Role of Aggregatibacter actinomycetemcomitans-induced autophagy in inflammatory response

Hyun Ah Lee,*‡ Mi Hee Park,*†‡ Yuri Song,*† Hee Sam Na,*† and Jin Chung*†

*
Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan 50162,

Korea

†
Oral Genomics Research Center, Pusan National University, Yangsan 50162, Korea

‡
The first two authors contributed equally to this work and its authorship.

Correspondence to: Jin Chung, Department of Oral Microbiology, School of Dentistry, Pusan

National University, Yangsan 50162, Korea. Tel: +82-51-510-8245, Fax: +82-51-510-8246, E-mail:

jchung@pusan.ac.kr

Authors Contribution

All authors have made substantial contributions to conception and design of the study. Hyun Ah Lee

and Mi Hee Park have been involved in data collection and analysis. Hyun Ah Lee, Mi Hee Park, Yuri

Song, Hee Sam Na, and Jin Chung have been involved in data interpretation, drafting the manuscript

and revising it critically and have given final approval of the version to be published

Running Title: A.a-induced autophagy reduced inflammation.

Summary sentence: A. actinomycetemcomitans-induced autophagy enhanced the bacterial

internalization and limited the release of IL-1

This is the author manuscript accepted for publication and has undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/JPER.19-0639.

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Abstract

· Background: Aggressive periodontitis is characterized by the early-onset and rapid progression of

periodontal destruction, and is closely associated with Aggregatibacter actinomycetemcomitans.

Autophagy is a conserved process that is critical for removing damaged proteins, organelles, and even

intracellular pathogens. Therefore, this study examined whether A. actinomycetemcomitans induces

autophagy. In addition, the relationship among autophagy, bacterial internalization, and inflammatory

molecules in periodontal aggressive inflammation was analyzed.

· Methods: The expression of autophagy-related proteins in human gingival tissue and THP-1 cells

was assessed by Western blot analysis. The formation of light chain 3 (LC3) puncta was examined by

confocal microscopy. The degree of bacterial internalization into the cells was determined by the

viable cell count. Phagocytosis and ROS production were measured using confocal microscopy and

flowcytometry.

· Results: When macrophages were infected with live A. actinomycetemcomitans, the autophagy

influx was activated by the increase in LC3-II, autophagy-related gene (ATG)5/12, and Beclin-1

expression through the Toll-like receptors (TLR) and extracellular signal-regulated kinase (ERK)

signaling pathways. The inhibition of A. actinomycetemcomitans-induced autophagy suppressed

bacterial internalization via phagocytosis into the macrophages and increased interleukin-1β (IL-1β)

production. Moreover, treatment with a ROS inhibitor inhibited these enhanced inflammatory

responses.

· Conclusions: A. actinomycetemcomitans-induced autophagy promotes bacterial internalization by

phagocytosis, which restricts the excessive inflammatory response by down-regulating IL-1β and

ROS production in macrophages. Thus, A. actinomycetemcomitans-induced autophagy and its role in

regulating the inflammatory response may play an important role in the aggressive periodontal

inflammatory process, and be a target for the development of new periodontal therapies.

Key words: Aggressive periodontitis, reactive oxygen species, inflammation and innate immunity,

pathogenesis of periodontal diseases

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Introduction

Periodontal disease is an inflammatory disease that is caused by exaggerated inflammatory

responses against periodontopathogens, which lead to the destruction of tooth-supporting tissues and a

loss of dentition.1 Periodontitis is classified into three types: chronic, aggressive, and necrotizing

periodontitis.2 The prevalence of chronic periodontitis increases with age, but most early onset forms

of periodontitis are typically aggressive and rapidly processing, classified as aggressive periodontitis.3

Aggressive periodontitis is characterized by major features, which are noncontributory medical

history, rapid attachment loss and bone destruction, and familial aggregation of cases.4

Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis are

strongly associated with the progression of periodontal disease and unsuccessful therapy.5 Among

these pathogens, A. actinomycetemcomitans is the causative pathogen found in localized aggressive

periodontitis.6 A. actinomycetemcomitans produces leukotoxin, a major virulence factor that is

cytotoxic to immune cells. Leukotoxin stimulates caspase-1 activation, which in turn, induces the

maturation and secretion of interleukin-1β (IL-1β) and IL-18.6

Autophagy is a conserved intracellular catabolic pathway that is important for cellular homeostasis

in response to a range of environmental and cellular stresses.7 Autophagy sequesters substrates into

autophagosomes, which fuse with lysosomes for the degradation of substrates.8 The formation of

autophagosomes is controlled by the ULK1 protein kinase complex, Vps34 lipid kinase complex,

which forms a complex with Beclin-1, and two ubiquitin-like conjugation systems, in which one

system is formed by autophagy-related gene 5 (ATG5) and ATG12, and the other is formed by light

chain 3 (LC3).9 Increasing evidence suggests that the dysregulation of autophagy is associated with

many human diseases, such as cancer, myopathies, neurodegenerative disorders, and inflammatory

diseases.10, 11 Several studies of crosstalk between autophagy and inflammation showed that

autophagy modulates the immune response, such as antigen presentation, cytokine secretion, and cell

