Immune response profiling of primary monocytes and oral keratinocytes to different Tannerella forsythia strains and their cell surface mutants

The oral pathogen Tannerella forsythia possesses a unique surface (S‐) layer with a complex O‐glycan containing a bacterial sialic acid mimic in the form of either pseudaminic acid or legionaminic acid at its terminal position. We hypothesize that different T. forsythia strains employ these stereoisomeric sugar acids for interacting with the immune system and resident host tissues in the periodontium. Here, we show how T. forsythia strains ATCC 43037 and UB4 displaying pseudaminic acid and legionaminic acid, respectively, and selected cell surface mutants of these strains modulate the immune response in monocytes and human oral keratinocytes (HOK) using a multiplex immunoassay. When challenged with T. forsythia, monocytes secrete proinflammatory cytokines, chemokines and vascular endothelial growth factor (VEGF) with the release of interleukin‐1β (IL‐1β) and IL‐7 being differentially regulated by the two T. forsythia wild‐type strains. Truncation of the bacteria's O‐glycan leads to significant reduction of IL‐1β and regulates macrophage inflammatory protein‐1. HOK infected with T. forsythia produce IL‐1Ra, chemokines and VEGF. Although the two wild‐type strains elicit preferential immune responses for IL‐8, both truncation of the O‐glycan and deletion of the S‐layer result in significantly increased release of IL‐8, granulocyte‐macrophage colony‐stimulating factor and monocyte chemoattractant protein‐1. Through immunofluorescence and confocal laser scanning microscopy of infected HOK we additionally show that T. forsythia is highly invasive and tends to localize to the perinuclear region. This indicates, that the T. forsythia S‐layer and attached sugars, particularly pseudaminic acid in ATCC 43037, contribute to dampening the response of epithelial tissues to initial infection and hence play a pivotal role in orchestrating the bacterium's virulence.     

Characterization and immunostimulatory activity of extracellular vesicles from Filifactor alocis

Filifactor alocis, a gram‐positive, obligate anaerobic rod, is an emerging periodontal pathogen that is frequently isolated from patients with periodontitis, peri‐implantitis, and apical periodontitis. Recent studies have shown that extracellular vesicles (EVs) from gram‐negative periodontal pathogens, so‐called outer membrane vesicles (OMVs), harbor various effector molecules responsible for inducing host inflammatory responses. However, there are no reports of EVs from F. alocis. In this study, we purified and characterized the protein profiles of EVs from F. alocis and investigated their immunostimulatory activity on human monocytic THP‐1 and human oral keratinocyte HOK‐16B cell lines. Highly pure EVs were obtained from F. alocis using density gradient ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy showed that F. alocis EVs were between 50 and 270 nm in diameter. Proteome analysis identified 28 proteins, including lipoproteins, autolysins, F. alocis complement inhibitor (FACIN), transporter‐related proteins, metabolism‐related proteins, and ribosomal proteins. Human cytokine array analysis showed that F. alocis EVs remarkably induced the expression of CCL1, CCL2, MIP‐1, CCL5, CXCL1, CXCL10, ICAM‐1, IL‐1β, IL‐1ra, IL‐6, IL‐8, MIF, SerpinE, and TNF‐α in THP‐1 cells and CXCL1, G‐CSF, GM‐CSF, IL‐6, and IL‐8 in HOK‐16B cells. The immunostimulatory activity of F. alocis EVs was similar to that of the whole bacterial cells. Our findings provide new insight into the role of EVs from gram‐positive oral bacteria in periodontal diseases.     

