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Year : 2006  |  Volume : 17  |  Issue : 3  |  Page : 114-6
Expression of Toll-like receptors 2 and 4 in gingivitis and chronic periodontitis.

Department of Periodontics, Sri Ramachandra Medical College & Research Institute, Porur, Chenai, India

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Periodontal disease is the major cause of adult tooth loss and is commonly characterized by a chronic inflammation caused by infection by oral bacteria. Members of Toll-like receptor (TLR) family recognize conserved microbial structures, such as bacterial lipopolysaccharides, and activate signaling pathways that result in immune responses against microbial infections. The aim of the present study was to assess the mRNA expression of TLR-2 and TLR-4 in gingivitis and chronic periodontitis. Gingival tissue samples were collected from patients with chronic periodontitis, gingivitis, and healthy controls. Total RNA was extracted and RT-PCR was done for TLR-2 and TLR-4. The results showed that TLR-2 was significantly increased in gingivitis compared to TLR-4 expression and decreased in chronic periodontitis.

Keywords: TLR-2. TLR-4, chronic periodontitis, gingivitis

How to cite this article:
Sarah S M, Tamilselvan S, Kamatchiammal S, Suresh R. Expression of Toll-like receptors 2 and 4 in gingivitis and chronic periodontitis. Indian J Dent Res 2006;17:114

How to cite this URL:
Sarah S M, Tamilselvan S, Kamatchiammal S, Suresh R. Expression of Toll-like receptors 2 and 4 in gingivitis and chronic periodontitis. Indian J Dent Res [serial online] 2006 [cited 2019 Oct 21];17:114. Available from:

   Introduction Top

Periodontal disease consists of a group of infections that lead to inflammation of gingiva to destruction of periodontal tissues [1]. The current disease paradigm for the manifestation of periodontitis, involves both the bacterial and host components. In particular, evidence implicating a pathogenic role for a group of gram negative anaerobes including the species Porphyromonas gingivali has accumulated [2]. The immune response to microbial pathogens relies on both innate and adaptive components. A primary challenge to the innate immune system is the discrimination of a large number of receptors which has been met by the evolution of a variety of receptors that recognize conserved motifs on pathogens called the Toll-like receptors (TLR), homologous of the fruit fly Drosophila toll and belong to the interleukin-1 receptor family [4]. To date, 10 members of the TLR family have been identified in mammals and each TLR recognizes specific PAMP. For example, the lipopolysaccharide of gram negative bacteria is a TLR-4 ligand [5]. One of the central features of this system of microbial recognition is that TLRs activate signaling pathways that are critical for induction of the immune response to the given microbial challenge as periodontal pathology is initiated by gram positive organism and progressed by gram negative pathogen. This study was undertaken to determine the corresponding level of expression of TLR-2 and' TLR-4 in healthy gingiva, gingivitis and chronic periodontitis.

   Materials and methods Top

Gingival sample were obtained from eight chronic periodontitis and nine gingivitis (3 mild; 3 moderate; 3 severe) patients. Eight control samples were also obtained from healthy individuals who have undergone orthodontic treatement. Gingival tissues were collected at the time of respective surgery from patients. The patients with chronic periodontitis were diagnosed based on the criteria of American Academy of Periodontology classification and they did not suffer any other systemic diseases. All the diseased samples expressed clinical signs of inflammation and were taken from sites with a pocket depth greater than 6mm with evidence of bleeding on probing. The control subjects were systemically and periodontology healthy without gingival inflammation and showed no evidence of bleeding on probing with a pocket depth less than 4mm. All the patients and healthy subjects were nonsmokers from enquiry on general history. These patients had consented to the participation in this study with full knowledge of its content and implications.

The total RNA was isolated from the gingival tissues by single step, acid guanidiun thicyanate phenol chloroform extraction method [6]. The total RNA was transcribed to cDNA using first strand cDNA synthesis kit (Qbiogene, supplied by imperial BioMedics, Chandigarh, India) for RT-PCR according to manufacturer instructions. The TLRs primers were designed fromthe known sequences of TLR-2 and TLR-4 as: TLR-2 5' -GCCAAAGTCTTGATTGATTGG- 3' (sense) and 5'­TTGAAGTTCTCCAGCTCCTG-3' (antisense), TLR-4 5'­TCCCTCCAGGTTCTTGATTA-3' (sense) and 5'­GTAGTGAAGGCAGAGCTGAAA-3' (antisense) which amplifies a 347 and 495 base pair DNA fragment respectively. As a positive control 13-actin designed as 5' AAGGATTCCTATGTGGGC-3' (sense) and 5'- CATCTCTTGCTCGAAGTC-3' (antisense) which were predicted to amplify a 300 base pair DNA fragment. The amplication profile was as follows: denaturing at 94°C for 1 min; annealing at optimal temperature for TLR-2, TLR-4 and 13-actin gene from control as the control. The relative expression of each TLR-2 and TLR-4 gene from control, gingivitis and chronic periodontitis tissues were compared. The differences in the levels of expression of TLR-2 and TLR­4 from control, gingivitis and chronic periodontitis tissues were analyzed using t-test for comparison between groups and ANOVAfor comparison within groups.