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3
death.12-14 Furthermore, autophagy dysregulates inflammasome activation and inhibits excessive

inflammation.10 Recently, the immunological functions to target bacteria for autophagy have been

reported and a number of studies in this area have increased dramatically.15-17

Autophagy plays critical roles in protecting the host from numerous infectious diseases by

providing a mechanism for the clearance of bacterial pathogens.9 Nevertheless, bacteria modulate the

autophagy through the secretion of virulence-associated proteins during infection, even though

autophagy promotes the degradation of effectors/toxins secreted by bacteria.18 Autophagy is involved

in inflammatory disease, but much less so in periodontitis.10 Increased levels of autophagy gene

expression are found in the peripheral blood mononuclear cells from periodontitis patients, and

lipopolysaccharides (LPS) from P. gingivalis induces autophagy by enhancing the production of ROS

in human gingival fibroblasts.19 Although several studies have shed some light on the relationship

between autophagy and periodontitis,19, 20 the precise role of periodontopathogen-induced autophagy

is not completely understood. Furthermore, most of these studies focused on the relationship between

autophagy and chronic periodontitis;19, 21, 22 hence, the role of autophagy in aggressive periodontitis is

largely unknown.

This study examined the role of autophagy induced by A. actinomycetemcomitans in periodontal

inflammation. A. actinomycetemcomitans induced autophagy by upregulating the autophagic signaling

molecules in the gingival samples of aggressive patients and THP-1-derived macrophages. In

addition, A. actinomycetemcomitans-induced autophagy increased bacterial internalization into the

cells via phagocytosis, leading to a suppression of the inflammatory response in THP-1-derived

macrophages.

Materials and Methods

Gingival tissue sampling, cell lines, and compounds

The gingival tissues were obtained from healthy controls (n=4; aged 28 to 38 years; mean age: 33

years) and patients (n=5; aged 35 to 42 years; mean age: 39 years) who were scheduled to undergo

treatment at the Department of Periodontology in Pusan National University (South Korea). The
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clinical examination was performed by a trained examiner. The aggressive periodontitis tissue was

obtained from sites showing specific signs of gingival inflammation and a probing depth of ≥ 5 mm.

All patients met the following inclusion criteria: they had not taken antibiotics or anti-inflammatory

drugs in the previous six months, they were not affected by immunodeficiency, and had undergone no

previous periodontal treatment. Exclusion criteria were pregnancy, lactation, smoking, previous

subgingival periodontal therapy, and any systemic condition that could affect the progression of

periodontal disease (e.g. diabetes and immunological disorders) in the previous six months. All

donors provided written informed consent, and their rights were protected according to the protocol

that was reviewed and approved by the Institutional Review Board of Pusan National University (IRB

No. PNUDH-2017-023) and was conducted following the guidelines of the Helsinki Declaration of

1975, as revised in 2013. Gingival samples were collected from both healthy and periodontitis

patients after administering the appropriate local anesthetic. The gingival tissue specimens were

rinsed thoroughly with a sterile PBS, transferred to microtubes, and stored at -80°C until examined.

Human THP-1 monocytic cells were cultured in RPMI 1640 medium* with 10% heat-inactivated

fetal bovine serum (FBS)* and 1% antibiotic-antimycotic.* Human embryonic kidney 293 (HEK293)

cells were cultured in DMEM* with 10% heat-inactivated FBS and 1% antibiotic-antimycotic. THP-1

cells were differentiated into macrophage-like cells with 50 ng/ml of phorbol 12-mystristate 13-

acetate (PMA).† The PMA-differentiated THP-1-derived macrophages were pretreated with 2 mM of

3-methyladenine (3-MA)† for 30 minutes and infected with A. actinomycetemcomitans for 24 hours.

Bacterial culture

A. actinomycetemcomitans was grown in Tryptic soy broth‡ with a 1% yeast extract.‡ An OD of 0.25

(650 nm) was found to correlate with 109 CFU/mL. The bacteria were washed and resuspended in

RPMI media to infect the PMA-differentiated THP-1-derived macrophages at a MOI (multiplicity of

infection) of 1:1, 10, 50, or 100.

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5
Western blot analysis

The cells were lysed in RIPA buffer§ and the total cell lysates were analyzed by immunoblot.

