JBC Papers in Press. Published on November 1, 2019 as Manuscript RA119.

010648
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.010648

Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4

phosphorylation

Peng Zhang1,2, Qian Wang1,2, Lulingxiao Nie1,2, Rui Zhu 1,2, Xinyi Zhou1,2, Pengfei
Zhao1,2, Ning Ji1, Xing Liang1,2, Yi Ding1,3, Quan Yuan1,4, Qi Wang1,2*

1 State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral
Diseases, West China Hospital of Stomatology, Sichuan University
2 Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University
3 Department of Periodontology, West China Hospital of Stomatology, Sichuan University
4 Department of Oral Implantology, West China Hospital of Stomatology, Sichuan
University

*Correspondence:
Dr. Qi Wang,
Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University,

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3rd Section S Renmin Road, 14#, Chengdu, China, 610041
wqinno8751@gmail.com
Fax: +86 28 85503503
Email: wqinno8751@gmail.com

Running Title: Inflamm-aging and gingival senescence

Keywords: inflamm-aging; cellular senescence; SASP; NLRC4; hyperglycemia; gingiva

Abstract mice. Simultaneously, hyperglycemia

Inflamm-aging was recently affiliated with
the progression of diabetic complications.
Local cellular senescence together with
senescence-associated secretory
phenotype (SASP) are the main
contributors to inflamm-aging. However,
little is known about their involvement in
diabetic periodontitis. Gingiva is the first
line of host defense in the periodontium,
and macrophages are key SASP-carrying
cells. Here, we explored the molecular
mechanism by which hyperglycemia drives
the inflamm-aging in the gingival tissue of
diabetic mice and macrophages. We
demonstrated that hyperglycemia
increased the infiltrated macrophage
senescence in gingival tissue of diabetic
elevated the local burden of senescent
cells in gingival tissue and induced the
serum secretion of SASP factors in vivo.
Moreover, in vitro, high glucose induced
macrophage senescence and SASP
factors secretion through phosphorylation
of NLRC4, which further stimulated the NF-
κB/Caspase-1 cascade via IRF8-
dependent pathway. Deletion of NLRC4 or
IRF8 abolished hyperglycemia-induced
cellular senescence and SASP in
macrophages. In addition, we found that
treatment with metformin inhibited NLRC4
phosphorylation and remarkably
decreased cellular senescence and SASP
in the context of hyperglycemia. Our data
demonstrated that hyperglycemia induces
the development of inflamm-aging in
gingival tissue and suggested that NLRC4
is a potential target for treatment of
diabetes-associated complications.
Introduction
Diabetes mellitus is a heterogeneous
group of disorders that affect millions of
people1. Hyperglycemia, the hallmark of
diabetes mellitus, is associated with a
range of complications, including
2 19
periodontitis . Diabetes increases the
susceptibility to periodontitis and leads to
more severe inflammation3. Recently,
inflamm-aging was linked to the etiology of
diabetic complications. Cellular
senescence and senescence-associated
secretory phenotype (SASP) are the key
contributors to inflamm-aging4. Evidence
indicates that senescent cell burden
increases in tissues that undergo
functional damage in diabetes, such as the
skin, pancreas, and kidney5-9. Cellular
senescence is crucial in the acquisition of
the SASP, which also mediates tissue
dysfunction through a chronic secretion of
proinflammatory cytokines, leading to
diabetic complications10-12. The SASP
factors inversely affect the surrounding
cells and accelerate cellular senescence13.
In periodontium, the gingiva plays an
important role as a mechanical barrier
against bacterial invasion and as a part of
the innate immune response to infectious
inflammation14. The senescent cell burden
in tissue and SASP may directly contribute
to diabetes-related gingival dysfunction
and susceptibility to periodontal pathogens
infection. However, the underlying
pathogenesis has not yet been elucidated.
In vivo data have suggested that
macrophages are key SASP-carrying
cells13, 15. Concurrently, macrophages are
innate immune cells in gingiva16 that may
be key to SASP factors secretion. However,
how high glucose induces cell senescence
and SASP in macrophages remains
elusive.
The main pathways involved in cellular
senescence regulation, including the
nucleotide-binding and oligomerization
domain (NOD)-like receptor (NLR) family
inflammasomes and interleukin (IL)-1β
pathway, are master modulators of aging17-
. The inflammasomes are cytosolic
multiprotein complexes that recruit and
activate Caspase-1, a key protease that
triggers secretion of the inflammatory
cytokine IL-1β20. The inflammasomes can
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be activated by many danger signals,
including hyperglycemia21. Recent
publications provided new evidence that
the NLR family caspase activation and
recruitment domain (CARD)-containing 4
(NLRC4) correlates with inflamm-aging22.
Moreover, NLRC4-driven IL-1β production
is critical for the progression of diabetic
nephropathy23. NLRC4 is also reported to
play a vital role in the progression of brain
inflamm-aging24. Hence, the activation of
NLRC4 in diabetes may induce cell
senescence and SASP in gingiva.
In this study, a mouse model of
hyperglycemia was used to investigate the
local burden of senescent cells in gingiva
and SASP in serum. Moreover, we utilized
RAW264.7 macrophage cell lines to
dissect the mechanistic role of NLRC4 in
the progression of hyperglycemia-induced
cell senescence and SASP in vitro. In
addition, treatment with metformin in vivo
and in vitro allowed to gain preliminary
insights into therapeutics for ameliorating
the high local burden of senescent cells
and SASP in gingiva.
Result
1. Hyperglycemia increases senescent
cell burden in gingival tissue and
induces SASP in serum of diabetic
mice.
To eliminate the influence of age and
focus on the effect of glucose only, 4-week-
old mice were rendered hyperglycemic by
successive injections of streptozotocin
(STZ) (Fig 1A). As shown in Fig. 1B, the
blood glucose of the diabetic mice
significantly increased between the 7th and
11th week and stabilized at around 30 mM
between the 11th and 17th week. Western
blot analysis confirmed that p16 and p21
expression levels gradually increased in
gingival tissue of diabetic mice from 7th to
11th week alongside the rise of blood
glucose, and elevated significantly from
13th to 17th week compared to the control
mice (Fig. 1C). Immunohistochemical (IHC)
staining of gingival tissue revealed that the
p16-positive and p21-positive cells
gradually increased from 9th to 17th week
and significantly increased from 11th to 17th
week under about 30 mM blood glucose in
