Effect of Tumor Necrosis Factor Alpha (Tnf-A) And Interleukin-10

(II-10) Levels of Aggressive Periodontitis In Rats (Rattus Norvegicus)

Induced by Agrregitibacter Actinomycetemcomitans

Dwi Leni Yuliana

, Yoes Prijatna Dachlan

and Heny Arwati

Post graduate school Universitas Airlangga, Surabaya, Indonesia

Faculty of Medicine Universitas Airlangga, Surabaya, Indonesia

Departement parasitology faculty of medicine Universitas Airlangga, Surabaya, Indonesia

Keywords: Aggregatibacter actinomycetemcomitans, Tumor Necrosis Factor Alpha (TNF-α), Interleukin 10 (IL-10),

Agressive Periodontitis.

Abstract: Aggregatibacter actinomycetemcomitans (A.actinomycetemcomitans) is a gram negative and a major

bacterial agent associated with aggressive periodontitis in young adults. These bacteria are an important

factor in pathogenesis of aggressive periodontitis. A. actinomycetemcomitans possesses fimbriae with an

adhesin protein that was is the first bacterial molecules to make physical contact with host. A complex

network of pro- and anti-inflammatory cytokines act in inflamed periodontal tissues. Among other

cytokines, interleukin-10 (IL-10) is an important multifunctional cytokine. An increase or decrease in IL-10

levels caused by bacterial infection is critical for the individual control of balance between inflammatory,

humoral and microbial challenges. Tumor necrosis factor-α (TNF-α) plays an important role in periodontal

inflammation as it has substantial potential to increase bone resorption and is involved in connective tissue

degradation by stimulating prostaglandin-E2 and colagenase. The purpose of this research was to analyze

TNF-α and IL-10 levels of aggressive periodontitis in rats (Rattus norvegicus) induced by A.

actinomycetemcomitans. This research was a true experimental study with Post Test Only Control Group

Design. Rats were divided into 4 groups for 0,25; 0.5 and 0.75 CFU/mL, and negative control. Each group

contained 5 rats. Aggressive periodontitis in rats was induced by injecting A.actinomycetemcomitans, at 48

hrs and 96 hrs post injection the inflammatory signs were observed, thee days later plasma were then

collected to measure TNF-α dan IL-10 level by ELISA. Analysis of Variance (ANOVA) showed

significantly increase levels of TNF-α in the infected group compared with that of the control group.

Aggressive periodontitis in rats showed by redness, abscess and tooth mobility. This condition indicated that

A.actinomycetemcomitans has the ability to adhere and invade the periodontal tissue further producing a

colony that caused periodontitis. High plasma level of TNF was seen in rats infected with 0.75 CFU/mL OF

ac, while IL-10 was low as seen in rats infected with 0.5 CFU/mL OF Ac.

1 INTRODUCTION

Periodontal disease and dental caries are the most

prevalent infections affecting the human dentition

(Brown et al., 1996). Periodontal disease is a chronic

bacterial infection characterized by persistent

inflammation, connective tissue breakdown and

alveolar bone destruction (Yamamoto et al., 2011).

Periodontitis, which is bacterially induced, can be

defined as a chronic inflammatory disease initiated

by dental plaque biofilm and perpetuated by a

deregulated immune response (Suvan et al., 2011)

usually accompanied by gingivitis resulting in

irreversible destruction of the connective tissues that

support the tooth, including the alveolar bone

(Yamamoto et al., 2011).

The gingiva, periodontium, alveolar bone and

cementum are structures that provide support to the

tooth. Any pathological process affecting

periodontium is defined as periodontitis. For a long

time, it was thought that gingivitis and periodontal

disease appeared as a result of aging of the

periodontal tissues that gave rise to inflammation

and recession of the gingival tissues bone and finally

Yuliana, D., Dachlan, Y. and Arwati, H.

