Toshihisa Kawai

Pathogens and host immunity in the ancient human oral cavity

Calcified dental plaque (dental calculus) preserves for millennia and entraps biomolecules from all domains of life and viruses. We report the first, to our knowledge, high-resolution taxonomic and protein functional characterization of the ancient oral microbiome and demonstrate that the oral cavity has long served as a reservoir for bacteria implicated in both local and systemic disease. We characterize (i) the ancient oral microbiome in a diseased state, (ii) 40 opportunistic pathogens, (iii) ancient human–associated putative antibiotic resistance genes, (iv) a genome reconstruction of the periodontal pathogen Tannerella forsythia, (v) 239 bacterial and 43 human proteins, allowing confirmation of a long-term association between host immune factors, red complex pathogens and periodontal disease, and (vi) DNA sequences matching dietary sources. Directly datable and nearly ubiquitous, dental calculus permits the simultaneous investigation of pathogen activity, host immunity and diet, thereby extending direct investigation of common diseases into the human evolutionary past.

Selective blockade of voltage-gated potassium channels reduces inflammatory bone resorption in experimental periodontal disease

The effects of the potassium channel (Kv1.3) blocker kaliotoxin on T‐cell‐mediated periodontal bone resorption were examined in rats. Systemic administration of kaliotoxin abrogated the bone resorption in conjunction with decreased RANKL mRNA expression by T‐cells in gingival tissue. This study suggests a plausible therapeutic approach for inflammatory bone resorption by targeting Kv1.3.

Etiological analysis of initial colonization of periodontal pathogens in oral cavity.

ABSTRACT It is unclear when the initial colonization by periodontal pathogens occurs in the oral cavity. Therefore, we report here the association between specific age groups and the time when the initial colonization by periodontal pathogens occurs in the oral cavity in such groups. Findings are based on an epidemiological analysis of the prevalence of five periodontal pathogens in the oral cavities of a wide range of age populations, from newborn to elderly, who were randomly selected in a geographic region of Brazil. These periodontal pathogens include Campylobacter rectus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Tannerella forsythia and were analyzed in the bacterial samples isolated from gingival sulcus, the dorsum of the tongue, and cheek mucosa of diverse age groups, using a bacterial DNA-specific PCR method. Results indicated that there are distinct age-related groups where initial colonization by the five periodontal pathogens examined in this study can be detected and that the presence of teeth is a permissive factor for colonization by P. gingivalis, P. intermedia, and T. forsythia. Although it remains unclear exactly how or when target pathogens colonize healthy subjects, an understanding of age-related groups does provide a potentially useful tool in the early detection and prevention of periodontitis in healthy individuals.

Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease

Abstract Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, we discuss earlier studies of immune intervention, ultimately leading to the identification of bacteria-reactive lymphocytes as the cellular source of osteoclast-induction factor lymphokine (now called RANKL) in the context of periodontal bone resorption. Next, we consider 1 the effects of periodontal bacteria on RANKL production from a variety of adaptive immune effector cells, as well as fibroblasts, in inflamed periodontal tissue and 2 the bifunctional roles (upregulation vs. downregulation) of LPS produced from periodontal bacteria in a RANKL-induced osteoclast-signal pathway. Future studies in these two areas could lead to new therapeutic approaches for the management of PD by down-modulating RANKL production and/or RANKL-mediated osteoclastogenesis in the context of host immune responses against periodontal pathogenic bacteria.

Diminished forkhead box P3/CD25 double-positive T regulatory cells are associated with the increased nuclear factor-kB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease

Periodontal disease involves multi‐bacterial infections accompanied by inflammatory bone resorption lesions. The abundant T and B lymphocyte infiltrates are the major sources of the osteoclast differentiation factor, receptor activator for nuclear factor‐kB ligand (RANKL) which, in turn, contributes to the development of bone resorption in periodontal disease. In the present study, we found that the concentrations of RANKL and regulatory T cell (Treg)‐associated cytokine, interleukin (IL)‐10, in the periodontal tissue homogenates were correlated negatively, whereas RANKL and proinflammatory cytokine, IL‐1β, showed positive correlation. Also, according to the fluorescent‐immunohistochemistry, the frequency of forkhead box P3 (FoxP3)/CD25 double‐positive cells was diminished strikingly in the bone resorption lesion of periodontal disease compared to healthy gingival tissue, while CD25 or FoxP3 single positive cells were still observed in lesions where abundant RANKL+ lymphocytes were present. Very importantly, few or no expressions of FoxP3 by the RANKL+ lymphocytes were observed in the diseased periodontal tissues. Finally, IL‐10 suppressed both soluble RANKL (sRANKL) and membrane RANKL (mRANKL) expression by peripheral blood mononuclear cells (PBMC) activated in vitro in a bacterial antigen‐specific manner. Taken together, these results suggested that FoxP3/CD25 double‐positive Treg cells may play a role in the down‐regulation of RANKL expression by activated lymphocytes in periodontal diseased tissues. This leads to the conclusion that the phenomenon of diminished CD25+FoxP3+ Treg cells appears to be associated with the increased RANKL+ T cells in the bone resorption lesion of periodontal disease.

