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이학 사 학 논

The effect of Toll-like receptor 2 activation on the non-opsonic

phagocytosis of oral bacteria and concomitant ROS production by human

neutrophils

Toll 사 용체 2 (TLR2) 가 인간

구에 한 구강 균 식균작용과

그에 하는 산소 생 에 미치는

효과

2016 2월

울 학 학원

치 과학과 면역 분자미생 공

갑 열

Abstract

The effect of Toll-like receptor 2 activation on the

non-opsonic phagocytosis of oral bacteria and

concomitant ROS production by human

neutrophils

Kap Youl Kim

Department of Dental Science

Major in Immunology and Molecular Microbiology in Dentistry

Graduate School, Seoul National University

(Supervised by Youngnim Choi, . D.D.S., Ph. D)

Background

Periodontitis is a chronic inflammatory disease that results in major damage

to periodontal tissues, especially the gingiva and underlying alveolar bones,

leading to eventual tooth loss. Chronic/cyclic neutropenia, leukocyte adhesion

deficiency syndrome, Papillon-Lefèvre syndrome and Chédiak-Higashi

syndrome are associated with severe periodontitis, suggesting the importance

of neutrophils in the maintenance of periodontal health. It is known that

various Toll-like receptor (TLR) ligands stimulate neutrophil function,

including FcR-mediated phagocytosis. The aim of this study was to

investigate whether the stimulation of TLR2 can enhance the non-opsonic

phagocytosis of oral bacteria and subsequent killing in neutrophils.

Methods

As the source of human neutrophils, peripheral blood polymorphonuclear

cells were isolated by the ficoll-hypaque method and subsequent lysis of red

blood cells using hypotonic NaCl solution. Streptococcus sanguinis 804

(NCTC 10904) and Porphyromonas gingivalis ATCC 49417 were used after

fixation with 3.7 % paraformaldehyde, except for the intracellular survival

assay where live bacteria were used. Bacteria were stained with 5[and-6]-

carboxyfluorescein diacetate succinimidyl ester (CFSE) for fluorescence

application. Neutrophils were incubated with S. sanguinis or P. gingivalis in

the presence of various concentrations of Pam3CSK4 and subjected to the

measurement of phagocytosis and reactive oxygen species (ROS) by flow

cytometry and enhanced chemiluminescence, respectively. To examine the

intracellular killing of live S. sanguinis and P. gingivalis within neutrophils,

antibiotic protection assay was performed.

Results

Pam3CSK4 significantly increased the phagocytosis of both bacterial species

in a dose-dependent manner. Pam3CSK4 alone induced ROS production from

neutrophils and also increased concomitant ROS production induced by

bacteria. Interestingly, incubation with P. gingivalis and Pam3CSK4 decreased

the amounts of ROS compared with those produced by Pam3CSK4 alone,

indicating a possibility that P. gingivalis survives within neutrophils.

However, neutrophils efficiently both phagocytosed species even in the

absence of Pam3CSK4. Stimulation with Pam3CSK4 increased the adhesion of

P. gingivalis, but not that of S. sanguinis, to neutrophils, suggesting that

TLR2-assisted phagocytosis in neutrophils works through different

mechanisms depending on the target bacterial species.

Key words : periodontitis, neutrophils, phagocytosis, reactive oxygen

species (ROS), TLR2

Student Number : 2008 - 22036

CONTENTS

Abstract

I. Introduction 1

II. Materials and Methods 4

2.1. Bacterial strains and growth conditions 4

2.2. Preparation of fixed bacteria samples for the phagocytosis

assay and measurement of reactive oxygen species (ROS)

production

4

2.3. Isolation of neutrophils 5

2.4. Phagocytosis assay 6

2.5. Enhanced chemiluminescence assay for ROS measurement 6

2.6. Antibiotic protection assay 7

2.7. Measurement of bacterial adherence to neutrophils 8

2.8. Statistical analysis 8

III. Results 9

3.1. Pre-stimulation of neutrophils with Pam3CSK4 enhances the

phagocytosis of S. sanguinis and concomitant ROS

production.