Synergistic effects of lipopolysaccharides from periodontopathic bacteria on pro‐inflammatory cytokine production in an ex vivo whole blood model

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly associated with chronic periodontitis. This disease is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. The secretion of high levels of inflammatory cytokines by those cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of whole blood from periodontitis patients following challenges with whole cells of P. gingivalis, T. denticola, and T. forsythia or their lipopolysaccharides (LPS), individually and in combination. Whole blood collected from seven periodontitis patients was stimulated with whole cells or LPS and the production of interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor alpha (TNF‐α) were quantified by enzyme‐linked immunosorbent assays. The mono and mixed challenges with whole bacterial cells or LPS induced the secretion of high amounts of IL‐1β, IL‐6, IL‐8, and TNF‐α by the mixed leukocyte population from periodontitis patients. In addition, P. gingivalis LPS, T. denticola LPS, and T. forsythia LPS acted in synergy to induce high levels of IL‐1β and TNF‐α. This study suggests that P. gingivalis, T. denticola, and T. forsythia may contribute to the immunodestructive host response characteristic of periodontitis through synergistic effects of their LPS on the inflammatory response induced by a mixed population of leukocytes.     

Inflammatory response of uric acid produced by Porphyromonas gingivalis gingipains

Uric acid is a potential metabolite that serves as a danger‐associated molecular pattern (DAMP) and induces inflammatory responses in sterile environments. Porphyromonas gingivalis is a keystone periodontopathogen, and its gingipain proteases play a critical role in the pathogenesis of periodontitis. In this study, we demonstrate that P. gingivalis gingipains play a role in THP‐1 macrophage uric acid production by increasing the expression and activity of xanthine oxidoreductase (XOR). Uric acid sodium salt induces caspase‐1 activation, cell death, and the expression of proinflammatory cytokines, including IL‐1α, IL‐6, and IL‐8, in the human keratinocyte HOK‐16B cell line. Our results suggest that gingipain‐induced uric acid can mediate inflammation in periodontal tissue cells.     

The fate of Treponema denticola within human gingival epithelial cells

Treponema denticola is one of the major pathogens associated with chronic periodontitis. Bacterial invasion into gingival tissues is a critical process in the pathogenesis of periodontal disease. We recently reported that T. denticola can invade gingival epithelial cells. The aim of this study is to determine the fate of internalized T. denticola in gingival epithelial cells. Immortalized human gingival epithelial HOK‐16B cells were infected with 5‐ (and 6‐) carboxy‐fluorescein diacetate succinimidyl ester (CFSE)‐labeled live or heat‐killed T. denticola for 24 h, and the presence of bacteria inside the cells was confirmed by confocal microscopy. Live T. denticola, but not heat‐killed bacteria, invaded HOK‐16B cells. Confocal microscopy also revealed that internalized T. denticola rarely colocalized with either endosomes or lysosomes. Transmission electron microscopy of infected cells showed that intracellular T. denticola was localized inside endosome‐like structures. Although a culture‐based antibiotics protection assay could not detect viable intracellular T. denticola 12 h after infection, a substantial number of bacteria were observed by confocal microscopy and weak expression of bacterial 16S ribosomal RNA was detected 48 h after infection. In addition, flow cytometric analysis of HOK‐16B cells infected with CFSE‐labeled T. denticola showed no loss of fluorescence over 48 h. Collectively, T. denticola invades gingival epithelial cells and remains within the host cells for many hours by resisting endolysosomal degradation. These findings may provide new insight into the role of T. denticola in the pathogenesis of periodontitis.     

Azithromycin may inhibit interleukin-8 through suppression of Rac1 and a nuclear factor-kappa B pathway in KB cells stimulated with lipopolysaccharide

Background: Recent studies have shown that the 15‐member macrolide antibiotic azithromycin (AZM) not only has antibacterial activity, but also results in the role of immunomodulator. Interleukin (IL)‐8 is an important inflammatory mediator in periodontal disease. However, there have been no reports on the effects of AZM on IL‐8 production from human oral epithelium. Therefore, we investigated the effects of AZM on IL‐8 production in an oral epithelial cell line. Methods: KB cells were stimulated by Escherichia coli or Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS) with or without AZM. IL‐8 mRNA and protein expression and production in response to LPS were analyzed by quantitative polymerase chain reaction, flow cytometry, and enzyme‐linked immunosorbent assay. The activation of nuclear factor‐kappa B (NF‐κB) and Rac1, which is important for IL‐8 expression, was analyzed by enzyme‐linked immunosorbent assay and Western blotting, respectively. Results: IL‐8 mRNA expression, IL‐8 production, and NF‐κB activation in LPS‐stimulated KB cells were inhibited by the addition of AZM. LPS‐induced Rac1 activation was also suppressed by AZM. Conclusions: This study suggests that AZM inhibits LPS‐induced IL‐8 production in an oral epithelial cell line, in part caused by the suppression of Rac1 and NF‐κB activation. The use of AZM might provide possible benefits in periodontal therapy, with respect to both its antibacterial action and apparent anti‐inflammatory effect.     

