The role of Toll‐like receptors in periodontitis

Periodontitis is a common infectious disease. Recent studies have indicated that the progression of periodontitis may be regulated by interactions between host immunity and periodontopathic bacteria. Although periodontopathic bacteria can destroy periodontal tissue, a dysfunctional host immune response triggered by the bacteria can lead to more severe and persistent destruction. Toll‐like receptors (TLRs), a type of pattern recognition receptor (PRR) that recognizes pathogens, have been implicated in host innate immune responses to periodontopathic bacteria and in the activation of adaptive immunity. TLR‐targeted drugs may hold promise to treat periodontal disease. This review summarizes recent studies on the role of TLRs in periodontitis and discusses areas needing further research. We believe TLRs may be an effective biomarker for the prevention, diagnosis, and treatment of periodontitis in the near future.     

Single nucleotide polymorphisms of pattern recognition receptors and chronic periodontitis

Sahingur SE, Xia X‐J, Gunsolley J, Schenkein HA, Genco RJ, De Nardin E. Single nucleotide polymorphisms of pattern recognition receptors and chronic periodontitis. J Periodont Res 2011; 46: 184–192. © 2010 John Wiley & Sons A/S Background and Objective: Periodontitis is a multifactorial disease influenced partly by genetics. Activation of pattern recognition receptors (PRRs) can lead to the up‐regulation of inflammatory pathways, resulting in periodontal tissue destruction. Hence, functional polymorphisms located in PRRs can explain differences in host susceptibility to periodontitis. This study investigated single nucleotide polymorphisms of PRRs including toll‐like receptor (TLR)2 (G2408A), TLR4 (A896G), TLR9 (T1486C), TLR9 (T1237C) and CD14 (C260T) in patients with chronic periodontitis and in periodontally healthy subjects. Methods: One‐hundred and fourteen patients with chronic periodontitis and 77 periodontally healthy subjects were genotyped using TaqMan® allelic discrimination assays. Fisher’s exact test and chi‐square analyses were performed to compare genotype and allele frequencies. Results: The frequency of subjects with the CC genotype of CD14 (C260T) (24.6% in the chronic periodontitis group vs. 13% in the periodontally healthy group) and those expressing the T allele of CD14 (C260T) (CT and TT) (75.4% in the chronic periodontitis group vs. 87% in the periodontally healthy group) was statistically different among groups (p = 0.04). Homozygocity for the C allele of the CD14 (C260T) polymorphism (CC) was associated with a two‐‐fold increased susceptibility to periodontitis (p = 0.04; odds ratio, 2.49; 95% confidence interval, 1.06–6.26). Individuals with the CC genotype of TLR9 (T1486C) (14.9% in the chronic periodontitis group vs. 28.6% in the periodontally healthy group) and those expressing the T allele of TLR9 (T1486C) (CT and TT) (85.1% in the chronic periodontitis group vs. 71.4% in the periodontally healthy group) were also significantly differently distributed between groups without adjustment (p = 0.03). Further analysis of nonsmokers revealed a significant difference in the distribution of genotypes between groups for TLR9 (T1486C; p = 0.017) and CD14 (C260T; p = 0.03), polymorphisms again without adjustment. Conclusion: The CC genotype of CD14 (C260T) is related to susceptibility to chronic periodontitis in Caucasians. In addition, differences observed in the distribution of TLR9 (T1486C) genotypes between groups warrant further investigation.     

Immunohistochemical localization of Toll‐like receptors 2 and 4 in gingival tissue from patients with periodontitis

The present study investigated the expression of Toll‐like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2‐, TLR4‐, CD14‐ and CD1a‐positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2‐positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4‐positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14‐positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down‐regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.     

In vivo expression of Toll‐like receptor 2, Toll‐like receptor 4, CSF2 and LY64 in Chinese chronic periodontitis patients

Oral Diseases (2010) 16 , 343–350 Objective: Toll‐like receptors (TLRs) are the essential components in the innate and adaptive immune systems. Colony stimulating factor 2 (CSF2) is a cytokine that may prevent endotoxin tolerance, and LY64 has the ability to interfere with the recognition of bacteria via TLR4. The aim of this study was to explore the in vivo expressions of TLR2, TLR4, CSF2 and LY64 in Chinese chronic periodontitis patients. Methods: Gingival biopsies were collected from 24 chronic periodontitis patients and 19 healthy controls. The gene expression profiles of TLR2, TLR4, CSF2 and LY64 were investigated by real‐time polymerase chain reaction, and the protein expressions of TLR2 and TLR4 were detected by immunohistochemistry. In addition, the levels of CSF2 in gingival crevicular fluid (GCF) were determined by ELISA. Results: The higher mRNA expressions of TLR2, TLR4 and CSF2, and the lower mRNA expression of LY64 were detected in chronic periodontitis patients. And the increased protein expressions of TLR2 and TLR4 were confirmed by immunohistochemistry. In addition, the increase of total amount of CSF2 in GCF was observed in chronic periodontitis patients. Conclusions: Our results suggest that TLR2 and TLR4 may play a role in periodontal pathogenesis. In addition, CSF2 and LY64 may contribute to the regulation of inflammatory response and maintaining periodontal homeostasis.     