9

3.2. Simultaneous stimulation with Pam3CSK4 also enhances

phagocytosing activity and concomitant ROS production in

neutrophils.

11

3.3. Neutrophils efficiently kill phagocytosed bacteria even in the

absence of a TLR2 stimulator 14

3.4. Stimulation with Pam3CSK4 increases the adhesion of P.

gingivalis, but not that of S. sanguinis, to neutrophils. 16

IV. Discussion 18

V. References 20

국 26

1

I. Introduction

Periodontitis is a chronic inflammatory disease that results in major damage to

periodontal tissues, especially the gingiva and underlying alveolar bones,

leading to eventual tooth loss. It is a common oral disease which affects

globally more than half of the adult population [1-4]. Periodontitis is initiated

by subgingival plaque biofilms. According to Socransky's classification,

subgingival plaque bacteria are classified into six major complexes - red,

orange, green, yellow, purple, and Actinomyces [4]. It is known that these

bacteria colonize in a certain order: yellow, green, and violet complexes, and

Actinomyces are early colonizers; the red complex is a late colonizer, and the

orange complex acts like a bridge between the early and late colonizers, called

a bridging colonizer [4]. Therefore, the red complex bacteria are not detected

in the absence of orange complex bacteria or early colonizers. The red

complex bacteria Porphyromonas gingivalis, Treponema denticola and

Tannerella forsythia are highly associated with chronic periodontitis and

distinctly more pathogenic than the others [5].

In the gingival sulcus, two major defense mechanisms regulate

plague bacteria and protect from the invasion of pathogens into gingival

tissues. One is antimicrobial peptides, such as human cathelicidin LL-37,

hBD-1 and HBD-2 that directly kill bacteria. The other is phagocytosis by

macrophages and neutrophils [6]. In addition, a genetic deficiency of the

immune systems impairs their response. Chronic/cyclic neutropenia,

2

leukocyte adhesion deficiency syndrome, Papillon-Lefèvre syndrome and

Chédiak-Higashi syndrome are associated with severe periodontitis,

suggesting the importance of neutrophils in the maintenance of periodontal

health [7-10].

In the gingival sulcus, the host cells, such as epithelial cells and

neutrophils, initiate immune responses to the plaque bacteria by recognizing

the microbial-associated molecular patterns (MAMPs) through known as

pattern recognition receptors (PRRs). The family of Toll-like receptors (TLRs)

is a class of PRRs [11, 12]. TLRs can be classified into two types depending

on their subcellular localization. Whereas TLR4, TLR5, TLR11 and

heterodimers of TLR2/1, TLR2/6 are located on the cell surface, TLR3, TLR9,

TLR13 and heterodimer TLR7/TLR8 are located in enodosomes [11-14].

Each of TLRs recognizes various microbial structures. For example, TLR2

recognizes the peptidoglycans and TLR4 recognizes bacterial

lipopolysaccharides. Neutrophils express TLR1, 2, 4, 5, 6, 7, 8, 9 and 10

except TLR3 [14-17]. Ligation of TLRs with their cognate ligands leads to

intracellular signaling transduction and activation of NF-κB, AP-1, and IRFs

that govern the inflammatory and immune responses [18]. As a result,

proinflamatory cytokine and chemokines are produced, which recruit

leukocytes to eliminate or neutralize pathogens eventually [19, 20]

Over the years, researchers have investigated how to prevent

pathogenic bacteria from invading periodontal tissues and how to effectively

eliminate the pathogens in the subgingival plaque. Periodontal pathogens have

diverse mechanisms to evade host defense machinery in the gingival sulcus.

3

First, there is interleukin (IL)-8 degradation. IL-8 is induced in epithelial cells

by invading pathogenic bacteria and serve as a key chemokine for neutrophils

[21]. It has been reported that P. gingivalis does not block the mRNA

expression of IL-8 but instead degrades IL-8 with its proteases [22, 23].

