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Metabolic Remodeling, Inflammasome Activation, and Pyroptosis in Macrophages Stimulated by Porphyromonas Gingivalis and Its Outer Membrane Vesicles

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Metabolic Remodeling, Inflammasome Activation, and Pyroptosis in Macrophages Stimulated by Porphyromonas Gingivalis and Its Outer Membrane Vesicles ORIGINAL RESEARCH published: 04 August 2017 doi: 10.3389/fcimb.2017.00351 Metabolic Remodeling, Inflammasome Activation, and Pyroptosis in Macrophages Stimulated by Porphyromonas gingivalis and Its Outer Membrane Vesicles Andrew J. Fleetwood 1*, Man K.S. Lee 2, William Singleton 3, Adrian Achuthan 1, Ming-Chin Lee 1, Neil M. O’Brien-Simpson 3, Andrew D. Cook 1, Andrew J. Murphy 2, Stuart G. Dashper 3, Eric C. Reynolds 3 and John A. Hamilton 1 1 Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville, VIC, Australia, 2 Haematopoiesis and Leukocyte Biology, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia, 3 Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, University of Melbourne, VIC, Australia 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 Edited by: products, so we sought to directly compare the response of macrophages to P.gingivalis Georgios N. Belibasakis, or its purified OMVs. Macrophages stimulated with OMVs produced large amounts of Karolinska Institute (KI), Sweden TNFα, IL-12p70, IL-6, IL-10, IFNβ, and nitric oxide compared to cells infected with Reviewed by: Özlem Yilmaz, P. gingivalis, which produced very low levels of these mediators. Both P. gingivalis Medical University of South Carolina, and OMVs induced a shift in macrophage metabolism from oxidative phosphorylation United States Peyman Kelk, (OXPHOS) to glycolysis, which was supported by enhanced lactate release, decreased Umeå University, Sweden mitochondrial oxygen consumption with reduced spare respiratory capacity, as well as *Correspondence: increased mitochondrial reactive oxygen species (ROS) production. Corresponding to Andrew J. Fleetwood this metabolic shift, gene expression analysis of macrophages infected with P. gingivalis andrew.fl[email protected] or stimulated with OMVs revealed a broad transcriptional upregulation of genes critical Received: 03 May 2017 to glycolysis and a downregulation of genes associated with the TCA cycle. Upon Accepted: 21 July 2017 examination of inflammasome signaling and pyroptosis it was found that P. gingivalis Published: 04 August 2017 did not activate the inflammasome in macrophages as the mature forms of caspase-1, Citation: Fleetwood AJ, Lee MKS, Singleton W, IL-1β, and IL-18 were not detected and there was no extracellular release of lactate Achuthan A, Lee M-C, dehydrogenase (LDH) or 7-AAD staining. In comparison, macrophages stimulated with O’Brien-Simpson NM, Cook AD, OMVs potently activated caspase-1, produced large amounts of IL-1β, IL-18, released Murphy AJ, Dashper SG, Reynolds EC and Hamilton JA (2017) Metabolic LDH, and were positive for 7-AAD indicative of pyroptotic cell death. These data directly Remodeling, Inflammasome quantitate the distinct effects of P. gingivalis and its OMVs on macrophage inflammatory Activation, and Pyroptosis in Macrophages Stimulated by phenotype, mitochondrial function, inflammasome activation, and pyroptotic cell death Porphyromonas gingivalis and Its that may have potential implications for their roles in chronic periodontitis. Outer Membrane Vesicles. Front. Cell. Infect. Microbiol. 7:351. Keywords: macrophages, metabolism, inflammasome, P. gingivalis, vesicles, pyroptosis, cytokines and doi: 10.3389/fcimb.2017.00351 inflammation, glycolysis and oxidative phosphorylation Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 August 2017 | Volume 7 | Article 351 Fleetwood et al. Activation of Macrophages by P. gingivalis INTRODUCTION inflammatory conditions (Hajishengallis, 2015). Pyroptosis is a programmed form of proinflammatory cell death that allows Porphyromonas gingivalis is recognized as a keystone pathogen the elimination of intracellular pathogens (Franchi et al., 2012; (Hajishengallis et al., 2012) and is one of the bacterial biofilm Aachoui et al., 2013). Pyroptosis occurs following activation of species isolated from subgingival plaque most strongly associated the cytosolic inflammasome signaling complex, which generates with clinical indicators of periodontitis, including increased active caspase-1 leading to pore formation and the release pocket depth and bleeding on probing (Socransky et al., 1998; of cytosolic contents (e.g., LDH) and production of the Komiya et al., 2000). A common feature of Gram-negative inflammatory cytokines IL-1β and IL-18 (Shi et al., 2015). There bacteria, like P. gingivalis, is the biogenesis of outer membrane is conflicting evidence as to whether P. gingivalis