Effect of Tumor Necrosis Factor Alpha (Tnf-A) And Interleukin-10 (II-10) Levels of Aggressive Periodontitis In Rats (Rattus Norvegicus) Induced by Agrregitibacter Actinomycetemcomitans.

DOI: 10.5220/0007542603430350

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 343-350

ISBN: 978-989-758-348-3

343

tooth loss. However, several studies have indicated

that this is not just an adult disease, but also appears

frequently in children (Escudero et al., 2008).

Gingiva is part of the mucosa of the oral cavity

that covers the alveolar bone and serves to protect

the underlying tissue. Normal gingiva has a pink

color, a supple consistency and a stippling texture or

orange peel. Periodontal ligaments are the

connective tissues that surround the teeth and bind

them to the bone. Periodontal ligaments serve to

protect blood vessels and nerves, tooth attachment to

bone and hard impact resistance due to occlusal

stress. Alveolar bone is a hard tissue composed of

layers of bone that serves as a support for teeth. The

cementum is the part that envelops the tooth root, is

hard, has no vena and serves as a periodontal

ligament adhesion (Carranza et al., 2006).

Periodontitis comes from interactions between

certain sub-gingival microorganisms, inflammation

and immune responses. Aggressive periodontitis is

predominantly the bacterium

A.actinomycetemcomitans which is the cause of

periodontal disease with progressive damage.

Bacteria A.actinomycetemcomitans release several

virulence factors as endotoxins and leukotoxins, and

infection factors by localized and systemic humoral

immune response (Carranza et al., 2002).

Bacteria A.actinomycetemcomitans have a

number of virulence factors that help the progression

of disease. Virulence determines the strength of the

pathogenic potential and also means the relative

capacity (quantity and quality) of the bacteria that

cause damage to the host and its ability to control

the body's defenses. Such bacterial virulence

includes its capacity in tissue destruction, invasive

bacterial levels, and the ability to avoid host defense

responses (Carranza et al., 2006).

Bacteria present in the plaque, including

lipopolysaccharide (LPS) and lipoteichoic acid,

interact with toll-like receptors in epithelial cells,

macrophages, leucocytes and fibroblasts, stimulating

the production of cytokines such as TNF-α, IL-1β,

IL-6, IL-8 and prostaglandin E2 (PGE2). To

facilitate leukocyte infiltration, fibroblasts

stimulated by TNF-α and IL-1β secrete matrix

metalloproteinase (MMPs), which degrade

extracellular matrix molecules including collagen.

The inflammatory response of periodontal tissue can

lead to tissue destruction and alveolar bone

resorption (Suvan et al., 2011).

Tumor necrosis factor-α (TNF-α) plays an

important role in periodontal inflammation. TNF-α

is primarily produced by activated macrophages.

TNF-α has a strong potential factor to increase bone

resorption and is involved in degradation of

connective tissue by stimulating PGE2 and

collagenase (Moore et al., 1994).

The complex network of pro- and anti-

inflammatory cytokines works on inflammatory

periodontal tissues. Among other cytokines,

interleukin-10 (IL-10) is an important

multifunctional cytokine. Increased or decreased

levels of IL-10 host are essential for balance control

between inflamed individuals (Gray, 2000).

Interleukin-10 is an anti-inflammatory cytokine,

produced by T-helper2 (Th2) cells, macrophages and

B cells, which inhibit the synthesis of pro-

inflammatory cytokines such as TNF-α, interleukin-

1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),

interleukin-8 (IL-8), and interferon-γ (IFNγ). IL-10

suppresses the production of metalloproteinase,

while increasing the synthesis of metalloproteinase

inhibitors in macrophages. In addition, it stimulates

the production of osteoprotegrin, which

consequently inhibits bone resorption by preventing

the involvement of the Receptor Activator of

Nuclear Factor Kappa-B Ligand (RANK-RANKL).

