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.

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.