Porphyromonas gingivalis-derived RgpA-Kgp Complex Activates the Macrophage Urokinase Plasminogen Activator System: IMPLICATIONS FOR PERIODONTITIS.

Urokinase plasminogen activator (uPA) converts plasminogen to plasmin, resulting in a proteolytic cascade that has been implicated in tissue destruction during inflammation. Periodontitis is a highly prevalent chronic inflammatory disease characterized by destruction of the tissue and bone that support the teeth. We demonstrate that stimulation of macrophages with the arginine- and lysine-specific cysteine protease complex (RgpA-Kgp complex), produced by the keystone pathogen Porphyromonas gingivalis, dramatically increased their ability to degrade matrix in a uPA-dependent manner. We show that the RgpA-Kgp complex cleaves the inactive zymogens, pro-uPA (at consensus sites Lys(158)-Ile(159) and Lys(135)-Lys(136)) and plasminogen, yielding active uPA and plasmin, respectively. These findings are consistent with activation of the uPA proteolytic cascade by P. gingivalis being required for the pathogen to induce alveolar bone loss in a model of periodontitis and reveal a new host-pathogen interaction in which P. gingivalis activates a critical host proteolytic pathway to promote tissue destruction and pathogen virulence.

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.

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).

Protease-activated receptor 2 has pivotal roles in cellular mechanisms involved in experimental periodontitis.

The tissue destruction seen in chronic periodontitis is commonly accepted to involve extensive upregulation of the host inflammatory response. Protease-activated receptor 2 (PAR-2)-null mice infected with Porphyromonas gingivalis did not display periodontal bone resorption in contrast to wild-type-infected and PAR-1-null-infected mice. Histological examination of tissues confirmed the lowered bone resorption in PAR-2-null mice and identified a substantial decrease in mast cells infiltrating the periodontal tissues of these mice. T cells from P. gingivalis-infected or immunized PAR-2-null mice proliferated less in response to antigen than those from wild-type animals. CD90 (Thy1.2) expression on CD4(+) and CD8(+) T-cell-receptor beta (TCRbeta) T cells was significantly (P < 0.001) decreased in antigen-immunized PAR-2-null mice compared to sham-immunized PAR-2-null mice; this was not observed in wild-type controls. T cells from infected or antigen-immunized PAR-2-null mice had a significantly different Th1/inflammatory cytokine profile from wild-type cells: in particular, gamma interferon, interleukins (interleukin-2, -3, and -17), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha demonstrated lower expression than wild-type controls. The absence of PAR-2 therefore appears to substantially decrease T-cell activation and the Th1/inflammatory response. Regulation of such proinflammatory mechanisms in T cells and mast cells by PAR-2 suggests a pivotal role in the pathogenesis of the 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.