Oral Pathobiont-Induced Changes in Gut Microbiota Aggravate the Pathology of Nonalcoholic Fatty Liver Disease in Mice.

Background & Aims: Periodontitis increases the risk of nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanisms are unclear. Here, we show that gut dysbiosis induced by oral administration of Porphyromonas gingivalis, a representative periodontopathic bacterium, is involved in the aggravation of NAFLD pathology. Methods: C57BL/6N mice were administered either vehicle, P. gingivalis, or Prevotella intermedia, another periodontopathic bacterium with weaker periodontal pathogenicity, followed by feeding on a choline-deficient, l-amino acid-defined, high-fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD60). The gut microbial communities were analyzed by pyrosequencing the 16S ribosomal RNA genes. Metagenomic analysis was used to determine the relative abundance of the Kyoto Encyclopedia of Genes and Genomes pathways encoded in the gut microbiota. Serum metabolites were analyzed using nuclear magnetic resonance-based metabolomics coupled with multivariate statistical analyses. Hepatic gene expression profiles were analyzed via DNA microarray and quantitative polymerase chain reaction. Results: CDAHFD60 feeding induced hepatic steatosis, and in combination with bacterial administration, it further aggravated NAFLD pathology, thereby increasing fibrosis. Gene expression analysis of liver samples revealed that genes involved in NAFLD pathology were perturbed, and the two bacteria induced distinct expression profiles. This might be due to quantitative and qualitative differences in the influx of bacterial products in the gut because the serum endotoxin levels, compositions of the gut microbiota, and serum metabolite profiles induced by the ingested P. intermedia and P. gingivalis were different. Conclusions: Swallowed periodontopathic bacteria aggravate NAFLD pathology, likely due to dysregulation of gene expression by inducing gut dysbiosis and subsequent influx of gut bacteria and/or bacterial products.

Rice peptide with amino acid substitution inhibits biofilm formation by Porphyromonas gingivalis and Fusobacterium nucleatum.

OBJECTIVE: Rice peptide has antibacterial properties that have been tested in planktonic bacterial culture. However, bacteria form biofilm at disease sites and are resistant to antibacterial agents. The aim of this study was to clarify the mechanisms of action of rice peptide and its amino acid substitution against periodontopathic bacteria and their antibiofilm effects. DESIGN: Porphyromonas gingivalis and Fusobacterium nucleatum were treated with AmyI-1-18 rice peptide or its arginine-substituted analog, G12R, under anaerobic conditions. The amount of biofilm was evaluated by crystal violet staining. The integrity of the bacteria cytoplasmic membrane was studied in a propidium iodide (PI) stain assay and transmission electron microscopy (TEM). RESULTS: Both AmyI-1-18 and G12R inhibited biofilm formation of P. gingivalis and F. nucleatum; in particular, G12R inhibited F. nucleatum at lower concentrations. However, neither peptide eradicated established biofilms significantly. According to the minimum inhibitory concentration and minimum bactericidal concentration against P. gingivalis, AmyI-1-18 has bacteriostatic properties and G12R has bactericidal activity, and both peptides showed bactericidal activity against F. nucleatum. PI staining and TEM analysis indicated that membrane disruption by G12R was enhanced, which suggests that the replacement amino acid reinforced the electostatic interaction between the peptide and bacteria by increase of cationic charge and α-helix content. CONCLUSIONS: Rice peptide inhibited biofilm formation of P. gingivalis and F. nucleatum, and bactericidal activity via membrane destruction was enhanced by amino acid substitution.

A bacterial metabolite induces Nrf2-mediated anti-oxidative responses in gingival epithelial cells by activating the MAPK signaling pathway.