The IL-10 cytokine can be a protective cytokine in

periodontal disease and regulate pro-inflammatory

cytokines, including those involved in alveolar bone

loss. Individuals who are high IL-10 level producers

are more protected from periodontitis due to the

anti-inflammatory role of IL-10. Therefore, elevated

anti-inflammatory cytokine IL-10, will play a role in

regulating immune response against

periodontopatogenic bacteria (Bage, 2013).

The pathogenesis of periodontitis is initiated by

bacteria that release LPS. LPS then activates

inflammatory cells, resulting in the release of

cytokines and local factors. At the same time, the

bacterial components and inflammatory mediators

react directly to the osteoblast or progenitor,

resulting in a decrease in osteoblast function, and

then the loss of adhesions of periodontal and dental

tissue, including the alveolar bone and connective

tissue. Periodontitis is an inflammation that extends

through the gingiva and causes tissue damage

through tooth attachment. The dominant bacteria in

periodontitis are the gram negative ones that release

the LPS by activating the host mechanism primarily

that causes bone damage in periodontitis. Significant

periodontal damage is clinically known as

aggressive periodontitis (Carranza et al., 2006).

ICPS 2018 - 2nd International Conference Postgraduate School

344

2 BACKGROUND

Aggressive periodontitis generally affects

systemically healthy individuals less than 30 years

of age, though patients may be older. Aggressive

periodontitis is distinguished from chronic

periodontitis by the age of onset, the rapid rate of

destruction, composition of the subgingival

microflora, alteration in the host immune response,

familial aggregation of diseased individuals, and a

strong racial influence (Joshipura, 2015).

Disease of the periodontium occurring in an

otherwise healthy adolescent is characterized by

rapid loss of alveolar bone about more than one

tooth of the permanent dentition. The amount of

destruction is not commensurate with the amount of

local irritants (Albandar, 2014).

Key diagnostic criteria of this disease include an:

− Early age of onset, involvement of multiple teeth

with a distinctive pattern of clinical attachment

loss and radiographic bone loss.

− A relatively high rate of disease progression and

the absence of systemic diseases that

compromise the host's response to infection.

− Although in some patients the disease may start

before puberty, in most patients the age of onset

is during, or somewhat after, the circumpubertal

period. A typical patient shows disease onset at

an early age (i.e., before 25 years of age),

although identification of the affected patient

usually occurs after disease commencement.

− Initially, the periodontal lesions show a

distinctive pattern, depicted radiographically as

vertical bone loss at the proximal surfaces of

posterior teeth, and the bone loss usually occurs

bilaterally. In advanced cases of aggressive

periodontitis the periodontal lesions may be

depicted radiographically as a horizontal loss of

bone. The primary teeth may also be affected,

although early exfoliation of these teeth is not

common.

− Aggressive periodontitis may be localized or

generalized. In localized aggressive periodontitis

(LAP), tissue loss usually starts at the permanent

first molars and incisors, and with increasing

patient age the disease may progress to involve

the adjacent teeth. The generalized form of

aggressive periodontitis involves most or all of

the permanent teeth.

A.actinomycetemcomitans is a perio-pathogenic

bacteria that has long been associated with localized

aggressive periodontitis. The mechanisms of its

pathogenicity have been studied in humans and pre-

clinical experimental models. Although different

serotypes of A. actinomycetemcomitans have

differential virulence factor expression,

A.actinomycetemcomitans cytolethal distending

toxin (CDT), leukotoxin, and lipopolysaccharide

(LPS) have been most extensively studied in the

context of modulating the host immune response.

Following colonization and attachment in the oral

cavity, A.actinomycetemcomitans employs CDT,

leukotoxin, and LPS to evade host innate defense

mechanisms and drive a pathophysiologic

inflammatory response. This supra-physiologic

immune response state perturbs normal periodontal

tissue remodeling/turnover and ultimately has

catabolic effects on periodontal tissue homeostasis

(Herbert, 2016).

Figure 1 : A.actinomycetemcomitans colonizes the

gingival sulcus by attachment to the sulcular/junctional

epithelium cells (Herbert et al., 2016).