diabetic mice compared to the control (Fig.
1D). Then, we determined whether
hyperglycemia induced SASP in diabetic
mice. We identified typical SASP factors in
the serum of mice using Luminex Assay
Customization Tool. As shown in Fig. 1E,
expression of numerous cytokines
changed to varying degrees. Pro-
inflammatory cytokines, such as IL-1β,
tumor necrosis factor (TNF)-α, IL-6, matrix
metalloproteinase (MMP)-2, MMP-8, and
advanced glycation end products (AGEs),
gradually increased in the diabetic mice.
However, anti-inflammatory cytokines like
IL-10 and adiponectin were significantly
lower in diabetic than in control mice.
Moreover, we investigated whether
macrophages in the gingiva of diabetic
mice were senescent. As shown in Fig. 1F,
immunofluorescence staining revealed
increased infiltration of macrophages
(F4/80-positive cell) in gingival tissue of
diabetic mice, and significantly higher rate
of p16-positive cells among these
macrophages.
2. High glucose induces cellular
senescence and SASP in macrophage.
In vivo data revealed higher infiltration
of senescent macrophages in gingival
tissue of diabetic mice. Next, we
investigated whether high glucose induced
cellular senescence and SASP in vitro
using the macrophage cell line RAW 264.7.
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The activity of senescence-associated β-
galactosidase (SA-β-gal), a canonical
marker of cell senescence25, gradually
increased in macrophages exposed to 10-
30 mM glucose for 24 hours (Fig. 2A).
Concomitantly, western blot also showed
that p16 and p21 were gradually
upregulated with increasing glucose
concentrations (Fig. 2B). The rate of SA-β-
gal-positive (blue-stained) cells and the
expression levels of p16 and p21 under 30
mM glucose for 24 hours were significantly
higher than under 5 mM glucose for 24
hours and under 30 mM glucose for 6
hours (#p<0.05, ##p<0.01 and *p<0.05,
**p<0.01, respectively) (Fig. 2A & B). In
addition, Luminex analysis (Fig. 2C)
revealed that, within the SASP factors, pro-
and anti-inflammatory cytokines
significantly increased and decreased,
respectively, after high glucose stimulation.
The trends were consistent with the SASP
of diabetic mice in vivo. We concluded that
30 mM glucose treatment for 24 hours
might be the optimal condition for high
glucose-induced cellular senescence and
SASP in macrophages. Then, we
determined whether the growth of
senescent macrophages was arrested.
Using 5-Ethynyl-2-deoxyuridine (EdU) as a
cell-proliferation marker, macrophages
exposed to 30 mM glucose for 24 hours
show significantly less EdU-positive cells
than those exposed to 5 mM glucose for 24
hours and 30 mM glucose for 6 hours
(##p<0.01 and **p<0.01, respectively; Fig.
2D).
3. High glucose activates the
phosphorylation of NLRC4.
NLRC4 may be a key factor in the
regulation of inflamm-aging18,19,22,24 and
IRF8 is required for activation of NLRC4
inflammasome26. Hence, we determined
the activation of NLRC4 under
hyperglycemia-induced cellular
senescence microenvironment in vivo and
in vitro by analysis of the target proteins.
Western blot results revealed that IRF8
and NLRC4 levels were higher in gingival
tissue of diabetic than control mice
between the 9th and the 17th week (Fig. 3A).
IHC staining also showed significantly
higher rate of IRF8- and NLRC4-positive
cells in gingival tissue of diabetic compared
to control mice in the 17th week (Fig. 3B).
Interestingly, phosphorylation of NLRC4 is
critical for inflammasome activation27,28.
Western blot results revealed that the
phosphorylation of NLRC4 significantly
increased in gingival tissue of diabetic mice
compared to control mice between the 9th
and 17th week (Fig. 3 A). In vitro, western
blot showed that the levels of IRF8, p-
NLRC4 and NLRC4 gradually increased in
macrophages exposed to 10-30 mM for 24
hours (Fig. 3C). Accordingly, IRF8, p-
NLRC4, and NLRC4 levels under 30 mM
glucose for 24 hours were significantly
higher than under 5 mM glucose for 24
hours and under high glucose for 6 hours
(#p<0.05, ##p<0.01 and *p<0.01, **p<0.01,
respectively). Immunofluorescence
staining revealed that, in macrophages
exposed to 30 mM glucose for 24 hours,
IRF8 and NLRC4 increasingly
accumulated in the cytoplasm and formed
a ring-like structure (Fig. 3D).
Simultaneously, NLRC4 could recruit and
activate Caspase-120. As shown in Fig. 3E,
diabetic mice have significantly higher
levels of Caspase-1 p45 and p20 than
control mice in the 17th week. In vitro, 30
mM glucose for 24 hours could significantly
increase the expression of Caspase-1 p45
and p20 compared to 5 mM glucose for 24
hours and 30 mM glucose for 6 hours
(#p<0.05, ##p<0.01 and *p<0.01, **p<0.01,
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respectively) (Fig. 3F). As reported, SASP
is mostly induced by NF-κB29, 30.
Consistently, western blot analysis
revealed that high glucose could increase
the expression of NF-κB in vivo (Fig. 3E)
and in vitro (Fig. 3F).
4. Deletion of IRF8 or NLRC4 abolishes
high glucose-induced cell senescence
and SASP in macrophages.
To pinpoint the contribution of NLRC4
to hyperglycemia-induced cell senescence
and SASP, we knocked out NLRC4 in
macrophages with the CRISPR/Cas9
system. Confirming previous results, the
24-hour treatment with 30 mM glucose
increased the rate of SA-β-gal-positive
cells (Fig. 4A) and accumulated p16 and
p21 in the cytoplasm of macrophages (Fig.
4B). However, the same treatment in
NLRC4-/- macrophages distinctly
downregulated the activity of SA-β-gal (Fig.
4A) and decreased the cytoplasmic
expression of p16 and p21 (Fig. 4B).
Among the SASP factors, pro- and anti-
inflammatory cytokines significantly
decreased and increased, respectively
(Fig. 4C). Simultaneously, the expression
of Caspase-1 p45, Caspase-1 p20, and
NF-κB was downregulated (Fig. 4D).
Interestingly, knockout of NLRC4
decreased the expression level of IRF8
that is recognized as upstream of NLRC426
(Fig. 4D). Furthermore, among IRF8-/-
macrophages, cells showing positive SA-
β-gal signal (Fig. 4A) and cytoplasmic
expression of p16 and p21 (Fig. 4B) also
significantly decreased. In addition, IRF8
knockout dephosphorylated NLRC4 and
decreased the expression levels of NLRC4,
Caspase-1 p45, Caspase-1 p20, and NF-
κB (Fig. 4D). Meanwhile, the SASP factors
showed similar changes as in NLRC4-/-
macrophages exposed to 30 mM for 24
hours (Fig. 4C). Thus, high glucose could
not induce cellular senescence and SASP
in IRF8-/- and NLRC4-/- macrophages.
5. Metformin alleviated senescent cell
burden in gingival tissue and SASP
factors secretion in serum of diabetic
mice.
Metformin was recently revealed to
reduce the risk and progression of
inflamm-aging31. Here, we examined
whether metformin could attenuate
hyperglycemia-induced cellular
senescence and SASP. In vivo, metformin