Another mechanism is resistance to LL-37. LL-37 is produced by epithelial

cells and neutrophils as a part of the chemical barrier of the epithelia at the

gingival sulcus. According to several studies, the late colonizers are more

resistant to LL-37 than other bacteria [24-27]. A third mechanism is survival

within macrophages. P. gingivalis can survive in macrophages through

crosstalk among several immune receptors, such as TLR2, CXC-chemokine

receptor 4 (CDCR4), CR3 and complement 5a receptor (C5aR) [28, 29]. A

fourth mechanism is resistance to phagocytosis by neutrophils that also occurs

by red complex bacteria [27, 30]. Therefore, the enhancement of neutrophil

function could be a good target to prevent of periodontitis. It is known that

various Toll-like receptor (TLR) ligands stimulate neutrophil function,

including FcR-mediated phagocytosis [31]. The aim of this study was to

investigate whether the stimulation of TLR2 can enhance the non-opsonic

phagocytosis of oral bacteria and subsequent killing in neutrophils.

4

II. Materials and Methods

2.1. Bacterial strains and growth conditions

All bacteria used in this study were obtained from the ATCC (American Type

Culture Collection, Bethesda, MD, USA). P. gingivalis ATCC 49417 was

cultured in brain heart infusion broth medium (BHI; BD Biosciences, Franklin

Lakes, NJ, USA) with 5 µg/ml Hemin (Sigma, St Louis, MO, USA) and 10

µg/ml Vitamin K (Sigma, St Louis, MO, USA) under anaerobic condition (5%

H2, 10% CO2 and 85% N2). Streptococcus sanguis 804 (NCTC 10904) was

cultured in brain heart infusion (BHI) broth under aerobic condition. Bacteria

in the log phase were harvested and washed twice with phosphate-buffered

saline (PBS; Gibco, NY, USA). Bacterial concentration was determined by

flow cytometry as previously described [32].

2.2. Preparation of fixed bacteria samples for the phagocytosis

assay and measurement of reactive oxygen species (ROS)

production

To prepare fixed bacterial samples, bacteria were collected by centrifugation

at 8,000 rpm for 5 minutes at room temperature, and then bacteria pellets were

washed twice and suspended in PBS and then incubated with 3.7 %

paraformaldehyde for 20 minute at room temperature. Finally, the samples

5

were washed once and suspended. For phagocytosis assay, bacteria were

stined with 5 mM 5[and-6]-carboxyfluorescein diacetate succinimidyl ester

(CFSE; Molecular Probes, Eugene, OR, USA) at room temperature for 30

minutes. The bacteria were sonicated (5 cycle of 10 seconds at the low power

with sonicators, Fisher Scientific, Fair Lawn, U.S.A) to dissociate aggregates

and counted with a FACSCalibur (BD Bioscience) to determine concentration.

2.3. Isolation of neutrophils

The use of human materials were approved by the Institutional Review Board

at the SNU School of Dentistry (S-D2009005). Leftover human peripheral

blood samples were provided by the Department of Diagnostic Medicine,

Seoul National University Hospital. To minimize inter-subject variation, a

mixture of 10 blood samples (1 ml each) was diluted in 1X Dulbecco's

phosphate-buffered saline (DPBS; Gibco, NY, USA) at 1:1, and layered in a

10 ml FICOLL-PAQUE™ plus (Amersham BioSchiencem Uppsala, Sweden)

at 720 x g for 40 min. After centrifugation, the pellet that contains the red

blood cells (RBCs) and polymorphonuclear cells (PMNs) was obtained, and

the PMNs were purified by lysing RBCs using hypotonic NaCl solutions. The

purified cells were suspended in RPMI 1640 medium (HyClone, UT, USA)

with 10% fetal bovine serum (FBS; HyClone, UT, USA) and 100 U/ml

penicillin and 100 µg/ml streptomycin (Gibco) and incubated on ice.