OBJECTIVE: Oxidative stress, which is defined as an imbalance between pro-oxidant and antioxidant systems, has been implicated in the development and/or progression of several inflammatory diseases, including periodontal disease. The reactive oxygen species (ROS) are the primary inducers of oxidative stress. In the induction of cytoprotective enzymes, the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling in antioxidant systems takes a main role. Notably, 10-oxo-trans-11-octadecenoic acid (KetoC), known as a bioactive metabolite generated by intestinal microorganisms, has been reported to have beneficial effects on several biological responses. Therefore, we investigated the antioxidant effect of KetoC on gingival epithelial cells (GECs) in this present study. METHODS: An SV40-T antigen-transformed human gingival epithelial cell line (Epi4) was used for experiments. The alteration of anti-oxidative stress related genes was analyzed by qPCR. The cellular ROS levels were evaluated by flow cytometry. To explore its molecular mechanisms, ARE promotor activity was analyzed by luciferase assay; the involvement of mitogen-activated protein kinase (MAPK) and G protein-coupled receptor 120 (GPR120) were evaluated by Western blotting and luciferase assay, respectively. RESULTS: KetoC significantly increased the expression of antioxidant-related genes in GECs. The level of ROS was significantly inhibited by the pretreatment of KetoC. Extracellular signal-regulated kinase (ERK) phosphorylation by KetoC promoted both the nuclear translocation of Nrf2 and its binding to the ARE in GECs. Further, GPR120 regulated the activation of KetoC induced-Nrf2-ARE signaling. CONCLUSION: KetoC exerts a protective function against the oxidative stress in GECs through GPR120-dependent ERK-Nrf2-ARE signaling.

Experimental arthritis and Porphyromonas gingivalis administration synergistically decrease bone regeneration in femoral cortical defects.

Porphyromonas gingivalis infection can lead to periodontitis and dysbiosis, which are known risk factors for rheumatoid arthritis (RA). We investigated whether P. gingivalis administration affected bone regeneration in mice with or without arthritis. We administered P. gingivalis to male DBA/1 J mice that were or were not sensitised to type II collagen-induced arthritis (CIA). All mice underwent drilling of bilateral femurs. We histologically evaluated new bone regeneration (bone volume of the defect [BVd]/tissue volume of the defect [TVd]) using micro-computed tomography (micro-CT), osteoclast number/bone area, and active osteoblast surface/bone surface (Ob.S/BS). We measured serum cytokine levels and bone mineral density of the proximal tibia using micro-CT. CIA resulted in significantly reduced bone regeneration (BVd/TVd) at all time-points, whereas P. gingivalis administration showed similar effects at 2 weeks postoperatively. CIA resulted in higher osteoclast number/bone area and lower Ob.S/BS at 2 and 3 weeks postoperatively, respectively. However, P. gingivalis administration resulted in lower Ob.S/BS only at 2 weeks postoperatively. During later-stage bone regeneration, CIA and P. gingivalis administration synergistically decreased BVd/TVd, increased serum tumour necrosis factor-α, and resulted in the lowest bone mineral density. Therefore, RA and dysbiosis could be risk factors for prolonged fracture healing.

Gingival epithelial barrier: regulation by beneficial and harmful microbes.

The gingival epithelium acts as a physical barrier to separate the biofilm from the gingival tissue, providing the first line of defense against bacterial invasion in periodontal disease. Disruption of the gingival epithelial barrier, and the subsequent penetration of exogenous pathogens into the host tissues, triggers an inflammatory response, establishing chronic infection. Currently, more than 700 different bacterial species have been identified in the oral cavity, some of which are known to be periodontopathic. These bacteria contribute to epithelial barrier dysfunction in the gingiva by producing several virulence factors. However, some bacteria in the oral cavity appear to be beneficial, helping gingival epithelial cells maintain their integrity and barrier function. This review aims to discuss current findings regarding microorganism interactions and epithelial barrier function in the oral cavity, with reference to investigations in the gut, where this interaction has been extensively studied.

Indirect regulation of PCSK9 gene in inflammatory response by Porphyromonas gingivalis infection.

