Extracellular vesicles from periodontal pathogens regulate hepatic steatosis via Toll-like receptor 2 and plasminogen activator inhibitor-1.

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.

Proteome and immune responses of extracellular vesicles derived from macrophages infected with the periodontal pathogen Tannerella forsythia.

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.

Activation of bone marrow-derived dendritic cells and CD4+ T cell differentiation by outer membrane vesicles of periodontal pathogens.

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-1ß, 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.

Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation.

Dysbiosis of the oral microbiota plays an important role in the progression of periodontitis, which is characterized by chronic inflammation and alveolar bone loss, and associated with systemic diseases. Bacterial extracellular vesicles (EVs) contain various bioactive molecules and show diverse effects on host environments depending on the bacterial species. Recently, we reported that EVs derived from Filifactor alocis, a Gram-positive periodontal pathogen, had osteoclastogenic activity. In the present study, we analysed the osteoclastogenic potency and immunostimulatory activity of EVs derived from the Gram-negative periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia, the oral commensal bacterium Streptococcus oralis, and the gut probiotic strain Lactobacillus reuteri. Bacterial EVs were purified by density gradient ultracentrifugation using OptiPrep (iodixanol) reagent. EVs from P. gingivalis, T. forsythia, and S. oralis increased osteoclast differentiation and osteoclstogenic cytokine expression in osteoclast precursors, whereas EVs from L. reuteri did not. EVs from P. gingivalis, T. forsythia, and S. oralis preferentially activated Toll-like receptor 2 (TLR2) rather than TLR4 or TLR9, and induced osteoclastogenesis mainly through TLR2. The osteoclastogenic effects of EVs from P. gingivalis and T. forsythia were reduced by both lipoprotein lipase and polymyxin B, an inhibitor of lipopolysaccharide (LPS), while the osteoclastogenic effects of EVs from S. oralis were reduced by lipoprotein lipase alone. These results demonstrate that EVs from periodontal pathogens and oral commensal have osteoclastogenic activity through TLR2 activation by lipoproteins and/or LPS.

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-1ß, 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.

Rapamycin-induced autophagy restricts porcine epidemic diarrhea virus infectivity in porcine intestinal epithelial cells.

Porcine epidemic diarrhea virus (PEDV) invades porcine intestinal epithelial cells (IECs) and causes diarrhea and dehydration in pigs. In the present study, we showed a suppression of PEDV infection in porcine jejunum intestinal epithelial cells (IPEC-J2) by an increase in autophagy. Autophagy was activated by rapamycin at a dose that does not affect cell viability and tight junction permeability. The induction of autophagy was examined by LC3I/LC3II conversion. To confirm the autophagic-flux (entire autophagy pathway), autophagolysosomes were examined by an immunofluorescence assay. Pre-treatment with rapamycin significantly restricted not only a 1 h infection but also a longer infection (24 h) with PEDV, while this effect disappeared when autophagy was blocked. Co-localization of PEDV and autophagosomes suggests that PEDV could be a target of autophagy. Moreover, alleviation of PEDV-induced cell death in IPEC-J2 cells pretreated with rapamycin demonstrates a protective effect of rapamycin against PEDV-induced epithelial cell death. Collectively, the present study suggests an early prevention against PEDV infection in IPEC-J2 cells via autophagy that might be an effective strategy for the restriction of PEDV, and opens up the possibility of the use of rapamycin in vivo as an effective prophylactic and prevention treatment.

Mutagenicity and Immune Toxicity of Emulsion-type Sausage Cured with Plasma-treated Water.

Cold plasma has been developed to reduce microbial contamination and to improve safety of food and medical products. In addition, the technology can be used in the manufacture of sausages without addition of nitrite. To be applied in food industry commercially, the new technology should be safe and efficient. However, toxicological test of plasma-treated food is limited. Therefore, the purpose of this study was to determine the mutagenicity and immune toxicity of the meat products cured with plasma-treated water (PTW) as a nitrite source. Emulsion sausages were prepared with no nitrite (control), sodium nitrite (SCS), and PTW (SCP). For a mutagenicity test, the Ames test was performed with the sausage samples. For immune toxicity test, 8-wk-old female Balb/c mice were given free access to the sausages in order to evaluate the tumor necrosis factor (TNF)-α level. As a result, no mutagenicity was detected in the sausages by the Ames test. The serum TNF-α values were less than 10 pg/mL in mice after feeding control and treated samples for 32 d, indicating that no inflammatory response was occurred by feeding the sausages made by PTW. Therefore, the present study opens the possibility of using plasma-treated water as a nitrite source without any toxicity.