Peripheral memory T-cell profile is modified in patients undergoing periodontal management.

AIMS: T-cells are known to have a role in periodontitis, however, the effect of periodontal therapy on peripheral memory T-cells is unclear. This study evaluated variation in peripheral memory T-cells and red complex bacteria in sub-gingival plaque in patients undergoing periodontal management. METHODS: Peripheral blood mononuclear cells and sub-gingival plaque were collected from 54 periodontitis patients at baseline, 3-, 6- and 12-months post-therapy and 40 healthy controls. Periodontitis patients were divided into treatment outcome (TxO) groups based on prevalence of sites with probing depth ≥5 mm as good (<10% of sites), moderate (10-20%) or poor (>20%) at study conclusion. Naïve (TN -CCR7+ CD45RA+ ), central memory (TCM -CCR7+ CD45RA- ), effector memory (TEM -CCR7- CD45RA- ) and effector memory T-cells re-expressing CD45RA (TEMRA -CCR7- CD45RA+ ) were phenotyped using flow cytometry in CD4+ , CD8+ , CD4+ CD8+ and CD4- CD8- T-cells and red complex bacteria were quantified using qPCR. RESULTS: At baseline, periodontitis subjects had significantly greater mean probing depths and Porphyromonas gingivalis proportions, lower TN but higher CD4+ TCM , CD8+ TCM , CD4+ CD8+ TEM and CD4- CD8- TEM cell proportions compared to health. Periodontal therapy decreased mean probing depths, P. gingivalis proportions, TEM and CD4+ and CD8+ TCM cells, but increased TN and CD4+ and CD8+ TEMRA cells. The T-cell profile in the good TxO group showed therapy-related changes in CD4+ TEM , and CD8+ TN and TEM cells, whereas, no changes were observed in the poor TxO group. CONCLUSION: Management and the reduction in red complex bacteria were associated with changes in peripheral memory T-cells in periodontitis.

Celogentin mimetics as inhibitors of tubulin polymerization.

Bicyclic analogues of celogentin C have been synthesized in which the side chain-side chain cross-links are replaced by thioether bonds. Several of the simplified bicyclic peptides displayed potent inhibition of tubulin polymerization.

Porphyromonas gulae Activates Unprimed and Gamma Interferon-Primed Macrophages via the Pattern Recognition Receptors Toll-Like Receptor 2 (TLR2), TLR4, and NOD2.

Porphyromonas gulae is an anaerobic, Gram-negative coccobacillus that has been associated with periodontal disease in companion animals. The aims of this study were to analyze the ligation of pattern recognition receptors by P. gulae and the subsequent activation of macrophages. Exposure of HEK cells transfected with Toll-like receptors (TLRs) or NOD-like receptors to P. gulae resulted in the ligation of TLR2, TLR4, and NOD2. The effects of this engagement of receptors were investigated by measuring the synthesis of nitric oxide (NO), CD86 expression, and inflammatory cytokine production by wild-type, TLR2-/-, and TLR4-/- macrophages. The addition of P. gulae to unprimed and gamma interferon (IFN-γ)-primed (M1 phenotype) macrophages significantly increased the surface expression of CD86, but only M1 macrophages produced nitric oxide. P. gulae-induced expression of CD86 on unprimed macrophages was dependent on both TLR2 and TLR4, but CD86 expression and NO production in M1 macrophages were only TLR2 dependent. P. gulae induced an increase in secretion of interleukin-1α (IL-1α), IL-1ß, IL-6, IL-12p70, IL-13, tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α) by M1 macrophages compared to that by unprimed controls. Among these cytokines, secretion of IL-6 and TNF-α by M1 macrophages was dependent on either TLR2 or TLR4. Our data indicate that TLR2 and TLR4 are important for P. gulae activation of unprimed macrophages and that activation and effector functions induced in M1 macrophages by P. gulae are mainly dependent on TLR2. In conclusion, P. gulae induces a strong TLR2-dependent inflammatory M1 macrophage response which may be important in establishing the chronic inflammation associated with periodontal disease in companion animals.