In vivo expression of proteases and protease inhibitor,a serpin,by periodontal pathogens at teeth and implants

Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK‐proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri‐implant health and disease conditions. Biofilm and gingival crevicular fluid (GCF)/ peri‐implant sulcular fluid (PISF) samples were taken from healthy tooth and implant sites (n = 10), gingivitis and mucositis sites (n = 12), and periodontitis and peri‐implantitis sites (n = 10). Concentration of interleukin‐8 (IL‐8), IL‐1β and IL‐10 in GCF was determined by enzyme‐linked immunosorbent assay. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by quantitative PCR, whereas gene expression was estimated by quantitative RT‐PCR. The presence of P. gingivalis and T. forsythia, as well as the level of IL‐8 and IL‐1β, were associated with disease severity in the periodontal and peri‐implant tissues. In biofilm samples harboring T. forsythia, genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK‐protease genes and miropin. At the same time, detectable mRNA expression of individual genes ranged from 20.7% to 58.6% of samples (for forsylisin and miropsin‐1, respectively). In comparison with the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK‐proteases and especially miropin, might play a role in pathogenesis of both periodontal and peri‐implant diseases.     

DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines

Toll‐like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP‐1). THP‐1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA‐initiated cytokine production was determined either by blocking TLR9 signaling in THP‐1 cells with chloroquine or by measuring IL‐8 production and nuclear factor‐κB (NF‐κB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL‐1β, IL‐6, and TNF‐α) was increased significantly in bDNA‐stimulated cells compared with controls. Chloroquine treatment of THP‐1 cells decreased cytokine production, suggesting that TLR9‐mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9‐transfected cells demonstrated significantly increased IL‐8 production (P < 0.001) and NF‐κB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF‐κB genes in response to periodontal bDNA in THP‐1 cells, suggesting that cytokine induction is through NF‐κB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.     

Pathogenic potential of Tannerella forsythia enolase

Although enolases are cytosolic enzymes involved in the glycolytic pathway, they can also be secreted or expressed on the surface of a variety of eukaryotic cells and bacteria. Surface‐exposed enolases of eukaryotes and bacteria can function as plasminogen receptors. Furthermore, antibodies raised against bacterial enolases can react with host enolases, suggesting molecular mimicry between bacterial and host enzymes. In this study, we analyzed an enolase of the major periodontopathogen Tannerella forsythia, which is either secreted or present on the cell surface, via matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry and immunofluorescence, respectively. The T. forsythia enolase retained the enzymatic activity converting 2‐phosphoglycerate to phosphoenolpyruvate and showed plasminogen binding and activating ability, which resulted in the degradation of fibronectin secreted from human gingival fibroblasts. In addition, it induced proinflammatory cytokine production, including interleukin‐1β (IL‐1β), IL‐6, IL‐8, and tumour necrosis factor‐α (TNF‐a) in the human THP‐1 monocytic cell line. Taken together, our results demonstrate that T. forsythia enolase plays a role in pathogenesis in the host by plasminogen activation and proinflammatory cytokine induction, which has the potential to exaggerate inflammation in periodontitis.     

