Porphyromonas gingivalis Vesicles Control Osteoclast–Macrophage Lineage Fate

<i>Porphyromonas gingivalis</i> (<i>Pg</i>), a keystone pathogen of chronic periodontitis, releases outer membrane vesicles (OMVs) that act as nanoscale vehicles to disseminate virulence factors within periodontal tissues and systemically beyond the oral cavity. Although <i>Pg</i>-OMVs are increasingly recognized as critical mediators of host-pathogen interactions, their effects on the differentiation and function of monocyte-macrophage/osteoclast lineage cells remain unclear. Here, we examined the impact of <i>Pg</i>-OMVs on the differentiation of RAW264.7 monocyte/macrophage-like cells into osteoclasts (OC) and/or macrophages (MΦ) in the presence of receptor activator of nuclear factor-κB ligand (RANKL). OMVs were isolated from <i>Pg</i> W83 and applied to RANKL-primed RAW264.7 cells using three distinct stimulation schedules: (1) simultaneous treatment with <i>Pg</i>-OMVs and RANKL at Day 0; (2) RANKL priming at Day 0 followed by <i>Pg</i>-OMV stimulation at Day 1; and (3) RANKL priming at Day 0 followed by <i>Pg</i>-OMV stimulation at Day 3. In all schedules, cells were cultured for 7 days from the initial RANKL exposure. Remarkably, simultaneous exposure to <i>Pg</i>-OMVs and RANKL (Schedule 1) markedly suppressed osteoclastogenesis (OC-genesis) while promoting M1 macrophage polarization. In contrast, delayed <i>Pg</i>-OMV stimulation of RANKL-primed cells (Schedules 2 and 3) significantly enhanced OC-genesis while reducing M1 polarization. These schedule-dependent effects were consistent with altered expression of osteoclastogenic markers, including <i>dc-stamp</i>, <i>oc-stamp</i>, <i>nfatc1</i>, and <i>acp5</i>. Importantly, a monoclonal antibody against OC-STAMP counteracted the <i>Pg</i>-OMV-induced upregulation of OC-genesis in Schedules 2 and 3. Furthermore, levels of <i>Pg</i>-OMV phagocytosis were inversely correlated with osteoclast formation. Finally, co-stimulation with RANKL and <i>Pg</i>-OMVs (Schedule 1) enhanced macrophage migratory capacity, whereas delayed stimulation with <i>Pg-</i>OMVs (Schedules 2 and 3) did not. Collectively, these findings indicate that <i>Pg</i>-OMVs exert stage-specific effects on the OC/MΦ lineage: stimulation at early stages of RANKL priming suppresses OC-genesis and promotes M1 polarization, whereas stimulation at later stages enhances OC-genesis without inducing M1 differentiation. Thus, <i>Pg</i>-OMVs may critically influence the fate of the OC/MΦ unit in periodontal lesions, contributing to disease progression and tissue destruction.