Determination of Active Phagocytosis of Unopsonized Porphyromonas gingivalis by Macrophages and Neutrophils Using the pH-Sensitive Fluorescent Dye pHrodo.

Phagocytosis of pathogens is an important component of the innate immune system that is responsible for the removal and degradation of bacteria as well as their presentation via the major histocompatibility complexes to the adaptive immune system. The periodontal pathogen Porphyromonas gingivalis exhibits strain heterogeneity, which may affect a phagocyte's ability to recognize and phagocytose the bacterium. In addition, P. gingivalis is reported to avoid phagocytosis by antibody and complement degradation and by invading phagocytic cells. Previous studies examining phagocytosis have been confounded by both the techniques employed and the potential of the bacteria to invade the cells. In this study, we used a novel, pH-sensitive dye, pHrodo, to label live P. gingivalis strains and examine unopsonized phagocytosis by murine macrophages and neutrophils and human monocytic cells. All host cells examined were able to recognize and phagocytose unopsonized P. gingivalis strains. Macrophages had a preference to phagocytose P. gingivalis strain ATCC 33277 over other strains and clinical isolates in the study, whereas neutrophils favored P. gingivalis W50, ATCC 33277, and one clinical isolate over the other strains. This study revealed that all P. gingivalis strains were capable of being phagocytosed without prior opsonization with antibody or complement.

Macrophage depletion abates Porphyromonas gingivalis-induced alveolar bone resorption in mice.

The role of the macrophage in the immunopathology of periodontitis has not been well defined. In this study, we show that intraoral inoculation of mice with Porphyromonas gingivalis resulted in infection, alveolar bone resorption, and a significant increase in F4/80(+) macrophages in gingival and submandibular lymph node tissues. Macrophage depletion using clodronate-liposomes resulted in a significant reduction in F4/80(+) macrophage infiltration of gingival and submandibular lymph node tissues and significantly (p < 0.01) less P. gingivalis-induced bone resorption compared with controls in BALB/c and C57BL/6 mice. In both mouse strains, the P. gingivalis-specific IgG Ab subclass and serum cytokine [IL-4, IL-10, IFN-γ, and IL-12 (p70)] responses were significantly (p < 0.01) lower in the macrophage-depleted groups. Macrophage depletion resulted in a significant reduction in the level of P. gingivalis infection, and the level of P. gingivalis infection was significantly correlated with the level of alveolar bone resorption. M1 macrophages (CD86(+)), rather than M2 macrophages (CD206(+)), were the dominant macrophage phenotype of the gingival infiltrate in response to P. gingivalis infection. P. gingivalis induced a significant (p < 0.01) increase in NO production and a small increase in urea concentration, as well as a significant increase in the secretion of IL-1ß, IL-6, IL-10, IL-12 (p70), eotaxin, G-CSF, GM-CSF, macrophage chemoattractant protein-1, macrophage inflammatory protein-α and -ß, and TNF-α in isolated murine macrophages. In conclusion, P. gingivalis infection induced infiltration of functional/inflammatory M1 macrophages into gingival tissue and alveolar bone resorption. Macrophage depletion reduced P. gingivalis infection and alveolar bone resorption by modulating the host immune response.

Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors.

Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.