It subsequently invades through the epithelium

via pro-apoptotic virulence mechanisms and

penetrates into the subgingival connective tissue

where is stimulates epithelial cells and fibroblasts to

secrete pro-inflammatory cytokines (I). Neutrophils

and monocytes are thereby recruited to the local site

of infection and perpetuate the host inflammatory

response. Subsequently, B and T cells are recruited

to the diseased periodontium from the circulation

(II). T cells secrete pro-resorptive factors that drive

osteoclast (OC) formation and drive bone resorption.

A.actinomycetemcomitans simultaneously impairs

osteoblast (OB) function, perturbing bone

remodeling processes, which ultimately results in

catabolic alveolar bone loss (III).

A.actinomycetemcomitans virulence factors

interact with host cells to initiate an aberrant

inflammatory response in the periodontal gingival

tissues. While it has been reported that trans-

epithelial migration of polymorphonuclear

Effect of Tumor Necrosis Factor Alpha (Tnf-A) And Interleukin-10 (II-10) Levels of Aggressive Periodontitis In Rats (Rattus Norvegicus)

Induced by Agrregitibacter Actinomycetemcomitans

345

leukocytes (PMNs) into the gingival sulcus results in

a formed pseudo-barrier, which is several cell layers

thick between the plaque and junctional/sulcular

epithelium surface (Garant, 1976), this review

considers the gingival epithelium to be the initial

barrier to A.actinomycetemcomitans. First

responders are non-hematopoietic resident cells:

gingival fibroblasts and epithelium cells.

A.actinomycetemcomitans stimulates the host

responses via exotoxic and endotoxic virulence

factors, activating superficial epithelial cells and

underlying fibroblast cells.

A.actinomycetemcomitans can effectively migrate

through the gingival epithelium and once

A.actinomycetemcomitans bypasses these initial

barriers, a host inflammatory response is initiated.

Once A.actinomycetemcomitans penetrates deeper

in the subgingival tissues, a broader host immune

response is activated (Fives-Taylor et al., 1999).

A.actinomycetemcomitans immune stimulation

in the periodontal microenvironment elicits a

pathophysiologic pro-inflammatory state, which

disrupts normal periodontal tissue remodeling

processes ultimately promoting collateral tissue

damage. In periodontal disease, the supra-

physiologic level of pro-inflammatory and pro-

resorptive cytokines favors alveolar bone resorption

by monocyte or defined osteoclast progenitor

(dOCP) derived osteoclasts, versus alveolar bone

formation by mesenchymal derived osteoblastic

cells. When bone resorption exceeds bone

formation, an unbalanced bone remodeling process

having catabolic effects on alveolar bone

homeostasis, ultimately results in net alveolar bone

loss (Baron et al, 1978). Bone-lining tartrate

resistant acid phosphatase (TRAP) positive

multinucleated osteoclasts secrete bone degradation

enzymes, including matrix metalloproteinases

(MMPs) and cathepsin K in the acidic sealing zone

microenvironment, via integrin protein adherence to

the bone surface (McCauley et al, 2002; Teitelbaum

et al, 1997).

A.actinomycetemcomitans has been shown to

induce osteoclast formation and bone loss in rodent

animal models. Pro-inflammatory cytokines with

potent pro-resorptive actions, including tumor

necrosis factor (TNF)-α, IL-1, and IL-6 are highly

upregulated by A.actinomycetemcomitans and thus

promote osteoclast formation and bone resorption

(Hotokezaka et al., 2007). In humans,

A.actinomycetemcomitans positive patients had

significantly greater periodontal bone loss than the

A.actinomycetemcomitans negative subjects,

supporting A.actinomycetemcomitans’s remarkable

impact on periodontal disease associated alveolar

bone loss (Fine et al., 2007).

