Hajime Sasaki

IL-10, But Not IL-4, Suppresses Infection-Stimulated Bone Resorption In Vivo

Periapical bone resorption occurs following infection of the dental pulp and is mediated mainly by IL-1α in the murine model. The production and activity of IL-1α is modulated by a network of regulatory cytokines, including those produced by Th1 (pro-inflammatory) and Th2 (anti-inflammatory) subset T cells. This study was designed to assess the functional role of the Th2-type cytokines IL-4 and IL-10 in infection-stimulated bone resorption in vivo. The dental pulps of the first molars were exposed and infected with a mixture of four common endodontic pathogens, and bone destruction was determined by micro-computed tomography at sacrifice on day 21. The results demonstrate that IL-10−/− mice had significantly greater infection-stimulated bone resorption in vivo compared with wild-type mice (p < 0.001), whereas IL-4−/− exhibited no increased resorption. IL-10−/− had markedly elevated IL-1α production within periapical inflammatory tissues (>10-fold) compared with wild type (p < 0.01), whereas IL-4−/− exhibited decreased IL-1α production (p < 0.05). IL-10 also suppressed IL-1α production by macrophages in a dose-dependent fashion in vitro, whereas IL-4 had weak and variable effects. We conclude that IL-10, but not IL-4, is an important endogenous suppressor of infection-stimulated bone resorption in vivo, likely acting via inhibition of IL-1α expression.

Toll-like receptor 4-deficient mice have reduced bone destruction following mixed anaerobic infection

ABSTRACT C3H/HeJ mice have an impaired ability to respond to lipopolysaccharide (LPS) due to a mutation in the gene that encodesToll-like receptor 4 (TLR4). The effect of TLR4 deficiency on host responses to endodontic infections is unknown. In the present study, we compared periapical bone destruction, sepsis, and inflammatory cytokine production in LPS-hyporesponsive C3H/HeJ and wild-type control C3H/HeOuJ mice. The mandibular first molars of both strains were subjected to pulpal exposure and infection with a mixture of four anaerobic pathogens, Prevotella intermedia,Fusobacterium nucleatum, Streptococcus intermedius, and Peptostreptococcus micros. At sacrifice on day 21, TLR4-deficient C3H/HeJ mice had significantly reduced periapical bone destruction compared to wild-type C3H/HeOuJ mice (P < 0.001). The decreased bone destruction in C3H/HeJ correlated with reduced expression of the bone resorptive cytokines interleukin 1α (IL-1α) (P < 0.01) and IL-1β (P < 0.05) as well as the proinflammatory cytokine IL-12 (P < 0.05). No significant differences were seen in the levels of gamma interferon, tumor necrosis factor alpha (TNF-α), or IL-10 between the two strains. The expression of IL-1α, IL-1β, TNF-α, IL-10, and IL-12 were all significantly reduced in vitro in macrophages from both TLR4-deficient C3H/HeJ and C57BL/10ScNCr strains, compared to wild-type controls. Notably, the responses of TLR4-deficient macrophages to both gram-positive and gram-negative bacteria were similarly reduced. Neither C3H/HeJ nor C3H/HeOuJ mice exhibited orofacial abscess development or infection dissemination as determined by splenomegaly or cachexia. We conclude that intact TLR function mediates increased proinflammatory responses and bone destruction in response to mixed anaerobic infections.