GM-CSF and uPA are required for Porphyromonas gingivalis-induced alveolar bone loss in a mouse periodontitis model.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and urokinase-type plasminogen activator (uPA) can contribute to the progression of chronic inflammatory diseases with possible involvement of macrophages. In this study, we investigated the role of both GM-CSF and uPA in Porphyromonas gingivalis-induced experimental periodontitis using GM-CSF-/- and uPA-/- mice. Intra-oral inoculation of wild-type (WT) C57BL/6 mice with P. gingivalis resulted in establishment of the pathogen in plaque and a significant increase in alveolar bone resorption. The infected mice also exhibited a CD11b(+) CD86(+) macrophage infiltrate into the gingival tissue, as well as P. gingivalis-specific pro-inflammatory cytokine and predominantly IgG2b antibody responses. In comparison, intra-oral inoculation of P. gingivalis did not induce bone resorption and there was significantly less P. gingivalis recovered from plaque in GM-CSF-/- and uPA-/- mice. Furthermore, P. gingivalis did not induce a macrophage gingival infiltrate or activate isolated peritoneal macrophages from the gene-deficient mice. Pro-inflammatory P. gingivalis-specific T-cell cytokine responses and serum interferon-gamma (IFN-γ) and IgG2b concentrations were significantly lower in GM-CSF-/- mice. In uPA-/- mice, T-cell responses were lower but serum IFN-γ and IgG2b levels were comparable with WT mice levels. These results suggest that GM-CSF and uPA are both involved in the progression of experimental periodontitis, possibly via a macrophage-dependent mechanism(s).

Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors.

Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

Porphyromonas gingivalis lipopolysaccharide weakly activates M1 and M2 polarized mouse macrophages but induces inflammatory cytokines.

Porphyromonas gingivalis is associated with chronic periodontitis, an inflammatory disease of the tooth's supporting tissues. Macrophages are important in chronic inflammatory conditions, infiltrating tissue and becoming polarized to an M1 or M2 phenotype. As responses to stimuli differ between these phenotypes, we investigated the effect of P. gingivalis lipopolysaccharide (LPS) on M1 and M2 macrophages. M1 and M2 polarized macrophages were produced from murine bone marrow macrophages (BMMϕ) primed with gamma interferon (IFN-γ) or interleukin-4 (IL-4), respectively, and incubated with a low or high dose of P. gingivalis LPS or control TLR2 and TLR4 ligands. In M1-Mϕ, the high dose of P. gingivalis LPS (10 µg/ml) significantly increased the expression of CD40, CD86, inducible nitric oxide synthase, and nitric oxide secretion. The low dose of P. gingivalis LPS (10 ng/ml) did not induce costimulatory or antibacterial molecules but did increase the secretion of IL-1α, IL-6, IL-12p40, IL-12p70, and tumor necrosis factor alpha (TNF-α). P. gingivalis LPS marginally increased the expression of CD206 and YM-1, but it did enhance arginase expression by M2-Mϕ. Furthermore, the secretion of the chemokines KC, RANTES, eotaxin, and MCP-1 from M1, M2, and nonpolarized Mϕ was enhanced by P. gingivalis LPS. TLR2/4 knockout macrophages combined with the TLR activation assays indicated that TLR2 is the main activating receptor for P. gingivalis LPS and whole cells. In conclusion, although P. gingivalis LPS weakly activated M1-Mϕ or M2-Mϕ compared to control TLR ligands, it induced the secretion of inflammatory cytokines, particularly TNF-α from M1-Mϕ and IL-10 from M2-Mϕ, as well as chemotactic chemokines from polarized macrophages.

The Potential of Modified and Multimeric Antimicrobial Peptide Materials as Superbug Killers.

Antimicrobial peptides (AMPs) are found in nearly all living organisms, show broad spectrum antibacterial activity, and can modulate the immune system. Furthermore, they have a very low level of resistance induction in bacteria, which makes them an ideal target for drug development and for targeting multi-drug resistant bacteria 'Superbugs'. Despite this promise, AMP therapeutic use is hampered as typically they are toxic to mammalian cells, less active under physiological conditions and are susceptible to proteolytic degradation. Research has focused on addressing these limitations by modifying natural AMP sequences by including e.g., d-amino acids and N-terminal and amino acid side chain modifications to alter structure, hydrophobicity, amphipathicity, and charge of the AMP to improve antimicrobial activity and specificity and at the same time reduce mammalian cell toxicity. Recently, multimerisation (dimers, oligomer conjugates, dendrimers, polymers and self-assembly) of natural and modified AMPs has further been used to address these limitations and has created compounds that have improved activity and biocompatibility compared to their linear counterparts. This review investigates how modifying and multimerising AMPs impacts their activity against bacteria in planktonic and biofilm states of growth.