Subsequently, 50 μg of the protein samples were separated using 15% sodium dodecyl sulfate-

polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The

blots were incubated with the specific primary antibodies as follows: LC3-I/II, ATG5/12, Beclin-1,

Toll-like receptors 2 (TLR2), TLR4, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), -actin;‖

mitogen-activated protein kinase (MAPK), phospho-MAPK, apoptosis-associated speck-like protein

containing a caspase recruitment domain (ASC), absent in melanoma 2 (AIM2);§ nucleotide-binding

oligomerization domain-like receptor containing pyrin domain 3 (NLRP3).¶ Enhanced

chemiluminescence was used for signal detection, and the signals were visualized using a Super

Signal West Femto maximum sensitivity substrate# on a LAS-4000 Immuno-Image Analyzer.**

ImageJ software was used to quantify the band densities relative to those of GAPDH or -actin.

RNA interference assay

Human small interfering RNAs (siRNAs) for TLR2, TLR4, and non-targeting control

oligonucleotides were purchased from Qiagen.†† The sequences of each siRNA oligonucleotide in this

pool are as follows: TLR2 siRNA, SI00050029; TLR4 siRNA, SI04951149; and non-targeting control

siRNA oligonucleotides, 1027281. For the siRNA experiments in THP-1 cells, the cells were seeded

in 6-well plates at a density of 1ⅹ106 cells per well, and transfected with each siRNA oligonucleotide

for 24 hours using the Attractene transfection reagent†† in 1 mL of RPMI. The transfected cells were

differentiated in the presence of 50 nM PMA for 24 hours. The siRNA-transfected cells were infected

with A. actinomycetemcomitans for 18 hours.

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Autophagy analysis by LC3 monitoring

To monitor the formation of LC3 puncta, the HEK293 cells were seeded in 8-well-chamber slides

and transfected with the fluorescent-fusion plasmid (mCherry-GFP-LC3) using polyethylenimine.†

After 24 hours, the cells were stimulated with A. actinomycetemcomitans for 24 hours, washed three

times with PBS, and fixed with 4% paraformaldehyde for 10 minutes at room temperature. After

washing, the chamber slides were mounted using a Prolong Gold antifade reagent‡‡ with DAPI‡‡ and

monitored by confocal laser-scanning microscopy.§§ The autophagic vacuoles were quantified by

counting the number of mCherry positive/GFP negative LC3 puncta.

Viable Cell Count

A viable cell count was measured by previous methods with slight modification.23 A.

actinomycetemcomitans was harvested, washed, and resuspended in RPMI without antibiotics. For

bacterial adhesion analysis, THP-1-derived macrophages were exposed to A. actinomycetemcomitans

(MOI 100) in the presence or absence of 3-MA for 90 minutes. The A. actinomycetemcomitans

associated with macrophages was quantified by washing the cells three times with cold PBS to

remove the non-adherent bacteria and lysing them in sterile distilled water for 20 minutes. To count

the number of internalized bacteria, the cells were infected with A. actinomycetemcomitans in the

presence or absence of 3-MA for 90 minutes and cultured for 15 minutes with 200 μg/mL

metronidazole† and 200 μg/mL gentamicin.† The cells were washed and lysed in sterile distilled water.

To determine the intracellular killing of A. actinomycetemcomitans, the cells were infected with A.

actinomycetemcomitans for 90 minutes. The cells were washed and treated with 200 μg/mL

metronidazole and 200 μg/mL gentamicin in the presence or absence of 3-MA for 15 minutes. The

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7
cells were then washed, lysed in sterile distilled water, and the bacterial numbers were counted

(Fig.3A).

Phagocytosis assay

For CFSE staining, A. actinomycetemcomitans was labeled with 10 μM CellTrace

carboxyfluorescein diacetate succinimidyl ester (CFSE)‡‡ in PBS containing 0.1% BSA for 13

minutes. For the confocal microscopic examination, the THP-1 cells were placed on 8-well chamber

slides. A. actinomycetemcomitans was labeled with CellTrace CFSE and used to infect the THP-1

cells for 24 hours. After infection, the cells were fixed with 4% paraformaldehyde for 10 minutes and

permeabilized for 5 minutes with PBS containing with 0.1% Triton X-100. The cells were stained

with Alexa Fluor 635 phalloidin‡‡ to selectively label F-actin for one hour at room temperature. The

cells were then mounted with a Prolong Gold antifade reagent with DAPI and monitored by Confocal

Laser-Scanning Microscopy.

For flow cytometry analysis, the THP-1 cells were seeded at 4.0×105 cells/well in a 12-well plate

and treated with FluoSpheres® 1.0 μm polystyrene microsphere‡‡ or infected with CFSE-labeled A.

actinomycetemcomitans for 45 minutes. After infection, the cells were washed with PBS and analyzed

using a FACSVerse flow cytometer.‖‖

Measurement of IL-1β

The IL-1β levels in the cell supernatant were measured using an enzyme-linked immunosorbent

assay (ELISA) kit¶¶ according to the manufacturer’s instruction.

