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 Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

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.     

Correlation of Toll‐Like Receptor 4, Interleukin‐18, Transaminases,and Uric Acid in Patients With Chronic Periodontitis and Healthy Adults

Background: Because of the potential association between periodontal disease and inflammation, the purpose of the present study is to examine the level of Toll‐like receptor 4 (TLR‐4), interleukin‐18 (IL‐18), and uric acid as markers of the inflammatory host response in the plasma and saliva of healthy individuals and patients with periodontitis. In addition, routine biochemical parameters such as fasting glucose, insulin, total cholesterol, high‐density lipoprotein (HDL) cholesterol, low‐density lipoprotein (LDL) cholesterol, triglycerides, alanine transaminase (ALT), and aspartate transaminase (AST) were measured. The authors also wanted to check whether patients with chronic periodontitis (CP) exhibit different modulations in salivary and/or plasma concentrations of these parameters compared with clinically healthy individuals. Methods: Saliva and plasma samples were collected from 40 patients with CP and 20 healthy individuals. TLR‐4 and IL‐18 measurements were done using commercially available enzyme‐linked immunosorbent assay kits. Total, HDL, and LDL cholesterol; triglycerides; fasting glucose; AST; and ALT levels were analyzed on a biochemistry analysis system using specific kits. Non‐parametric tests were used for certain parameters in the statistical analyses because the data did not follow Gaussian distribution. Results: Significant differences were observed in plasma and salivary TLR‐4 and IL‐18 levels, along with clinical measurements such as plaque index and probing depth, in patients with CP (P <0.001). The plasma level of TLR‐4 was found to be increased from 0.99 to 3.28 ng/mL in patients with CP. Salivary TLR‐4 levels also showed a slightly higher increase in the diseased state (12.44 to 29.97 ng/mL). A significant increase of ≈46% was recorded in the plasma IL‐18 level. However, salivary IL‐18 levels rose up to >5‐fold in the patients with CP compared with healthy individuals. The level of plasma uric acid was found to be highly significantly increased compared with control individuals. HDL cholesterol and triglyceride also showed significant differences (P <0.02 and P <0.03, respectively). Plasma glucose, total cholesterol, LDL cholesterol, and insulin levels did not show any significant difference. There was only a slight increase in plasma AST and ALT levels between diseased and healthy states (22.55 versus 25.50 IU/L and 12.35 versus 15.95 IU/L, respectively). However, salivary AST and ALT levels showed a ≈6‐fold rise in the patients with CP compared with the healthy individuals. Cross‐correlation analysis in the periodontitis disease group showed a significant association of plasma AST, salivary AST, and salivary ALT with uric acid level. Conclusions: Based on this study, the authors believe that TLR‐4, IL‐18, and uric acid could have a role in the inflammatory pathology of periodontitis. These parameters are suggested to be useful in the prognosis and diagnosis of CP. However, the mechanistic association of these parameters with inflammatory pathology of patients with periodontitis needs to be further elucidated in a higher number of samples.     

LncRNA MAFG‐AS1 regulates human periodontal ligament stem cell proliferation and Toll‐like receptor 4 expression

LncRNA MAFG‐AS1 is predicted to interact with miR‐146a, which can target Toll‐like receptor 4 (TLR4), a key player in periodontitis. This study aimed to investigate the roles of MAFG‐AS1 in periodontitis. It was observed that MAFG‐AS1 was downregulated in the human periodontal ligament stem cells (PDLSCs) derived from periodontitis‐affected teeth. Dual‐luciferase assay revealed that co‐transfection of MAFG‐AS1 expression vector and miR‐146a mimic showed significantly lower relative luciferase activity comparing to co‐transfection of MAFG‐AS1 expression vector and negative control (NC) miRNA. However, MAFG‐AS1 and miR‐146a failed to affect each other. Interestingly, MAFG‐AS1 overexpression led to the upregulated TLR4. In addition, MAFG‐AS1 overexpression also led to the inhibited proliferation of PDLSCs. Therefore, MAFG‐AS1 may regulate the proliferation of PDLSCs and the expression of TLR4 to participate in periodontitis.     

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.     