Tumor Associated Macrophages: Origin, Recruitment, Phenotypic Diversity, and Targeting.

The tumor microenvironment (TME) is known to have a strong influence on tumorigenesis, with various components being involved in tumor suppression and tumor growth. A protumorigenic TME is characterized by an increased infiltration of tumor associated macrophages (TAMs), where their presence is strongly associated with tumor progression, therapy resistance, and poor survival rates. This association between the increased TAMs and poor therapeutic outcomes are stemming an increasing interest in investigating TAMs as a potential therapeutic target in cancer treatment. Prominent mechanisms in targeting TAMs include: blocking recruitment, stimulating repolarization, and depletion methods. For enhancing targeting specificity multiple nanomaterials are currently being explored for the precise delivery of chemotherapeutic cargo, including the conjugation with TAM-targeting peptides. In this paper, we provide a focused literature review of macrophage biology in relation to their role in tumorigenesis. First, we discuss the origin, recruitment mechanisms, and phenotypic diversity of TAMs based on recent investigations in the literature. Then the paper provides a detailed review on the current methods of targeting TAMs, including the use of nanomaterials as novel cancer therapeutics.

The multi-copper-ion oxidase CueO of Salmonella enterica serovar Typhimurium is required for systemic virulence.

Salmonella enterica serovar Typhimurium possesses a multi-copper-ion oxidase (multicopper oxidase), CueO (also known as CuiD), a periplasmic enzyme known to be required for resistance to copper ions. CueO from S. Typhimurium was expressed as a recombinant protein in Escherichia coli, and the purified protein exhibited a high cuprous oxidase activity. We have characterized an S. Typhimurium cueO mutant and confirmed that it is more sensitive to copper ions. Using a murine model of infection, it was observed that the cueO mutant was significantly attenuated, as indicated by reduced recovery of bacteria from liver and spleen, although there was no significant difference in recovery from Peyer's patches and mesenteric lymph nodes. However, the intracellular survival of the cueO mutant in unprimed or gamma-interferon-primed murine macrophages was not statistically different from that of wild-type Salmonella, suggesting that additional host factors are involved in clearance of the cueO mutant. Unlike a cueO mutant from E. coli, the S. Typhimurium cueO mutant did not show greater sensitivity to hydrogen peroxide and its sensitivity to copper ions was not affected by siderophores. Similarly, the S. Typhimurium cueO mutant was not rescued from copper ion toxicity by addition of the branched-chain amino acids and leucine.

Multifunctional Antimicrobial Polypeptide-Selenium Nanoparticles Combat Drug-Resistant Bacteria.

Antibiotic-resistant bacteria are a severe threat to human health. The World Health Organization's Global Antimicrobial Surveillance System has revealed widespread occurrence of antibiotic resistance among half a million patients across 22 countries, with Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae being the most common resistant species. Antimicrobial nanoparticles are emerging as a promising alternative to antibiotics in the fight against antimicrobial resistance. In this work, selenium nanoparticles coated with the antimicrobial polypeptide, ε-poly-l-lysine, (Se NP-ε-PL) were synthesized and their antibacterial activity and cytotoxicity were investigated. Se NP-ε-PL exhibited significantly greater antibacterial activity against all eight bacterial species tested, including Gram-positive, Gram-negative, and drug-resistant strains, than their individual components, Se NP and ε-PL. The nanoparticles showed no toxicity toward human dermal fibroblasts at the minimum inhibitory concentrations, demonstrating a therapeutic window. Furthermore, unlike the conventional antibiotic kanamycin, Se NP-ε-PL did not readily induce resistance in E. coli or S. aureus. Specifically, S. aureus began to develop resistance to kanamycin from ∼44 generations, whereas it took ∼132 generations for resistance to develop to Se NP-ε-PL. Startlingly, E. coli was not able to develop resistance to the nanoparticles over ∼300 generations. These results indicate that the multifunctional approach of combining Se NP with ε-PL to form Se NP-ε-PL is a highly efficacious new strategy with wide-spectrum antibacterial activity, low cytotoxicity, and significant delays in development of resistance.

