Outer Membrane Vesicles From Fusobacterium nucleatum Switch M0-Like Macrophages Toward the M1 Phenotype to Destroy Periodontal Tissues in Mice

Abstract

Periodontitis is a chronic inflammatory oral disease that affects nearly 50% of all adults. Fusobacterium nucleatum (F. nucleatum) is known to be involved in the formation and development of periodontitis. Outer membrane vesicles (OMVs) harboring toxic bacterial components are continuously released during F. nucleatum growth and regulate the extent of the inflammatory response by controlling the functions of immune and non-immune cells in tissues. Macrophages are important immune cells in periodontal tissue that resist pathogen invasion and play an important role in the pathophysiological process of periodontitis. However, the role of the interaction between F. nucleatum OMVs and macrophages in the occurrence and development of periodontitis has not been studied. The purpose of this study was to clarify the effect of F. nucleatum OMVs on the polarization of macrophages and the roles of this specific polarization and F. nucleatum OMVs in the pathophysiology of periodontitis. The periodontitis model was established by inducing ligation in C57BL/6 mice as previously described. Micro-CT, RT-qPCR, hematoxylin-eosin (H&E) and tartrate acid phosphatase (TRAP) staining assays were performed to analyze the periodontal tissue, alveolar bone loss, number of osteoclasts and expression of inflammatory factors in gingival tissue. The changes in the state and cytokine secretion of bone marrow-derived macrophages (BMDMs) stimulated by F. nucleatum OMVs were observed in vivo by confocal microscopy, flow cytometry, Western blot and ELISA. Mouse gingival fibroblasts (MGFs) were isolated and then cocultured with macrophages. The effects of F. nucleatum OMVs on the proliferation and apoptosis of MGFs were analyzed by flow cytometry and lactate dehydrogenase (LDH) assays. The periodontitis symptoms of mice in the F. nucleatum OMVs + ligation group were more serious than those of mice in the simple ligation group, with more osteoclasts and more inflammatory factors (IL-1β, IL-6, and TNF-α) being observed in their gingival tissues. M0 macrophages transformed into M1 macrophages after the stimulation of BMDMs with F. nucleatum OMVs, and the M1 macrophages then released more inflammatory cytokines. Analysis of the coculture model showed that the MGF apoptosis and LDH release in the inflammatory environment were increased by F. nucleatum OMV treatment. In conclusion, F. nucleatum OMVs were shown to aggravate periodontitis, alveolar bone loss and the number of osteoclasts in an animal model of periodontitis. F. nucleatum OMVs promoted the polarization of macrophages toward the proinflammatory M1 phenotype, and the inflammatory environment further aggravated the toxicity of F. nucleatum OMVs on MGFs. These results suggest that M1 macrophages and F. nucleatum OMVs play roles in the occurrence and development of periodontitis.

Transforming Intratumor Bacteria into Immunopotentiators to Reverse Cold Tumors for Enhanced Immuno-chemodynamic Therapy of Triple-Negative Breast Cancer

“…First, OMVs were isolated from the liquid medium containing F. nucleatum culture by gradient centrifugation according to the previously reported protocols. , Their morphology was observed by transmission electron microscopy (TEM) (Figure A). Dynamic light scattering (DLS) data showed that the average diameter of the OMVs was 126 nm (Figure F).…”

“…The antigens and damage-associated molecular patterns released by dead cancer cells significantly enhanced immunogenicity. Meanwhile, noncellular OMVs containing a range of immunostimulatory components inherited from their parent cells served as a powerful and safe immune modulator. − More importantly, dead F. nucleatum was transformed into a potentiator, which used released pathogen-associated molecular patterns to further facilitate the maturation of dendritic cells (DCs) and subsequent T-cell infiltration.…”

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

“…The pathogenic role of EVs from T. forsythia ‐infected macrophages and T. forsythia ‐derived OMVs needs to be investigated in animal periodontitis models. Recently, there have been several trials aiming to induce periodontitis and assess inflammatory responses in the periodontal tissues using silk thread ligation with OMVs derived from periodontal pathogens, including Fusobacterium nucleatum and P. gingivalis (Chen et al., 2022 ; Fan et al., 2023 ). Although we did not evaluate the inflammatory responses of EVs on the periodontal tissues, we believe that the EVs released from immune cells and periodontal pathogens of inflamed periodontal tissues may freely disseminate to systemic sites and induce inflammation.…”

Bacterial membrane vesicles: orchestrators of interkingdom interactions in microbial communities for environmental adaptation and pathogenic dynamics

“…The MVs of Burkholderia cepacia ATCC 7966TM can induce the overexpression of proinflammatory cytokines, promoting the activation and apoptosis of mononuclear lymphocytes ( 98 ). On the other hand, MVs from Fusobacterium nucleatum can polarize macrophages toward an M1-like phenotype in a murine model of periodontal disease, promoting the development of periodontitis ( 99 ). During its growth, pathogenic bacteria continuously release MVs containing toxic bacterial components, which regulate the degree of inflammatory response by controlling the functions of immune and non-immune cells in the tissue ( 100 ).…”