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

Abstract

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, <i>Porphyromonas gingivalis, Treponema denticola</i>, and <i>Tannerella forsythia</i>. In this study, we investigated the immune-modulatory effects of <i>P. gingivalis, T. denticola</i>, and <i>T. forsythia</i> OMVs on monocytes and differentiated macrophages. All of the bacterial OMVs were phagocytosed by monocytes, M(naïve) and M(IFNγ) macrophages in a dose-dependent manner. They also induced NF-κB activation and increased TNFα, IL-8, and IL-1β cytokine secretion. <i>P. gingivalis</i> 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. <i>P. gingivalis, T. denticola</i>, and <i>T. forsythia</i> OMVs all activated inflammasome complexes, as monitored by IL-1β 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 <i>P. gingivalis</i> OMVs primed and activated macrophage inflammasomes <i>in vivo</i> 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.