Tumor necrosis factor-a (TNF-α) plays a role in

periodontal inflammation. TNF-α is mainly

produced by activated macrophages. TNF-α has

strong potential to increase bone resorption and be

involved in tissue degradation with prostaglandin-E2

and collagenase (Morimoto et al., 2008). Several

studies have reported that there is an increase in

TNF-α levels in crevicular gingival fluid (CGF) and

gingival tissue in patients with periodontitis (Peggie

et al., 2015). Pro- and anti-inflammatory cytokine

tissue works on periodontal tissues that experience

inflammation. Among other cytokines, interleukin-

10 (IL-10) is an important multifunctional cytokine.

Increased or decreased levels of IL-10 host are very

important for controlling balance between

individuals who experience inflammation (Gonzales

et al., 2002).

Interleukin-10 is an anti-inflammatory cytokine,

produced by T-helper 2 (Th2) cells, macrophages

and B cells, which inhibit the synthesis of pro-

inflammatory cytokines such as interleukin-1 (IL-1),

interleukin-2 (IL-2), interleukin-6 (IL-6),

interleukin-8 (IL-8), tumor necrosis factor-a (TNF-

α) and interferon-γ (IFNγ). IL-10 production of radio

metalloproteinase also increases the inhibitory of

metalloproteinase tissue in macrophages (Lacraz et

al., 1995). In addition, production of the

osteoprotegrin hormone is a result of bone resorption

by preventing the Kappa-B Ligan Factor Nuclear

Receiver Activator (RANK-RANKL). Cytokines IL-

10 can be protective cytokines in periodontal disease

and regulate pro-inflammatory cytokines, including

those involved in lost alveolar bone. Individuals who

are high IL-10 producers are more protected from

periodontitis due to IL-10 anti-inflammatory.

Therefore, an increase in IL-10 anti-inflammatory

cytokines will play an immune response to

periodontopathogenic bacteria.

3 METHODS

This research was a true experimental study with

Post Test Only Control Group Design. Rats were

divided into 4 groups for 0.25; 0.5 and 0.75

CFU/mL, and negative control. Each group

contained 5 rats. How to obtain

A.actinomycetemcomitans bacteria with diluted

culture using 700 μl PBS with 2% Sodium

Carboxymethyl cellulose. Aggressive periodontitis

in rats was induced by injecting

A.actinomycetemcomitans, at 48 hrs and 96 hrs, post

ICPS 2018 - 2nd International Conference Postgraduate School

346

injection the inflamatory signs were observed, three

days later. Blood was centrifuged at 3000 rpm for 15

minutes to separate blood cells and plasma. Plasma

is stored in a freezer of -80

C then examination of

TNF-α and IL-10 levels. Plasma were then collected

to measure TNF-α dan IL-10 level by ELISA.

After obtaining data from the examination

results, the data is processed in several stages. The

data obtained is collected and checked whether there

are writing errors, results mismatches, etc. so that it

must be corrected. Data that has passed the editing

process is coded so that it is easily displayed in the

results table and it is analyzed. The data that has

been coded is displayed in the table of the results of

the examination so that it is easy to analyze and

interpret its meaning. After tabulation, the data were

analyzed according to the research objectives. In this

study, the results of TNF-α and IL-10 ELISA test

data from the patient's plasma were associated with

the degree of blood of patients with normal control

of plasma TNF-α and IL-10 ELISA test results. The

relationship data for each observed group is

described as looking for its meaning. Then the

ELISA test result will be analyzed using ANOVA.

4 RESULT

Analysis of Variance (ANOVA) showed

significantly increased levels of TNF-α in the

infected group compared with that of the control

group. Aggressive periodontitis in rats showed by

redness, abscess and tooth mobility. This condition

indicated that A. actinomycetemcomitans has the

ability to adhere and invade the periodontal tissue

further producing a colony that caused periodontitis.

Table 1. The mean and standard deviation of TNF-α levels

in rats (Rattus norvegicus) with localised aggressive

periodontitis.

