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 .     

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.     

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.     

Analysis of Subgingival Plaque Ability to Stimulate Toll‐Like Receptor 2 and 4

Background: It has been shown that toll‐like receptor (TLR) 2‐ and TLR4‐stimulating abilities of supragingival plaque (SPP) are associated with periodontal conditions. It is hypothesized that SPP might affect the periodontium through its influence on subgingival plaque (SBP). This study investigates relationships between TLR2‐ and TLR4‐stimulating abilities of SBP and periodontal conditions. Methods: One hundred thirteen SBP samples were collected from the deepest pockets in patients with chronic periodontitis. TLR2‐ and TLR4‐stimulating abilities were measured using genetically engineered nuclear factor‐kappa B reporter cells. Numbers of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in each plaque sample were determined by real‐time polymerase chain reaction. Peripheral blood mononuclear cells (PBMCs) were stimulated with SBP samples in presence or absence of TLR4 or TLR2 inhibitor. Production of tumor necrosis factor (TNF)‐α and interleukin (IL)‐8 was analyzed by enzyme‐linked immunosorbent assay. Results: TLR4‐stimulating ability of SBP was associated with plaque index (PI), but not with other clinical parameters at sampling sites. TLR2‐stimulating ability of SBP was associated with none of the parameters. Number of P. gingivalis and A. actinomycetemcomitans in each plaque sample was not associated with TLR2‐ or TLR4‐stimulating ability of SBP. PBMCs stimulated with SBP samples produced TNF‐α and IL‐8, which was inhibited by TLR4 but not by TLR2 inhibitor. Conclusion: TLR4‐ but not TLR2‐stimulating ability of SBP is associated with PI. Enhanced TLR4‐stimulating ability at sites with accumulated plaque may mediate gingival inflammation.     

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.     

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.     

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.     

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell‐surface components through the CD14/Toll‐like receptor system

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin‐8 (IL‐8) responses of the cells to Salmonella lipopolysaccharide, a water‐soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll‐like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR‐related molecules, i.e. TLR2, TLR4, MD‐2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL‐8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL‐8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2‐ and TLR4‐independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti‐CD14 MAb.     

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.     

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.