had a remarkable hypoglycemic effect for
diabetic mice (Fig. 5B). IHC revealed that
p16- and p21-positive cells decreased in
gingival tissue of diabetic mice treated with
metformin (Fig. 5C). Accordingly, western
blot analysis in these mice showed
significant downregulation of p16 and p21
in gingival tissue (Fig. 5F). Simultaneously,
metformin could markedly ameliorate
proinflammatory cytokines IL-1β, IL-6,
TNF-α, MMP-2, MMP-8, and AGEs, and
increase the levels of IL-10 and
adiponectin (Fig. 5D). As shown in Fig. 5E,
immunofluorescence staining indicated
that metformin decreased the infiltration of
senescent macrophages (F4/80/p16-
positive cells) in gingival tissue of diabetic
mice. Interestingly, metformin could also
dephosphorylate and inhibit the expression
of NLRC4 (Fig. 5F). Finally, metformin
treatment also downregulated IRF8,
Caspase-1 p45, Caspase-1 p20, and NF-
κB in gingival tissue (Fig. 5F).
6. Metformin attenuated hyperglycemia-
induced cellular senescence and
SASP through the dephosphorylation
of NLRC4 in macrophage.
In vitro, co-treatment with 10 mM
metformin and 30 mM glucose for 24 hours
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in macrophages decreased SA-β-gal-
positive cells (Fig. 6A) and reduced the
expression and accumulation of p16 and
p21 in the cytoplasm (Fig. 6B). Meanwhile,
metformin altered the SASP factors by
decreasing the pro-inflammatory and
increasing the anti-inflammatory cytokines
(Fig. 6C). Consistently with the in vivo
results, metformin decreased the
phosphorylation of NLRC4 and the
expression levels of IRF8, NLRC4,
Caspase-1 p45, Caspase-1 p20, and NF-
κB in macrophages exposed to high
glucose for 24 hours (Fig. 6D). Finally, as
shown in Fig. 6E, metformin also
decreased the accumulation of IRF8 and
NLRC4 in the cytoplasm.
Discussion
In this study, we investigated the
biological mechanism by which
hyperglycemia aggravated the inflamm-
aging of macrophages in gingival tissue via
NLRC4 phosphorylation and induced a
high local burden of senescent cells and
the secretion of SASP factors. The
activation of NLRC4 stimulated the NF-
κB/Caspase-1 cascade via IRF8-
dependent signaling. Moreover, we found
that metformin modulated hyperglycemia-
induced cellular senescence and SASP
through a NLRC4-dependent pathway.
Therefore, we identified a NLRC4-
mediated causal link between
hyperglycemia and the inflamm-aging in
gingival tissue and macrophages.
Periodontitis is one of the major
complications of diabetes mellitus and
hyperglycemia increases the susceptibility
to and severity of this condition32. In the
development of this disease, the gingiva is
a critical component of innate immunity
and provides protection as a mechanical
barrier against microbial invasion14.
Growing epidemiological evidence
demonstrated that the accumulation of
senescent cells in tissues was associated
with diabetic complications33. Meanwhile,
senescent cells secrete a number of
extracellular factors leading to SASP, that
can reinforce senescence and amplify
inflammation13,34. Cellular senescence and
SASP could cause the chronic, low-grade,
systemic inflammation occurring in the
absence of overt infection (sterile
inflammation) defined as “inflamm-
aging”4,35,36, that recently emerged as a
possible pathogenesis of tissue
9,33
dysfunction . Macrophages are
important immune cells in gingiva 16,37 as
13,15
SASP-promoters . When stimulated by
internal environmental factors including
high glucose, macrophages may be
induced senescence, then oversecrete
various SASP factors and propagate
senescence 13,38,39. Hyperglycemia may
play an important role in the accumulation
of senescent cells and secretion of SASP
in gingival tissue, thus causing gingival
dysfunction. However, the role of cellular
senescence and SASP in the
pathogenesis of diabetic complications
have not been fully explored.
In our study, we established the
diabetic mouse model using 4-week-old
mice to eliminate the effect of age and
explore the single effect of hyperglycemia.
We found that the senescent cells
significantly increased in gingival tissue of
diabetic mice from 9th week to 17th week.
This indicated that hyperglycemia induced
a high local burden of senescent cells in
gingival tissue that may consequently
damage its barrier function33.
Simultaneously, we found that
hyperglycemia significantly increased the
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infiltration of senescent macrophages in
gingival tissue of diabetic mice. In the
serum of diabetic mice, we observed that
TNF-α, IL-1β, IL-6, MMP-2, MMP-8, and
AGEs significantly increased compared to
control mice. These are the pivotal
proinflammatory cytokines involved in
aggravating inflammation and tissue
dysfunction in diabetic periodontitis40-42.
Conversely, we found decreased secretion
of IL-10 and adiponectin, which play a
central role in limiting inflammation in
diabetic periodontitis43,44. As the key
SASP-carrying cells13,15, senescent
macrophages could secrete numerous
SASP factors in gingival microenvironment
to reinforce and propagate senescence,
amplifying the senescent cell burden and
SASP factors secretion. Gingival crevicular
fluid (GCF), the gingival microenvironment,
is mainly originated from the serum45.
Despite the discrepancy in cytokine
concentrations and the lack of correlation
among different biological matrices, an
overall high agreement in the detection of
cytokine levels was observed in serum and
GCF46. SASP in serum of diabetic mice
could reflect the average inflammatory
level of gingival microenvironment.
Macrophages may be the principle source
of SASP factors in gingival
microenvironment, and induce a high local
burden of senescent cells in gingival tissue.
Moreover, in vitro, 30 mM glucose for 24
hours could increase p16 and p21
expression and SA-β-gal activity, and
reduce cell proliferation in macrophages,
demonstrating that high glucose could
accelerate macrophage senescence.
SASP changes in the supernatant of
macrophages exposed to high glucose
were consistent with those observed in
vivo. These results implied that
hyperglycemia could trigger the inflamm-
aging in macrophages, and suggesting a
potential new contributor to the
development of low-grade inflammation
and possibly diabetic periodontitis.
The molecular mechanism of
hyperglycemia-induced cellular
senescence and SASP is not clear. The
NLRC4 inflammasome has recently been
implicated in inflamm-aging18,19,22,24.
Although the inflammasome can be
activated by different danger signals,
including high glucose21, the only NLRC4
activators identified to date are bacterial
flagellin and components of bacterial
secretion systems27. Previous studies
revealed that Salmonella typhimurium
could induce the phosphorylation of