Sample Mean

± SD

Median Max-

Min

Control 777,4

164,6

784,7 961,1-

531

0,0075

P1 (Bakteri

Aa 0,25

CFU/ml)

90 ±

41,84

886,9 948,7-

832,7

P2 (Bakteri

Aa 0,5

CFU/ml)

1043

67,24

1032 1137-

976,9

P3 (Bakteri

Aa 0,75

CFU/ml)

1213

96,87

1178 1347-

1116

Table 2. The mean and standard deviation of IL-10 levels

in rats (Rattus norvegicus) with localised aggressive

periodontitis.

Sample Mean

± SD

Median Max-

Min

Control 576 ±

327

628,3 953,3-

224,2

0,0051

(Bakteri

Aa 0,25

CFU/ml)

391,8

135,9

402,7 556,1-

178,8

(Bakteri

Aa 0,5

CFU/ml)

125,4

55,8

137 187,5-

37,7

(Bakteri

Aa 0,75

CFU/ml)

198,3

52,1

224,2 248,5-

137,1

Table 3. Ratio between TNF-α levels and IL-10

Mean Ratio

TNF-α IL-10

Control 777,4 576 1,3

0,25 CFU/ml 890 391,8 2,3

0,5 CFU/ml 1043 125,4 8,3

0,75 CFU/ml 1213 198,3 6,3

The highest ratio was found in the sample with

the treatment of injection of

A.actinomycetemcomitans bacteria with a

concentration of 0.5 CFU / ml because it had the

lowest IL-10 levels compared to the control group

(K), P1 and P3.

5 DISCUSSION

In periodontal health the oral cavity is colonized by

the oral commensal (non-pathogenic) flora. Under

physiological states the normal oral flora stimulates

the innate immune defense system in the

periodontium, which controls bacterial colonization

of periodontal tissues in close proximity to the

gingival sulcus. In this periodontal

microenvironment, A.actinomycetemcomitans

infection induces a supra-physiological immune

inflammatory response state, which disrupts normal

periodontal tissue homeostasis in the gingiva,

periodontal ligament (PDL), cementum, and alveolar

bone, ultimately promoting tooth loss.

A.actinomycetemcomitans virulence factors interact

with host cells to initiate an aberrant inflammatory

response in the periodontal gingival tissues. While it

Effect of Tumor Necrosis Factor Alpha (Tnf-A) And Interleukin-10 (II-10) Levels of Aggressive Periodontitis In Rats (Rattus Norvegicus)

Induced by Agrregitibacter Actinomycetemcomitans

347

has been reported that trans-epithelial migration of

polymorphonuclear leukocytes (PMNs) into the

gingival sulcus results in a formed pseudo-barrier,

which is several cell layers thick between the plaque

and junctional/sulcular epithelium surface (Garant,

1976). A.actinomycetemcomitans stimulates the host

responses via exotoxic and endotoxic virulence

factors, activating superficial epithelial cells and

underlying fibroblast cells.

A.actinomycetemcomitans can effectively migrate

through the gingival epithelium and once

A.actinomycetemcomitans bypasses these initial

barriers, a host inflammatory response is initiated.

Once A.actinomycetemcomitans penetrates deeper

in the subgingival tissues, a broader host immune

response is activated (Ahmed et al., 2001).

Figure 2. Effect of A.actinomycetemcomitans on human

blood cells causing periodontal inflammation and tissue

destruction (Malik et al., 2015).

One of the most studied virulence factors of

A.actinomycetemcomitans is leukotoxin. This toxin

is a 116 kDa protein produced by 56% of strains

isolated from LJP patients. The mechanism of

virulence leukotoxin is not only species specific but

also cell specific. The toxin binds to neutrophils,

monocytes, and a subset of lymphocytes; and forms

pores in the membranes of these target cells

overwhelming their ability to sustain osmotic

homeostasis, resulting in cell death. Interaction is

with polymorphonuclear leukocytes (PMNs).

Leukotoxin has shown to efficiently cause death of

human PMNs through extra-cellular release of

proteolytic enzymes from both primary and

secondary granules, along with activation and

release of matrix metalloproteinase-8, which can

contribute to periodontal tissue destruction (Malik et

al., 2015).

