Abstract
Porphyromonas gingivalis is one of the bacterial species most closely associated with periodontitis and can shed large numbers of outer membrane vesicles (OMVs), which are increasingly thought to play a significant role in bacterial virulence and pathogenicity. Macrophages are amongst the first immune cells to respond to bacteria and their products, so we sought to directly compare the response of macrophages to P. gingivalis or its purified OMVs. Macrophages stimulated with OMVs produced large amounts of TNFα, IL-12p70, IL-6, IL-10, IFNβ, and nitric oxide compared to cells infected with P. gingivalis, which produced very low levels of these mediators. Both P. gingivalis and OMVs induced a shift in macrophage metabolism from oxidative phosphorylation (OXPHOS) to glycolysis, which was supported by enhanced lactate release, decreased mitochondrial oxygen consumption with reduced spare respiratory capacity, as well as increased mitochondrial reactive oxygen species (ROS) production. Corresponding to this metabolic shift, gene expression analysis of macrophages infected with P. gingivalis or stimulated with OMVs revealed a broad transcriptional upregulation of genes critical to glycolysis and a downregulation of genes associated with the TCA cycle. Upon examination of inflammasome signaling and pyroptosis it was found that P. gingivalis did not activate the inflammasome in macrophages as the mature forms of caspase-1, IL-1β, and IL-18 were not detected and there was no extracellular release of lactate dehydrogenase (LDH) or 7-AAD staining. In comparison, macrophages stimulated with OMVs potently activated caspase-1, produced large amounts of IL-1β, IL-18, released LDH, and were positive for 7-AAD indicative of pyroptotic cell death. These data directly quantitate the distinct effects of P. gingivalis and its OMVs on macrophage inflammatory phenotype, mitochondrial function, inflammasome activation, and pyroptotic cell death that may have potential implications for their roles in chronic periodontitis.
Highlights
Porphyromonas gingivalis is recognized as a keystone pathogen (Hajishengallis et al, 2012) and is one of the bacterial biofilm species isolated from subgingival plaque most strongly associated with clinical indicators of periodontitis, including increased pocket depth and bleeding on probing (Socransky et al, 1998; Komiya et al, 2000)
To compare cytokine production induced by the pathogen and its components, murine bone-marrow-derived macrophages (BMM) were infected with viable P. gingivalis or its purified outer membrane vesicles (OMVs) for 2 h, after which the culture medium was removed and replaced with fresh medium containing antibiotics
BMM infected with P. gingivalis produced only modest amounts of TNFα, IL-12p70, IL-6, IL-10, and IFNβ compared to those stimulated with OMVs, which produced significantly (p < 0.05) higher levels of all of these mediators (Figure 1)
Summary
Porphyromonas gingivalis is recognized as a keystone pathogen (Hajishengallis et al, 2012) and is one of the bacterial biofilm species isolated from subgingival plaque most strongly associated with clinical indicators of periodontitis, including increased pocket depth and bleeding on probing (Socransky et al, 1998; Komiya et al, 2000). LPSinduced glycolysis enables dendritic cell maturation (Everts et al, 2014) whilst glycolysis is involved in inflammasome activation (Masters et al, 2010; Tannahill et al, 2013; Moon et al, 2015) and promotion of antibacterial responses in macrophages (Cordes et al, 2016; Lampropoulou et al, 2016) Much of this important information has been generated with purified LPS (reviewed in O’Neill et al, 2016) with relatively few studies (Garaude et al, 2016; Gleeson et al, 2016) addressing the impact of viable bacteria on cellular metabolism
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