6

2.4. Phagocytosis assay

To examine the phagocytosing ability of neutrophils, purified neutrophils (1 x

105/assay) were incubated with CFSE-labeled bacteria at a cell to bacteria

ratio of 1:25 in the presence of various concentrations of Pam3CSK4

(Invivogen, San Diego, CAL, USA) for 60 minutes at 37℃. Incubated

neutrophils were washed with DPBS. After quenching the fluorescence of the

extracellular bacteria with 500 µl trypan blue (Gibco, Grand Island, N.Y.,

USA), the cells were analyzed with FACSCalibur. A negative control was

prepared with fixed cells treated with 3.7 % formaldehyde and then treated

with the same amount of CFSE-labeled bacteria. The phagocytosing ability of

cells was solely analyzed for live cells that were gated based on the forward

scatter and FL-3 fluorescence of trypan blue.

2.5. Enhanced chemiluminescence assay for ROS measurement

In 96-well plates, purified neutrophils (1 x 105/assay/100 μl) were incubated

with unlabeled fixed bacteria at a cell to bacteria ratio of 1:25 in the presence

of 0.5 mM luminol and various concentrations of Pam3CSK4 at 37℃ for 100

minutes. All the cells and reagents were resuspended in RPMI medium

supplemented with 10 % heat inactivated FBS and 2 mM L-glutamine. As a

7

negative and positive control, neutrophils were stimulated with medium alone

and 0.1 µM Phorbol 12-myristate 13-acetate (PMA; Sigma, St Louis, MO,

USA), respectively. After plating, the plate was immediately placed in a

microplate luminometer (BMGLabtech, Ortenberg, Germany), and light

emission in relative light units was recorded every 10 minutes during a 100

minutes incubation at 37℃.

2.6. Antibiotic protection assay

To examine the intracellular killing of live S. sanguinis and P. gingivalis

within neutrophils, purified neutrophils (1 x 105 cells/ 500 µl per FACS tube)

were incubated with those bacteria at a cell to bacteria ratio of 1:25 in the

presence of various concentrations of Pam3CSK4 at 37℃ for 30 minutes. Then,

the extracellular bacteria were killed by incubating the neutrophils in

antibiotic (gentamicin 30 µg/ml, amphotericin 15 µg/ml and amoxicillin 5

µg/ml)-containing fresh medium for 30 minutes. The antibiotic-treated

neutrophils were washed 3 times with cold DPBS and further cultured in fresh

medium without antibiotics at 37℃ for 1, 2, or 4 hours. The neutrophils were

lysed with sterile distilled water for 10 minutes to liberate intracellular

bacteria. The lysates were plated onto agar plates and incubated under the

appropriate condition until colonies were observed by the naked eye.

8

2.7. Measurement of bacterial adherence to neutrophils

In order to determine the effect of TLR2 activation on the bacterial adherence

to neutrophils, neutrophils were stimulated with various doses of Pam3CSK4

for 1 hour at 37℃. The stimulated neutrophils were incubated with CFSE-

labeled bacteria at a cell to bacteria ratio of 1:25 for 30 minutes on ice. The

neutrophils were washed twice with cold DPBS and analyzed immediately by

FACSCalibur (BD bioscience) while keeping them at cold conditions.

2.8. Statistical analysis

All experiments were performed at least three times in triplicate. Statistical

difference between the untreated control and the treated sample was analyzed

with t-test. A p value of <0.05 was considered statistically significant.

9

III. Results

3.1. Pre-stimulation of neutrophils with Pam3CSK4 enhances the

phagocytosis of S. sanguinis and concomitant ROS production.