Pro-protein convertase subtilisin/kexin type 9 (PCSK9), a secreted serine protease, regulates serum low-density lipoprotein (LDL) cholesterol levels by targeting the degradation of LDL receptor (LDLR) in the liver. Although previous reports describe elevated levels of PCSK9 in patients with periodontitis, the mechanisms that trigger this increase in serum PCSK9 levels and induce the related inflammatory response remain unclear. In an unc93b1-deficient mouse of Porphyromonas gingivalis infection, nucleic acid antigen recognition via Toll-like receptors was found to promote PCSK9 production, suggesting an indirect role for tumor necrosis factor-α as an inducer of PCSK9 in contrast to that reported in previous studies. Furthermore, PCSK9 production was independent of the TIR domain-containing adapter-inducing interferon-ß-dependent signaling pathway. These results indicate that changes in LDLR expression precede an increase in the serum PCSK9 level in the context of an infectious disease such as periodontitis.

An orally administered oral pathobiont and commensal have comparable and innocuous systemic effects in germ-free mice.

BACKGROUND AND OBJECTIVES: We recently proposed a novel mechanism linking periodontitis and systemic diseases, in which orally administered Porphyromonas gingivalis affects gut microbiota composition and subsequently leads to systemic inflammation. However, the mechanism by which P. gingivalis generates systemic effects from the gut is unknown. MATERIAL AND METHODS: Six-week-old germ-free mice were orally administered with either an oral pathobiont P. gingivalis or an oral commensal Lactobacillus salivarius twice a week for 5 weeks. Control mice were administered with vehicle only. Alveolar bone resorption was evaluated histologically. The expression profile of various genes was analyzed in gingival tissue, liver, small intestine and large intestine using real-time polymerase chain reaction. Sera were analyzed for antibody, endotoxin and interleukin (IL)-6 levels. Antibody levels were also analyzed for culture supernatant of cells from mesenteric lymph nodes and spleens. A proportion of T-helper 17 and Treg in the cells from mesenteric lymph nodes and spleens was analyzed by flow cytometry. The level of IL-6 and IL-17 in the cell culture supernatants was analyzed by enzyme-linked immunosorbent assay. RESULTS: P. gingivalis administration did not induce alveolar bone resorption. Although P. gingivalis elicited systemic antibody response in germ-free mice, unlike in specific pathogen-free mice, P. gingivalis did not induce an inflammatory response in gingiva, liver and intestinal tissue, or alter the proportion of T-helper 17 and Treg. However, IL-6 and IL-17 productions were significantly elevated and tended to be elevated, respectively, in the cells from mesenteric lymph nodes of P. gingivalis-administered mice. Interestingly, the expression of IL-10 and tight junction protein in the gingiva and intestine, respectively, was significantly upregulated in P. gingivalis-treated mice. Administration of L. salivarius elicited almost similar effects as P. gingivalis. CONCLUSION: The oral pathobiont P. gingivalis did not induce any detectable pathogenic changes or any major host responses when administered to germ-free mice. There may be indirect mechanisms for gut-mediated systemic effects by P. gingivalis.

Periodontal manifestations of systemic diseases and developmental and acquired conditions: Consensus report of workgroup 3 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.