The ability of leukotoxin to induce apoptosis in

lymphocytes might impair the acquired immune

response of periodontal infections. A shift in the

balance between Th-1 and Th-2 subsets of T-cells is

found in periodontal inflammation, with the Th-2

cells to associate with chronic periodontitis. Its

ability to affect the lymphocytes also indicates a

possible role of this molecule in Th-1/Th-2/Th-17

differentiation, important in inflammatory

pathogenesis. Leukotoxin causes the activation of

caspase-1, which is a cytosolic cysteine proteinase

that specifically induces activation and secretion of

the pro-inflammatory cytokines interleukin-1 and

18, which result in monocyte/macrophage lysis by

incorporation in a cytosolic multimer complex

named the inflammasome (Malik et al., 2015).

TNF-α is a strong pro-inflammatory cytokine,

secreted by mononuclear leukocytes or macrophages

in the early stages of the inflammatory response.

TNF-α can stimulate osteoclasts, which lead to

alveolar bone resorption, and can increase the

release of matrix metalloproteinases (MMPs), which

leads to the destruction of the extracellular matrix of

periodontal tissue (Alexander et al., 1994). TNF-α is

strongly associated with the pathogenesis and

severity of periodontitis (Graves et al., 2003).

Compared with healthy periodontal, there was an

increase in TNF-α levels in Gingival crevicular fluid

(GCF) periodontitis and in inflammatory periodontal

tissues (Kennedy et al., 1990).

TNF-α affects cell migration by inducing and

regulating adhesion molecules to encourage

neutrophil turnover and adhesion to the vessel wall,

which can cause extravasation. It also stimulates

chemokine production, which is involved in the

migration of infected cells and inflammation

(Wajant et al., 2003). TNF-α also correlates with

extracellular matrix degradation and bone resorption

through the secretion of MMPs and RANKL

(Graves et al., 2008).

Periodontitis is a disease that involves bone

damage that can cause tooth loss. Therefore, anti-

inflammatory cytokines are needed to inhibit bone

resorption and increase alveolar bone regeneration

which is also associated with the role of IL-10 in

bone remodeling inhibiting bone resorption as well

as reducing inflammation (Cochran, 2008). IL-10 is

an anti-inflammatory cytokine that suppresses the

immunoproliferative and inflammatory responses.

As a factor produced by T helper2 cells (Th2), IL-10

inhibits cytokine production by Th1 cells. It is

known that IL-10 is also produced by many other

cell types, including B cells, mast cells, eosinophils,

macrophages, and dendritic cells (DC), and a large

number of T cell subsets such as CD8

T cells and

CD4

T-regulation cells (Petska, 2004).

IL-10 can reduce the synthesis of pro-

inflammatory cytokines and chemokines, such as IL-

ICPS 2018 - 2nd International Conference Postgraduate School

348

1, IL-6, and TNF-α. This can also reduce the

synthesis of nitric oxide, gelatinase, and collagenase.

IL-10 succeeded in increasing the neutralization of

synthesis of IL-1 and TNF-α. Therefore, IL-10 is

also considered an important regulator of

periodontal tissue homeostasis, in homeostatic and

inflammatory conditions (Lee et al., 2009). IL-10

can directly inhibit osteoclast formation. The

inhibitory effect of IL-10 on osteoclast formation is

by direct action on osteoclast precursors. The

molecular mechanism of this inhibition shows that

IL-10 increases the expression of osteoprotegerin

(OPG) but decreases the expression of NF-κB ligand

receptor activator (RANKL) and colony stimulating

factor-1 (CSF-1) (Liu et al., 2006).