To investigate the effect of TLR2 activation on the phagocytic function of

neutrophils, neutrophils were pre-stimulated with Pam3CSK4, a synthetic

TLR2 ligand, and incubated with CFSE-labeled S. sanguinis. Pre-stimulation

with Pam3CSK4 increased not only the percentage of neutrophils that

phagocytosed S. sanguinis but also the amount of S. sanguinis phagocytosed

by each neutrophil (Fig.1A left and right panel). Upon phagocytosis of the

microbes, neutrophils produce ROS, one of the important mechanisms to kill

phagocytosed microbes [33]. Therefore, the effect of pre-stimulation with

Pam3CSK4 on ROS production was also evaluated. Non-stimulated

neutrophils produced ROS upon exposure to S. sanguinis as expected. Pre-

stimulation with Pam3CSK4 increased the ROS production by neutrophils

more than twice (Fig. 1B). These results suggest that Pam3CSK4 increases the

phagocytosing ability and ROS production.

10

Figure 1. Pre-stimulation of neutrophils with Pam3CSK4 enhances the

phagocytosis of S. sanguinis and concomitant ROS production.

(A) Neutrophils were prestimulated with various concentrations of Pam3CSK4

at 37℃ for 60 minutes and then incubated with CFSE-labeled fixed S.

sanguinis at 37℃ for 60 minutes. The cells were analyzed by flow cytometry

after quenching the fluorescence of extracellular bacteria bound on the cell

surface with trypan blue. Phagocytosis was expressed by percentage of

phagocytosing cells and the mean fluorescence intensity (MFI) of total cells.

(B) Neutrophils were prestimulated with various concenturations of

Pam3CSK4 at 37℃ for 60 minutes. Cells were then incubated with luminol

and unlabeled fixed S. sanguinis at 37℃ for 100 minutes. ROS production

was measured every 10 min and a column graph represents the maximal peak

level during measurement period. *P < 0.05, compared with cells in the

absence of Pam3CSK4.

11

3.2. Simultaneous stimulation with Pam3CSK4 also enhances

phagocytosing activity and concomitant ROS production in

neutrophils.

The effect of simultaneous stimulation with Pam3CSK4 on the phagocytosis of

S. sanguinis or P. gingivalis was also examined. Simultaneous stimulation

with Pam3CSK4 also enhanced the ability of neutrophils to phagocytose both S.

sanguinis and P. gingivalis, presenting the maximal effect by 1 mg/ml

Pam3CSK4. However, the enhancing effect was greater for S. sanguinis than

for P. gingivalis (Fig. 2A). Stimulation with Pam3CSK4 induced ROS

production by itself and increased the bacteria-induced ROS production for

both S. sanguinis and P. gingivalis in a dose-dependent manner (Fig. 2B Left

panel). Interestingly, P. gingivalis attenuated the Pam3CSK4 (1 and 10 µg/ml)-

induced ROS production (Fig. 2B Right panel).

12

Figure 2. Simultaneous stimulation of neutophils with Pam3CSK4 also

enhances phagocytosing activity and concomitant ROS production in

neutrophils.

(A) Neutrophils were incubated with various concentrations of Pam3CSK4 and

CFSE-labeled fixed bacteria at 37℃ for 60 minutes and analyzed by flow

cytometry after quenching the fluorescence of extracellular bacteria bound on

13

the cell surface with trypan blue. Phagocytosis was expressed by percentage

of phagocytosing cells and MFI of total cells. *P < 0.05, compared with cells

in the absence of Pam3CSK4. (B) Neutrophils were incubated with various

concentrations of Pam3CSK4 and unlabeled bacteria in the presence of

luminol at 37℃ for 100 minutes. ROS production was measured every 10

minutes and a column graph represents the maximal peak level during

measurement. *, #, ǂ, P < 0.05, compared with cells in the absence of

Pam3CSK4 in each group. ψ, P < 0.05, compared with cells with Pam3CSK4

alone.

14

3.3. Neutrophils efficiently kill phagocytosed bacteria even in the

absence of a TLR2 stimulator

The increased ROS production induced by Pam3CSK4 may assist intracellular

killing of phagocytosed bacteria in neutrophils. To determine the effect of

Pam3CSK4 stimulation on the intracellular killing of bacteria, antibiotic

protection assay was performed. Right after phagocytosis and killing of

extracellular bacteria (0 h), the enhancement of phagocytosis by Pam3CSK4

was evident for both bacterial species. After 4 hours of further culture, all P.

gingivalis and 80 % of S. sanguinis were killed even in the absence of

Pam3CSK4 stimulation. The presence of 1 mg/ml Pam3CSK4 increased the

phagocytosis of S. sanguinis three times, 89 % of which was eliminated in 4

hours (Fig. 3).

