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Plasminogen activator inhibitor‐1 (PAI‐1) is associated with nonalcoholic fatty liver disease (NAFLD) by lipid accumulation in the liver. In this study, we showed that extracellular vesicles (EVs) from the periodontal pathogens Filifactor alocis and Porphyromonas gingivalis induced steatosis by inducing PAI‐1 in the liver and serum of mice fed a low‐fat diet. PAI‐1 induction was not observed in TLR2−/− mice. When tested using HEK‐Blue hTLR2 cells, human TLR2 reporter cells, the TLR2‐activating ability of serum from NAFLD patients (n = 100) was significantly higher than that of serum from healthy subjects (n = 100). Correlation analysis confirmed that PAI‐1 levels were positively correlated with the TLR2‐activating ability of serum from NAFLD patients and healthy subjects. Amphiphilic molecules in EVs were involved in PAI‐1 induction. Our data demonstrate that the TLR2/PAI‐1 axis is important for hepatic steatosis by EVs of periodontal pathogens. [ABSTRACT FROM AUTHOR]

Periodontitis is a chronic inflammatory disease caused by periodontal pathogens in subgingival plaque and is associated with systemic inflammatory diseases. Extracellular vesicles (EVs) released from host cells and pathogens carry a variety of biological molecules and are of interest for their role in disease progression and as diagnostic markers. In the present study, we analysed the proteome and inflammatory response of EVs derived from macrophages infected with Tannerella forsythia, a periodontal pathogen. The EVs isolated from the cell conditioned medium of T. forsythia‐infected macrophages were divided into two distinct vesicles, macrophage‐derived EVs and T. forsythia‐derived OMVs, by size exclusion chromatography combined with density gradient ultracentrifugation. Proteome analysis showed that in T. forsythia infection, macrophage‐derived EVs were enriched with pro‐inflammatory cytokines and inflammatory mediators associated with periodontitis progression. T. forsythia‐derived OMVs harboured several known virulence factors, including BspA, sialidase, GroEL and various bacterial lipoproteins. T. forsythia‐derived OMVs induced pro‐inflammatory responses via TLR2 activation. In addition, we demonstrated that T. forsythia actively released OMVs when T. forsythia encountered macrophage‐derived soluble molecules. Taken together, our results provide insight into the characterisation of EVs derived from cells infected with a periodontal pathogen. [ABSTRACT FROM AUTHOR]

Outer membrane vesicles (OMVs) of bacteria harbor physiologically active molecules, and quorum sensing inhibitors (QSIs) are expected to regulate bacterial virulence. In this study, we analyzed the proinflammatory activity of OMVs of the periodontal pathogen Tannerella forsythia treated with d‐arabinose and d‐galactose as QSIs, which inhibit the biofilm formation of periodontal pathogens and autoinducer 2 activity. Compared to OMVs of nontreated T. forsythia (TF OMVs), OMVs released from QSI‐treated T. forsythia, designated TF ara‐OMVs and TF gal‐OMVs, showed reduced production of TNF‐α, IL‐1β, IL‐6, and IL‐8 in THP‐1 monocytes through decreased activation of NF‐κB/MAPKs. Using a human NF‐κB reporter cell line and bone marrow‐derived macrophages from TLR2−/− mice, TF ara‐OMVs and TF gal‐OMVs showed less activation of TLR2 than TF OMVs. These results demonstrated that QSIs provide a dual advantage against bacterial infection by inhibiting bacterial biofilm formation and generating OMVs with reduced proinflammatory activity. [ABSTRACT FROM AUTHOR]