can activate the vesicles (OMVs), which are spherical membrane structures that inflammasome in macrophages, which in large part seems to be are increasingly thought to play a significant role in microbial due to differences in cell populations studied (Taxman et al., 2006, virulence and not simply by-products of bacterial cell wall 2012; Slocum et al., 2014). Another complication has been the damage or lysis (Kuehn and Kesty, 2005; Ellis and Kuehn, gingipain-mediated degradation of the major readouts used to 2010). P. gingivalis OMVs are enriched for the pathogen’s determine inflammasome activation and pyroptosis (Jung et al., major virulence factors such as gingipains (Arg- and Lys-specific 2015). P. gingivalis evasion of inflammasome activation would proteolytic enzymes) and lipopolysaccharide (LPS) (Veith et al., provide an intracellular niche for the pathogen to survive and 2014). Due to the small size of OMVs (50–70 nm in diameter) disseminate to distant sites. they spread more readily in tissues than their larger parent cells P. gingivalis OMVs can also persist within the lysosomes (Kuehn and Kesty, 2005; Darveau, 2010). As a result, P. gingivalis of host cells (Gui et al., 2016), are potent activators of OMVs are highly immunogenic and have been found to induce Toll-like receptors (TLRs) (Cecil et al., 2016) and can deliver infiltration of neutrophils in connective tissue (Srisatjaluk et al., virulence factors deep into host tissues (Mashburn-Warren 1999) and promote macrophage foam cell formation (Qi et al., and Whiteley, 2006; Darveau, 2010). It is currently unknown 2003). whether P. gingivalis OMVs activate the inflammasome or induce Recently, metabolic reprogramming in host immune cells, pyroptotic cell death. The host response to OMVs is likely particularly in macrophages and dendritic cells has been to be different to the parent bacterium as their phospholipid, implicated in regulating their phenotype and function (O’Neill gingipain and LPS profiles are distinct (Veith et al., 2014; Ho and Pearce, 2016). Macrophages activated with LPS and IFNγ et al., 2015; Xie, 2015; Gui et al., 2016). It has been speculated that (so called M1 macrophages) shift their glucose metabolism P. gingivalis OMVs act as a virulence factor secretion system that from oxidative phosphorylation (OXPHOS) to glycolysis and contributes to pathogenicity partly by supporting evasion of host this metabolic shift is central to their production of mediators immune defense mechanisms (Gui et al., 2016). In agreement associated with an M1 phenotype (e.g., NO) (Tannahill et al., with this a recent study found that the ability of P. gingivalis to 2013). Likewise the commitment of IL-4 stimulated macrophages subvert a local immune response was due to OMVs rendering (so called M2 macrophages) to OXPHOS to generate ATP is monocytes unresponsive to live P. gingivalis (Waller et al., 2016). critical to their adoption of a M2 phenotype (Vats et al., 2006; To better understand the immunostimulatory capabilities of Huang et al., 2014). A detailed comparison of metabolism in M1 OMVs, we recently developed a high-sensitivity flow cytometry vs. M2 macrophages identified specific metabolic pathways in method to enumerate P. gingivalis-derived OMVs (Cecil et al., both cell types that were critical in governing their polarization 2016). This method allows removal of non-OMV associated (Jha et al., 2015). Many recent studies have examined the links material (e.g., fimbriae and secreted proteins; Nakao et al., between glycolysis and cell effector function. For example, LPS- 2014) to yield highly purified single lipid-bilayer vesicles. We induced glycolysis enables dendritic cell maturation (Everts et al., found that the immunogenicity of vesicles purified from three 2014) whilst glycolysis is involved in inflammasome activation periodontal pathogens (as measured by TLR activation) could (Masters et al., 2010; Tannahill et al., 2013; Moon et al., 2015) and be better differentiated when standardized using vesicle number promotion of antibacterial responses in macrophages (Cordes compared to protein concentration (Cecil et al., 2016). To our et al., 2016; Lampropoulou et al., 2016). Much of this important knowledge there have been no studies directly comparing the information has been generated with purified LPS (reviewed immune responses elicited by P. gingivalis or its purified OMVs. in O’Neill et al., 2016) with relatively few studies (Garaude By utilizing our protocol for enumerating vesicles (Cecil et al., et al., 2016; Gleeson et al., 2016) addressing the impact of viable 2016) we were able to directly compare the effect of P. gingivalis bacteria on cellular metabolism. and OMVs at identical infection ratios on macrophage biology. P. gingivalis has been shown
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