ASC pyroptosome quantitation in live cells

PMA-differentiated THP-1/ASC-GFP cells were pretreated with 3-MA and challenged further with

A. actinomycetemcomitans. ASC pyroptosome formation was observed by confocal laser-scanning

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microscopy. The percentage of cells with ASC pyroptosome was calculated by dividing the number of

cells with ASC pyroptosome by the total number of cells counted.

Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

The total RNA was extracted using the TRIzol reagent‡‡ according to the manufacturer's instructions,

and the cDNA was synthesized using a reverse transcription system.## For qRT-PCR, the cDNA was

amplified using SYBR green PCR master mix*** on an ABI 7500 real-time PCR system*** with the

following programs: 10-minute pre-incubation at 95°C and 40 cycles of 15 seconds at 95°C and 1

minute at 62°C. The data were normalized to those of GAPDH, which was used as an endogenous

control. The primer sequences used for PCR analysis were as follows: AIM2, 5’-

TCAAGCTGAAATGAGTCCTGC-3’ (forward) and 5’-CTTGGGTCTCAAACGTGAAGG-3’

(reverse); NLRP3, 5’-CGTGAGTCCCATTAAGATGGAGT-3’ (forward) and 5’-

CCCGACAGTGGATATAGAACAGA-3’ (reverse).

Measurement of intracellular reactive oxygen species (ROS)

The THP-1 cells were pretreated with 3-MA and infected further with A. actinomycetemcomitans for

24 hours. The cells were loaded with H2DCFDA (10 μM)‡‡ in PBS for 60 minutes at 37°C and then

recovered with growth medium for 30 minutes. The cells were then washed and analyzed by flow

cytometry using FACSverse‖‖ and confocal laser-scanning microscopy.

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9
Statistics

All results are expressed as the mean ± standard deviation (SD) and an unpaired, one-tailed

Student’s t test was performed to compare within the groups using SPSS version 14.††† P values <

0.01 were considered significant.

Results

Autophagy was induced in A. actinomycetemcomitans-infected THP-1-derived macrophages.

Initially, this study examined whether autophagy-related proteins were elevated in aggressive

periodontitis patients. The levels of LC3-II, ATG5/12 conjugate, and Beclin-1 expression in the

gingival tissues of aggressive periodontitis patients were higher than those of healthy gingival tissues

(Fig. 1A). The expression levels of the autophagy-related proteins following an A.

actinomycetemcomitans infection were measured to examine the effects of A. actinomycetemcomitans

on autophagy in THP-1-derived macrophages. The infection with A. actinomycetemcomitans

increased the levels of LC3-II, ATG5/12 conjugate, and Beclin-1 expression in a MOI and time-

dependent manner (Fig. 1B and 1C). The induction of autophagy was confirmed using monomeric red

fluorescent protein (mCherry)-GFP tandem fluorescent-tagged LC3 (tfLC3). The GFP-LC3 punctate

signals were not detectable in the lysosomes because the fluorescent signal of GFP was quenched by

the low pH of lysosome, whereas the mCherry-LC3 punctate signals were still detectable.24 An

infection with live A. actinomycetemcomitans increased the transition of the mCherry-GFP/LC3

positive cells significantly to mCherry-positive and GFP-negative cells in mCherry-GFP/LC3-

transfected cells (Fig. 1D). The results suggest that live A. actinomycetemcomitans induces autophagy

in THP-1-derived macrophages as in the gingival tissues of aggressive periodontitis patients.

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TLR and ERK signaling was involved in A. actinomycetemcomitans-induced autophagy

To examine the molecular mechanisms of the signaling pathway involved in A.

actinomycetemcomitans-induced autophagy, this study checked TLR signaling, three types of

MAPKs, which are extracellular signal-regulated kinase (ERK), p38, and c-Jun-N-terminal kinase

(JNK), and NF-B expression in A. actinomycetemcomitans-infected THP-1-derived macrophages. A.

actinomycetemcomitans induced TLR2 and TLR4 signaling and the phosphorylation of ERK, JNK,

and NF-B (Fig. 2A and 2B). The roles of TLR2 and TLR4 in autophagy influx were confirmed using

the specific siRNAs. The knockdown of TLR2 and/or TLR4 inhibited the A. actinomycetemcomitans-

induced LC3-II expression. When the cells were pretreated with the specific MAPK inhibitors, A.

actinomycetemcomitans-induced autophagy was suppressed significantly only with an ERK inhibitor

(PD98059), not with the other inhibitors of p38 (SB203580), JNK (SP600125), and NF-B (PDTC)

(Fig. 2C). Next, this study examined whether the internalization of A. actinomycetemcomitans into

THP-1-derived macrophages was involved in the activation of autophagy. The PMA-differentiated

THP-1-derived macrophages were pretreated with inhibitors of internalization, including inhibitors of

endocytosis (monodansylcadaverine, MDC; methyl-β-cycloextrin, MβCD) or phagocytosis

(cytochalasin D, CytoD).† The inhibitors of endocytosis or phagocytosis did not affect the A.

actinomycetemcomitans-induced autophagy (Fig. 2D and 2E). Overall, these results suggest that TLR

and the ERK-mediated signaling pathway are involved in the A. actinomycetemcomitans-induced

autophagy independently of bacterial internalization in THP-1-derived macrophages.