NLRC4 Ser53327,28. Unlike wild-type
NLRC4, Ser533A mutant could not
activate Caspase-1 in NLRC4-/-
macrophages in response to S.
typhimurium, indicating that Ser533
phosphorylation is critical for NLRC4
function27,28. In our study, hyperglycemia
upregulated the expression of NLRC4 and
induced its phosphorylation on Ser533 in
the gingival tissue of diabetic mice, while
also activating Caspase-1. Similarly, in
macrophages, high glucose
microenvironment also induced the
expression and phosphorylation of NLRC4
and activated Caspase-1. In turn, activated
Caspase-1 stimulated IL-1β productions in
vivo and in vitro. However, the mechanism
underlying NLRC4 phosphorylation
remains unclear. We suggest that NLRC4
phosphorylation could be promoted by a
hyperglycemia-associated protein with
homologous function to the known NLRC4-
activating bacterial components23,47.
Recent research revealed that IRF8 is an
upstream regulator of NLR family
apoptosis inhibitory proteins (NAIPs),
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ultimately contributing to NLRC4 activity,
but is dispensable for the activation of
other inflammasome proteins, including
NLRP326. In our research, we revealed that
IRF8 levels increased consistently with p-
NLRC4 and NLRC4 both in vivo and in vitro.
Moreover, in diabetic mice and
macrophages, high glucose also
upregulated NF-κB. Evidences indicated
that NF-κB regulated the SASP by
switching of inflammatory
pathways 29,30,48,49. In turn, NF-κB may be
activated by IL-1β binding to IL-1
receptors50. In addition, NLRC4 may
activate TAK1 through CRAD-CARD
domains to increase the expression of NF-
κB51. Therefore, the activation of NLRC4
may regulate the SASP through NF-κB.
To determine the link between NLRC4
activation and inflamm-aging in the context
of high glucose, we knocked out NLRC4 in
macrophages and observed that cellular
senescence and SASP improved in high
glucose microenvironment. Interestingly,
IRF8 was downregulated after knockout of
NLRC4, indicating a feedback regulation
between NLRC4 and IRF8. As expected,
knockout of IRF8 downregulated the
expression and phosphorylation of NLRC4
while simultaneously decreasing the
expression of Caspase-1 and NF-κB. As
IRF8 is required for phosphorylation of
NLRC4, we speculate it may be a
homologue to the NLRC4-activating
bacterial components. Finally, knockout of
IRF8 also ameliorated cellular senescence
and SASP. Together, these results
indicated that NLRC4 may be the key
target of high glucose-induced cellular
senescence and SASP.
Metformin was shown to possess an
anti-inflammatory and anti-aging function
besides its hypoglycemic effect52,53.
Louroab TM et al. found that metformin
treatment in type 2 diabetic mice could
reduce serum levels of proinflammatory
cytokines and improve the diabetic
nephropathy inflammatory response54. The
“Metformin, Anti-aging” clinic trial
supported by the U.S. Food and Drug
Administration (FDA) was considered a
landmark of aging research55. Concurrently,
metformin was revealed to inhibit the
expression of multiple inflammatory
cytokines during cellular senescence and
block NF-κB activity56. Our findings provide
a rationale to develop specific therapeutics
for treating hyperglycemia-induced cellular
senescence and SASP. In diabetic mice,
metformin could decrease the infiltration of
senescent macrophages while also
alleviating the burden of senescent cells in
gingiva and the SASP in serum.
Interestingly, in vitro, metformin treatment
could attenuate high glucose-induced
cellular senescence and SASP in
macrophages. This suggests that
metformin may directly dephosphorylate
NLRC4 in high glucose microenvironment;
however, the detailed mechanism should
be further studied. Overall, our results
indicated that metformin may have and
anti-inflamm-aging effect through direct
inhibition of NLRC4 and indirect
hypoglycemic effect.
Collectively, we observed that
hyperglycemia-induced macrophages
inflamma-ging accelerated gingival
senescence through IRF8/NLRC4
signaling (Fig. 7), which was in turn
amplified by NF-κB. We further
demonstrated that metformin ameliorated
hyperglycemia-induced senescence by
inhibiting the phosphorylation of NLRC4,
highlighting the pivotal role of this post-
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translational modification in inflamm-aging.
Currently, the treatment of diabetic
periodontitis is limited to blood glucose
control and periodontal basic treatment.
Identification of the underlying
mechanisms will allow us to better
understand the pathogenesis of diabetic
periodontitis and help to develop the novel
targeting-NLRC4 anti-inflamm-aging
therapeutic strategies.
Materials and Methods
1. Animals
Four-week-old male C57BL/6 wild-type
mice were obtained from the Model Animal
Research Center of Nanjing University
(Nanjing, China). Animal treatment was
approved by the institutional committee for
animal use and care in Experimental
Animal Center of West China Second
University Hospital. Mice were given free
access to a standard laboratory diet and
tap water and caged individually under
controlled temperature (23±2°C) and
humidity (55±5%) with an artificial light
cycle. They were randomly divided into
control mice (N group, n=40), diabetic mice
(D group, n=40), and diabetic mice treated
with metformin (DM group, n=8). At 6
weeks of age, the mice in D and DM
groups were intraperitoneally injected with
STZ (Sigma–Aldrich, USA) at a dose of 55
mg/kg for 5 consecutive days. At 8 weeks
of age, DM group was intragastrically
infused with metformin (Sigma–Aldrich,
USA) at a dose of 300 mg/kg every day
until sacrifice. Eight mice in N and D group
were sacrificed every two weeks from 9th to
17th week of age, while DM group was
sacrificed at 17th week age.
2. Cell cultures and stimulation
RAW 264.7 murine macrophage cell
line was obtained from American Type
Culture Collection (ATCC, Rockville, MD,
USA). The cells were grown in DMEM
(Gibco, USA) supplemented with 10% fetal
bovine serum (FBS; Gibco, USA) and 1%
penicillin-streptomycin (HyClone, USA)
and cultured at 37°C in a humidified
incubator in an atmosphere of 5% CO2.
The cells were incubated with different
concentrations of glucose (5-30 mM) and
metformin (10 Mm; Sigma, USA) for 6 or
24 hours respectively.
3. CRISPR / Cas9 KO Plasmid
To obtain knockout cells, we used the
NLRC4 (sc-418306; Santa Cruz, USA) or
IRF8 (sc-421016; Santa Cruz, USA)
CRISPR/Cas9 KO Plasmid sequences to
target the NLRC4 or IRF8 DNA following
the manufacturer’s recommended protocol.
In a 6-well tissue culture plate, 1.5 - 2.5 x
105 cells were seeded in 3 ml of antibiotic-
free standard growth medium per well.
Twenty-four hours prior to transfection,
cells were grown to 80% confluency. Cells
were then transfected with control, NLRC4
or IRF8 CRISPR / Cas9 KO Plasmid and