Antigen direction of specific T‐cell clones into gingival tissues

This study was performed to investigate T‐cell traffic to periodontal tissues during infection with a periodontal pathogen Actinobacillus actinomycetemcomitans (Aa). Rowett rat T‐cell clones, A3 (CD4+ CD8−, αβTCR+, NKRP‐1−, specific to Aa) and G2 (CD4− CD8−, αβTCR+, NKRP‐1+, which reacts to Aa, Gram‐negative and ‐positive bacteria), both expressed the same prominent adhesion molecules (LFA‐1, VLA‐4) to the same extent. Binding of both T‐cell clones to rat endothelial cells in vitro was blocked by antibody to VLA‐4. Rowett rats were infected with Aa and infused with Aa‐stimulated, isogenic T‐clone lymphocytes that had been labelled in vitro with 125IUdR. Radioactivity associated with recovery of clone A3, but not G2, was significantly elevated in the gingivae of infected rats, suggesting migration to infected animals’ gingival tissues. Migration of radioactive Aa‐specific A3 clone cells traced by autoradiography reached a maximum at 24u2003hr (1·2% of total lymphocytes as radiolabelled cells in infected gingiva versus 0·6% in non‐infected), indicating an apparent antigen‐directed retention in infected rats’ gingival tissues. The G2 clone was not retained in the gingival tissues (0·20% of total lymphocytes as radiolabelled cells in infected gingiva versus 0·26% in non‐infected). However, the possibility of A3 retention directed by inflammation or tissue‐selective homing could not be excluded. In further experiments, other adoptively transferred T‐clone lymphocytes [clones G23 (Th1) and F13 (Th2)] with specificity for the 29u2003000u2003MW outer membrane protein of Aa with the same prominent adhesion molecules could be recovered from rat gingivae previously challenged with this antigen. However, transferred T‐clone lymphocytes [clone G26 (Th1)] with specificity for a different Aa antigen were not recovered. Therefore, the dynamics of cell entry into periodontal lesions vary for activated T lymphocytes with different antigenic specificities, indicating the significance of antigen in lymphocyte traffic to periodontal tissues.

A Role of Oral Bacteria in Bisphosphonate-induced Osteonecrosis of the Jaw

No consensus has yet been reached to associate oral bacteria conclusively with the etio-pathogenesis of bisphosphonate-induced osteonecrosis of the jaw (BONJ). Therefore, the present study examined the effects of oral bacteria on the development of BONJ-like lesions in a mouse model. In the pamidronate (Pam)-treated mice, but not control non-drug-treated mice, tooth extraction followed by oral infection with Fusobacterium nucleatum caused BONJ-like lesions and delayed epithelial healing, both of which were completely suppressed by a broad-spectrum antibiotic cocktail. Furthermore, in both in vitro and in vivo experiments, the combination of Pam and Fusobacterium nucleatum caused the death of gingival fibroblasts (GFs) and down-regulated their production of keratinocyte growth factor (KGF), which induces epithelial cell growth and migration. Therefore, in periodontal tissues pre-exposed to bisphosphonate, bacterial infection at tooth extraction sites caused diminished KGF expression in GFs, leading to a delay in the epithelial wound-healing process that was mitigated by antibiotics.

Fluoxetine inhibits inflammatory response and bone loss in a rat model of ligature-induced periodontitis

BACKGROUNDnFluoxetine, a selective serotonin reuptake inhibitor, has been found recently to possess anti-inflammatory properties. The present study investigates the effects of fluoxetine on inflammatory tissue destruction in a rat model of ligature-induced periodontal disease.nnnMETHODSnThirty male Wistar rats were randomly assigned into three groups (n = 10 animals per group): 1) control rats (without ligature); 2) rats with ligature + placebo (saline; oral gavage); and 3) rats with ligature + fluoxetine (20 mg/kg/day in saline; oral gavage). Histologic analyses were performed on the furcation region and mesial aspect of mandibular first molars of rats sacrificed at 15 days after ligature-induced periodontal disease. Reverse transcription-polymerase chain reaction and zymography were performed to analyze the mRNA expression of interleukin (IL)-1β, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9 and inducible nitric oxide synthase and the MMP-9 activity, respectively, in gingival tissues samples.nnnRESULTSnCompared to the ligature + placebo group, alveolar bone loss was reduced in the fluoxetine group (P <0.05), and the amount of collagen fibers in the gingival tissue was maintained. Moreover, in gingival tissue sampled 3 days after ligature attachment, fluoxetine administration reduced IL-1β and COX-2 mRNA expression. Fluoxetine downregulated MMP-9 activity, without affecting MMP-9 mRNA expression induced by ligature, compared to the ligature + placebo group (P <0.05). These data suggest that fluoxetine suppressed proinflammatory responses, as well as proteolytic enzyme activity, induced by ligature.nnnCONCLUSIONnIn the present study, fluoxetine suppresses the inflammatory response and protects against periodontal bone resorption and destruction of collagen fibers, suggesting that fluoxetine can constitute a promising therapeutic approach for periodontal diseases.

Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo.

Tetracycline antibiotics, including doxycyclie (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.

Structures and biological activities of novel phosphatidylethanolamine lipids of Porphyromonas gingivalis

The Gram-negative periodontal pathogen Porphyromonas gingivalis synthesizes several classes of novel phosphorylated complex lipids, including the recently characterized phosphorylated dihydroceramides. These sphingolipids promote the interleukin-1 (IL-1)-mediated secretion of inflammatory mediators from fibroblasts, including prostaglandin E2 and 6-keto prostaglandin F2α, and alter gingival fibroblast morphology in culture. This report demonstrates that one additional class of phosphorylated complex lipids of P. gingivalis promotes IL-1-mediated secretory responses and morphological changes in cultured fibroblasts. Structural characterization identified the new phospholipid class as 1,2-diacyl phosphatidylethanolamine, which substituted predominantly with isobranched C15:0 and C13:0 fatty acids. The isobranched fatty acids, rather than unbranched fatty acids, and the phosphoethanolamine head group were identified as the essential structural elements required for the promotion of IL-1-mediated secretory responses. These structural components are also observed in specific phosphorylated sphingolipids of P. gingivalis and likely contribute to the biological activity of these substances, in addition to the phosphatidylethanolamine lipids described in this report.