A. actinomycetemcomitans-induced autophagy promotes the phagocytosis-mediated

internalization of A. actinomycetemcomitans

To determine the role of autophagy in an A. actinomycetemcomitans infection, the bacterial

adhesion, internalization, and intracellular killing of A. actinomycetemcomitans were determined

using the viable cell count (VCC). Treatment of 3-MA, a selective autophagy inhibitor, did not affect

the bacterial adhesion to THP-1-derived macrophages and the intracellular bacterial killing activity of

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11
the cells, but decreased the internalization of A. actinomycetemcomitans into THP-1-derived

macrophages significantly (Fig. 3A). Further analysis by confocal microscopy showed that higher A.

actinomycetemcomitans bacteria were internalized into the THP-1-derived macrophages and their

internalization was suppressed significantly after the 3-MA treatment (Fig. 3B). To examine the

effects of autophagy on the phagocytosis of A. actinomycetemcomitans, THP-1-derived macrophages

with or without a 3-MA pretreatment were challenged with CFSE-stained A. actinomycetemcomitans

and a phagocytosis assay was performed. The 3-MA treatment decreased the phagocytosis activity of

THP-1-derived macrophages against A. actinomycetemcomitans (Fig. 3C).

To determine the internalization pathway further, the THP-1-derived macrophages were pretreated

with the inhibitors of endocytosis (MDC and MβCD) or phagocytosis (CytoD). Interestingly, only

CytoD and 3-MA reduced the internalization of A. actinomycetemcomitans into the cells (Fig. 3D)

and the uptake ability of CFSE-stained A. actinomycetemcomitans or latex beads (Fig. 3E and 3F).

These results suggest that A. actinomycetemcomitans-induced autophagy promotes the internalization

of A. actinomycetemcomitans into the cells via phagocytosis.

A. actinomycetemcomitans-induced autophagy prevents excessive inflammatory reactions in

THP-1-derived macrophages.

Because autophagy is involved in the regulation of IL-1β secretion,25 the effects of autophagy on the

A. actinomycetemcomitans-induced inflammatory response were investigated. A.

actinomycetemcomitans-induced IL-1β production and ASC pyroptosome formation were increased

significantly after the 3-MA treatment (Fig. 4A and 4B). A previous study reported that A.

actinomycetemcomitans promoted IL-1β maturation, caspase-1 activity, and ASC pyroptosome

formation via AIM2 inflammasome activation.23 Interestingly, the 3-MA pretreatment decreased the

level of AIM2 expression significantly, whereas NLRP3 expression was increased significantly

compared to that observed after an A. actinomycetemcomitans infection (Fig. 4C and 4D). ROS

production in A. actinomycetemcomitans-infected cells was measured because ROS activates NLRP3

expression26. The A. actinomycetemcomitans infection weakly induced ROS production, whereas the

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3-MA pretreatment increased the level of ROS production significantly (Fig. 4E). The effects of ROS

on NLRP3 and IL-1β expression after an A. actinomycetemcomitans infection were analyzed using N-

acetylcysteine (NAC), a ROS scavenger. The NAC treatment suppressed 3-MA-induced NLRP3

expression and IL-1β production significantly in A. actinomycetemcomitans-infected cells (Fig. 4F).

These results suggest that the A. actinomycetemcomitans-induced autophagy negatively regulates IL-

1β production via the modulation of ROS production, NLRP3 expression, and ASC pyroptosome

formation.

Discussion

Chronic and aggressive periodontitis have a range of similarities and differences; the cytokine

profiles are similar in both.27 Although different host cellular responses are clearly involved in chronic

and aggressive periodontitis,28 many studies of the relationship between autophagy and periodontitis

focused mainly on chronic periodontitis.19, 21, 22 In particular, the role of periodontopathogen-induced

autophagy in aggressive periodontitis is not completely understood. Therefore, this study investigated

the involvement of A. actinomycetemcomitans, a major causative agent of aggressive periodontitis, in

the activation of autophagy and the effects of autophagy on inflammation in THP-1-derived

macrophages infected with A. actinomycetemcomitans. In this study, live A. actinomycetemcomitans

was used to mimic the actual in vivo infection instead of the bacterial components, such as

lipopolysaccharide. These results showed that A. actinomycetemcomitans activated the influx of

autophagy by increasing the expression of autophagy-related molecules, which was mediated by the

TLR and ERK signaling pathways. Moreover, blocking the autophagy flux with 3-MA pretreatment

inhibited the bacterial internalization by suppressing the phagocytic activity of macrophages and

enhanced the A. actinomycetemcomitans-stimulated inflammatory response by modulating the

production of ROS.

