Metabolic Remodeling, Inflammasome Activation, and Pyroptosis in Macrophages Stimulated by Porphyromonas gingivalis and Its Outer Membrane Vesicles.
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 TNF?, IL-12p70, IL-6, IL-10, IFN?, 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-1?, 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-1?, 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.
Project description: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(naïve) and M(IFN?) macrophages in a dose-dependent manner. They also induced NF-?B activation and increased TNF?, IL-8, and IL-1? 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? 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.
Project description:Porphyromonas gingivalis, a major periodontopathogen, is involved in the pathogenesis of periodontitis. Interleukin-1? (IL-1?), a proinflammatory cytokine, regulates innate immune responses and is critical for the host defense against bacterial infection. However, excessive IL-1? is linked to periodontal destruction. IL-1? synthesis, maturation, and secretion are tightly regulated by Toll-like receptor (TLR) signaling and inflammasome activation. We found much higher levels of inflammasome components in the gingival tissues from patients with chronic periodontitis than in those from healthy controls. To investigate the molecular mechanisms by which P. gingivalis infection causes IL-1? secretion, we examined the characteristics of P. gingivalis-induced signaling in differentiated THP-1 cells. We found that P. gingivalis induces IL-1? secretion and inflammatory cell death via caspase-1 activation. We also found that P. gingivalis-induced IL-1? secretion and pyroptic cell death required both NLRP3 and AIM2 inflammasome activation. The activation of the NLRP3 inflammasome was mediated by ATP release, the P2X7 receptor, and lysosomal damage. In addition, we found that the priming signal via TLR2 and TLR4 activation precedes P. gingivalis-induced IL-1? release. Our study provides novel insight into the innate immune response against P. gingivalis infection which could potentially be used for the prevention and therapy of periodontitis.
Project description:Emerging evidence suggests a role for purinergic signaling in the activation of multiprotein intracellular complexes called inflammasomes, which control the release of potent inflammatory cytokines, such as interleukin (IL) -1? and -18. Porphyromonas gingivalis is intimately associated with periodontitis and is currently considered one of the pathogens that can subvert the immune system by limiting the activation of the NLRP3 inflammasome. We recently showed that P. gingivalis can dampen eATP-induced IL-1? secretion by means of its fimbriae in a purinergic P2X7 receptor-dependent manner. Here, we further explore the role of this purinergic receptor during eATP-induced IL-1? processing and secretion by P. gingivalis-infected macrophages. We found that NLRP3 was necessary for eATP-induced IL-1? secretion as well as for caspase 1 activation irrespective of P. gingivalis fimbriae. Additionally, although the secretion of IL-1? from P. gingivalis-infected macrophages was dependent on NLRP3, its adaptor protein ASC, or caspase 1, the cleavage of intracellular pro-IL-1? to the mature form was found to occur independently of NLRP3, its adaptor protein ASC, or caspase 1. Our in vitro findings revealed that P2X7 receptor has a dual role, being critical not only for eATP-induced IL-1? secretion but also for intracellular pro-IL-1? processing. These results were relevant in vivo since P2X7 receptor expression was upregulated in a P. gingivalis oral infection model, and reduced IFN-? and IL-17 were detected in draining lymph node cells from P2rx7(-/-) mice. Furthermore, we demonstrated that P2X7 receptor and NLRP3 transcription were modulated in human chronic periodontitis. Overall, we conclude that the P2X7 receptor has a role in periodontal immunopathogenesis and suggest that targeting of the P2X7/NLRP3 pathway should be considered in future therapeutic interventions in periodontitis.