Bone resorption is largely induced by the

production of pro-inflammatory cytokines, such as

TNF-a and IL-1. These cytokines can act by directly

increasing the proliferation and activity of cells in

osteoclasts or indirectly affecting the production of

osteoclast differentiation factors such as RANKL

and OPG through osteoblasts or stroma cells. IL-10

has been recognized to have strong anti-

inflammatory activity for a long time, and this has

proven to be an important endogenous suppressor of

bone resorption which means IL-10 can suppress

osteoclastic differentiation through the above aspects

(Boyle et al., 2003).

Gingival epithelial invasion and apoptosis. In the

in vitro model, A.actinomycetemcomitans was able

to migrate through the gingival layer and epithelial

cells by increasing the production of pro-

inflammatory cytokines TNF-a, IL-1β, IL-6, IL-8

and increasing cell apoptosis (Dickinson et al.,

2011). A study using A.actinomycetemcomitans in

model mice found that apoptosis of

A.actinomycetemcomitans was mediated by gingival

epithelial cells via the caspase3 and caspase7

pathways (Kang et al., 2012). At the subcellular

level, A.actinomycetemcomitans stimulates

phosphorylation via TGFβR1 which signals gingival

epithelial cells causing caspase3 activity to divide

and subsequent cell apoptosis (Yoshimoto et al.,

2014). This finding shows that A.

actinomycetemcomitans bacteria have the potential

to cross the gingival epithelium through the

mechanism of pro-apoptosis. Disruption of gingival

epithelial results in the induction of a periodontal

pathological inflammatory microenvironment that

supports recruitment of hematopoietic immune

response cells derived to subgingival tissues

(Yoshimoto et al., 2014).

Macrophages have been shown to play a role in

the pathogenesis of periodontitis caused by

A.actinomycetemcomitans. Monocytes enter the

tissue which results in local infection with

diapedesis from circulation where it can differentiate

into activated macrophages or osteoclasts. Llike

receptor toll receptors (TLRs) recognize pathogenic

constituents and have been extensively studied in

macrophage function. A.actinomycetemcomitans has

strong endotoxic LPS received by TLR2, TLR4, and

TLR5, with TLR4 currently being considered as the

main receptor for LPS, which clearly illustrates the

function of TLR2 and TLR4 signaling in

macrophage interactions with

A.actinomycetemcomitans (Park et al., 2014).

When bone marrow macrophages and TLR2 and

TLR4 were stimulated with

A.actinomycetemcomitans, TLR2 and TLR4

macrophages were attenuated and showed pro-

inflammatory production of TNF-α and IL-6

cytokines. MyD88 is an adapter protein for all TLRs

(except for TLR3), IL-1R, and IL-18R. MyD88

deficiency further reduces cytokine production by

A.actinomycetemcomitans, suggesting that while

TLR2 and TLR4 are critical regulators of

A.actinomycetemcomitans, which induce the

production of inflammatory cytokines, other

receptors also spread TNF-α and IL-6 production.

A.actinomycetemcomitans also stimulates the

production of IL-12p40 (IL-12B) in macrophages,

which mainly depend on MyD88. This finding

clearly shows that TLR signaling is very important

for the formation of inflammatory cytokines, but it

has not been explained how TLR4 and TLR2

interact with A.actinomycetemcomitans to modulate

their periopathogenesis (Park et al., 2014).

6 CONCLUSION

The induction of A.actinomycetemcomitans bacteria

on periodontal tissues can cause periodontitis by

being characterized when one of them is at high

levels of TNF-α and low levels of IL-10 in rats

(Rattus Norvegicus). In this case if TNF-α levels

rise and IL-10 levels fall, the severity of infection

and the prognosis of periodontitis in rats (Rattus

Norvegicus) is getting worse.

High plasma level of TNF was seen in rats

infected with 0.75 CFU/mL OF ac, while IL-10 was

low as seen in rats infected with 0.5 CFU/mL OF

Ac.

Effect of Tumor Necrosis Factor Alpha (Tnf-A) And Interleukin-10 (II-10) Levels of Aggressive Periodontitis In Rats (Rattus Norvegicus)

Induced by Agrregitibacter Actinomycetemcomitans

349

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