BACKGROUND: A variety of systemic diseases and conditions can affect the course of periodontitis or have a negative impact on the periodontal attachment apparatus. Gingival recessions are highly prevalent and often associated with hypersensitivity, the development of caries and non-carious cervical lesions on the exposed root surface and impaired esthetics. Occlusal forces can result in injury of teeth and periodontal attachment apparatus. Several developmental or acquired conditions associated with teeth or prostheses may predispose to diseases of the periodontium. The aim of this working group was to review and update the 1999 classification with regard to these diseases and conditions, and to develop case definitions and diagnostic considerations. METHODS: Discussions were informed by four reviews on 1) periodontal manifestions of systemic diseases and conditions; 2) mucogingival conditions around natural teeth; 3) traumatic occlusal forces and occlusal trauma; and 4) dental prostheses and tooth related factors. This consensus report is based on the results of these reviews and on expert opinion of the participants. RESULTS: Key findings included the following: 1) there are mainly rare systemic conditions (such as Papillon-Lefevre Syndrome, leucocyte adhesion deficiency, and others) with a major effect on the course of periodontitis and more common conditions (such as diabetes mellitus) with variable effects, as well as conditions affecting the periodontal apparatus independently of dental plaque biofilm-induced inflammation (such as neoplastic diseases); 2) diabetes-associated periodontitis should not be regarded as a distinct diagnosis, but diabetes should be recognized as an important modifying factor and included in a clinical diagnosis of periodontitis as a descriptor; 3) likewise, tobacco smoking - now considered a dependence to nicotine and a chronic relapsing medical disorder with major adverse effects on the periodontal supporting tissues - is an important modifier to be included in a clinical diagnosis of periodontitis as a descriptor; 4) the importance of the gingival phenotype, encompassing gingival thickness and width in the context of mucogingival conditions, is recognized and a novel classification for gingival recessions is introduced; 5) there is no evidence that traumatic occlusal forces lead to periodontal attachment loss, non-carious cervical lesions, or gingival recessions; 6) traumatic occlusal forces lead to adaptive mobility in teeth with normal support, whereas they lead to progressive mobility in teeth with reduced support, usually requiring splinting; 7) the term biologic width is replaced by supracrestal tissue attachment consisting of junctional epithelium and supracrestal connective tissue; 8) infringement of restorative margins within the supracrestal connective tissue attachment is associated with inflammation and/or loss of periodontal supporting tissue. However, it is not evident whether the negative effects on the periodontium are caused by dental plaque biofilm, trauma, toxicity of dental materials or a combination of these factors; 9) tooth anatomical factors are related to dental plaque biofilm-induced gingival inflammation and loss of periodontal supporting tissues. CONCLUSION: An updated classification of the periodontal manifestations and conditions affecting the course of periodontitis and the periodontal attachment apparatus, as well as of developmental and acquired conditions, is introduced. Case definitions and diagnostic considerations are also presented.

Periodontal manifestations of systemic diseases and developmental and acquired conditions: Consensus report of workgroup 3 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.

BACKGROUND: A variety of systemic diseases and conditions can affect the course of periodontitis or have a negative impact on the periodontal attachment apparatus. Gingival recessions are highly prevalent and often associated with hypersensitivity, the development of caries and non-carious cervical lesions on the exposed root surface and impaired esthetics. Occlusal forces can result in injury of teeth and periodontal attachment apparatus. Several developmental or acquired conditions associated with teeth or prostheses may predispose to diseases of the periodontium. The aim of this working group was to review and update the 1999 classification with regard to these diseases and conditions, and to develop case definitions and diagnostic considerations. METHODS: Discussions were informed by four reviews on 1) periodontal manifestions of systemic diseases and conditions; 2) mucogingival conditions around natural teeth; 3) traumatic occlusal forces and occlusal trauma; and 4) dental prostheses and tooth related factors. This consensus report is based on the results of these reviews and on expert opinion of the participants. RESULTS: Key findings included the following: 1) there are mainly rare systemic conditions (such as Papillon-Lefevre Syndrome, leucocyte adhesion deficiency, and others) with a major effect on the course of periodontitis and more common conditions (such as diabetes mellitus) with variable effects, as well as conditions affecting the periodontal apparatus independently of dental plaque biofilm-induced inflammation (such as neoplastic diseases); 2) diabetes-associated periodontitis should not be regarded as a distinct diagnosis, but diabetes should be recognized as an important modifying factor and included in a clinical diagnosis of periodontitis as a descriptor; 3) likewise, tobacco smoking - now considered a dependence to nicotine and a chronic relapsing medical disorder with major adverse effects on the periodontal supporting tissues - is an important modifier to be included in a clinical diagnosis of periodontitis as a descriptor; 4) the importance of the gingival phenotype, encompassing gingival thickness and width in the context of mucogingival conditions, is recognized and a novel classification for gingival recessions is introduced; 5) there is no evidence that traumatic occlusal forces lead to periodontal attachment loss, non-carious cervical lesions, or gingival recessions; 6) traumatic occlusal forces lead to adaptive mobility in teeth with normal support, whereas they lead to progressive mobility in teeth with reduced support, usually requiring splinting; 7) the term biologic width is replaced by supracrestal tissue attachment consisting of junctional epithelium and supracrestal connective tissue; 8) infringement of restorative margins within the supracrestal connective tissue attachment is associated with inflammation and/or loss of periodontal supporting tissue. However, it is not evident whether the negative effects on the periodontium are caused by dental plaque biofilm, trauma, toxicity of dental materials or a combination of these factors; 9) tooth anatomical factors are related to dental plaque biofilm-induced gingival inflammation and loss of periodontal supporting tissues. CONCLUSION: An updated classification of the periodontal manifestations and conditions affecting the course of periodontitis and the periodontal attachment apparatus, as well as of developmental and acquired conditions, is introduced. Case definitions and diagnostic considerations are also presented.