Interleukin-6 deficiency increases inflammatory bone destruction

ABSTRACT Periapical bone destruction occurs as a consequence of pulpal infection. In previous studies, we showed that interleukin-1 (IL-1) is the primary stimulator of bone destruction in this model. IL-6 is a pleiotropic cytokine that is induced in these infections and has both pro- and anti-inflammatory activities. In the present study, we determined the role of IL-6 in regulating IL-1 expression and bone resorption. The first molars of IL-6 knockouts (IL-6−/−) and wild-type mice were subjected to surgical pulp exposure and infection with a mixture of four common pulpal pathogens, includingPrevotella intermedia, Fusobacterium nucleatum,Peptostreptococcus micros, and Streptococcus intermedius. Mice were killed after 21 days, and bone destruction and cytokine expression were determined. Surprisingly, bone destruction was significantly increased in IL-6−/− mice versus that in wild-type mice (by 30%; P < 0.001). In a second experiment, the effects of chronic (IL-6−/−) IL-6 deficiency and short-term IL-6 deficiency induced by in vivo antibody neutralization were determined. Both IL-6−/− (30%;P < 0.001) and anti-IL-6 antibody-treated mice (40%;P < 0.05) exhibited increased periapical bone resorption, compared to wild-type controls. The increased bone resorption in IL-6-deficient animals correlated with increases in osteoclast numbers, as well as with elevated expression of bone-resorptive cytokines IL-1α and IL-1β, in periapical lesions and with decreased expression of the anti-inflammatory cytokine IL-10. These data demonstrate that endogenous IL-6 expression has significant anti-inflammatory effects in modulating infection-stimulated bone destruction in vivo.

MIP-1γ Promotes Receptor Activator of NF-κB Ligand-Induced Osteoclast Formation and Survival

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes, and have also been reported to modulate osteoclast differentiation from hemopoietic precursor cells of the monocyte-macrophage lineage. In this study, we examined the effect of MIP-1γ, a C-C chemokine family member, on receptor activator of NF-κB ligand (RANKL)-stimulated osteoclast differentiation, survival, and activation. RANKL induced osteoclasts to dramatically increase production of MIP-1γ and to also express the MIP-1γ receptor CCR1, but had only minor effects on the related C-C chemokines MIP-1α and RANTES. Neutralization of MIP-1γ with specific Ab reduced RANKL-stimulated osteoclast differentiation by 60–70%. Mature osteoclasts underwent apoptosis within 24 h after removal of RANKL, as shown by increased caspase 3 activity and DNA fragmentation. Apoptosis was reduced by the addition of exogenous MIP-1γ or RANKL, both of which increased NF-κB activation in osteoclasts. Neutralization studies showed that the prosurvival effect of RANKL was in part dependent on its ability to induce MIP-1γ. Finally, osteoclast activation for bone resorption was stimulated by MIP-1γ. Taken together, these results demonstrate that MIP-1γ plays an important role in the differentiation and survival of osteoclasts, most likely via an autocrine pathway.

T Cell Response Mediated by Myeloid Cell-Derived IL-12 Is Responsible for Porphyromonas gingivalis-Induced Periodontitis in IL-10-Deficient Mice

Periodontal disease is a chronic inflammatory disease in the oral cavity, which culminates in alveolar bone loss. Porphyromonas gingivalis is a consensus periodontal pathogen that has been implicated in adult forms of periodontitis. We previously demonstrated that IL-10-deficient mice exhibit a hyperinflammatory phenotype and are highly susceptible to P. gingivalis-induced periodontitis, indicating an important anti-inflammatory effect of IL-10 in suppressing bone loss. In this study, we analyzed the pathway(s) by which IL-10 deficiency leads to severe P. gingivalis-induced periodontitis. Because Stat3 is essential in IL-10 signaling, immune cell-specific Stat3-deficient mice were subjected to P. gingivalis infection to identify the key IL-10-responsive cells in preventing periodontitis. Myeloid cell-specific Stat3-deficient mice exhibited increased periodontal bone loss (p < 0.001), whereas T cell- and B cell-specific Stat3 mice were resistant, suggesting that macrophages (MP) and/or polymorphonuclear leukocytes are the key target cells normally suppressed by IL-10. Myeloid cell-specific Stat3-deficient mice exhibited elevated gingival CD40L gene expression in vivo compared with wild-type controls (p < 0.01), and Stat3-deficient MPs exhibited vigorous P. gingivalis-stimulated IL-12 production in vitro and induced elevated Ag-specific T cell proliferation compared with wild-type MPs (p < 0.01). Of importance, both IL-12p40/IL-10 and T cell/IL-10 double-deficient mice were resistant to P. gingivalis-induced periodontitis, demonstrating roles for both IL-12p40 and T cells in pathogenesis in a hyperinflammatory model of disease. These data demonstrate that P. gingivalis-induced periodontitis in IL-10-deficient mice is dependent upon IL-12p40-mediated proinflammatory T cell responses.