Project description:OBJECTIVE:Periodontitis is a microbe-induced chronic inflammatory disease. Previous exposure of the host to bacteria or their virulence factors leads to refractory responses to further stimuli, which is called tolerance. Porphyromonas gingivalis (P. gingivalis) is one of the most important pathogenic microorganisms associated with periodontitis, and is a potent inducer of pro- and anti-inflammatory cytokines. The aim of this study was to explore the roles and possible mechanisms of tolerance induced by P. gingivalis. METHODS:THP-1-derived macrophages were pretreated with 1x108 colony-forming units/ml P. gingivalis ATCC 33277 or 21 clinical isolates from moderate to severe chronic periodontitis patients (24 h), washed (2 h) and treated with P. gingivalis ATCC 33277 or the same clinical isolates again (24 h). Levels of pro-inflammatory cytokines TNF-? and IL-1? and anti-inflammatory cytokine IL-10 in supernatants were detected by ELISA. Moreover, to identify the possible mechanisms for the changes in cytokine secretion, Toll-like receptor 2 (TLR2) and TLR4 protein expressions were explored in these cells by flow cytometry. RESULTS:After repeated challenge with P. gingivalis ATCC 33277 or clinical isolates, production of TNF-? and IL-1? in macrophages was decreased significantly compared with that following a single stimulation (p<0.05), while only comparable levels of IL-10 were detected in P. gingivalis ATCC 33277 or clinical isolate-tolerized cells (p>0.05). In addition, there was interstrain variability in the ability to induce IL-1? and IL-10 production after repeated P. gingivalis stimulation. However, no significant changes in TLR2 or TLR4 were detected in macrophages that were repeatedly treated with P. gingivalis ATCC 33277 or clinical isolates compared with those stimulated with P. gingivalis only once (p>0.05). CONCLUSIONS:Repeated P. gingivalis stimulation triggered tolerance, which might contribute to limiting periodontal inflammation. However, tolerance induced by P. gingivalis might develop independently of TLR2 and TLR4 and be related to molecules in signaling pathways downstream of TLR2 and TLR4.
Project description:Periodontitis is a chronic inflammatory disease caused by gram-negative anaerobic bacteria. Monocytes and macrophages stimulated by periodontopathic bacteria induce inflammatory mediators that cause tooth-supporting structure destruction and alveolar bone resorption. In this study, using a DNA microarray, we identified the enhanced gene expression of thrombospondin-1 (TSP-1) in human monocytic cells stimulated by Porphyromonas gingivalis lipopolysaccharide (LPS). TSP-1 is a multifunctional extracellular matrix protein that is upregulated during the inflammatory process. Recent studies have suggested that TSP-1 is associated with rheumatoid arthritis, diabetes mellitus, and osteoclastogenesis. TSP-1 is secreted from neutrophils, monocytes, and macrophages, which mediate immune responses at inflammatory regions. However, TSP-1 expression in periodontitis and the mechanisms underlying TSP-1 expression in human monocytic cells remain unknown. Here using real-time RT-PCR, we demonstrated that TSP-1 mRNA expression level was significantly upregulated in inflamed periodontitis gingival tissues and in P. gingivalis LPS-stimulated human monocytic cell line THP-1 cells. TSP-1 was expressed via Toll-like receptor (TLR) 2 and TLR4 pathways. In P. gingivalis LPS stimulation, TSP-1 expression was dependent upon TLR2 through the activation of NF-?B signaling. Furthermore, IL-17F synergistically enhanced P. gingivalis LPS-induced TSP-1 production. These results suggest that modulation of TSP-1 expression by P. gingivalis plays an important role in the progression and chronicity of periodontitis. It may also contribute a new target molecule for periodontal therapy.
Project description:Canonical inflammasome activation induces a caspase-1/gasdermin D (Gsdmd)-dependent lytic cell death called pyroptosis that promotes antimicrobial host defense but may contribute to sepsis. The nature of the caspase-1-dependent change in plasma membrane (PM) permeability during pyroptotic progression remains incompletely defined. We assayed propidium(2+) (Pro(2+)) influx kinetics during NLRP3 or Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDMs) as an indicator of this PM permeabilization. BMDMs were characterized by rapid Pro(2+) influx after initiation of NLRP3 or Pyrin inflammasomes by nigericin (NG) or Clostridium difficile toxin B (TcdB), respectively. No Pro(2+) uptake in response to NG or TcdB was observed in Casp1(-/-) or Asc(-/-) BMDMs. The cytoprotectant glycine profoundly suppressed NG and TcdB-induced lysis but not Pro(2+) influx. The absence of Gsdmd expression resulted in suppression of NG-stimulated Pro(2+) influx and pyroptotic lysis. Extracellular La(3+) and Gd(3+) rapidly and reversibly blocked the induced Pro(2+) influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome assembly and caspase-1 activation. Thus, caspase-1-driven pyroptosis requires induction of initial prelytic pores in the PM that are dependent on Gsdmd expression. These PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca(2+) Although lanthanides and Gsdmd deletion both suppressed PM pore activity and pyroptotic lysis, robust IL-1? release was observed in lanthanide-treated BMDMs but not in Gsdmd-deficient cells. This suggests roles for Gsdmd in both passive IL-1? release secondary to pyroptotic lysis and in nonlytic/nonclassical IL-1? export.