Toll‐like receptor 2‐mediated modulation of growth and functions of regulatory T cells by oral streptococci

This study was designed to determine whether oral streptococci modulate the growth and functions of regulatory T cells. Heat‐killed cells of wild‐type strains of Streptococcus gordonii and Streptococcus mutans induced the Toll‐like receptor 2 (TLR2) ‐mediated nuclear factor‐κB (NF‐κB) activation, but their lipoprotein‐deficient strains did not. Stimulation with these streptococci resulted in a significant increase in the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in splenocytes derived from both TLR2^+/+ and TLR2^?/? mice, but the level of increase in TLR2^+/+ splenocytes was stronger than that in TLR2^?/? splenocytes. Both strains of S. gordonii enhanced the proliferation of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells isolated from TLR2^+/+ mice at the same level as those from TLR2^?/? mice in an interleukin‐2‐independent manner. However, wild‐type and lipoprotein‐deficient strains of both streptococci did not enhance the suppressive activity of the isolated regulatory T cells in vitro, but rather inhibited it. TLR ligands also inhibited the suppressive activity of the regulatory T cells. Inhibition of the suppressive activity was recovered by the addition of anti‐IL‐6 antibody. Pretreatment of antigen‐presenting cells with the NF‐κB inhibitor BAY11‐7082 enhanced the suppressive activity of the regulatory T cells. These results suggested that interleukin‐6 produced by antigen‐presenting cells inhibits the suppressive activity of the regulatory T cells. Wild‐type strain, but not lipoprotein‐deficient strain, of S. gordonii reduced the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in the acute infection model, whereas both strains of S. gordonii increased it in the chronic infection model mice. Hence, this study suggests that oral streptococci are capable of modulating the growth and functions of regulatory T cells in vitro and in vivo.     

Toll样受体2和4在细胞成骨向分化中的作用

Toll样受体（TLR）4是人类发现的第一个TLR相关蛋白质,主要介导格兰阴性细菌的免疫过程。TLR2具有相对广泛的配体特异性,可识别多种病原体相关分子模式,协助TLR4参与人体内对脂多糖的反应。研究显示, TLR2和TLR4参与多种细胞成骨向分化的调控。譬如TLR2和TLR4即可通过促进主动脉瓣间质细胞的成骨向分化参与主动脉瓣钙化过程,也可通过上调冠状动脉内皮细胞中骨形态发生蛋白2的表达参与冠状动脉粥样硬化,还可参与骨髓间质干细胞的成骨向分化。在牙髓组织中,有TLR4表达的成牙本质细胞样细胞可向成牙本质细胞受损处迁移并形成修复性牙本质;而TLR2和TLR4作为参与牙髓防御反应的成员,在牙髓组织受损时,不仅可调节炎症因子的表达,还参与牙髓细胞成牙本质向分化的调控。由此可见,TLR2和TLR4在细胞成骨向分化中的作用机制值得进一步的探讨。     

Lack of Toll‐like receptor 4 decreases lipopolysaccharide‐induced bone resorption in C3H/HeJ mice in vivo

Introduction: Few in vivo studies have demonstrated whether Toll‐like receptor 4 (TLR4) is indispensable for lipopolysaccharide (LPS)‐induced bone resorption and little is known about the receptor activator of nuclear factor‐κB ligand (RANKL) and osteoprotegerin (OPG) expression induced by LPS under conditions of lack of TLR4. Methods: We compared bone resorption histomorphometrically in C3H/HeN and C3H/HeJ mice that were repeatedly injected with Actinobacillus actionmycetemcomitans LPS into their gingiva every 48 h. RANKL‐, interleukin‐1β‐ and OPG‐positive cells in the connective tissue were also compared immunohistochemically. Results: Bone resorption in C3H/HeJ mice in the fourth, seventh, and tenth injection groups was significantly less than that C3H/HeN mice (P < 0.05). The number of RANKL‐positive cells in C3H/HeJ mice in the 10th injection group was significantly smaller than that in C3H/HeN mice (P < 0.05). The numbers of interleukin‐1β‐positive cells in C3H/HeJ mice in the seventh and tenth injection groups were significantly decreased compared with those in C3H/HeN mice (P < 0.05). The numbers of OPG‐positive cells in C3H/HeN and C3H/HeJ mice gradually increased, but there was no significant difference between the two strains of mice. Conclusion: TLR4 is indispensable for LPS‐induced bone resorption in vivo.     

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.     