   Results and discussion Top

The results showed that both the TLRs were expressed in control and the expression of TLR-2 was increased compared to TLR-4 (not significant). The expression of TLR-2 and TLR-4 was significantly elevated in tissues of gingivitis and chronic periodontitis compared to control [Figure - 1]. In gingivitis, TLR-2 expression was increased compared to TLR-4 (P<0.01). The expression of TLR-4 was significantly increased in chronic periodontitis compared to TLR-2 [Figure - 2]. The discovery of mammalian TLRs has provided new insight into mechanism of inmate immunity to microbial pathogens. Activation of TLRs

leads to the induction of antimicrobial pathways central to irmate defense as well as the up regulation of antigen presentation molecules and secretion of cytokines that influence the nature of adaptive immune response [7].The periodontium is integrally composed of the alveolar bone. Periodontal disease is initiated by microbial colonization of the superficially placed gingiva leading to inflammation of this tissue. This initial colonization is of a gram positive nature which later by means of microbial succession turns into a gram negative flora. The deeper structures like the periodontal ligament getting inflamed in untreated conditions with the predominance of gram negative is an ideal situation to study the amount of expression of TLR-2 and TLR-4.

TLR-2 and TLR-4 were found to be expressed in healthy human gingival tissues [8].The bacteria associated with periodontal health are primarily gram positive facultative species and small proportions of gram negative species are also found [9]. Studies have shown that human gingival fibroblasts expressed TLR-2 and TLR-4, which turned out to be significantly elevated in inflammation [10]. In our study, TLR-2 was expressed more in control than TLR-4, but the increase was not statistically significant.

As expected, we found a significant increase in TLR-2 expression in gingivitis as compared to control. The microbiota of gingivitis consists of gram positive rods, gram positive cocci and gram negative cocci [11]. Wang et al found increased expression of TLR-2 in human gingival fibroblasts of inflammatory gingiva than those in healthy group [12]. We observed that the increased expression of TLR-2 was statistically significant compared to TLR-4 in gingivitis and we also found more expression of TLR-2 in severe gingivitis tissues compared to mild and moderate gingivitis tissue samples. Gingival epithelial cells are central components of the barrier between oral micro flora and internal tissue. Human gingival epithelial cells predominantly expressed TLR-2 but not TLR-4 [13]. Contrary to our findings, Mori et al investigated the expression of TLR-2 and TLR-4 in human periodontal disease and showed higher TLR-2 positive cells in mild group and higher TLR-4 positive cells in severe gingivitis [14]. The bacteria found in severe gingivitis consist of roughly equal proportion of gram-positive (56%) and gram-negative (44%) species [15]. Though gram-negative bacteria were present in severe gingivitis, grain-positive bacteria were also found, confirming the increased expression of TLR-2 in severe gingivitis.

Interestingly, in periodontitis, TLR-4 was significantly more expressed than TLR-2. The structure of sub gingival plaque in periodontitis consists of, dense layer of gram positive organisms between this layer and pocket epithelium; spirochetes and other motile forms at the base of the pocket [16]. It is presently accepted that periodontal disease is initiated and perpetuated by specific gram negative bacteria, among which the black pigmented anaerobeP gingivalis has been implicated as the putative pathogen of periodontal disease. P gingivalis LPS has been considered to be an important pathogenic component in the initiation and development of periodontal disease, because bacterial LPS are known to be a potent stimulator of inflammatory cytokine production and bone resorption [17]. Human gingiva is exposed to a high density and diversity of gram positive and gram negative bacteria It is infiltrated by large number of TLR-2 and TLR-4 positve cells and that their numbers increase significantly in chronic periodontitis, relative to health [18].

TLR-4 is the dominant receptor for LPS, whereas TLR-2 is not required for LP S recognition [19]. TLR-2 mediates signals from other bacterial components including lipoteichoic acid, peptidoglycan and lipoprotein/ lipopoptides [20]. TLR-2 was significantly increased in chronic periodontitis compared to gingivitis. Bacteroids forsythus is a gram negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. It was found that signaling by B forsythus lipoprotein was mediated by TLR-2 but not TLR-4 [21]. In addition, TLR-2 participates, at least partly, in the signaling pathway to induce chemokine production against Pgingivalis components, probably other than LPS [22]. Studies have shown that human gingival fibroblasts expressed TLR-4 constitutively and that stimulation with P gingivalis LPS increased their levels of expression [23]. Hatakeyama et al reported that human gingival fibroblasts and human periodontal ligament fibroblasts expressed TLR-2 more strongly than human gingival fibroblasts [8]. From these findings, it was concluded that TLR-2 and' TLR-4 on gingival tissues recognize different bacterial cell wall component. So this pilot study of TLR-2 and TLR-4 expression in gingivitis and chronic periodontitis pave way for future studies on changes in the ratio of TLR-2 to TLR-4 in different stages gingivitis and periodontitis.

