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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, 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]