A bacterial metabolite ameliorates periodontal pathogen-induced gingival epithelial barrier disruption via GPR40 signaling.

Several studies have demonstrated the remarkable properties of microbiota and their metabolites in the pathogenesis of several inflammatory diseases. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a bioactive metabolite generated by probiotic microorganisms during the process of fatty acid metabolism, has been studied for its protective effects against epithelial barrier impairment in the intestines. Herein, we examined the effect of HYA on gingival epithelial barrier function and its possible application for the prevention and treatment of periodontal disease. We found that GPR40, a fatty acid receptor, was expressed on gingival epithelial cells; activation of GPR40 by HYA significantly inhibited barrier impairment induced by Porphyromonas gingivalis, a representative periodontopathic bacterium. The degradation of E-cadherin and beta-catenin, basic components of the epithelial barrier, was prevented in a GPR40-dependent manner in vitro. Oral inoculation of HYA in a mouse experimental periodontitis model suppressed the bacteria-induced degradation of E-cadherin and subsequent inflammatory cytokine production in the gingival tissue. Collectively, these results suggest that HYA exerts a protective function, through GPR40 signaling, against periodontopathic bacteria-induced gingival epithelial barrier impairment and contributes to the suppression of inflammatory responses in periodontal diseases.

Randomized Placebo-Controlled and Controlled Non-Inferiority Phase III Trials Comparing Trafermin, a Recombinant Human Fibroblast Growth Factor 2, and Enamel Matrix Derivative in Periodontal Regeneration in Intrabony Defects.

We investigated the efficacy, safety, and clinical significance of trafermin, a recombinant human fibroblast growth factor (rhFGF)-2, for periodontal regeneration in intrabony defects in Phase III trials. Study A, a multicenter, randomized, double-blind, placebo-controlled study, was conducted at 24 centers. Patients with periodontitis with 4-mm and 3-mm or deeper probing pocket depth and intrabony defects, respectively, were included. A total of 328 patients were randomly assigned (2:1) to receive 0.3% rhFGF-2 or placebo, and 323 patients received the assigned investigational drug during flap surgery. One of the co-primary endpoints, the percentage of bone fill at 36 weeks after drug administration, was significantly greater in the rhFGF-2 group at 37.131% (95% confidence interval [CI], 32.7502 to 41.5123; n = 208) than it was in the placebo group at 21.579% (95% CI, 16.3571 to 26.8011; n = 100; p < 0.001). The other endpoint, the clinical attachment level regained at 36 weeks, was not significantly different between groups. Study B, a multicenter, randomized, blinded (patients and evaluators of radiographs), and active-controlled study was conducted at 15 centers to clarify the clinical significance of rhFGF-2. Patients with 6-mm and 4-mm or deeper probing pocket depth and intrabony defects, respectively, were included. A total of 274 patients were randomly assigned (5:5:2) to receive rhFGF-2, enamel matrix derivative (EMD), or flap surgery alone. A total of 267 patients received the assigned treatment during flap surgery. The primary endpoint, the linear alveolar bone growth at 36 weeks, was 1.927 mm (95% CI, 1.6615 to 2.1920; n = 108) in the rhFGF-2 group and 1.359 mm (95% CI, 1.0683 to 1.6495; n = 109) in the EMD group, showing non-inferiority (a prespecified margin of 0.3 mm) and superiority of rhFGF-2 to EMD. Safety problems were not identified in either study. Therefore, trafermin is an effective and safe treatment for periodontal regeneration in intrabony defect, and its efficacy was superior in rhFGF-2 compared to EMD treatments.