Retinoic acid‐inducible gene‐I is induced in gingival fibroblasts by lipopolysaccharide or poly IC: possible roles in interleukin‐1β, ‐6 and ‐8 expression

Retinoic acid‐inducible gene‐I (RIG‐I) is a member of the DExH family of proteins, and little is known of its biological function in the oral region. We previously reported that interleukin 1β (IL‐1β) induced RIG‐I expression in gingival fibroblasts. In this study, we studied the mechanism of RIG‐I expression induced by lipopolysaccharide (LPS) or double‐stranded RNA (dsRNA) in gingival fibroblasts. We also addressed the role of RIG‐I in the expression of IL‐1β, IL‐6 and IL‐8 in gingival fibroblasts stimulated with LPS or dsRNA. We stimulated cultured human gingival fibroblasts with LPS or dsRNA, and examined the expression of RIG‐I mRNA and protein. The effect of cycloheximide, a protein synthesis inhibitor, on RIG‐I induction by these stimuli was examined. The expression of IL‐1β, IL‐6 and IL‐8 in gingival fibroblasts transfected with RIG‐I cDNA stimulated with LPS or dsRNA was examined. LPS or dsRNA induced the expression of mRNA and protein for RIG‐I in concentration‐ and time‐dependent manners. We also examined the localization of RIG‐I, and found that it was expressed in cytoplasm. Cycloheximide did not suppress the LPS or dsRNA‐induced RIG‐I expression. Introduction of RIG‐I cDNA into gingival fibroblasts resulted in enhanced expression of IL‐1β, IL‐6 and IL‐8; moreover, overexpression of RIG‐I stimulated with LPS or dsRNA synergistically increased expression of IL‐1β, IL‐6 and IL‐8. RIG‐I may have important roles in the innate immune response in the regulation of IL‐1β, IL‐6 and IL‐8 expression in gingival fibroblasts in response to LPS and dsRNA.     

Divergence of the systemic immune response following oral infection with distinct strains of Porphyromonas gingivalis

Periodontitis is a polymicrobial oral infection characterized by the destruction of tooth‐supporting structures that can be linked to systemic diseases such as cardiovascular disease, diabetes or rheumatoid arthritis. Porphyromonas gingivalis, a bacterium implicated in the etiology of periodontitis, has shown variation in inducing T‐cell responses among different strains. Therefore, in this study we investigated the strain‐specific immune response using a murine experimental model of periodontitis. Periodontitis was induced by P. gingivalis strains A7A1‐28, W83 and W50, and later confirmed by the presence of P. gingivalis in the oral microflora and by alveolar bone resorption. Splenocytes were evaluated for gene expression, cellular proteins and cytokine expression. Dendritic cells were stimulated in vitro for T helper cell–cytokine profiling. Results showed that P. gingivalis had the ability to alter the systemic immune response after bacterial exposure. Strains W50 and W83 were shown to induce alveolar bone loss, whereas the A7A1‐28 strain did not significantly promote bone resorption in mice. Splenocytes derived from mice infected with strains W50 and W83 induced expression of high levels of interleukin‐4 (IL‐4) but A7A1‐28 stimulated increased IL‐10. Stimulation of dendritic cells in vitro showed a similar pattern of cytokine expression of IL‐12p40, IL‐6 and transforming growth factor‐β among strains. A distinct systemic response in vivo was observed among different strains of P. gingivalis, with IL‐10 associated with the least amount of alveolar bone loss. Evaluation of pathogen‐driven systemic immune responses associated with periodontal disease pathogenesis may assist in defining how periodontitis may impact other diseases.     

Rosuvastatin Inhibits Interleukin (IL)‐8 and IL‐6 Production in Human Coronary Artery Endothelial Cells Stimulated With Aggregatibacter actinomycetemcomitans Serotype b