Autophagy is essential for the survival of cells during nutrient starvation and is involved in cellular

remodeling during cell development and differentiation.29 Furthermore, autophagy is well known for

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13
the protein degradation process, and the autophagic substrates are surrounded by double-membrane

structures known as autophagosomes. Cytoplasmic bacteria can also be targeted for the autophagic

pathway.10, 30 For example, Mycobacterium tuberculosis is targeted for autophagy when the

macrophages are stimulated with the autophagy activator, rapamycin or IFN.11 Intracellular Shigella

flexneri and Salmonella typhimurium are recognized by autophagy and then degraded by

autolysosomes.31, 32 Listeria monocytogenes is targeted for degradation by autophagy during a primary

infection, which limits the onset of early bacterial growth.33 Therefore, the anti-microbial function of

autophagy provides a mechanism for the elimination of invading microorganisms in the host cells. A

previous study reported that P. gingivalis induces autophagy and macrophages eliminate P. gingivalis

through an autophagic response, leading to the restriction of an excessive inflammatory.21 Despite the

reported crosstalk between several periodontal pathogens and autophagy, there is limited available

data showing the role of autophagy in the survival of A. actinomycetemcomitans. This study examined

whether the autophagy was induced in the gingival samples of aggressive patients and clearly by an A.

actinomycetemcomitans infection in THP-1-derived macrophages.

Phagocytosis is a hallmark of the host defense against bacteria and is involved in degrading and

killing bacteria.34 Autophagy and phagocytosis are highly conserved processes involved in the

destruction of organisms in the cytosol and extracellular region, respectively, and TLR signaling in

macrophages links those defense mechanisms.35 The bacterial internalization into phagosomes

restricts microbial replication and plays an important role not only in killing microbes but also in

activating innate immune signaling pathway during the maturation of phagosomes.36 This study

indicates that A. actinomycetemcomitans-induced autophagy promotes bacterial internalization by

enhancing phagocytosis, which in turn increases the elimination of A. actinomycetemcomitans and

decreases IL-1 production, leading to the prevention of an excessive inflammatory response. On the

other hand, these results showed that A. actinomycetemcomitans-induced autophagy did not contribute

to the intracellular killing activity of A. actinomycetemcomitans.

Several studies reported that autophagy is involved in the removal of pathogens, including

Mycobacterium tuberculosis or Toxoplasma gondii, by modulating phagosome maturation.37, 38 TLR

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signaling-mediated autophagy, as A. actinomycetemcomitans-induced autophagy from the present

results, triggered phagosome maturation in a manner that depends on the autophagy pathway proteins

and led to the death of the ingested organisms.35 Some ATG proteins are involved in phagocytosis by

modulating phagosome maturation and in the exocytosis of signal-peptide-lacking substrates.8 The

survival of Saccharomyces cerevisiae increased markedly in ATG7-deficient macrophages, and these

processes were associated with double-membrane structures, which are characteristic of conventional

autophagosomes.35 Furthermore, 3-MA decreased oridonin-induced phagocytosis, whereas it

increased caspase-1-mediated IL-1release in U937 cells.39 3-MA, a popular inhibitor of the

autophagic pathway, has been reported to inhibit the activity of PI3-kinase and block the formation of

autophagosome and autophagic vacuoles.40 Another study showed that 3-MA abolished the capability

of phagocytosis of bacteria in macrophages.41 Similar to these reports, 3-MA decreased the

internalization of A. actinomycetemcomitans into cells dramatically, which provided the linkage

between autophagy, phagocytosis, and A. actinomycetemcomitans internalization. These results

suggest that autophagy is a part of the feedback loop to control the degree of phagocytosis and

bacterial internalization.

Increasing evidence shows that autophagy is crucial for inflammation by regulating IL-1β

production via its degradation.42 Atg16L1-deficient macrophages showed a significant increase in the

IL-1 family cytokines, IL-1β and IL-18, by LPS.43 Recently, several studies reported a relationship

between autophagy and inflammasomes.44, 45 The plasminogen activator inhibitor-1 (PAI-1) enhances

autophagy, resulting in NLRP3 degradation during the inflammatory response.44 The activation of

autophagy by inflammatory signals is dependent on the AIM2 and NLRP3 inflammasomes, which

helps limit the excessive production of IL-1β by targeting the ubiquitinated inflammasomes for

destruction.45 Inflammasome complexes are also critical for recognizing the microbial products by the

direct binding of ligands and indirect pathways and are considered to be the key multimolecular

complex for the secretion of IL-1β via the activation of caspase-1 in the response to pathogenic

stimuli.12 In these experiments, the inhibition of A. actinomycetemcomitans-induced autophagy by 3-

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15
MA suppressed the AIM2 inflammasomes and increased NLRP3 expression and IL-1β production

significantly. The AIM2 inflammasome is activated by dsDNA of a viral and bacterial origin via a

direct interaction.46 During an A. actinomycetemcomitans infection, the dsDNA of A.

actinomycetemcomitans may be released into the cytosol, which can be sensed by the AIM2 molecule.