Th2 cytokines efficiently stimulate periostin production in gingival fibroblasts but periostin does not induce an inflammatory response in gingival epithelial cells.

OBJECTIVES: This study aims to clarify whether gingival fibroblasts produce periostin in response to Th2 cytokines which are elevated in periodontitis lesion and, if so, whether periostin affects the inflammatory response and matrix-protein metabolism. DESIGN: Human gingival fibroblasts, periodontal ligament cells and the gingival epithelial cell line epi4 were stimulated with interleukin-4 (IL-4), IL-13, tumour necrosis factor-α (TNF-α) and Porphyromonas gingivalis lipopolysaccharide (LPS). Periostin expression was analysed by real-time polymerase chain-reaction (PCR) and Western blotting. The expression of the IL-4 receptor α-chain was evaluated by immunocytochemistry. The effect of periostin on the production of inflammatory cytokines and the expression of matrix protein-related genes was analysed by real-time PCR and enzyme-linked immunosorbent assay (ELISA). RESULTS: While IL-4 and IL-13 significantly induced periostin production in gingival fibroblasts and periodontal ligament cells, no effect was observed in epi4 cells. No stimulatory effect of TNF-α or P. gingivalis LPS on the production of periostin was observed. The effect of periostin on the production of inflammatory cytokines was weak in gingival fibroblasts; however, little or no effect was observed on periodontal ligament cells or epi4 cells. No significant effect of periostin on the expression of matrix protein-related genes was found. CONCLUSION: The results suggest that gingival fibroblasts may be a source of periostin in periodontitis lesions but periostin has only a limited role either in the inflammatory response or in matrix-protein metabolism. Thus, the role of periostin in the cellular interaction between epithelial and mesenchymal cells in gingiva may be distinct from that of skin.

Porphyromonas gingivalis lipopolysaccharide induces miR-146a without altering the production of inflammatory cytokines.

Lipopolysaccharide (LPS) from Porphyromonas gingivalis, an oral Gram-negative bacterium, acts as a virulence factor for periodontal disease. Although P. gingivalis LPS does not induce proinflammatory cytokines as strongly as Escherichia coli LPS, it is still able to exploit negative Toll-like receptor (TLR) regulatory pathways and facilitate pathogen persistence. Recent reports suggest that microRNAs (miRNAs) are also involved in the regulation of TLR signaling. Here, we demonstrate that P. gingivalis LPS strongly induces miRNA-146a expression in THP-1 cells and THP-1-derived macrophages. However, the inhibition or overexpression of miR-146a, through the transfection of a specific inhibitor or precursor, respectively, had little effect on cytokine production in macrophages stimulated with P. gingivalis LPS. Moreover, the expression of interleukin-1 associated-kinase-1 (IRAK-1) and tumor-necrosis factor (TNF) receptor-associated factor-6 (TRAF6), potential target molecules of miR-146a, were not affected by the stimulation with P. gingivalis LPS. Because TLR signaling induces various negative regulators, these results call into question the role of miR-146a in cells stimulated with TLR ligands.

Increased serum PCSK9 concentrations are associated with periodontal infection but do not correlate with LDL cholesterol concentration.

BACKGROUND: Periodontal disease increases the risk of atherothrombotic disease, and high concentrations of low density lipoprotein (LDL) cholesterol are considered to be involved; however, the underlying mechanisms are largely unknown. Recent studies demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in circulating LDL cholesterol concentrations. The aim of the present study is to analyze serum PCSK9 concentrations and their relation to lipoprotein concentrations in periodontitis patients. METHODS: Sera were obtained from 40 periodontitis patients and 30 control subjects. PCSK9 concentrations, high-sensitivity C-reactive protein (hs-CRP), IL-6, TNF-α and Porphyromonas gingivalis antibodies were measured by ELISA, and lipid profiles were determined by a commercial laboratory. RESULTS: Periodontitis patients demonstrated significantly higher serum antibody titer to P. gingivalis and hs-CRP concentrations than control subjects, suggesting infection with P. gingivalis and a systemic inflammatory response. PCSK9 concentrations in periodontitis patients were significantly higher than those in control subjects. However, the concentrations of total and LDL cholesterols were not significantly different between periodontitis patients and control subjects. Moreover, no correlations were observed between PCSK9 concentrations and lipid profiles. CONCLUSION: Periodontal infection upregulates PCSK9 production. However, further studies are required to elucidate how periodontal infection affects PCSK9 concentrations and subsequent lipid metabolism.