Engineering highly effective antimicrobial selenium nanoparticles through control of particle size.

The overuse of antibiotics has induced the rapid development of antibiotic resistance in bacteria. As a result, antibiotic efficacy has become limited, and infection with multidrug-resistant bacteria is considered to be one of the largest global human health threats. Consequently, new, effective and safe antimicrobial agents need to be developed urgently. One promising candidate to address this requirement is selenium nanoparticles (Se NPs), which are made from the essential dietary trace element Se and have antimicrobial activity against Gram-positive bacteria. The size of nanomaterials can strongly affect their biophysical properties and functions; however, the effects of the size of Se NPs on their antibacterial efficacy has not been systematically investigated. Therefore, in this work, spherical Se NPs ranging from 43 to 205 nm in diameter were fabricated, and their mammalian cytotoxicity and antibacterial activity as a function of their size were systematically studied. The antibacterial activity of the Se NPs was shown to be strongly size dependent, with 81 nm Se NPs showing the maximal growth inhibition and killing effect of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). The Se NPs were shown to have multi-modal mechanisms of action that depended on their size, including depleting internal ATP, inducing ROS production, and disrupting membrane potential. All the Se NPs were non-toxic towards mammalian cells up to 25 µg mL-1. Furthermore, the MIC value for the 81 nm particles produced in this research is 16 ± 7 µg mL-1, significantly lower than previously reported MIC values for Se NPs. This data illustrates that Se NP size is a facile yet critical and previously underappreciated parameter that can be tailored for maximal antimicrobial efficacy. We have identified that using Se NPs with a size of 81 nm and concentration of 10 µg mL-1 shows promise as a safe and efficient way to kill S. aureus without damaging mammalian cells.

Spatially resolved force spectroscopy of bacterial surfaces using force-volume imaging.

Force spectroscopy using the atomic force microscope (AFM) is a powerful technique for measuring physical properties and interaction forces at microbial cell surfaces. Typically for such a study, the point at which a force measurement will be made is located by first imaging the cell using AFM in contact mode. In this study, we image the bacterial cell Shewanella putrefaciens for subsequent force measurements using AFM in force-volume mode and compare this to contact-mode images. It is known that contact-mode imaging does not accurately locate the apical surface and periphery of the cell since, in contact mode, a component of the applied load laterally deforms the cell during the raster scan. Here, we illustrate that contact-mode imaging does not accurately locate the apical surface and periphery of the cell since, in contact mode, a component of the applied load laterally deforms the cell during the raster scan. This is an artifact due to the deformability and high degree of curvature of bacterial cells. We further show that force-volume mode imaging avoids the artifacts associated with contact-mode imaging due to surface deformation since it involves the measurement of a grid of individual force profiles. The topographic image is subsequently reconstructed from the zero-force height (the contact distance between the AFM tip and the surface) at each point on the cell surface. We also show how force-volume measurements yield applied load versus indentation data from which mechanical properties of the cell such as Young's modulus, cell turgor pressure and elastic and plastic energies can be extracted.

Architectural Effects of Star-Shaped "Structurally Nanoengineered Antimicrobial Peptide Polymers" (SNAPPs) on Their Biological Activity.

