Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo

Outer membrane vesicles (OMVs) are proteoliposomes blebbed from the surface of Gram-negative bacteria. Chronic periodontitis is associated with an increase in subgingival plaque of Gram-negative bacteria, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. In this study, we investigated the immune-modulatory effects of P. gingivalis, T. denticola, and T. forsythia OMVs on monocytes and differentiated macrophages. All of the bacterial OMVs were phagocytosed by monocytes, M(naive) and M(IFN gamma) macrophages in a dose-dependent manner. They also induced NF-kappa B activation and increased TNF alpha, IL-8, and IL-1 beta cytokine secretion. P. gingivalis OMVs were also found to induce anti-inflammatory IL-10 secretion. Although unprimed monocytes and macrophages were resistant to OMV-induced cell death, lipopolysaccharide or OMV priming resulted in a significantly reduced cell viability. P. gingivalis, T. denticola, and T. forsythia OMVs all activated inflammasome complexes, as monitored by IL-1 beta secretion and ASC speck formation. ASC was critical for OMV-induced inflammasome formation, while AIM2-/- and Caspase-1-/- cells had significantly reduced inflammasome formation and NLRP3-/- cells exhibited a slight reduction. OMVs were also found to provide both priming and activation of the inflammasome complex. High-resolution microscopy and flow cytometry showed that P. gingivalis OMVs primed and activated macrophage inflammasomes in vivo with 80% of macrophages exhibiting inflammasome complex formation. In conclusion, periodontal pathogen OMVs were found to have significant immunomodulatory effects upon monocytes and macrophages and should therefore influence pro-inflammatory host responses associated with disease.