Outer membrane vesicles (OMVs) released from gram-negative bacteria harbor diverse molecules to communicate with host cells. In this study, we evaluated the OMVs of periodontal pathogens for their effects on the activation of dendritic cells and CD4+ T cell differentiation. OMVs of Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 33521, and Tannerella forsythia ATCC 43037 ('red complex' pathogens) were isolated by density gradient ultracentrifugation. Mouse bone marrow-derived dendritic cells (BMDCs) were treated with OMVs, and OMV-primed BMDCs were cocultured with naïve CD4+ T cells to analyze the polarization of effector helper T cells. The OMVs upregulated maturation markers, including MHC class II, CD80, CD86, and CD40, on BMDCs. OMVs of P. gingivalis and T. forsythia induced the expression of the proinflammatory cytokines IL-1β, IL-6, IL-23, and IL-12p70 in BMDCs. In T. denticola OMV-primed BMDCs, proinflammatory cytokines were poorly detected, which may be attributed to posttranslational degradation due to the highly proteolytic nature of OMVs. In cocultures of naïve CD4+ T cells with OMV-primed BMDCs, OMVs of P. gingivalis and T. denticola induced the differentiation of Th17 cells, whereas T. forsythia OMVs induced Th1 cell differentiation. These results demonstrate that OMVs derived from the 'red complex' periodontal pathogens induce maturation of BMDCs and differentiation of naïve CD4+ T cells to Th1 or Th17 cells. [ABSTRACT FROM AUTHOR]

The major surface protein (MspTL) of Treponema lecithinolyticum, associated with periodontitis and endodontic infections, has been reported to induce proinflammatory mediators such as intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-1[beta], IL-6 and IL-8. The purpose of this study was to examine the role of MspTL in cell adhesion/migration and to identify its proinflammatory domains. Using the human monocytic cell line THP-1 and human dermal microvascular endothelial cells (HMEC-1), it was demonstrated that MspTL increased adhesion of monocytes to endothelial cells and transendothelial migration. To analyse the proinflammatory domains of the protein, four gene constructs covering different regions of MspTL were designed and expressed in Escherichia coli using the expression vector pQE-30. Histidine-tagged recombinant proteins were purified using Ni-NTA agarose and polymyxin B agarose to remove LPS contamination. Recombinant truncated polypeptides were assessed for the ability to induce ICAM-1 and proinflammatory factors in THP-1 cells by real-time RT-PCR and ELISA. Of the four polypeptides, the one spanning the N-terminal 86 amino acids significantly induced ICAM-1, IL-1[beta], IL-6, IL-8, tumour necrosis factor-[alpha] (TNF-[alpha]), cyclooxygenase (COX)-2, and prostaglandin [E.sub.2] ([PGE.sub.2]). The results indicate that MspTL may induce cell adhesion and inflammation via its N-terminal region.

Treponema socranskii is one of the most frequently found oral spirochaetes in periodontitis and endodontic infections. LPS or glycolipids from bacteria are potent stimulators of innate immune and inflammatory systems. In this study the bioactivity of a phenol/water extract from T. socranskii subsp, socranskii (TSS-P) was analysed. TSS-P showed minimal endotoxicity and no inducing potential for proinflammatory cytokines (TNF-[alpha] and IL-8) or for intercellular adhesion molecule-1 (ICAM-1) in human monocyte cell line THP-1 cells and primary cultured human gingival fibroblasts. Rather, it inhibited ICAM-1 expression and IL-8 secretion from cells stimulated by the LPS of Escherichia coli and Actinobacillus actinomycetemcomitans, which are known to be Toll-like receptor 4 (TLR4) agonists. However, this antagonistic activity was not shown in cells stimulated by peptidoglycan or IL-1 [beta]. As its antagonistic mechanism, TSS-P blocked the binding of E. coli LPS to LPS-binding protein (LBP) and CD14, which are molecules involved in the recruitment of LPS to the cell membrane receptor complex TLR4-MD-2 for the intracellular signalling of LPS. TSS-P itself did not bind to MD-2 or THP-1 cells, but inhibited the binding of E. coil LPS to MD-2 or to the cells in the presence of serum (which could be replaced by recombinant human LBP and recombinant human CD14). The results suggest that TSS-P acts as an antagonist of TLR4 signalling by interfering with the functioning of LBP/CD14.