In this work, the effect of two key structural parameters, number of arms and arm length, of star-shaped "structurally nanoengineered antimicrobial peptide polymers" (SNAPPs) on their antimicrobial activity and biocompatibility, is investigated. A library of star-shaped SNAPPs is prepared, containing varying arm numbers and arm lengths. Antimicrobial assays are then performed to assess the capacity of the SNAPPs to disrupt the membrane, inhibit the growth, and kill pathogenic bacteria. A major finding of the study is that increasing arm number and length of SNAPPs enhanced antimicrobial activity, which can be respectively attributed to the higher local concentrations of polypeptide arms and increased α-helical content. SNAPP architecture is shown to affect the bacteria membrane state and therefore mechanism of killing. Two more potent structures with up to twice the antimicrobial activity of the previously reported SNAPP are discovered in this process. Toxicities of the SNAPPs also increase with arm number and arm length, however therapeutic index calculations identified a 16-arm SNAPP and an easier to prepare 4-arm SNAPP as the best therapeutic agents. The biocompatibility of the SNAPP with the best biological activity is also evaluated in vivo, showing no markers of systemic damage in mice.

Biocompatibility and Osteogenic/Calcification Potential of Casein Phosphopeptide-amorphous Calcium Phosphate Fluoride.

INTRODUCTION: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and CPP-ACP with fluoride (CPP-ACFP) have been shown to provide bioavailable ions to promote mineralization. Hence, the aim of this study was to evaluate the materials' biocompatibility and osteogenic/calcification potential for endodontic applications. METHODS: Human and mouse osteoblast-like and fibroblast-like cell lines were incubated with 0.05%-3.0% w/v CPP-ACP and CPP-ACFP, and toxicity, proliferation, alkaline phosphatase, interleukin (IL)-1α, and IL-6 production, collagen type I, osteocalcin, and osteopontin production, and mineralization/calcification were determined. RESULTS: CPP-ACP and CPP-ACFP were non-toxic and had no significant effect on proliferation or production of the inflammatory cytokine IL-1α. Alkaline phosphatase activity of the osteoblast-like cells was significantly increased (P < .05) by CPP-ACP and CPP-ACFP, as was the production of the osteotropic cytokine IL-6, the formation of calcium mineral deposits, and the secretion of mineralization-related proteins (collagen type I and osteocalcin). CONCLUSIONS: CPP-ACP and CPP-ACFP are biocompatible and have the potential to induce osteoblastic differentiation and mineralization. Potential applications include apexification, perforation repair, vital pulp therapy, and regenerative endodontic procedures.

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(naïve) and M(IFNγ) macrophages in a dose-dependent manner. They also induced NF-κB activation and increased TNFα, IL-8, and IL-1ß 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-1ß 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.

Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis.

Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Tissue macrophages are amongst the first immune cells to respond to bacteria and depending on the cytokine profile at the infection site, macrophages are primed to react to infection in different ways. Priming of naive macrophages with IFN-γ produces a classical pro-inflammatory, antibacterial M1 macrophage after TLR ligation, whereas priming with IL-4 induces an anti-inflammatory tissue-repair M2 phenotype. Previous work has shown that M1 are preferentially generated in gingival tissue following infection with P. gingivalis. However, few studies have investigated the interactions of macrophage subsets with P. gingivalis cells. The aim of this study was to determine the ability of naive, M1 and M2 macrophages to phagocytose P. gingivalis and investigate how this interaction affects both the bacterial cell and the macrophage. M1 and M2 macrophages were both found to have enhanced phagocytic capacity compared with that of naive macrophages, however only the naive and M1 macrophages were able to produce a respiratory burst in order to clear the bacteria from the phagosome. P. gingivalis was found to persist in naive and M2, but not M1 macrophages for 24 hours. Phagocytosis of P. gingivalis also induced high levels of TNF-α, IL-12 and iNOS in M1 macrophages, but not in naive or M2 macrophages. Furthermore, infection of macrophages with P. gingivalis at high bacteria to macrophage ratios, while inducing an inflammatory response, was also found to be deleterious to macrophage longevity, with high levels of apoptotic cell death found in macrophages after infection. The activation of M1 macrophages observed in this study may contribute to the initiation and maintenance of a pro-inflammatory state during chronic periodontitis.

Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

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.