Background: Rosuvastatin exhibits anti‐inflammatory effects and reduces periodontal diseases and atherosclerosis; however, its role in regulating periodontopathogen‐induced endothelial proinflammatory responses remains unclear. The purpose of this study is to determine whether rosuvastatin can reduce the proinflammatory response induced by Aggregatibacter actinomycetemcomitans (Aa) in human coronary artery endothelial cells (HCAECs). Methods: HCAECs were stimulated with purified Aa serotype b lipopolysaccharide (LPS) (Aa‐LPS), heat‐killed (HK) bacteria (Aa‐HK), or live bacteria. Expression of Toll‐like receptors and cellular adhesion molecules were evaluated by fluorometric enzyme‐linked immunosorbent assay. Endothelial cell activation was evaluated by quantifying nuclear factor (NF)‐kappa B‐p65 and cytokine expression levels by quantitative polymerase chain reaction and flow cytometry. Effect of rosuvastatin in expression of the atheroprotective factor Krüppel‐like factor 2 (KLF2) and cytokines were also studied using similar approaches. Results: HCAECs showed increased interleukin (IL)‐6, IL‐8, intercellular adhesion molecule 1, and platelet endothelial cell adhesion molecule 1 expression when stimulated with Aa‐LPS or Aa‐HK. NF‐κB‐p65 activation was induced by all antigens. Aa‐induced IL‐6 and IL‐8 production was inhibited by rosuvastatin, particularly at higher doses. Interestingly, reduced IL‐6 and IL‐8 levels were observed in HCAECs stimulated with Aa in the presence of higher concentrations of rosuvastatin. This anti‐inflammatory effect correlated with a significant increase of rosuvastatin‐induced KLF2. Conclusions: These results suggest Aa‐induced proinflammatory endothelial responses are regulated by rosuvastatin in a mechanism that appears to involve KLF2 activation. Use of rosuvastatin to prevent cardiovascular disease may reduce risk of endothelial activation by bacterial antigens.     

Gingipain‐dependent augmentation by Porphyromonas gingivalis of phagocytosis of Tannerella forsythia

In the pathogenesis of periodontitis, Porphyromonas gingivalis plays a role as a keystone pathogen that manipulates host immune responses leading to dysbiotic oral microbial communities. Arg‐gingipains (RgpA and RgpB) and Lys‐gingipain (Kgp) are responsible for the majority of bacterial proteolytic activity and play essential roles in bacterial virulence. Therefore, gingipains are often considered as therapeutic targets. This study investigated the role of gingipains in the modulation by P. gingivalis of phagocytosis of Tannerella forsythia by macrophages. Phagocytosis of T. forsythia was significantly enhanced by coinfection with P. gingivalis in a multiplicity of infection‐dependent and gingipain‐dependent manner. Mutation of either Kgp or Rgp in the coinfecting P. gingivalis resulted in attenuated enhancement of T. forsythia phagocytosis. Inhibition of coaggregation between the two bacterial species reduced phagocytosis of T. forsythia in mixed infection, and this coaggregation was dependent on gingipains. Inhibition of gingipain protease activities in coinfecting P. gingivalis abated the coaggregation and the enhancement of T. forsythia phagocytosis. However, the direct effect of protease activities of gingipains on T. forsythia seemed to be minimal. Although most of the phagocytosed T. forsythia were cleared in infected macrophages, more T. forsythia remained in cells coinfected with gingipain‐expressing P. gingivalis than in cells coinfected with the gingipain‐null mutant or infected only with T. forsythia at 24 and 48 h post‐infection. Collectively, these results suggest that P. gingivalis, mainly via its gingipains, alters the clearance of T. forsythia, and provide some insights into the role of P. gingivalis as a keystone pathogen.     

MAPKs, activator protein-1 and nuclear factor-κB mediate production of interleukin-1β-stimulated cytokines, prostaglandin E₂ and MMP-1 in human periodontal ligament cells

