Augmented Osteoclastogenesis from Committed Osteoclast Precursors by Periodontopathic Bacteria Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis

치주염 유발 세균 Aggregatibacter actinomycetemcomitans와 Porphyromonas gingivalis 에 의한 committed osteoclast precursor 분화 증가

박옥진¹, 권영각¹, 윤철희², 한승현¹*

1서울대학교치의학대학원구강미생물

·

2서울대학교농업생명과학대학농생명공학부동물생명공학전공

Received _: August 31, 2016 / Revised _: September 20, 2016 / Accepted _: September 21, 2016

서 론

치주질환은만성염증성질환으로심할경우치조골을파 괴시켜성인치아상실의주된원인이 된다

^[11].

^,

^,

^{[4, 20].}

^1,000

의 세균 중

Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans

^{[14, 15].}

P. gingivalis

A. actinomycetemcomitans

(Chronic periodontitis)

(Localized aggressive periodontitis)

관되어있다

^{[3, 14].}

질다당체

(Lipopolysaccharide; LPS)

^-

^/

^,

^,

[7, 12].

Augmented Osteoclastogenesis from Committed Osteoclast Precursors by Periodontopathic Bacteria Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis

Ok-Jin Park

, Yeongkag Kwon

, Cheol-Heui Yun

, and Seung Hyun Han

*

1

Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry,

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis are gram-negative bacteria frequently found in lesions from patients with periodontitis manifesting alveolar bone loss. Lipopolysac- charides are a major virulence factor of gram-negative bacteria. Bone resorption is known to be regulated by bacteria and their virulence factors. In the present study, we investigated the effects of A. actinomyce- temcomitans and P. gingivalis on bone resorption. Heat-killed A. actinomycetemcomitans (HKAa) and heat- killed P. gingivalis (HKPg) induced bone loss in the femurs of mice after intraperitoneal administration.

HKAa and HKPg augmented the differentiation of committed osteoclast precursors into osteoclasts, while they inhibited the differentiation of bone marrow-derived macrophages into osteoclasts. Concordant with the effects of the heat-killed whole cells, LPS purified from A. actinomycetemcomitans and P. gingivalis also augmented osteoclast differentiation from committed osteoclast precursors but attenuated it from bone marrow-derived macrophages. Taken together, these results suggest that the whole cells and lipopolysac- charides of A. actinomycetemcomitans and P. gingivalis induce the differentiation of committed osteoclast precursors into osteoclasts, potentially contributing to bone resorption in vivo.

Keywords: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, lipopolysaccharide, osteoclast, bone loss

*Corresponding author

Tel _: +82-2-880-2310, Fax: +82-2-743-0311 E-mail: [email protected]

© 2016, The Korean Society for Microbiology and Biotechnology

기하게된다

^[17].

^,

^.

골항상성

(Bone homeostasis)

(Osteoclast)

(Osteoblast)

의활성과분화의적절한균형에의해유지된다

^[16].

세포에서 기원한 파골세포는 핵심 사이토카인인

^M-CSF (Macrophage-colony stimulating factor)

RANKL (Receptor activator of nuclear factor kappa B ligand)

(Multinucleated giant cell)

^[1, 8].

^,

^,

^[2].

^,

^,

^{[20, 22].}

^{, LPS,}

(Lipoteichoic acid)

^(Pathogen- associated molecular pattern; PAMP)

(Pattern-recognition receptor;

PRR)

^[9].

한패턴인식수용체들을발현하고있어세균과세균병독력인 자는골항상성을조절하게된다

^{[18, 21].}

본연구는 치주질환을 일으키는 대표적 구강세균인

^P.

gingivalis

A. actinomycetemcomitans

^,

^(micro- CT)

^/

^.

^,

P. gingivalis

^A.

actinomycetemcomitans,

^,

LPS

^.

재료 및 방법

재료

Brain heart infusion (BHI)

BD Biosciences (USA)

. Vitamin K

^hemine

^Sigma- Aldrich Chemical Inc. (USA)

^{. Fetal} bovine serum (FBS)

^-MEM

Gibco (USA)

^, Trypsin-EDTA

penicillin/streptomycin

Hyclone (USA)

. Recombinant mouse M-CSF

R&D Systems (USA)

, recombinant murine RANKL

PeproTech (USA)

^.

Sigma-Aldrich Chemical Inc.

^.