Toll‐like receptors 2 and 5 in human gingival epithelial cells co‐operate with T‐cell cytokine interleukin‐17

Background/aim: Periodontitis begins as the result of perturbation of the gingival epithelial cells caused by subgingival bacteria interacting with the epithelial cells via pattern recognition receptors. Toll‐like receptors (TLRs) have been shown to play an important role in the recognition of periodontal pathogens so we have studied the interaction of TLR ligands with TLR2 and TLR5 for cytokine production in the cultures of gingival epithelial cells. Methods: Immunohistochemistry was used for the localization of TLR2 and TLR5 in tissue specimens. Enzyme‐linked immunosorbent assays were performed to detect the levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), released from gingival epithelial cell cultures following stimulation with TLR ligand alone or in combination with IL‐17. Results: Both TLR2 and TLR5 were increased in periodontitis (2128 ± 159 vs. 449 ± 59 and 2456 ± 297 vs. 679 ± 103, respectively, P < 0.001) including gingival epithelial cells that stained strongly. Cultured gingival epithelial cells stimulated with their respective ligands (HKLM, a TLR2 ligand that is also found in Porphyromonas gingivalis, and flagellin, a TLR5 ligand that is also found in Treponema denticola) produced both IL‐1β and TNF‐α. To mimic T‐cell help, IL‐17 was added. This further greatly enhanced TLR ligand‐induced IL‐1β (P < 0.001) and TNF‐α (P < 0.01) production. Conclusions: These findings show how pathogen‐associated molecular patterns, shared by many different periodontopathogenic bacteria, stimulate the resident gingival epithelial cells to inflammatory responses in a TLR‐dependent manner. This stimulation may be particularly strong in periodontitis and when T helper type 17 cells provide T‐cell help in intercellular cooperation.     

Activation of toll‐like receptors 2 and 4 by gram‐negative periodontal bacteria

Background/aims: Periodontitis is a chronic infectious disease associated with a gram‐negative subgingival microflora. Bacterial components stimulate, among other receptors, Toll‐like receptor (TLR) 2 and/or TLR4. Accumulating evidence indicates that both qualitatively and quantitatively distinct immune responses result from the triggering of TLR2 as compared to TLR4 triggering. The aim was to study the interaction of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum and Veillonella parvula with TLR2 and TLR4. We investigated all known serotypes (K⁻, K1–K6) of P. gingivalis and A. actinomycetemcomitans serotype a–e strains for their potency to stimulate cytokine production. Methods: Human embryonic kidney (HEK) cells, stably transfected with CD14, CD14‐TLR2, or CD14‐TLR4 and whole blood were stimulated with bacterial sonicates. Cytokine production (interleukin‐6, ‐8, ‐10 and ‐12) was measured in the supernatant by enzyme‐linked immunosorbent assay. Results: All test bacteria stimulated HEK‐CD14‐TLR2, but only A. actinomycetemcomitans and V. parvula stimulated HEK‐CD14‐TLR4. No differences were found in the activation of HEK‐CD14‐TLR2/4, or cytokine production in whole blood between serotypes of P. gingivalis and A. actinomycetemcomitans. Conclusion: Gram‐negative periodontal bacteria predominantly stimulated TLR2, which may be of importance for the Th1/Th2 cell orientation of the immune response in periodontitis.     

Quantitative messenger RNA expression of Toll‐like receptors and interferon‐α1 in gingivitis and periodontitis

Introduction: In addition to bacteria, viruses have been reportedly implicated in periodontitis. However, the available data are confined to Toll‐like receptor 2 (TLR2) and TLR4, which recognize bacterial products in periodontitis. In the present study, we investigated the expression levels of TLR5, ‐7, and ‐9 messenger RNAs (mRNAs) in addition to those of TLR2 and ‐4, and compared gingivitis and periodontitis. Interferon‐α1 (IFN‐α1), which is important for the antiviral response, was also compared. Methods: Gene expression was analyzed using quantitative real‐time polymerase chain reaction for 59 periodontitis and 27 gingivitis tissue samples together with viral serology in some patients. The presence of plasmacytoid dendritic cells (pDCs), a robust producer of IFN‐α, was immunohistochemically analyzed in an additional seven periodontitis and two gingivitis specimens. Results: The expression levels of TLR2, ‐4, ‐7, and ‐9 were significantly higher in periodontitis lesions than gingivitis lesions. The expression level of TLR5 was comparable to levels of TLR2 and ‐4; however, no significant difference was found between gingivitis and periodontitis. Although the expression of IFN‐α1 mRNA was higher in periodontitis lesions compared with gingivitis lesions, the level was quite low. Only a few pDCs were found in some periodontitis specimens. No difference was found for antibody‐positivity between gingivitis and periodontitis. Conclusion: This is the first study to show that a variety of TLRs are up‐regulated in periodontitis lesions compared with gingivitis lesions, suggesting that diverse microbial and possibly viral antigens are involved in the pathogenic mechanisms for periodontal diseases. However, the ligands recognized by the various TLRs in periodontal lesions remain to be determined.     