incubated for 24 hours under normal
culture conditions. Media was replaced 24
hours post-transfection and cells were
stimulated with 5 and 30 mM glucose for
24 hours.
4. Western blot
Proteins were extracted from the RAW
264.7 cells or mouse mandible gingival
tissues following the recommended
protocol by Total Protein Extraction Kit
(Signalway Antibody LLC, USA). Fifteen
mg of protein from each sample was
subjected to 5% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-
PAGE) and transferred to nitrocellulose
membranes by electro-blotting. The
membranes were incubated overnight at
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4°C with primary antibodies against β-actin
(1:1000; ta-09; ZSGB-BIO, CN), p-NLRC4
(1:500; NM5491; ECMbiosciences),
NLRC4 (1:1000; ab99860; Abcam, USA),
IRF8 (1:1000; sc-365042; Santa Cruz,
USA), Caspase-1 (1:1000; ET1608-69;
HUABIO, CN), NF-κB p65 (1:1000; sc-
514451; Santa Cruz, USA), p16 (1:1000;
sc-166760; Santa Cruz, USA), and p21
(1:1000; sc-166630; Santa Cruz, USA)
followed by either secondary anti-rabbit
(1:5000; 1706515; BIO-RAD, CN) or anti-
mouse (1:5000; ZB-2305; ZSGB-BIO, CN)
antibody for 1 hour at room temperature.
The signals were revealed using a West
Pico Chemiluminescent Substrate System
(SuperSignal, BioSpectrum® 310 Imaging
System, USA).
5. SA-β-Gal staining
SA-β-Gal staining was performed
using the SA-β-Gal staining kit (C0602;
Beyotime Biotechnology, Shanghai, CN)
according to the manufacturer’s
instructions. Cells were washed three
times with phosphate-buffered saline (PBS)
and fixed with 4% (w/v) paraformaldehyde
(PFA) for 15 minutes at room temperature.
Then, cells were incubated overnight at
37°C in darkness with the working solution
containing 0.05 mg/ml X-gal. After being
rinsed with PBS, cells were observed at the
microscope for the development of the blue
coloration, at a magnification of 20×. The
percentage of SA-β-Gal-positive cells was
calculated from five random fields by
ImageJ software.
6. EdU staining
Cell proliferation was assessed using
an EdU Labeling/Detection Kit (C10310-1;
RiboBio, CN). RAW 264.7 cells were
cultured in 6-well plates at 1×106 cells per
well and transfected with 5 or 30 mM
glucose for 6 or 24 hours. Then, 50 mM
EdU labeling media was added to the 6-
well plates, and they were incubated for 2
hours at 37°C under 5% CO2. After
treatment with 4% PFA and 0.5% (v/v)
Triton X-100, cells were stained with anti-
EdU working solution. Hoechst 33342 was
used to label cell nuclei. The percentage of
EdU-positive cells was calculated from five
random fields by imageJ
7. Immunocytochemistry
After 15-min fixation in 4% PFA and 10-
min incubation in 0.1% Triton X-100 at
room temperature, the cells were blocked
in PBS containing 4% (v/v) goat serum and
1% (v/v) glycerol for 1 hour at room
temperature. Cells were then incubated
overnight at 4°C with primary antibodies
against p16 (1:100; sc-166760; Santa Cruz,
USA) and p21 (1:100; sc-166630; Santa
Cruz, USA), NLRC4 (1:100; sc-514658;
Santa Cruz, USA), and IRF8 (1:100; sc-
365042; Santa Cruz, USA) followed by
corresponding secondary antibodies, anti-
mouse Alexa Fluor 488 (1:200; ab150113;
Abcam, USA) for 1 hour at 37°C and 4′,6-
diamidino-2-phenylindole (DAPI) for 2
minutes at 37°C for nuclear staining . The
images were obtained by the Pannoramic
MIDI (3D HISTECH, Hungary). The
fluorescence intensity was calculated by
ImageJ.
8. Luminex assay
Mouse serum and supernatant of
RAW264.7 cells were applied on Luminex
Assay Customization Tool (R&D Systems,
Minneapolis, MN, USA) following the
manufacturer's instructions. Cytokine-
specific changes between cohorts were
visualized by row-specific Z-scores in a
heatmap.
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9. Immunohistochemical staining
Mouse maxillary samples were fixed
with 4% PFA for 24 hours, decalcified with
10% Ethylene Diamine Tetraacetic Acid
(EDTA) solution for 30 days, changing the
solution every other day. The samples
were then dehydrated with the different
ethanol concentrations and subjected to
paraffin acetone and chloroform washes,
both at 60°C for 30 minutes, and three 1-
hour paraffin infiltrations at 60°C. Paraffin-
embedded samples were coronally
sectioned (4 μm) and stained with
antibodies against NLRC4 (1:100;
ab99860; Abcam, USA), IRF8 (1:100; sc-
365042; Santa Cruz, USA), p16 (1:100; sc-
166760; Santa Cruz, USA), and p21 (1:100;
sc-166630; Santa Cruz, USA ). After
washing with PBS for 3 times, the sections
were incubated with goat anti-mouse IgG
H&L secondary antibody (1:4000;
ab205719; Abcam, USA) and goat anti-
rabbit IgG H&L secondary antibody
(1:4000; ab ab205718; Abcam, USA) for
30 minutes at room temperature.
Immunostaining was performed using a
DAB kit (Solarbio, CN), and counterstain
hematoxylin. The images were acquired by
Aperio Digital Pathology Slide Scanners
(Leica, Germany). The percentage of
positive cells was calculated by ImageJ.
10. Immunofluorescence staining
Mouse maxillary sections were de-
paraffinized for 2 h at 65°C, incubated in
xylene, and hydrated through a graded
series of alcohols. Heat-mediated antigen
retrieval was performed using citrate buffer
(pH 6.0). After washing three times for 5
minutes with PBS, the samples were
incubated for 15 minutes with PBS
containing 0.3% Triton and 2% goat serum,
washed again three times with PBS, and
incubated with PBS containing 10% goat
serum for 30 minutes. The sections were
incubated overnight at 4°C with antibodies
against F4/80 (1:200; sc-377009; Santa
Cruz, USA) and p16 (1:100; sc-166760;
Santa Cruz, USA). After washing three
times for 20 minutes, the sections were
incubated with a goat anti-rabbit Alexa
Fluor 488 (1:300; A-27034; Life
Technologies, USA) targeting F4/80
antibody, together with a goat anti-mouse
Alexa Fluor 568 (1:300; A-21134; Life
Technologies, USA) targeting p16 antibody.
After 1 hour of incubation, the sections
were washed and incubated with DAPI.
The images were obtained by the
Pannoramic MIDI (3D HISTECH, Hungary).
11. Statistical analysis
All tests were conducted in triplicate.
The data were analyzed as the means ±
standard errors of the mean (SEM).
Differences between data sets were
assessed by Dunnett's t-test or by the
student's t-test. A value of p<0.05 was
considered significant.
Funding
This work was supported by the
National Natural Science Foundation of
China (81870779), the International
Scientific Cooperation and Exchanges
Project of Sichuan Province
(2017HH0078), and the International
Cooperation Project of Chengdu Municipal
Science and Technology Bureau (2015-
GH02-00035-HZ).
Duality of Interest
The authors declare that they have no
potential conflicts of interest relevant to this
study.
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Fig legends