미세단층촬영기

모든동물실험은서울대학교동물실험윤리위원회에서승 인을받은후진행하였다

^.

⁶

^C57BL/6

^PBS

⁴

^,

⁷

^4%

paraformaldehyde

^.

(Skyscan 1172 scanner, Skyscan, Belgium)

^.

³

^,

(SkyScan CT analyzer)

^,

^,

^,

^.

열사멸 세균 준비

A. actinomycetemcomitans ATCC43718

P. gingivalis ATCC49417

American Type Culture Collection (USA)

. P. gingivalis

^BHI

¹⁰

^g/ml

^vitamin K

⁵

^g/ml

^hemine

³⁷

^(Whitley DG250 Workstation; Don Whitley Scientific, UK)

A. actinomycetemcomitans

^BHI

³⁷

^.

^mid-log

^{1 ×} 10

CFU/ml

⁶⁰

¹

^.

A. actinomycetemcomitans (HKAa)

P. gingivalis (HKPg)

^{1.5% agar}

^BHI

³⁷

¹

(data not shown).

LPS 추출

배양된

P. gingivalis

A. actinomycetemcomitans

11,068 × g

³⁷

거해주고

^{, PBS}

^.

^LPS

extraction kit (iNtRON Biotechnology, Korea)

^.

세포배양

6

^C57BL/6

^(Seongnam,

Korea)

^,

였다

^.

10% FBS, 100 U/ml penicillin

100

g/ml streptomycin

^-MEM

^{2 ng/ml} M-CSF

³⁷

^CO

¹

^,

20 ng/ml M- CSF

³

(Bone marrow-derived macrophage;

BMM)

^.

^BMM

20 ng/ml M-CSF

20 ng/ml RANKL

²

committed osteoclast precursor

^.

파골세포 분화 및 tartrate-resistant acid phosphatase

(TRAP) 염색

2 × 10

cells/ml

^BMM

96-well plate

^well

²⁰⁰

^l

^seeding

, 20 ng/ml M-CSF

20 ng/ml RANKL

LPS

²

^.

2 × 105 cells/ml

^committed osteoclast precursor

96-well plate

^well

²⁰⁰

^l

seeding

, 20 ng/ml M-CSF

^LPS

⁵

⁸

^.

(26% citrate, 66% acetone, 8% formaldehyde)

^TRAP

^(Sigma- Aldrich Chemical Inc.)

^.

적자색의

^TRAP

³

하여통계처리하였다

^.

모든실험은

³

^.

(two-tailed t test)

^{p <}

0.05

^.

결과 및 고찰

HKAa

치주염은심할경우치조골소실에의한성인치아상실을일

으키게된다

^[11].

적세균인

A. actinomycetemcomitans

P. gingivalis

^{. 6}

^C57BL/6

^HKAa

^HKPg

⁴

.

7

,

^.

^HKAa

^HKPg

(Fig. 1A). HKAa

^HKPg

^.

¹⁰

^CFU/ml

^HKPg

^,

^(Fig.

1B).

^HKAa

^HKPg

^,

A. actinomycetemcomitans

P. gingivalis

^{[5, 23]}

^.

^,

^-

^.

^,

A. actinomycetemcomitans

^P.

gingivalis

Fig. 1. HKAa and HKPg induced bone resorption in vivo. (A) Mice (n = 3 per group) were intraperitoneally administered with HKAa or HKPg, twice with a 4-day interval. At day 7 after the first administration, micro-CT images of the femurs were obtained by the CT analyzer. (B) BV/TV, Tb.Th, Tb.N, and Tb.Sp of the femurs were analyzed. BV/TV = trabecular bone volume; Tb.N = trabecular number;

Tb.Th = trabecular thickness; Tb.Sp = trabecular separation. *p < 0.05.

는치주질환의치조골소실에의한치아상실발생기전을뒷 받침하는결과라하겠다

^.

HKAa

생체외실험에서

A. actinomycetemcomitans

P. gingivalis

^{(Fig. 1),}

^.

^{, M-CSF}

³

^BMM

BMM

^M-CSF

^RANKL

²

(committed osteoclast precursor)

^{. HKAa}

^HKPg

20 ng/ml

^RANKL

^BMM

^{, HKAa}

HKPg

^TRAP-

였다

(Fig. 2A, B).

^HKAa

^HKPg

^RANKL-

^.