Murayama R, Kobayashi M, Takeshita A, Yasui T, Yamamoto M. MAPKs, activator protein‐1 and nuclear factor‐κB mediate production of interleukin‐1β‐stimulated cytokines, prostaglandin E ₂ and MMP‐1 in human periodontal ligament cells. J Periodont Res 2011; 46: 568–575. © 2011 John Wiley & Sons A/S Background and Objective: Determination of the interleukin‐1 (IL‐1) signaling cascades that lead to the production of various inflammatory mediators and catabolic factors may clarify attractive targets for therapeutic intervention for periodontitis. We comprehensively assessed the involvement of MAPKs, activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB) in IL‐1β‐induced production of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin E₂ (PGE₂) and MMP‐1 in human periodontal ligament cells. Material and Methods: Human periodontal ligament cells were pretreated with an inhibitor for each of the MAPKs or NF‐κB and subsequently treated with IL‐1β. Following treatment, phosphorylation of three types of MAPK (ERK, p38 MAPK and c‐Jun N‐terminal kinase), IκB kinase (IKK) α/β/γ and IκB‐α, as well as the DNA binding activity of AP‐1 and NF‐κB and the production of IL‐6, IL‐8, PGE₂ and MMP‐1, were determined by western blotting, a gel mobility shift assay and ELISA, respectively. Results: The three MAPKs, simultaneously activated by IL‐1β, mediated the subsequent DNA binding of AP‐1 at various magnitudes, while IKKα/β/γ, IκB‐α and NF‐κB were also involved in the IL‐1 signaling cascade. Furthermore, IL‐1β stimulated the production of IL‐6, IL‐8, PGE₂ and MMP‐1 via activation of the three MAPKs and NF‐κB, because inhibitors of these significantly suppressed the IL‐1β‐stimulated production of these factors. Conclusion: Our results strongly suggest that MAPK, AP‐1 and NF‐κB mediate the IL‐1β‐stimulated synthesis of IL‐6, IL‐8, PGE₂ and MMP‐1 in human periodontal ligament cells. Therefore, inhibition of activation of MAPK, AP‐1 and/or NF‐κB may lead to therapeutic effects on progression of periodontitis.     

Oral bacterial DNA differ in their ability to induce inflammatory responses in human monocytic cell lines

Background: Deoxyribonucleic acids (DNA) of periodontal pathogens, Porphyromonas gingivalis (Pg) and Tannerella forsythia, stimulate cytokine production in human monocytic cells (THP‐1) through Toll‐like receptor 9 (TLR‐9) and nuclear factor‐κB signaling. Fusobacterium nucleatum (Fn) is one of the most frequently isolated bacteria in periodontally diseased tissues and is reported to synergize with Pg, enhancing the pathogenicity. We investigate inflammatory mediator production in THP‐1 cells challenged with Fn and Streptococcus sanguinis (Ss) DNA, a non‐pathogenic oral bacteria, and further assess whether cytokines triggered by whole pathogens or Pg lipopolysaccharide (LPS) are affected by TLR‐9 signaling inhibitors (chloroquine). Methods: THP‐1 cells were stimulated with Pg‐DNA (100 ng/μL), Fn‐DNA (100 ng/μL), Ss‐DNA (100 ng/μL), Pg‐LPS (10 ng/μL), and heat‐killed whole bacteria (multiplicity of infection, 1:100) for 16 hours with or without chloroquine pretreatment (10 μg/mL). Interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor‐α levels were determined using enzyme‐linked immunosorbent assay. Statistical analyses included analysis of variance with multiple comparisons using Dunnett or Tukey methods and paired t test. A value of P <0.05 was significant. Results: Inflammatory mediator levels were increased in response to all the stimuli with the exception of Ss‐DNA (P <0.05). Chloroquine pretreatment significantly decreased cytokine production from THP‐1 cells with the exception of IL‐6 production triggered by whole Fn and Ss (P <0.05). Conclusions: Differences exist among oral bacterial DNA in inducing immune responses. By altering the conditions in cytosolic compartments, we can interfere with cellular responses triggered by extracellular receptor activation. Thus, alternative treatment approaches targeted to intracellular receptors might be of benefit in controlling periodontal inflammation.     

