This study aimed to investigate the effect of the periodontal bacterium Filifactor alocis on proinflammatory cytokine production by osteoblasts. Specifically, we examined the mechanisms underlying interleukin (IL)-6 production by osteoblasts stimulated with live and lyophilized F. alocis, and F. alocis-derived extracellular vesicles (EVs).

MC3T3-E1 mouse osteoblasts were stimulated with live bacteria of F. alocis and, as a comparative control, P. gingivalis. Furthermore, cells were treated with F. alocis lyophilized whole cells or EVs, and IL-6 production was quantified by ELISA; cytokine expression profiled using membrane-based array; and mRNA expression analyzed by RT-qPCR. Signaling pathways were analyzed using specific inhibitors. Statistical analyses were performed using non-parametric tests with significance set at p < 0.05.

F. alocis live bacteria, lyophilized whole cells, and F. alocis-derived EVs induced IL-6, leukemia inhibitory factor, and chemokines in MC3T3-E1 cells. Mechanistically, IL-6 production by F. alocis involved osteoblast activation via Toll-like receptor (TLR) 2 on the bacterial surface or released EVs. Lyophilized F. alocis induced IκB-ζ mRNA expression in osteoblasts. Moreover, F. alocis-derived EVs induced IL-6 production in osteoblasts via protein kinase C (PKC).

Live and lyophilized F. alocis and F. alocis-derived EVs induce the production of proinflammatory cytokines by osteoblasts, including IL-6. Additionally, the induction of IL-6 by F. alocis-derived EVs involves the activation of TLR2 and PKC. Conclusively, these findings suggest that F. alocis-induced cytokine production by osteoblasts may induce osteoclast differentiation, highlighting its role in the pathogenesis of periodontitis.