^{, HKAa}

^HKPg

committed osteoclast precursor

^{, HKPg}

HKAa

^TRAP-

유의적으로 증가하였다

(Fig. 2C, D).

^HKPg

HKAa

^.

한결과는

P. gingivalis

^RANKL-

소시키고

^{, RANKL}

가시킨다는 보고와 일치한다

[19]. P. gingivalis

^A.

actinomycetemcomitans

^,

^.

^, committed osteoclast precursor

^RANKL

^BMM

^,

^.

^[16].

^[6, 13].

^{, BMM}

^RANKL

committed osteoclast precursor

^{[6, 23].}

^,

^BMM

^,

committed osteoclast precursor

^.

AaLPS

LPS

Fig. 2. HKAa and HKPg attenuated osteoclast differentiation from BMMs, but induced differentiation of committed osteoclast precursors into osteoclasts. BMMs were treated with 20 ng/ml M-CSF and 20 ng/ml RANKL in the presence of HKAa at MOI of 0.5, 5, or 50 (A) and HKPg at MOI of 0.5, 5, or 50 (B) for 24 h. Committed osteoclast precursors were treated with 20 ng/ml M-CSF in the pres- ence of HKAa at MOI of 0.5, 5, or 50 (C) and HKPg at MOI of 0.5, 5, or 50 (D) for 8 h. The cells were fixed and subjected to TRAP staining.

TRAP-positive multinucleated cells (MNCs) with three or more nuclei were enumerated through microscopic analysis. *p < 0.05.

체로서다양한염증

^-

^{[7, 10].}

^HKAa

^HKPg

A. actinomycetemcomitans

P. gingivalis

^LPS

^.

^LPS

^BMM

committed osteoclast precursor

^.

^{, AaLPS}

^PgLPS

가처리된

^BMM

^TRAP-

으로감소하고

(Fig. 3A, B), AaLPS

^PgLPS

^committed osteoclast precursor

^TRAP-

(Fig. 3C, D).

^HKAa

^HKPg

^{, AaLPS}

^PgLPS

^RANKL-

committed osteoclast precursor

^.

Escherichia coli

O55:B5 LPS

^BMM

^,

committed osteoclast precursor

^[24].

^LPS

^.

^LPS

committed osteoclast precursor

^.

골소실을예측하기위해

committed osteoclast precursor

^.

요 약

치주질환은만성염증성질환으로치조골소실을일으켜 성인치아상실을유발하는요인중하나이다

^.

Aggregatibacter actinomycetemcomitans

Porphyromonas gingivalis

^.

(Lipopolysaccharide; LPS)

^.

^LPS

^.

A. actinomycetemcomitans

P. gingivalis

^,

A. actinomycetemcomitans, P. gingivalis, A.

actinomycetemcomitans

P. gingivalis

^LPS

^.

^A.

actinomycetemcomitans (HKAa)

P. gingivalis

(HKPg)

소된골량을보여주었다

^.

^, Fig. 3. AaLPS and PgLPS attenuated osteoclast differentiation from BMMs, but induced differentiation of committed osteoclast precursors into osteoclasts. BMMs were treated with 20 ng/ml M-CSF and 20 ng/ml RANKL in the presence of 10, 100, or 1000 pg/

ml AaLPS (A) and 10, 100, or 1000 pg/ml PgLPS (B) for 48 h. Committed osteoclast precursors were treated with 20 ng/ml M-CSF in the

presence of 10, 100, or 1000 pg/ml AaLPS (C) and 10, 100, or 1000 pg/ml PgLPS (D) for 5 h. The cells were fixed and subjected to TRAP

staining. TRAP-positive multinucleated cells (MNCs) with three or more nuclei were enumerated through microscopic analysis. *p < 0.05.

bone marrow-derived macrophage (BMM)

^RANKL-

, committed osteoclast precursor

^.

A. actinomycetemcomitans

P. gingivalis

^LPS

^RANKL-

, committed osteoclast precursor

^.

^A.

actinomycetemcomitans

P. gingivalis

^committed osteoclast precursor

^,

^LPS

^.

Acknowledgments

This work was supported by grants from the National Research Foundation of Korea, which is funded by the Korean government (NRF-2015R1A2A1A15055453, NRF-2015M2A2A6A01044894, and NRF-2015R1D1A1A09056592) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (HI14C0469), Republic of Korea.

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