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.