Treponema denticola in Disseminating Endodontic Infections

Treponema denticola is a consensus periodontal pathogen that has recently been associated with endodontic pathology. In this study, the effect of mono-infection of the dental pulp with T. denticola and with polymicrobial red-complex organisms (RC) (Porphyromonas gingivalis, Tannerella forsythia, and T. denticola) in inducing disseminating infections in wild-type (WT) and severe-combined-immunodeficiency (SCID) mice was analyzed. After 21 days, a high incidence (5/10) of orofacial abscesses was observed in SCID mice mono-infected with T. denticola, whereas abscesses were rare in SCID mice infected with the red-complex organisms or in wild-type mice. Splenomegaly was present in all groups, but only mono-infected SCID mice had weight loss. T. denticola DNA was detected in the spleen, heart, and brain of mono-infected SCID mice and in the spleen from mono-infected wild-type mice, which also had more periapical bone resorption. The results indicate that T. denticola has high pathogenicity, including dissemination to distant organs, further substantiating its potential importance in oral and linked systemic conditions.

Regulatory T cells in mouse periapical lesions.

INTRODUCTIONnT-regulatory (Treg, CD4+ FOXP3+) cells constitute a unique subpopulation of CD4+ T cells that inhibit T-cell responses and prevent disease development/exacerbation in models of autoimmunity. In the present study, we tested the hypothesis that Treg cells are induced in periapical lesions by dental pulp infection.nnnMETHODSnIn situ hybridization (ISH) was used to localize FOXP3+ cells on day 21 after pulp exposure of the first molar teeth and infection with bacteria from the oral environment. FOXP3/GFP knock-in transgenic mice were used to quantify FOXP3+ Treg cells that infiltrate into periapical lesions by flow cytometry on days 7, 14, and 21 after infection. Periodontal ligament from uninfected teeth served as a negative control.nnnRESULTSnISH showed strong signals that showed the presence of FOXP3+ cells mainly at the periphery of periapical lesions. In contrast, no positive cells were present in the periodontal ligament of uninfected controls. Flow cytometry showed an increase in the number of FOXP3+ Treg beginning between day 7 and day 14 (0.69% of the infiltrate) after infection and increased to day 21 (0.94%) (p < 0.05 and p < 0.001, respectively, vs uninfected controls). Treg were also increased in number in draining cervical lymph nodes after pulpal infection.nnnCONCLUSIONSnThese results show that Treg cells are induced to infiltrate into periapical lesions by pulpal infection and suggest that they increase in a time-dependent manner.

Bacteria-reactive immune response may induce RANKL-expressing T-cells in the mouse periapical bone loss lesion

INTRODUCTIONnThe present study investigated whether bacteria infecting the root canal can activate any infiltrating T cells to produce receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL).nnnMETHODSnUsing a mouse model of periapical lesion induced by artificial dental pulp exposure, the presence of RANKL-positive T cells and osteoclasts in the periapical lesion was examined by an immunohistochemical approach. The bacteria colonizing the exposed root canal were identified by 16S ribosomal RNA (rRNA) sequence analysis. The isolated endodontic bacteria were further immunized to normal mice, and soluble activator of NF-κB ligand (sRANKL) production by the T cells isolated from the immunized mice was evaluated by exxa0vivo culture system.nnnRESULTSnRANKL-positive T cells along with TRAP+ osteoclasts were identified in periapical bone resorption lesions. The gram-negative bacterium Pasteurella pnumotropica, which was most frequently detected from the root canal of exposed pulp, showed remarkably elevated serum immunoglobulin G (IgG)-antibody response in pulp-exposed mice compared with control nontreated mice. Immunization of mice with P. pneumotropica induced not only serum IgG-antibody but also primed bacteria-reactive T cells that produced sRANKL in response to exxa0vivo exposure to P. pneumotropica.nnnCONCLUSIONSnT cells infiltrating the periapical region express RANKL, and the endodontic bacteria colonizing the root canal appear to induce RANKL expression from bacteria-reactive T cells, suggesting the possible pathogenic engagement of the immune response to endodontic bacteria in the context of developing bone resorptive periapical lesions.