15

Figure 3. Neutrophils efficiently kill phagocytosed bacteria even in the

absence of a TLR2 stimulator. Neutrophils (1 x 105/500 ㎕) were incubated

with live bacteria (2.5 x 106) in the presence of various concentrations of

Pam3CSK4 at 37 ℃ for 30 minutes. The extracellular bacteria were killed by

incubation with antibiotics for 30 minutes on ice, and the neutrophils were

further cultured in fresh medium without antibiotics at 37℃ for indicated

time. The intracellular bacteria were liberated by lysing the neutrophils and

cultured on agar plates until colonies were observed by the naked eye.

16

3.4. Stimulation with Pam3CSK4 increases the adhesion of P.

gingivalis, but not that of S. sanguinis, to neutrophils.

To understand the underlying mechanisms for the increased phagocytosis by

TLR2 activation, the effect of Pam3CSK4 stimulation on the adhesion of

bacteria to neutrophils was examined. Whereas the adhesion of S. sanguinis

did not show significant change, the adhesion of P. gingivalis to neutrophils

was increased by stimulation with Pam3CSK4 in a dose-dependent manner

(Fig. 4).

17

Figure 4. Stimulation with Pam3CSK4 increases the adhesion of P.

gingivalis, but not that of S. sanguinis, to neutrophils.

Neutrophils (1 x 105/500 ㎕) were stimulated with various concentrations of

Pam3CSK4 at 37 ℃ for 60 minutes. After stimulation, cells were incubated

with CFSE-labeled bacteria for 30 minutes on ice. Cells were washed twice

with DPBS and analyzed by flow cytometry without quenching. *P < 0.05,

compared with cells in the absence of Pam3CSK4.

18

IV. Discussion

Phagocytosis is the primary defense mechanism against bacteria. It involves

several events including the recognition of pathogens through various cell

surface receptors, receptor-mediated uptake, fusion of the phagosomes,

generation of ROS, and elimination of pathogens in the phagosomes [34]. A

number of studies have shown that TLRs on the cell surface of neutrophils

recognize various microbial structures such as lipopolysaccharides (TLR4),

lipoproteins (TLR2), and bacterial DNA (TLR9), resulting in the production

of cytokines and chemokines or the inhibition of neutrophil apoptosis [16, 35,

36]. This study showed that TLR2 activation enhances the phagocytosing

ability and ROS production in neutrophils.

Stimulation of neutrophils with a TLR2 ligand increased

phagocytosis of both S. sanguinis and P. gingivalis. Compared to S. sanguinis,

P. gingivalis was resistant to phagocytosis by neutrophils even in the presence

of Pam3CSK4. It is similar to the fact that T. denticola was resistant to

phagocytosis by neutrophils compared to S. sanguinis, even after opsonization

with specific antibodies [37]. Although the effect of Pam3CSK4 stimulation

was not compared with the effect of opsonizing antibodies in parallel, 1 mg/ml

Pam3CSK4 increased the phagocytosis of S. sanguinis about 4 times, which

was comparable to the effect of specific opsonizing antibodies shown in the

previous study [37]. As the underlying mechanisms for the enhancement of

phagocytosis, TLR2 activation may increase the bacterial adhesion to

19

neutrophils by modulating cell surface receptors for bacteria. However, only

the adhesion of P. gingivalis, but not the adhesion of S. sanguinis, was

increased by Pam3CSK4 stimulation. These results suggest that TLR2-assisted

phagocytosis in neutrophils works through different mechanisms depending

on the target bacterial species.