In this study, however, 3-MA reduced the internalization of A. actinomycetemcomitans, which may

lead to the reduced release of dsDNA from A. actinomycetemcomitans, resulting in decreased

expression of the AIM2 sensor.

The NLRP3 inflammasomes were induced by a 3-MA treatment but not by an A.

actinomycetemcomitans infection. NLRP3 inflammasome is activated by numerous microbial,

environmental, and endogenous stimuli, including the ATP and K+ concentrations.12, 46 Recently,

several studies have reported that NLRP3 inflammasome activation is induced by ROS.47, 48

Moreover, the inhibition of autophagy enhanced NLRP3 inflammasome activation,49 and autophagy

could suppress NLRP3 inflammasome activation by modulating the production of ROS.50 Similar to

the above reports, these results showed that 3-MA enhanced ROS production, leading to an increase

in NLRP3 expression and IL-1β release in A. actinomycetemcomitans-infected cells. The NAC

treatment definitely reduced these effects. Therefore, these results suggest that ROS induction by the

inhibition of autophagy could contribute to NLRP3 inflammasome activation, leading to increased IL-

1β production.

Conclusion

An infection with A. actinomycetemcomitans triggers the activation of autophagy via the up-

regulation of Beclin-1, ATG5/12 conjugation, and LC3-I/II conversion in THP-1-derived

macrophages. Moreover, A. actinomycetemcomitans-induced autophagy enhanced the bacterial

internalization and limited the release of IL-1 induced by A. actinomycetemcomitans. Therefore, the

failure to clear bacteria and the enhancement of inflammasome activation by autophagy inhibition can

result in a hyper-inflammatory environment. Accordingly, this study provides key insights into the

relationship between A. actinomycetemcomitans infections and autophagy in the innate immune

This article is protected by copyright. All rights reserved.

response. Furthermore, targeting this pathway may provide new therapeutic interventions for

aggressive periodontitis.

Acknowledgment

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the

Korea government (MEST; NRF-2017M3A9B6062021). The authors wish to thank Juyeon Lee in

Pusan National University Dental Hospital for providing the gingival tissues used in this study.

Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication

of this article.

Footnotes

*
Gibco, Carlsbad, CA

†
Sigma Aldrich, St. Louis, MO

‡
Difco, Detroit, MI

§
Cell Signaling Technology, Beverly, MA

‖
Santa Cruz Biotechnology, Santa Cruz, CA

¶
Adipogen, San Diego, CA

#
Pierce Biotechnology, Rockford, IL

**
Fuji Film, Tokyo, Japan
This article is protected by copyright. All rights reserved.

17
††
Qiagen, Valencia, CA

‡‡
Invitrogen, Carlsbad, CA

§§
LSM 700, Carl Zeiss, Oberkochen, Germany

‖‖
BD, San Jose, CA

¶¶
eBioscience, San Diego, CA

##
Bioneer, Daejeon, South Korea

***
Applied Biosystems, Foster City, CA

†††
SPSS, Chicago, IL

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Figure legends

Figure 1. Induced autophagy was found in patients with aggressive periodontitis and A.

actinomycetemcomitans-infected THP-1 cells.

(A) Expression of LC3 I/II, ATG5/12, and Beclin-1 in gingival tissue of healthy and periodontitis

patients was analyzed by Western blot analysis. THP-1-derived macrophages were infected with A.

actinomycetemcomitans for 6 hours at different MOIs (B) or for different infection times at MOI 100

(C). The cell lysates were subjected to Western blot analysis. (D) HEK293 cells were transfected with

mCherry-GFP-LC-3. After 24 hours, the cells were infected with A. actinomycetemcomitans for 24

hours and analyzed by confocal microscopy (×200 magnification).

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25
Figure 2. TLR and ERK signaling mediated the A. actinomycetemcomitans-induced autophagy.