Summary This study describes a potential of Phytolaccaceae (Phytolacca americana var.) as an inhibitor of high glucose-stimulated production of extracellular matrix (ECM) proteins and TGF-β in cultured glomerular mesangial cells [...]

Filifactor alocis, an asaccharolytic anaerobic Gram-positive rod (AAGPR), is an emerging marker of periodontitis. Severe periodontitis causes destruction of the alveolar bone that supports teeth and can even lead to tooth loss. Based on our previous report that F. alocis-derived extracellular vesicles (FA EVs) contain various effector molecules and have immunostimulatory activity, we investigated the effect of FA EVs on osteogenesis using mouse bone-derived mesenchymal stromal cells (BMSCs). FA EVs dramatically inhibited bone mineralization similar to whole bacteria and reduced the expression levels of osteogenic marker genes. The osteogenic differentiation of TLR2-deficient BMSCs was not inhibited by FA EVs, suggesting that their inhibitory effect on osteogenesis is dependent on TLR2 signaling. FA EVs effectively activated TLR2 downstream signaling of the MAPK and NF-κB pathways. In addition, FA EVs regulated RANKL and OPG gene expression, increasing the RANKL/OPG ratio in BMSCs in a TLR2-dependent manner. Our study suggests that F. alocis-derived EVs interfere with bone metabolism via TLR2 activation, providing insight into the pathogenesis of bone loss associated with periodontitis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Research was conducted to clone and characterize a gene encoding the major sheath protein of Treponema maltophium ATCC 51939T. Cloning was carried out by screening a genomic library with an anti-outer membrane fraction antibody. The MspA gene formed a heat-modifiable, detergent- and trypsin-stable complex that is associated with the outer membrane. Results demonstrate that hybridization with an msp-A-specific probe yielded no cross-reactivity with the msp gene from Treponema denticola.

In situ hybridization (ISH) is useful in detecting and identifying bacteria in their natural environments. Fluorescence in situ hybridization (FISH) has been used on sections of plastic-embedded tissue to visualize uncultured treponemes in skin biopsies of cows with digital dermatitis. Debris covering the ulcers had a mixture of different microorganisms, with a layering of treponemal phylotypes deeper in the epidermis.

Periodontitis is an inflammatory disease induced by local infection in tooth‐supporting tissue. Periodontitis is associated with systemic bone diseases, but little is known about the mechanism of the causal effect of periodontitis on systemic bone resorption. Bacteria‐derived extracellular vesicles (EVs) act as natural carriers of virulence factors that are responsible for systemic inflammation. In this study, we investigated the role of EVs derived from Filifactor alocis, a Gram‐positive, anaerobic periodontal pathogen, in systemic bone loss and osteoclast differentiation. F. alocis EVs accumulated in the long bones of mice after intraperitoneal administration. These EVs induced proinflammatory cytokines, osteoclastogenesis, and bone resorption via Toll‐like receptor 2 (TLR2). The phase separation of F. alocis EVs showed that amphiphilic molecules were responsible for the induced bone resorption and osteoclastogenesis. The osteoclastogenic effects of F. alocis EVs were reduced by lipoprotein lipase. Proteomic analysis of the amphiphilic molecules identified seven lipoproteins. Our results indicate that lipoprotein‐like molecules in F. alocis EVs may contribute to systemic bone loss via TLR2. [ABSTRACT FROM AUTHOR]