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FIG1 Hyperglycemia increased the gingival senescent burden and induced serum SASP
in diabetic mice. A: All protocols were performed strictly according to the procedure. C57
mice were rendered diabetic by STZ injections and sacrificed every two weeks. B: Fasting
glucose levels were determined every two weeks from 5th week to 17th week. P value
between control mice (N group) and diabetic mice (D group) was shown (**p<0.01). C:
Western blot analysis showing p16 and p21 specific immunoreactivity in the gingival tissue
of N group and D group. Optical density (O.D.) values of p16 and p21 levels relative to β-
actin were represented in bar histograms. Data were mean ± SD; n = 3. *p<0.05, **p<0.01
vs. N group. Fig. 1C, Fig. 3A and Fig. 3E use the same band of β-actin because of the
identical protein sample in same western blot experiment. D: Immunohistochemistry using
antibody against p16 and p21 was analyzed in the gingival tissue of N group and D group.
Scale bar: 50 μm. The percentage of positive cells was represented in bar histograms.
Data were mean ± SD; n=3. *p<0.05, **p<0.01 vs. N group. E: The SASP factors in the
serum of N group and D group were determined every two weeks by Luminex Assay
Customization Tool and shown in the heatmap. F: Gingival tissues of N group and D group
were stained for immunofluorescence using an F4/80 antibody targeting macrophages (red)
and a p16 antibody (green). Nuclei were stained with DAPI (blue). Scale bar: 50 μm.