Induction of ROS production by TLR2 activation in neutrophils has

been already reported [16, 38]. Therefore, it is not surprising that neutrophils

exposed to bacteria produced increased amounts of ROS in the presence of

Pam3CSK4. The increase in ROS production may be necessary to handle the

increased amounts of phagocytosed bacteria. One interesting finding is

attenuation of the Pam3CSK4-induced ROS production by P. gingivalis, which

suggests the potential survival of P. gingivalis within neutrophils. However,

the small number of P. gingivalis phagocytosed by neutrophils did not survive

even in the absence of Pam3CSK4.

In summary, TLR2 activation increases phagocytosis of bacteria

and ROS production by neutrophils. Although the levels of phagocytosis of P.

gingivalis, a periodontal pathogen, are still low even by the TLR2-dependent

increase, a TLR2 ligand may be able to regulate the colonization of P.

gingivalis through efficient elimination of S. sanguinis an early colonizer in

subgingival biofilm.

20

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국

Toll 사 용체 2 (TLR 2) 가 인간

구에 한 구강 균 식균작용과

그에 하는 산소 생 에 미치는

효과

갑 열

울 학 학원

치 과학과 면역 분자 미생 공

(지도 : 님)

1. 목

치주염 만 염증 질 특히 치 과 치조골 같 구강

조직에 손상 이어 결국에는 치아 상실 가 다. 심각한 치

주염과 만 / 주 구 감소증 (chronic/cyclic

neutropenia), 구부착 결핍증 (leukocyte adhesion deficiency

syndrome), 피용-르페 르 증후군 (Papillon-Lefèvre), 그리고

아크-히가시 증후군 (Chédiak-Higashi) 등 식 포

27

(phagocytes)가 치주건강에 매우 요한 역할 담당하고 있다는

것 단 보여 다. 다양한 Toll 사 용체 리간드들

(Toll-like receptor ligands, TLRs) FcR 매개하는 식균작용

포함한 구 능 자극 한다. 본 연구는 TLR2 자극이

구에 한 구강 균 소닌 부재 식균작용과 식균 균 살

해를 증가 시킬 있는지 조사 하고자 하 다.

2. 법

인간 구 원천 는 말 액 다 핵 포를 ficoll hypaque

원심분리법 분리한 후 삼투합 NaCl용액 이용하여

구를 용해 시킴 써 분리하 다. Streptococcus sanguinis 804

(NCTC 10904) Porphyromonas gingivalis ATCC 49417 경우 3.7 %

라포름알데하이드 고 후 사용 하 고 포 내 생존 분

한 실험에는 생균 상태 사용하 다. 리아는 용

해 CFSE 염색 후 사용 었다. 구는 다양한 농도

Pam3CSK4 존재 하에 S. sanguinis P. gingivalis 함께

양하 다. 그리고 동 포분 (flow cytometry) 학 법

(chemiluminescence) 사용하여 식균작용과 산소 (ROS; reactive

oxygen species)를 측 하 다. 식균 S. sanguinis P. gingivalis

구 내 살해를 평가하 해 항생 보 분 (antibiotic

protection assay) 실행하 다.

3. 결 과

Pam3CSK4에 해 S. sanguinis P. gingivalis 식균작용이

모 농도 존 증가 하 고, 산소 생 도 농도

존 증가하는 결과를 얻었다. 미 운 P. gingivalis

28

Pam3CSK4를 함께 처리 하 산소 생 이 Pam3CSK4

단독 처리 시 증가 는 치보다 감소 결과를 보 다. 이것 P.

gingivalis 구 내 생존 가능 나타낸다. 하지만,

Pam3CSK4가 없어도 구는 식균 가지 균 모

효과 거 하 다. Pam3CSK4에 한 자극 P. gingivalis

부착 증가시 다. 하지만, S. sanguinis는 그 지 못하 다.

이것 구에 TLR2에 한 도움인 식균작용이 목 리아

종에 라 다른 커니즘 작용한다는 것 시한다.

키워드 : 치주염, 구, 식균작용, 산소, TLR2

학 번: 2008 - 22036