Focal adhesion kinase activation is required for TNF‐α‐induced production of matrix metalloproteinase‐2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts

Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF‐α) and its effects on interleukin (IL)‐6 and IL‐8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP‐2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real‐time PCR. Tumor necrosis factor alpha dose‐dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF‐α‐induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL‐6, IL‐8, and MMP‐2 in a dose‐dependent manner. Knockdown of FAK significantly suppressed TNF‐α‐induced expression of IL6 and IL8 mRNA and release of IL‐6 and IL‐8 protein in HPDLFs. Similarly, MMP‐2 down‐regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF‐α‐induced IL‐6, IL‐8, and MMP‐2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.     

Tannerella forsythia‐produced methylglyoxal causes accumulation of advanced glycation endproducts to trigger cytokine secretion in human monocytes

The periodontal pathogen Tannerella forsythia has the unique ability to produce methylglyoxal (MGO), an electrophilic compound which can covalently modify amino acid side chains and generate inflammatory adducts known as advanced glycation endproducts (AGEs). In periodontitis, concentrations of MGO in gingival‐crevicular fluid are increased and are correlated with the T. forsythia load. However, the source of MGO and the extent to which MGO may contribute to periodontal inflammation has not been fully explored. In this study we identified a functional homolog of the enzyme methylglyoxal synthase (MgsA) involved in the production of MGO in T. forsythia. While wild‐type T.forsythia produced a significant amount of MGO in the medium, a mutant lacking this homolog produced little to no MGO. Furthermore, compared with the spent medium of the T. forsythia parental strain, the spent medium of the T. forsythia mgsA‐deletion strain induced significantly lower nuclear factor‐kappa B activity as well as proinflammogenic and pro‐osteoclastogenic cytokines from THP‐1 monocytes. The ability of T. forsythia to induce protein glycation endproducts via MGO was confirmed by an electrophoresis‐based collagen chain mobility shift assay. Together these data demonstrated that T. forsythia produces MGO, which may contribute to inflammation via the generation of AGEs and thus act as a potential virulence factor of the bacterium.     

The Influences of Periodontal Status and Periodontal Pathogen Quantity on Salivary 8‐Hydroxydeoxyguanosine and Interleukin‐17 Levels

Background: Periodontitis is a biofilm‐initiated disease that is characterized by elevated inflammatory status. 8‐Hydroxydeoxyguanosine (8‐OHdG) and interleukin (IL)‐17 are highly associated with inflammation and bone resorption and therefore are regarded as potential biomarkers for periodontitis. In this study, the associations between salivary 8‐OHdG and IL‐17 levels and clinical and microbial parameters before and after non‐surgical treatment are investigated. Methods: Forty‐five patients with chronic periodontitis (CP) and 47 periodontally healthy volunteers were recruited for the study. Clinical parameters, including the probing depth (PD), clinical attachment level (CAL), sulcular bleeding index, and simplified oral hygiene index (OHI‐S), were examined for each participant. Microbial parameters including the quantities of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola in the subgingival plaque and saliva were determined by real‐time polymerase chain reaction at baseline and 1 and 3 months after the non‐surgical treatment. Salivary 8‐OHdG and IL‐17 levels were detected by enzyme‐linked immunosorbent assays. Results: Compared with healthy volunteers, CP group patients had significantly higher salivary 8‐OHdG and IL‐17 levels at baseline. Baseline salivary 8‐OHdG and IL‐17 levels were positively correlated with all clinical parameters as well as the quantities of T. forsythia and T. denticola. After non‐surgical treatment, baseline levels of salivary 8‐OHdG and IL‐17 were reduced significantly at both the 1‐ and 3‐month follow‐ups. The hierarchical linear model revealed that variations in the PD, CAL, and OHI‐S had significant positive effects on variation in the salivary 8‐OHdG level. However, variations in the PD; quantity of T. forsythia in the subgingival plaque; and quantities of P. gingivalis, T. forsythia, and T. denticola in saliva were associated significantly with variation in the salivary IL‐17 levels. Conclusions: There was a strong association between salivary 8‐OHdG and IL‐17 levels and periodontitis. Variation in the salivary 8‐OHdG level was correlated with variations in the clinical parameters, whereas variation in the IL‐17 level was correlated with variation in the microbial parameters.