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FIG2 High glucose induced cellular senescence and SASP in macrophage derived from
RAW 264.7 cell. A: The activity of SA-β-gal was determined in macrophage exposed to
5~30 mM glucose for 6 or 24 hours. Scale bar: 100 μm. The rate of SA-β-gal-positive (blue-
stained) cells was represented in bar histograms. Data were mean ± SD; n=3. **p<0.01 vs.
30mM glucose for 6 hours. ##p<0.01 vs. 30mM glucose for 24 hours. B: The expression
levels of p16 and p21 in macrophage were analyzed by Western blot. O.D. values of p16
and p21 levels relative to β-actin were represented in bar histograms. Data were mean ±
SD; n = 3. *p<0.05, **p<0.01 vs. 30mM glucose for 6 hours. #p<0.05, ##p<0.01 vs. 30mM
glucose for 24 hours. Fig. 2B, Fig. 3C and Fig. 3F use the same band of β-actin because
of the identical protein sample in same western blot experiment. C: The SASP factors in
the supernatant of macrophage were determined by Luminex Assay Customization Tool
and shown in the heat map. D: Cell proliferation was detected using EdU detection kits to
analyze the incorporation of EdU during DNA synthesis. Scale bar: 100 μm. The
percentage of proliferating cells was represented in bar histograms. Data are mean ± SD;
n=3. **p<0.01 vs. 30mM glucose for 6 hours. ##p<0.01 vs. 30mM glucose for 24 hours.
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FIG3 NLRC4-related pathway was activated in the gingival tissue of diabetic mice and
macrophage exposed to high glucose for 24 hours. A: Western blot analysis showing IRF8,
p-NLRC4 and NLRC4 specific immunoreactivity in the gingival tissue of N group and D
group. O.D. values of IRF8, p-NLRC4 and NLRC4 levels relative to β-actin were
represented in bar histograms. Data were mean ± SD; n = 3. *p<0.05, **p<0.01 vs. N group.
B: Representative of immunohistochemical staining of IRF8 and NLRC4 on the gingival
sections from N group and D group. Scale bar: 50 μm. The percentage of positive cells
was calculated and represented in bar histograms. Data were mean ± SD; n=3. *p<0.05,
**p<0.01 vs. N group. C: The expression levels of IRF8, p-NLRC4 and NLRC4 in
macrophage were analyzed by Western blot. O.D. values of IRF8, NLRC4 and p-NLRC4
levels relative to β-actin were represented in bar histograms. Data were mean ± SD; n = 3.
*p<0.05, **p<0.01 vs. 30mM glucose for 6 hours. #p<0.05, ##p<0.01 vs. 30mM glucose for
24 hours. The black vertical line represents the splicing border, because there is a protein
marker between the 6 hours- and 24 hours- samples in one band. D: IRF8 and NLRC4
levels were measured by immunofluorescence staining. Scale bar: 50 μm. The
fluorescence intensity was represented in bar histograms. Data were mean ± SD; n=3.
*p<0.05, **p<0.01 vs. 5mM glucose for 24 hours. E: The expression levels of Caspase-1,
cleaved Caspase-1 and NF-κB were analyzed by Western blot in vivo. O.D. values of
Caspase-1, cleaved Caspase-1 and NF-κB levels relative to β-actin were represented in
bar histograms. Data were mean ± SD; n = 3. *p<0.05, **p<0.01 vs. N group. Fig. 1C, Fig.
3A and Fig. 3E use the same band of β-actin because of the identical protein sample in
same western blot experiment. F: The expression levels of Caspase-1, cleaved Caspase-
1 and NF-κB were analyzed by Western blot in vitro. O.D. values of Caspase-1, cleaved
Caspase-1 and NF-κB levels relative to β-actin were represented in bar histograms. Data
were mean ± SD; n = 3. *p<0.05, **p<0.01 vs. 30mM glucose for 6 hours. #p<0.05, ##p<0.01
vs. 30mM glucose for 24 hours. The black vertical line represents the splicing border,
because there is a protein marker between the 6 hours- and 24 hours- samples in one