Effect of interleukin-17 on the expression of chemokines in gingival epithelial cells.

The role of interleukin (IL)-17 in cellular communication in inflammation has been well described, and a positive correlation between the severity of periodontitis and the level of IL-17 was reported. Although epithelial cells are a major target of IL-17, little is known about the effect of IL-17 on the production of chemokines by human gingival epithelial cells (HGECs). We evaluated the effects of IL-17 on the expression of CXCL8 and CCL2 by HGECs using quantitative real-time PCR and ELISA. In addition, the role of the nuclear factor (NF)-κB signalling pathway in the IL-17-mediated expression of chemokines was assessed using a specific inhibitor. Stimulation with IL-17 up-regulated the expression of CXCL8 mRNA but not of CCL2 mRNA in HGECs, whereas tumour necrosis factor-α (TNF-α) elevated the expression of mRNA for both chemokines. Stimulation with IL-17 up-regulated the secretion of CXCL8 protein, but not the secretion of CCL2 protein. The effect of IL-17 on CXCL8 production was suppressed using an anti-IL-17R Ig, suggesting a role for a specific receptor-ligand interaction. Inhibition of the NF-κB signalling pathway demonstrated that NF-κB activation is required for the CXCL8 expression in HGECs. In conclusion, IL-17 is involved in the regulation of the innate immune response in HGECs by inducing CXCL8 production.

Increased expression of C-reactive protein gene in inflamed gingival tissues could be derived from endothelial cells stimulated with interleukin-6.

BACKGROUND: Epidemiological studies have suggested periodontitis as a risk factor for ischemic heart disease. High sensitive C-reactive protein (hs-CRP), a predictor of cardiovascular risk, is elevated in periodontitis patients. Therefore, local infection-induced elevation of systemic CRP could account for the relationship between the 2 diseases. However, the underlying mechanism of CRP production in the periodontal tissues has not been fully elucidated. Therefore, the aim of the present study was to clarify the mechanism of CRP production in periodontal tissues. METHODS: Gene expression of CRP in gingival biopsies was analysed by quantitative PCR. Human gingival epithelial cells (HGECs), human gingival fibroblasts (HGFBs), and human coronary artery endothelial cells (HCAECs) were characterized for CRP-producing ability by incubating with interleukin (IL)-1ß, IL-6, soluble IL-6 receptor (sIL-6R), and Porphyromonas gingivalis strain W83. RESULTS: Gene expression of CRP is significantly elevated in periodontitis lesions compared with gingivitis lesions. HCAECs, but not HGECs and HGFBs, produced CRP in response to IL-6 and IL-1ß in the presence of sIL-6R. In contrast to IL-6, the effect of IL-1ß on CRP production was indirect via induction of IL-6. IL-1ß was produced by HGECs and HGFBs with stimulation of P. gingivalis antigens. CONCLUSION: These results suggest that CRP induced locally by periodontal infection may play another role in the pathogenesis of periodontal disease, and to a much lesser extent, has the potential to modulate systemic CRP level by extra-hepatic CRP production.

Single-strand conformation polymorphism analysis for the diagnosis of T-cell clonality in periodontal disease.