Innate Immune Receptor Expression in Peri‐Implant Tissues of Patients With Different Susceptibility to Periodontal Diseases

Background: Although inflammation mediates the pathogenesis of periodontal diseases, the effects of innate immune responses on implant therapies have not been evaluated. Innate immune receptors, including toll‐like‐receptors (TLRs) and the receptor for advanced glycated end‐products (RAGE), are upregulated within inflamed gingiva and are responsible for initiation of detrimental host responses. The aim of this study is to compare the expression of TLR2, TLR4, and RAGE in gingival tissues from participants susceptible to periodontitis and participants not susceptible to periodontitis before and after implant therapy. Methods: Periodontally healthy participants received implant therapy for non‐periodontal edentulism. Participants susceptible to periodontitis were diagnosed with chronic periodontitis prior to implant therapy. Gingival biopsies were collected from edentulous ridges before implant installation and from peri‐implant mucosa 2 months after treatment. Histology, real‐time PCR, and Western blot were used to evaluate levels of inflammatory infiltrate, TLR2, TLR4, and RAGE expression. Results: Before implant therapy, elevated levels of RAGE were detected in gingival tissues from participants susceptible to periodontitis when compared to those from participants with healthy periodontiums, whereas no differences in the expression of TLR2 or TLR4 were detected. After implant therapy, there was an upregulation of RAGE and TLR4 levels that coincided with a downregulation of TLR2 levels in biopsies from participants susceptible to periodontitis. Levels of RAGE and TLR4 remained unchanged in biopsies from participants with healthy periodontiums, whereas TLR2 levels were significantly upregulated. Histologically, post‐implant biopsies from participants susceptible to periodontitis displayed higher levels of inflammatory infiltrate. Conclusion: Elevated levels of inflammatory potential were found after implant therapy in participants susceptible to periodontitis.     

Ⅱ型糖尿病合并牙周炎患者龈下菌斑微生物群落分析

目的：本研究旨在比较Ⅱ型糖尿病合并慢性牙周炎患者与无糖尿病的牙周炎患者龈下菌斑微生物构成。方法：12名患者分为糖尿病合并慢性牙周炎组（T2DM＋CP组）与慢性牙周炎组（CP组）2组,各6人。记录所有患者基本信息及牙周临床参数,包括年龄、性别、探诊深度和附着丧失。根据探诊深度和附着丧失取患病位点的菌斑样本。PCR检测7种牙周可疑致病菌。采用DGGE分离扩增的16SrDNA片段。结果：两组结果显示两组牙周参数无显著差异。两组7种细菌检出率相似。所有对象中均检出牙龈卟啉单胞菌、福塞坦氏菌、齿垢密螺旋体和中间普氏菌,而具核梭杆菌在两组中均有一个样本未检出。变黑普氏菌在T2DM＋CP组的2个样本中检出,而CP组有4个样本检出。伴放线共聚菌在所有样本中均未检测到。DGGE分析结果示两组间条带数量及树状聚类分析均无显著差异。结论：Ⅱ型糖尿病合并牙周炎患者龈下菌斑的牙周可疑致病菌的检出情况以及DGGE分析与无糖尿病患者相似。     

Toll‐like receptor 5 activation promotes migration and invasion of salivary gland adenocarcinoma

J Oral Pathol Med (2011) 40 : 187–193 Background: Toll‐like receptor (TLR) signaling has been found to be closely associated with tumor development. The aim of this study was to examine whether activation of TLRs promote migration and invasion of salivary gland adenocarcinoma. Materials and Methods: TLR expression in SGT and HSG cells was examined by RT‐PCR. Wound scratch and chemotaxis cell migration assay were performed. Invasiveness was determined by Matrigel invasion assay. Results: All the tested TLRs including TLR1, TLR2, TLR4, and TLR5 and myeloid differentiation factor‐2 (MD‐2) were expressed on SGT and HSG cells. Treatment of flagellin, but not Pam₃CSK₄ and LPS, led to the production of IL‐6 and IL‐8, suggesting TLR5 is functional in both cells. Stimulation by flagellin also accelerated wound closure of SGT and HSG cells in a dose‐dependent manner. In addition, flagellin promoted migration and invasion ability of SGT cells. Blocking of TLR5 using antibody restored the promoting effect of flagellin on migration and invasion of SGT cells. Conclusion: These findings suggest that TLR5 activation by flagellin can promote migration and invasion of salivary gland adenocarcinoma.