(A) For siRNA experiments in THP-1 cells, the cells were seeded, and then transfected with each

siRNA oligonucleotide for 24 hours. The siRNA-transfected cells were infected with A.

actinomycetemcomitans for 24 hours. The cell lysates were collected and the protein expression of

TLR2 and TLR4 was measured by Western blot assay. (B) To determine the level of MAPK-related

protein expression, the THP-1-derived macrophages were infected with A. actinomycetemcomitans for

the indicated times. The cell lysates were subjected to Western blot analysis. (C) THP-1-derived

macrophages were pretreated with 20 M PD98059 (PD98), SB (SB203580), SP (SP600125) or 100

M PDTC for 30 minutes, and then infected with A. actinomycetemcomitans. After six hours, the cell

lysates were subjected to Western blot analysis. (D) THP-1-derived macrophages were pretreated with

10 M MDC, 0.025% MCD, or 0.5 M CytoD for 30 minutes, and then infected with A.

actinomycetemcomitans. After 24 hours, the cell lysates were subjected to Western blot analysis. (E)

HEK293 cells were transfected with mCherry-GFP-LC-3. After 24 hours, the cells were infected with

A. actinomycetemcomitans in the presence or absence of MDC, MβCD, or CytoD for 24 hours, and

analyzed by confocal microscopy (×400 magnification). The total number of mCherry-positive/GFP-

negative puncta per cell was counted. *, P < 0.001.

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This article is protected by copyright. All rights reserved.

27
Figure 3. A. actinomycetemcomitans-induced autophagy regulated internalization of A.

actinomycetemcomitans via phagocytosis.

(A) For the adhesion assay, THP-1-derived macrophages were pretreated with 2 mM 3-MA for 30

minutes prior to infection with live A. actinomycetemcomitans for 90 minutes. To determine the level

of bacterial adhesion, the cells were washed and lysed for 20 minutes, and then poured on plates. For

the internalization assay, THP-1-derived macrophages were pretreated with 3-MA for 30 minutes

prior to infection with live A. actinomycetemcomitans for 90 minutes. The cells were lysed after the

addition of cell-impermeable antibiotics for 15 minutes and poured on plates; the level of bacterial

internalization was determined. For the intracellular killing assay, THP-1-derived macrophages were

infected with live A. actinomycetemcomitans for 90 minutes. The cells were treated with cell-

impermeable antibiotics in the presence or absence of 3-MA for 15 minutes, lysed and then poured on

plates to determine the level of bacterial killing. *, P < 0.001. (B) After pretreatment with or without

3-MA for 30 minutes, THP-1-derived macrophages were infected with CFSE-labeled A.

actinomycetemcomitans for 24 hours. F-actin (red) and A. actinomycetemcomitans (green) were

observed by confocal microscopy (×400 and ×1600 magnification). (C) THP-1-derived macrophages

were infected with CFSE-labeled A. actinomycetemcomitans for 45 minutes. After incubation,

bacterial phagocytosis was assessed by flow cytometry. *, P < 0.001. (D) THP-1-derived

macrophages were pretreated with MDC, MCD, or CytoD for 30 minutes prior to infection with live

A. actinomycetemcomitans for 90 minutes. The cells were lysed after the addition of cell impermeable

antibiotics for 15 minutes and poured on plates to determine the level of bacterial internalization. **,

P < 0.01. After pretreatment with or without MDC, MCD, CytoD, or 3-MA for 30 minutes, THP-1-

derived macrophages were infected with CFSE-labeled A. actinomycetemcomitans (E), or treated with

FluoSpheres® 1.0 μm polystyrene microspheres (F) for 45 minutes. After incubation, the

phagocytosis ability was assessed by flow cytometry. *, P < 0.001.

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This article is protected by copyright. All rights reserved.

29
Figure 4. A. actinomycetemcomitans-induced autophagy modulated inflammatory response. (A) THP-

1-derived macrophages were pretreated with 3-MA for 30 minutes, then infected with A.

actinomycetemcomitans for 24 hours. The cell culture supernatant was assayed to detect the level of

IL-1using ELISA. (B) PMA-primed ASC-GFP stable expressing-THP-1 macrophages were infected

with A. actinomycetemcomitans for 24 hours in the presence or absence of 3-MA, and the ASC

pyroptosomes were observed and photographed by confocal microscopy (×200 magnification). The

graph shows the percentage of cells containing the ASC pyroptosomes, which was calculated by

dividing the number of cells with the ASC pyroptosome by the total number of cells counted. THP-1-

derived macrophages were pretreated with 3-MA for 30 minutes and infected with A.

actinomycetemcomitans for 24 hours. The cell lysates were subjected to real-time PCR (C) and

Western blot analysis (D). (E) THP-1 (top panel) or THP-1-derived macrophages (bottom panel) were

pretreated with 3-MA for 30 minutes, and infected with A. actinomycetemcomitans for 24 hours. The

total ROS was measured by flow cytometry (top panel) or confocal microscopy (bottom panel, ×200

magnification). THP-1-derived macrophages were pretreated with 3-MA or NAC (10 or 20 mM) for

30 minutes, and then infected with A. actinomycetemcomitans for 24 hours. The cell lysates were

analyzed by Western blot analysis (F) and the cell culture supernatant was assayed for human IL-

1using ELISA (G). *, P < 0.001.

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This article is protected by copyright. All rights reserved.

31