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band. Fig. 2B, Fig. 3C and Fig. 3F use the same band of β-actin because of the identical
protein sample in same western blot experiment.

FIG4 High glucose was incapable of inducing cellular senescence and SASP in in IRF8 -/-
or NLRC4-/- macrophage exposed to 30 mM glucose for 24 hours. A: The activity of SA-β-
gal were determined in control, NLRC4-/- and IRF8-/- macrophage exposed to 5mM and
30mM glucose for 24 hours. Scale bar: 100 μm. The rate of SA-β-gal-positive cells was
represented in bar histograms. Data were mean ± SD; n=3. **p<0.01 vs. Control
CRISPR/Cas9 Plasmid (30mM glucose). B: p16 and p21 levels were measured by
immunofluorescence staining. Scale bar: 50 μm. The fluorescence intensity was
represented in bar histograms. Data were mean ± SD; n=3. **p<0.01 vs. Control
CRISPR/Cas9 Plasmid (30mM glucose). C: The change of SASP-associated factors is
displayed in the heatmap. D: Western blot analysis showing IRF8, p-NLRC4, NLRC4,
Caspase-1, cleaved Caspase-1, NF-κB, p16 and p21 specific immunoreactivity. O.D.
values of these proteins’ levels relative to β-actin were represented in bar histograms. Data
were mean ± SD; n = 3. *p<0.05, **p<0.01 vs. Control CRISPR/Cas9 Plasmid (30mM
glucose).

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FIG5 Metformin ameliorated the burden of senescent cells in gingival tissue and the SASP
in serum of diabetic mice. A: All protocols were performed strictly according to the
procedure. The diabetic mice were treated with the metformin (300mg/kg body weight,
everyday) from 9th week to 17th week. B: Fasting blood glucose levels were determined at
sacrifice (17th weeks) among control mice (N group), diabetic mice (D group) and diabetic
mice treated with metformin (DM group). Data are mean ± SD; n = 3. *p<0.05, **p<0.01 vs.
D group. C: Representative of immunohistochemical staining of p16, p21, IRF8 and NLRC4
on the gingival sections from N, D and DM group. Scale bar: 50 μm. The percentage of
positive cells was calculated and represented in bar histograms. Data were mean ± SD;
n=3. *p<0.05, **p<0.01 vs. D group. D: The SASP factors in the serum of N, D and DM
group were determined at sacrifice (17th week) by Luminex Assay Customization Tool and
shown in the heatmap. E: The gingival tissues of N, D and DM group mice were stained
for immunofluorescence using an F4/80 antibody targeting macrophages (red) and a p16
antibody (green). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. F: IRF8, p-
NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16 and p21 in the gingival
tissue of N, D and DM group were analyzed by Western blot. O.D. values of these proteins’
levels relative to β-actin were represented in bar histograms. Data were mean ± SD; n = 3.
*p<0.05, **p<0.01 vs. D group.

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FIG6 Metformin attenuated high glucose-induced cellular senescence and SASP in
macrophage exposed to high glucose (30mM) for 24hours. A: The activity of SA-β-gal were
determined in macrophage treated with low glucose (5mM) (N), high glucose (30mM) (HG)
and high glucose (30mM) + metformin (10mM) (HGM). Scale bar: 100 μm. The rate of SA-
β-gal-positive cells was calculated and represented in bar histograms. Data were mean ±
SD; n=3. *p<0.05, **p<0.01 vs. HG. B & E: p16, p21, IRF8 and NLRC4 levels were
measured by immunofluorescence staining. Scale bar: 50 μm. The fluorescence intensity
was represented in bar histograms. Data were mean ± SD; n=3. **p<0.01 vs. HG. C: The
change of SASP factors are displayed in the heatmap. D: The expression levels of IRF8,
p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16 and p21 in N, HG and
HGM were analyzed by Western blot. O.D. values of these proteins’ levels relative to β-
actin were represented in bar histograms. Data were mean ± SD; n = 3. *p<0.05, **p<0.01
vs. HG.
 Downloaded from http://www.jbc.org/ by guest on November 4, 2019
FIG7 The potential mechanism of high glucose induced-inflamm-aging in gingival tissue.
Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4
phosphorylation
Peng Zhang, Qian Wang, Lulingxiao Nie, Rui Zhu, Xinyi Zhou, Pengfei Zhao, Ning Ji,
Xing Liang, Yi Ding, Quan Yuan and Qi Wang
J. Biol. Chem. published online November 1, 2019

Access the most updated version of this article at doi: 10.1074/jbc.RA119.010648

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