T cells recognize antigens via the T-cell receptor (TCR). Diversity in antigen recognition by T cells is generated in part by the recombination of V, (D), J, and C segments of the TCR. It is further enhanced by the N region, in addition to non-germline-encoded nucleotides at the V-(D)-J junction. It is generally believed that each T cell bears a distinct clonotype of TCR and that each clonotype is responsible for an antigen-specific T-cell response. T-cell clonal expansion has been detected in the peripheral blood or the disease-affected sites in patients with infections, autoimmune diseases, malignancy, and post-transplantation complications. Since antigen stimulation of T cells induces the proliferation of specific T cells, clonal T-cell expansion is considered to be a result of an antigen-specific immune response. For the analysis of such antigen-specific T cells, it is common to use their specific antigens if they are known. However, there are many diseases, such as periodontal diseases, in which there are a number of putative pathogenic antigens involved. In these circumstances, the detection of clonally expanded T cells is an effective method to evaluate whether antigen-specific immune responses are involved, since only a few clonally expanded T cells are detected in healthy individuals. In addition, the characterization of any clonally expanded T cells that are detected would further promote the understanding of the disease mechanisms. By using single-strand conformation polymorphism (SSCP) analysis, we demonstrated that oligoclonal T-cell accumulation was present in periodontitis lesions, in contrast to a heterogeneous T-cell population in the peripheral blood. SSCP is a powerful tool for analyzing specific T-cell responses both in vitro and in vivo.

Analysis of immunostimulatory activity of Porphyromonas gingivalis fimbriae conferred by Toll-like receptor 2.

Bacterial fimbriae are an important pathogenic factor. It has been demonstrated that fimbrial protein encoded by fimA gene (FimA fimbriae) of Porphyromonas gingivalis not only contributes to the abilities of bacterial adhesion and invasion to host cells, but also strongly stimulates host innate immune responses. However, FimA fimbriae separated from P. gingivalis ATCC 33277 using a gentle procedure showed very weak proinflammatory activity compared with previous reports. Therefore, in the present study, biological characteristics of FimA fimbriae were further analyzed in terms of proinflammatory activity in macrophages. Macrophages differentiated from THP-1 cells were stimulated with native, heat-denatured, or either proteinase- or lipoprotein lipase-treated FimA fimbriae of P. gingivalis ATCC 33277. Stimulating activities of these FimA fimbriae were evaluated by TNF-alpha-inducing activity in the macrophages. To clarify the mode of action of FimA fimbriae, anti-Toll-like receptor (TLR) 2 blocking antibody was added prior to stimulation. Weak stimulatory activity of native FimA fimbriae was enhanced by heat treatment and low-dose proteinase K treatment. Higher dose of proteinase K treatment abrogated this up-regulation. The activity of treated FimA fimbriae was suppressed by anti-TLR2 antibody, and more substantially by lipoprotein lipase treatment. These results suggest that lipoproteins or lipopeptides associated with FimA fimbriae could at least in part account for signaling via TLR2 and subsequent TNF-alpha production in macrophages.

Porphyromonas gingivalis antigens and interleukin-6 stimulate the production of monocyte chemoattractant protein-1 via the upregulation of early growth response-1 transcription in human coronary artery endothelial cells.

BACKGROUND: Individuals with periodontitis have elevated serum levels of IL-6 and C-reactive protein and have been reported to have a significantly increased risk of developing cardiovascular disease. The transcription factor early growth response factor 1 (Egr-1) has been shown to play an important role in the development and progression of atherosclerosis. However, it is not known whether periodontal infection affects the expression of Egr-1 and subsequent endothelial cells expression of monocyte chemoattractant protein (MCP)-1, a key molecule of leukocyte chemoattraction into vessels. METHODS: Human coronary artery endothelial cells (HCAECs) were stimulated with either sonicated extracts from Porphyromonas gingivalis strains 381 or SU63, or a combination of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R). The expression of Egr-1, and subsequently MCP-1, was then analyzed. The role of Egr-1 on MCP-1 expression was analyzed by siRNA transfection. RESULTS: Both P. gingivalis antigens and IL-6/sIL-6R stimulations upregulated the expression of Egr-1, with a more robust effect by IL-6/sIL-6R. Increased expression of Egr-1 coincided with MCP-1 production, and Egr-1 downregulation by siRNA suppressed this effect. CONCLUSION: These results clearly suggest that periodontal infection has the potential to affect HCAECs and hence contribute to the development of subsequent atherosclerosis.