PAMM: A Redox Regulatory Protein That Modulates Osteoclast Differentiation

The central role of reactive oxygen species (ROS) in osteoclast differentiation and in bone homeostasis prompted us to characterize the redox regulatory system of osteoclasts. In this report, we describe the expression and functional characterization of PAMM, a CXXC motif-containing peroxiredoxin 2-like protein expressed in bone marrow monocytes on stimulation with M-CSF and RANKL. Expression of wild-type (but not C to G mutants of the CXXC domain) PAMM in HEK293 cells results in an increased GSH/GSSG ratio, indicating a shift toward a more reduced environment. Expression of PAMM in RAW264.7 monocytes protected cells from hydrogen peroxide-induced oxidative stress, indicating that PAMM regulates cellular redox status. RANKL stimulation of RAW 264.7 cells caused a decrease in the GSH/GSSG ratio (reflecting a complementary increase in ROS). In addition, RANKL-induced osteoclast formation requires phosphorylation and translocation of NF-kappaB and c-Jun. In stably transfected RAW 264.7 cells, PAMM overexpression prevented the reduction of GSH/GSSG induced by RANKL. Concurrently, PAMM expression completely abolished RANKL-induced p100 NF-kappaB and c-Jun activation, as well as osteoclast formation. We conclude that PAMM is a redox regulatory protein that modulates osteoclast differentiation in vitro. PAMM expression may affect bone resorption in vivo and help to maintain bone mass.

18β-Glycyrrhetinic acid inhibits periodontitis via glucocorticoid-independent nuclear factor-κB inactivation in interleukin-10-deficient mice

BACKGROUND AND OBJECTIVEn18β-Glycyrrhetinic acid (GA) is a natural anti-inflammatory compound derived from licorice root extract (Glycyrrhiza glabra). The effect of GA on experimental periodontitis and its mechanism of action were determined in the present study.nnnMATERIAL AND METHODSnPeriodontitis was induced by oral infection with Porphyromonas gingivalis W83 in interleukin-10-deficient mice. The effect of GA, which was delivered by subcutaneous injections in either prophylactic or therapeutic regimens, on alveolar bone loss and gingival gene expressions was determined on day 42 after initial infection. The effect of GA on lipopolysaccharide (LPS)-stimulated macrophages, T cell proliferation and osteoclastogenesis was also examined in vitro.nnnRESULTSn18β-Glycyrrhetinic acid administered either prophylactically or therapeutically resulted in a dramatic reduction of infection-induced bone loss in interleukin-10-deficient mice, which are highly disease susceptible. Although GA has been reported to exert its anti-inflammatory activity via downregulation of 11β-hydroxysteroid dehydrogenase-2 (HSD2), which converts active glucocorticoids to their inactive forms, GA did not reduce HSD2 gene expression in gingival tissue. Rather, in glucocorticoid-free conditions, GA potently inhibited LPS-stimulated proinflammatory cytokine production and RANKL-stimulated osteoclastogenesis, both of which are dependent on nuclear factor-κB. Furthermore, GA suppressed LPS- and RANKL-stimulated phosphorylation of nuclear factor-κB p105 in vitro.nnnCONCLUSIONnThese findings indicate that GA inhibits periodontitis by inactivation of nuclear factor-κB in an interleukin-10- and glucocorticoid-independent fashion.