866. This remains to
be further elucidated.
To date, the contribution of iNAMPT to either general peri-
odontitis or RA-mediated periodontal inflammation has not
been established, although the possible role of eNAMPT in
periodontitis has been suggested. To elucidate the role of
eNAMPT, we treated the PDL cells with rNAMPT and
observed a slight but significant upregulation of MMP-1
expression. This finding is consistent with an observation by
Nokhbehsaim et al. (2013), who showed the stimulation of
MMP1 and CCL2 by NAMPT in PDL cells. However, we sug-
gest that eNAMPT is not sufficient for mediating periodontal
inflammation pathogenesis, at least in our experimental sys-
tem. This was demonstrated by the observation that the anti-
NAMPT antibody could not alter the gene expression regulated
by NAMPT overexpression. NAMPT enzymatic activity is
known to stimulate the synthesis of NAD+, which is an essen-
tial cofactor of SIRT protein deacetylases. To examine whether
the effects of iNAMPT in PDL cells were due to the NAD+-
SIRT axis, SIRT activity was inhibited by nicotinamide, a
SIRT inhibitor (Avalos et al. 2005), or by EX527, a SIRT1-
specific inhibitor (Solomon et al. 2006). Unlike the effects of
nicotinamide, EX527 had no effect on the expression of cata-
bolic factors upregulated by NAMPT overexpression (data not
shown). The mammalian SIRT family is composed of 7 mem-
bers, which are found in different subcellular locations. This
result suggests that other members of the SIRT family, rather
than SIRT1, are associated with NAMPT-induced gene expres-
sion in periodontal inflammation.
Herein we provide evidence suggesting that iNAMPT is an
essential catabolic factor involved in the pathogenesis of
RA-induced periodontal inflammation, which acts by regulat-
ing the expression of various catabolic genes, including ECM-
degrading enzymes. The etiology of RA-induced periodontal
inflammation has not yet been elucidated, and effective treat-
ment of periodontal inflammation by various stimuli remains a
significant unmet medical need. Hence, these results provide
new insights into the mechanisms by which NAMPT upregula-
tion contributes to RA-mediated periodontal inflammation and
suggest that NAMPT could be a potential therapeutic target for
new strategies to treat or prevent periodontal diseases.
Author Contributions
D. Kim, J. Ryu, contributed to conception, design, data acquisi-
tion, analysis, and interpretation, drafted the manuscript; G. Lee,
contributed to design, data acquisition, analysis, and interpreta-
tion, critically revised the manuscript; Y.H. Huh, contributed to
conception, data analysis, and interpretation, critically revised the
manuscript; S.Y. Lee, K.H. Park, contributed to data acquisition,
critically revised the manuscript; S. Kim, J. Kim, J. Koh, contrib-
uted to data analysis and interpretation, critically revised the man-
uscript. All authors gave final approval and agree to be accountable
for all aspects of the work.
Acknowledgments
This work was supported by grants from the National Research
Foundation of Korea (2011-0030121 and 2015R1D1A1A
01057870); the Korea Health Technology R&D Project through
the Korea Health Industry Development Institute (KHIDI), funded
by the Ministry of Health & Welfare (HI12C1606 and HI16C0287);
and Chonnam National University (2013-2899). The authors
declare no potential conflicts of interest with respect to the author-
ship and/or publication of this article.
References
Araújo VM, Melo IM, Lima V. 2015. Relationship between periodontitis
and rheumatoid arthritis: review of the literature. Mediators Inflamm.
2015:259074.
Avalos JL, Bever KM, Wolberger C. 2005. Mechanism of sirtuin inhibition by
nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme.
Mol Cell. 17(6):855–868.
Brand DD, Latham KA, Rosloniec EF. 2007. Collagen-induced arthritis. Nat
Protoc. 2(5):1269–1275.
Brennan FM, McInnes IB. 2008. Evidence that cytokines play a role in rheuma-
toid arthritis. J Clin Invest. 118(11):3537–3545.
Chi AC, Neville BW, Krayer JW, Gonsalves WC. 2010. Oral manifestations of
systemic disease. Am Fam Physician. 82(11):1381–1388.
Choi H, Jeong BC, Hur SW, Kim JW, Lee KB, Koh JT. 2015. The angiopoietin-1
variant COMP-Ang1 enhances BMP2-induced bone regeneration with recruit-
ing pericytes in critical sized calvarial defects. PLoS One. 10(10):e0140502.
Deschner J, Eick S, Damanaki A, Nokhbehsaim M. 2014. The role of adipokines
in periodontal infection and healing. Mol Oral Microbiol. 29(6):258–269.
Evans L, Williams AS, Hayes AJ, Jones SA, Nowell M. 2011. Suppression
of leukocyte infiltration and cartilage degradation by selective inhibition
of pre–B cell colony-enhancing factor/visfatin/nicotinamide phosphoribo-
syltransferase: apo866-mediated therapy in human fibroblasts and murine
collagen-induced arthritis. Arthritis Rheum. 63(7):1866–1877.
Fuggle NR, Smith TO, Kaul A, Sofat N. 2016. Hand to mouth: a systematic
review and meta-analysis of the association between rheumatoid arthritis
and periodontitis. Front Immunol. 7:80.