Interleukin-24 is a pleiotropic immunoregulatory cytokine and a member of the IL-20R subfamily of the IL-10 family. The aim of this study was to investigate the regulation of IL-24 in the human oral keratinocyte cell line HOK-16B following infection with Tannerella forsythia, a major periodontal pathogen. T. forsythia induced the expression of IL-24 mRNA and the secretion of glycosylated IL-24 in HOK-16B cells. Glycosylation of IL-24 is linked to its solubility and bioavailability. T. forsythia-stimulated reactive oxygen species (ROS) induced the expression of IL-24, which was regulated by IL-6. The ROS inhibitor N-acetylcysteine and MAPK inhibitors significantly reduced the expression of IL-6 and IL-24 induced by T. forsythia. Recombinant human IL-24 significantly enhanced the expression of IL-1α, IL-8, CXCL10, and MCP-1 in HOK-16B cells. Together, these results indicate that ROS, MAPKs, and IL-6 comprise the axis of IL-24 expression in HOK-16B cells stimulated with T. forsythia. Thus, IL-24 may be involved in inflammation in oral keratinocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Autoinducer (AI)-2 has an important role in biofilm formation in the oral environment. Mature biofilms formed as a result of the cell-to-cell communication make it difficult to overcome periodontitis with the use of antibiotics. Previous in vitro studies suggest that quorum-sensing inhibitors (QSIs) interfere with AI-2. This study compares the QSI effects resulting from an oral inoculation of Porphyromonas gingivalis in an experimental animal model.Methods: Forty-five male mice were divided into three groups (n = 15 each): 1) infection; 2) QSI; and 3) control. Infection and QSI groups received oral inoculation of P. gingivalis, whereas treatment with QSIs (furane compound and d-ribose) was only performed in the QSIs group. The control group was a negative control not receiving manipulation. After 42 days, mice were sacrificed, and the distance from the alveolar bone crest (ABC) to the cemento-enamel junction (CEJ) was measured by microcomputed tomography. P. gingivalis DNA was quantified in the soft and hard tissues around the molar teeth by real-time polymerase chain reaction.Results: Distance from ABC to CEJ was significantly increased in the P. gingivalis infection group compared with the control group (P = 0.02) and significantly decreased in the QSI group compared with the infection group (P = 0.02). The QSI group contained 31.64% of the bacterial DNA count of the infection group.Conclusion: Use of QSIs in the mice infection model showed a reduction of bone breakdown and a decrease in the number of bacteria in vivo, suggesting that QSIs can be a new approach to prevention and treatment of periodontitis. [ABSTRACT FROM AUTHOR]

This study aimed to verify, in in vivo settings, whether quorum‐sensing inhibition molecules could attenuate alveolar bone loss induced by Porphyromonas gingivalis/Fusobacterium nucleatum co‐infection and reduce the bacterial colonization of periodontal tissues. In BALB/c mice, periodontitis was induced through oral inoculation with P. gingivalis and F. nucleatum six times during a 42‐d period. Quorum sensing inhibitors (a furanone compound and D‐ribose) were administered simultaneously with bacterial infection. Linear and volumetric modifications of interproximal alveolar bone levels were compared between groups using micro‐computed tomography. Total bacteria, and P. gingivalis and F. nucleatum DNA in periodontal tissues, were quantified using real‐time PCR. Radiographic linear measurements demonstrated a significant reduction of alveolar bone loss, of approximately 40%, in mice treated with quorum sensing inhibitors when compared with the co‐infection group. This was confirmed by a significant increase of residual bone volume in the test group. While total bacterial genes in the treatment group significantly decreased by 93% in periodontal tissue samples when quorum sensing inhibitors were administered, no significant differences of P. gingivalis DNA were found. Quorum sensing inhibitors reduced periodontal breakdown and bacterial infection in periodontal tissues after co‐infection with P. gingivalis and F. nucleatum. [ABSTRACT FROM AUTHOR]

Quorum sensing (QS) is a chemical communication between bacteria, with which bacteria sense the population of their own species. Autoinducer-2 ( AI-2) is a class of universal quorum-sensing molecules, which is used by both Gram-negative and Gram-positive bacteria. The inhibition of AI-2-mediated QS has various practical applications, including the prevention of the formation of biofilm in dental gums. In this work, we develop a computational protocol for developing AI-2 inhibitors. A challenging aspect of such an endeavor is that the receptor undergoes a large conformational change upon ligand binding. We combine several methods such as molecular docking with multiple conformations, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann computation, in order to estimate binding affinity of candidate molecules to a quorum-sensing receptor. We apply our method to rank the substances in a chemical library. We indeed find a molecule that has a higher affinity than previously known ligands, thus showing the feasibility of the protocol for the development of quorum-sensing inhibitors. [ABSTRACT FROM AUTHOR]