   References Top

1.Haffajee AD and Socransky SS: Microbial etiological agents of destructive periodontal disease, Periodonto12000, 5: 78-111,1994.  Back to cited text no. 1    
2.Moore WEC and Moore LVH: The bacteria of periodontal diseases, Periodontology 2000, 5: 66­77,1994.  Back to cited text no. 2    
3.TakedaK,KaishoTandAkiraS:Toll-likereceptors, Anna Rev hnmunol, 21: 335-376, 2003.  Back to cited text no. 3    
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5.Lemaitre B, Nicolas E, Michut L, Reichhart JM and Hoffman JA: The Dorsoventral regulatory gene cassette spatzel/Toll/cactus controls the potent antifungal response in drosophila adults, Cell, 86: 973-983,1996.  Back to cited text no. 5    
6.Chomezynski P and Sacchi N: RNA isolation by single step of acid guanidium thiocyanate- phenol­chloroform extraction, Ann Biochem, 163-2: 156­159,1987.  Back to cited text no. 6    
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8.Hatakeyanmra J, Tamai R and Sugiyama S: Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell surface components through CD14/Toll-like receptor system oral Microbiol hrrmunol, 18: 14­23,2003.  Back to cited text no. 8    
9.Socransky SS and Haffajee AD: The bacterial etiology of destruction disease: Current concepts, J Periodontol, 63; 322,1992.  Back to cited text no. 9    
10.Uehara A, Sugawara S and Tahada H: Priming of human oral epithelial cells by interferon gamma to secrete cytokines in response to lipopoly saccharides, lipoteichoic acids and peptidolycans, J Med Microbiol, 51: 626-634,2002.  Back to cited text no. 10    
11.Theilade E, Wright Wh, Jensen SB and Leo H: Experimental gingivitis in man It. A longitudinal clinical and bacteriological investigation, J PeriodontolRes,1:1,1966.  Back to cited text no. 11    
12.Wang PL, Ohma K, Flrjii T, Oido-Mori M, Kowashi Y; Kikuchi M and et al : DNAmicro array analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues, Biochem Biophys Res Common, 305: 970-973, 2003.  Back to cited text no. 12    
13.Asai Y, Ohyama Y, Gen K and Ogawa T: Bacterial fimbriae and their peptides active human gingival epithlial cells through TLR-2, Infect Immun, 69: 7387-7395,2001.  Back to cited text no. 13    
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15.Slot J: Subgingival microflora and periodontal disease, J Clin Periodontol, 6: 35   Back to cited text no. 15    
16.Hamada S, Fujiwara T and Maihara J: Bacterial endotoxic substances and their effects on host cells. In: Molecular pathogenesis of periodontal disease. Genco R, I-Iamada S, Lehner J, McGhee J, Mergenhagen S, (Eds), Washington DC: ASM Press. pages 105-117,1994.  Back to cited text no. 16    
17.Muthukura M, Jotnani R and cutler CW: Oral muscosal endotoxin tolerance induction in chronic periodontitis, Infect human, 73; 387-694, 2005.   Back to cited text no. 17    
18.Brightbill HD, Libraty DH, Krutzik SR, Yang RB, Belisle JT, Bleharski JR and et al : Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors, Science, 285: 732-736, 1999.  Back to cited text no. 18    
19.Schwandner R, Dziarski R, WescheH, RotheM and Kirsching CJ: Peptidoglycan and lipoteichioc acid induced cell activation mediated by toll-like receptor 2,JBiolChem,274:17406-17406,1999.   Back to cited text no. 19    
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21.Hasebe A, Yoshimura S, Into T, Kataoka H, Tanaka S, Arakawa S and et al : Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease, Infechrrmun,72:1318-1325,2004.  Back to cited text no. 21    
22.Kusumoto Y, Hirano H, Saitoh K, Yamada S, Takedachi M, Nazaki T, et al : Human gingival epithelial cell produce chemotactic factors IL-8 and monocyte chemoattactant protein-1 after stimulation with Porphyromonas gingivalis via Toll-like receptor-2, J Periodontol, 75: 370-379, 2004.  Back to cited text no. 22    
23.Tabeta K, Yamazaki K, Akashi S, Miyake K, KumadaH. Umemoto T and et al : Toll like receptos confer responsiveness to lipopolysaccharide, from Phorphyromonas gingivalis in human gingival fibroblast, Infect fibroblast, Infect human, 68: 3731-3735,2000.  Back to cited text no. 23    

Correspondence Address:
S M Sarah
Department of Periodontics, Sri Ramachandra Medical College & Research Institute, Porur, Chenai
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.29879

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