Garlet GP. 2010. Destructive and protective roles of cytokines in periodontitis:
a re-appraisal from host defense and tissue destruction viewpoints. J Dent
Res. 89(12):1349–1363.
Graves DT, Cochran D. 2003. The contribution of interleukin-1 and tumor necro-
sis factor to periodontal tissue destruction. J Periodontol. 74(3):391–401.
Graves DT, Li J, Cochran DL. 2011. Inflammation and uncoupling as mecha-
nisms of periodontal bone loss. J Dent Res. 90(2):143–153.
Hajishengallis G. 2015. Periodontitis: from microbial immune subversion to
systemic inflammation. Nat Rev Immunol. 15(1):30–44.
Horai R, Saijo S, Tanioka H, Nakae S, Sudo K, Okahara A, Ikuse T, Asano
M, Iwakura Y. 2000. Development of chronic inflammatory arthropathy
resembling rheumatoid arthritis in interleukin 1 receptor antagonist-defi-
cient mice. J Exp Med. 191(2):313–320.
Imai S. 2009. Nicotinamide phosphoribosyltransferase (Nampt): a link between
NAD biology, metabolism, and diseases. Curr Pharm Des. 15(1):20–28.
Leech MT, Bartold PM. 2015. The association between rheumatoid arthritis and
periodontitis. Best Pract Res Clin Rheumatol. 29(2):189–201.
Marchesan JT, Gerow EA, Schaff R, Taut AD, Shin SY, Sugai J, Brand D,
Burberry A, Jorns J, Lundy SK, et al. 2013. Porphyromonas gingivalis oral
NAMPT Is an Essential Regulator of RA-Mediated Periodontal Inflammation 9
infection exacerbates the development and severity of collagen-induced
arthritis. Arthritis Res Ther. 15(6):R186.
Moudgil KD, Kim P, Brahn E. 2011. Advances in rheumatoid arthritis animal
models. Curr Rheumatol Rep. 13(5):456–463.
Neumann E, Junker S, Schett G, Frommer K, Müller-Ladner U. 2016.
Adipokines in bone disease. Nat Rev Rheumatol. 12(5):296–302.
Nokhbehsaim M, Eick S, Nogueira AV, Hoffmann P, Herms S, Fröhlich H,
Jepsen S, Jäger A, Cirelli JA, Deschner J. 2013. Stimulation of MMP-1 and
CCL2 by NAMPT in PDL cells. Mediators Inflamm. 2013:437123.
Otero M, Lago R, Gomez R, Lago F, Dieguez C, Gómez-Reino JJ, Gualillo
O. 2006. Changes in plasma levels of fat-derived hormones adiponec-
tin, leptin, resistin and visfatin in patients with rheumatoid arthritis. Ann
Rheum Dis. 65(9):1198–1201.
Park CH, Abramson ZR, Taba M, Jin Q, Chang J, Kreider JM, Goldstein SA,
Giannobile WV. 2007. Three-dimensional micro-computed tomographic
imaging of alveolar bone in experimental bone loss or repair. J Periodontol.
78(2):273–281.
Park JC, Su C, Jung IH, Choi SH, Cho KS, Kim CK, Park YB, Lee SK, Kim
CS. 2011. Mechanism of alveolar bone loss in a collagen-induced arthritis
model in mice. J Clin Periodontol. 38(2):122–130.
Payne JB, Golub LM, Thiele GM, Mikuls TR. 2015. The link between peri-
odontitis and rheumatoid arthritis: a periodontist’s perspective. Curr Oral
Health Rep. 2:20–29.
Pischon N, Pischon T, Kröger J, Gülmez E, Kleber BM, Bernimoulin JP, Landau
H, Brinkmann PG, Schlattmann P, Zernicke J, et al. 2008. Association
among rheumatoid arthritis, oral hygiene, and periodontitis. J Periodontol.
79(6):979–986.
Pradeep AR, Raghavendra NM, Prasad MV, Kathariya R, Patel SP, Sharma
A. 2011. Gingival crevicular fluid and serum visfatin concentration:
their relationship in periodontal health and disease. J Periodontol.
82(9):1314–1319.
Ryu JH, Chae CS, Kwak JS, Oh H, Shin Y, Huh YH, Lee CG, Park YW,
Chun CH, Kim YM, et al. 2014. Hypoxia-inducible factor-2α is an essen-
tial catabolic regulator of inflammatory rheumatoid arthritis. PLoS Biol.
12(6):e1001881.
Solomon JM, Pasupuleti R, Xu L, McDonagh T, Curtis R, DiStefano PS, Huber
LJ. 2006. Inhibition of SIRT1 catalytic activity increases p53 acetylation
but does not alter cell survival following DNA damage. Mol Cell Biol.
26(1):28–38.
Yang S, Ryu JH, Oh H, Jeon J, Kwak JS, Kim JH, Kim HA, Chun CH, Chun
JS. 2015. NAMPT (visfatin), a direct target of hypoxia-inducible factor-
2α, is an essential catabolic regulator of osteoarthritis. Ann Rheum Dis.
74(3):595–602.
Zhang LQ, Heruth DP, Ye SQ. 2011. Nicotinamide phosphoribosyltransferase
in human diseases. J Bioanal Biomed. 3:13–25.