Extracellular fibronectin (Fn) can activate pro-inflammatory pathways and serves as an endogenous danger signalling molecule; thus, it has been suggested as a biomarker for several diseases. In the present study, we found that pathogen-derived activators of the inflammasomes induce the expression and secretion of Fn in macrophages through a mechanism involving adenosine triphosphate and caspase-1 activation. We also found that plasma Fn induces caspase-1 activation and cell death in macrophages, epithelial cells, and fibroblasts. Together, these results indicate that Fn plays a critical role in inflammasome-activated cells by amplifying caspase-1 activation and inducing inflammatory cell death. [ABSTRACT FROM AUTHOR]

Objectives: This in vitro study was conducted: (i) to evaluate the effect of using cotton pellets soaked with chlorhexidine (CHX) on titanium surface roughness; (ii) to assess the removal of Porphyromonas gingivalis (P. gingivalis) from resorbable blast material (RBM) titanium surfaces using CHX pellets; and (iii) to evaluate the effects of additional brushing on bacterial removal efficiency.Materials and Methods: RBM titanium discs were treated with CHX-soaked cotton pellets, and change in surface roughness was measured using confocal microscopy. After the titanium discs were incubated with P. gingivalis for 2 days, the discs were cleaned with CHX pellets for 40 s. The quantity of remaining adherent bacteria was measured using crystal violet assay. Additional brushing was performed with dentifrice for a total of 40 s, and bacterial removal efficiency with brushing and dentifrice was evaluated using crystal violet assay and scanning electron microscopy.Results: The changes in surface roughness after treatment were observed by confocal microscopy. Statistically significant decrease in surface roughness was seen in CHX 40-s group (p < 0.05). Cleaning with CHX-soaked pellets resulted in significant decrease in remaining adherent bacteria. Brushing the bacteria-incubated discs with dentifrice reduced adhering bacteria. There were fewer bacteria left on the CHX-pre-treated discs compared with the brushing-only group, but there were no significant differences when compared with the brushing-only group (p > 0.05).Conclusions: This study clearly showed that burnishing with CHX influenced the RBM titanium surface, and burnishing with CHX pellets and brushing with dentifrice were efficient in removing bacteria from the contaminated titanium surface. [ABSTRACT FROM AUTHOR]

P orphyromonas gingivalis utilizes its major proteases, Arg gingipains ( RgpA and RgpB) and Lys gingipain ( Kgp), for dysregulation of host immune systems. The aim of this study was to investigate the roles of gingipains in caspase-1 activation and its sequelae in P . gingivalis-infected macrophages. Infection with P . gingivalis at low multiplicity of infections ( MOIs), but not at high MOIs, resulted in low levels of interleukin-1β and lactate dehydrogenase without detectable active caspase-1 in the culture supernatants. The proteins released from caspase-1-activated cells were rapidly degraded by gingipains. However, P . gingivalis with gingipains induced higher intracellular caspase-1 activity in the infected cells than the gingipain-null mutant, which was associated with ATP release from the infected cells. In addition, growing the gingipain-null mutant with gingipains enhanced caspase-1 activation by the mutant. In contrast, inhibition of the protease activity of Kgp or Rgps increased the caspase-1-activating potential of wild-type P . gingivalis, indicating an inhibitory effect of the collaborative action of Kgp and Rgps. These results illuminate the contradictory roles of gingipains in the manipulation of host defence systems by P . gingivalis, as they act by both stimulating and inhibiting innate immune responses. [ABSTRACT FROM AUTHOR]