Salivary and plasma levels of Toll-like receptor 2 and Toll-like receptor 4 in chronic periodontitis

Background: This cross‐sectional study was planned to investigate whether patients with chronic periodontitis exhibit different salivary or plasma concentrations of Toll‐like receptor (TLR) 2 and TLR4 compared to subjects who are clinically healthy. Methods: Whole saliva and plasma samples were obtained and full‐mouth clinical periodontal measurements were recorded from 22 otherwise healthy patients with chronic periodontitis and 21 systemically and periodontally healthy control subjects. Salivary and plasma TLR2 and TLR4 levels were determined by enzyme‐linked immunoassays. Data were tested statistically using Mann‐Whitney U test. Results: The healthy group exhibited significantly lower values in all clinical measurements (P <0.001). The salivary TLR2 levels were similar in the two study groups (P >0.05). The patients with chronic periodontitis exhibited significantly higher salivary TLR4 (P <0.01) and plasma TLR2 and TLR4 levels (P <0.05). Conclusion: The present findings support a hypothesis that inflammation increases expression of TLRs which leads to an increased detection of TLRs in saliva and plasma, which could be useful as a diagnostic test for periodontal diseases.     

Differential expression of Toll‐like receptors in chronic hyperplastic candidosis

Introduction: The first step in the host defense against oral candidosis is the recognition of Candida albicans through a set of germ‐encoded pathogen recognition receptors, e.g. Toll‐like receptors (TLRs). In man, 10 types of such receptors have been identified so far, of which only TLR2, TLR4, and TLR6 have been linked to mediating candidal ligands, e.g. zymosan. Methods: Biopsies from patients with chronic hyperplastic candidosis (n = 5), leukoplakia (n = 5), and healthy mucosa (n = 5) were immunohistochemically stained with antibodies to the TLRs (TLR1 to TLR9) to distinguish and compare the staining patterns of the epithelial layer in the three categories of tissues. Results: On analysis, the epithelium of all tissues was divided into three layers: basal, middle, and superficial. Two of the five chronic hyperplastic candidosis sections showed high numbers of hyphae compared to yeasts, which paralleled a decrease in the expression of TLR2 and an increase in the staining intensity of TLR4. Leukoplakia and healthy tissue sections demonstrated stronger immunostaining of TLRs, except TLR9 which showed weaker staining in some sections of the former, and in the basal layers of some sections of the latter. Discussion: This study supports the concept of negative regulation of TLRs that are either ligand‐bound (e.g. in chronic hyperplastic candidosis), or not stimulated (in healthy tissue). It also augments the opinion that C. albicans, through its hyphae rather than blastospore, may utilize TLRs, i.e. TLR2, to evade the immune system of the host. Leukoplakia seems to be more immunologically alert, which reduces the chances of worsening the already‐diseased tissue.     

Determination of salivary levels of mucin and amylase in chronic periodontitis patients

Sánchez GA, Miozza V, Delgado A, Busch L. Determination of salivary levels of mucin and amylase in chronic periodontitis patients. J Periodont Res 2011; 46: 221–227. © 2010 John Wiley & Sons A/S Background and Objective: Patients with periodontal disease show differences in the profile of proteins in whole saliva. This profile reflects the nature and amplitude of the host response to a periodontal microbial challenge. Since periodontitis is a chronic inflammatory disease with different progression stages, the aim of the study was to evaluate the host response in these different clinical stages by assessing salivary flow rate, the concentrations of proteins and mucin and the amylase activity. Material and Methods: Sixty adult subjects were clinically examined and distributed into four groups (n = 15) according to the periodontal status, namely, healthy, mild, moderate and severe periodontitis. Whole saliva was collected for 5 min, followed by a second 5 min sampling period with stimulation by chewing a paraffin block, and flow rate was determined. Salivary proteins, amylase and mucin were determined by colorimetric methods. Results: The concentrations of proteins, amylase and mucin increased in subjects with moderate and severe periodontal disease in unstimulated saliva, while flow rate decreased. A positive correlation was found between proteins and amylase or mucin concentrations among the different groups, indicating that the concentrations changed in the same way, being the response of salivary glands to the disease, possibly to enhance the protective potential of saliva. Mucin concentration was lower in the mild periodontitis group. Mechanical stimulation induced an increase in flow rate and output of proteins, amylase and mucin. Conclusion: Periodontitis induces an increase in the output of proteins, including mucin and amylase, thereby enhancing the protective potential of saliva, but this is accompanied by a decrease in flow rate.