Project description:Background: Oral commensals contribute to microbe-host symbiosis in periodontal homeostasis, and Porphyromonas gingivalis (P. gingivalis) as the keystone pathogen critically accounts for the shift of symbiosis to dysbiosis and periodontal destruction. Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome-mediated interleukin-1? (IL-1?) is significantly involved in periodontal diseases, and notably P. gingivalis enables to modulate the induction and expression of NLRP3. Whereas, the exact mechanism by which NLRP3 inflammasome is regulated in response to commensal and pathogenic bacteria remains unclear. Methods: To examine the expression of IL-1? and NLRPs inflammasome in tissues with severe chronic periodontitis, and further investigate how Caspase-4-dependent non-canonical NLRP3 inflammasome pathways functioned during the interactions of Streptococcus mitis (S. mitis) and P. gingivalis with human THP-1 cells. Results: IL-1? and NLRP3, NLRP6, NLRP12, and absent in melanoma 2 (AIM2) inflammasomes are highly expressed in gingival tissues with severe chronic periodontitis. In human THP-1 cells, P. gingivalis activates the synthesis and secretion of IL-1? to higher levels than S. mitis. Importantly, NLRP3-, Caspase-1-, and Caspase-4-siRNA knockdown THP-1 cells treated with P. gingivalis exhibited a lower expression level of IL-1? as compared to the control cells. In addition, silencing of either CASP4 or CASP1 can lead to a concurrent or reciprocal decrease in the expression of the other. Of note, the IL-1? induction is not affected in the S. mitis-treated THP-1 cells with the silence of NLRP3, Caspase-1, and Caspase-4 genes. Conclusion: NLRP3/Caspase-4 and NLRP3/Caspase-1 dependent IL-1? production may crucially contribute to the dysregulated immuno-inflammatory response in periodontal pathogenesis.
Project description:Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis.
Project description:Klebsiella pneumoniae is a Gram-negative bacterium responsible for severe cases of nosocomial pneumonia. During the infectious process, both neutrophils and monocytes migrate to the site of infection, where they carry out their effector functions and can be affected by different patterns of cell death. Our data show that clinical strains of K. pneumoniae have dissimilar mechanisms for surviving within macrophages; these mechanisms include modulation of microbicidal mediators and cell death. The A28006 strain induced high IL-1? production and pyroptotic cell death in macrophages; by contrast, the A54970 strain induced high IL-10 production and low IL-1? production by macrophages. Pyroptotic cell death induced by the A28006 strain leads to a significant increase in bacterial sensitivity to hydrogen peroxide, and efferocytosis of the pyroptotic cells results in efficient bacterial clearance both in vitro and in vivo. In addition, the A54970 strain was able to inhibit inflammasome activation and pyroptotic cell death by inducing IL-10 production. Here, for the first time, we present a K. pneumoniae strain able to inhibit inflammasome activation, leading to bacterial survival and dissemination in the host. The understanding of possible escape mechanisms is essential in the search for alternative treatments against multidrug-resistant bacteria.
Project description:The DNA sensor absent in melanoma 2 (AIM2) forms an inflammasome complex with ASC and caspase-1 in response to Francisella tularensis subspecies novicida infection, leading to maturation of IL-1? and IL-18 and pyroptosis. AIM2 is critical for host protection against F. novicida infection in vivo; however, the role of pyroptosis downstream of the AIM2 inflammasome is unknown. Recent studies have identified gasdermin D (GSDMD) as the molecule executing pyroptosis by forming pores on the plasma membrane following activation by inflammatory caspase-1 and -11. In this study, we report that GSDMD-deficient mice were susceptible to F. novicida infection compared with wild type mice. Interestingly, we observed that GSDMD is required for optimal caspase-1 activation and pyroptotic cell death in F. novicida-infected bone marrow-derived macrophages. Furthermore, caspase-1 activation was compromised in bone marrow-derived macrophages lacking GSDMD stimulated with other AIM2 inflammasome triggers, including poly(dA:dT) transfection and mouse CMV infection. Overall, our study highlights a function, to our knowledge previously unknown, for GSDMD in promoting caspase-1 activation by AIM2 inflammasome.