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Toshihisa Kawai, DDS, PhD

Published on 4/23/2019

Toshihisa Kawai, DDS, PhD

Toshihisa Kawai, DDS, PhD

Associate Member of the Staff
Department of Immunology

email: tkawai@forsyth.org

Hiroshima University, D.D.S., 1989, Dental Science
Osaka University, Ph.D., 1993, Immunology/Periodontology

Immune responses are an essential means by which the body defends itself from infectious agents. A critical part of this defense system depends upon the body’s ability to discriminate between self and non-self. This raises an interesting fundamental question as to how the body is able to recognize the difference between commensal bacteria, which are beneficial, or at least not harmful, and pathogenic microorganisms which must be eliminated. Clues to the elucidation of this phenomenon might lie in our understanding of mucosal tolerance. Mucosal tolerance is defined as the suppression, or down-regulation, of adaptive immune responses mediated by T- and B-cells via prior challenge with the antigen through the mucosal surface.

In contrast to the well-characterized mucosal tolerance in gastrointestinal systems, the tolerance systems in the oral mucosa remain unclear. However, many lines of evidence imply that the commensal host-bacteria relationship in the oral cavity is maintained by means of oral mucosal tolerance. For this reason, the Kawai Laboratory is investigating the mechanisms underlying the oral mucosal tolerance system that allow commensal organisms to flourish, as well as studying the consequences when such immune tolerance is disrupted. Our ultimate goal is to establish therapeutic approaches to the amelioration of periodontal disease and other systemic disorders where pathogenesis is associated with the loss or reduction of immune tolerance.

Gingival epithelial cells play a key role in educating regulatory dendritic cells (DCr) that can induce regulatory T cells (Tr). In the initial focus of our investigation, we are interested in understanding how cells lining the mucosal surface transfer information to immune regulatory cells in order to induce tolerance. Oral tolerance is characterized by lymphocyte hypo-responsiveness to both food antigens and commensal bacteria. The lymphocyte hypo-responsiveness appears to be controlled by regulatory T (Tr) cells that express a gene encoding Forkhead box protein P3 (FOXP3). A recent report has demonstrated that FOXP3 positive T cells are abundant in the gingival tissues. FOXP3 positive Tr cells produce the immune suppressive cytokine IL-10, which counter-regulates the production of tissue-destructive proinflammatory factors, such as IL-1, TNFå- and RANKL. The results from our experiments have indicated that the dendritic cells (DC) localized in gingival epithelial cells can control the differentiation of FOXP3+ Tr cells.

These dendritic cells (regulatory DC; DCr) present bacterial antigen to naïve T cells, and consequently induce differentiation of FOXP3+ Tr cells. Very interestingly, gingival epithelial cells (GEC) appear to possess a potential to educate peripheral blood monocytes so that the monocytes differentiate into DCr. After in vitro co-culture with GEC, but not with other types of epithelial cells, peripheral blood monocytes differentiated into DCr that produce prominent IL-10 and less IL-12. The DCr, developed via an in vitro "GEC education," induced the differentiation of FOXP3+/II-10+ Tr cells from peripheral blood naïve T cells. Additionally, the surface marker, Langerin (CD207), was expressed by the DCr as well as very distinct signal transduction molecules that are found in the gingival epithelial DCr.
The bone destructive factor, RANKL, is produced by activated lymphocytes in the bone resorptive lesion of periodontal disease.

A second focus of our investigation is the receptor activator of nuclear factor-kB (RANKL) which is a TNF-super family molecule that plays a pivotal role in homeostatic bone remodeling as well as in the development of pathogenic bone resorptive lesions. Our study on clinical gingival samples revealed that concentrations of soluble RANKL (sRANKL), but not its decoy receptor osteoprotegerin (OPG), were significantly elevated in gingival tissues with periodontal disease compared to healthy gingival tissues. Based on double-color confocal microscopic analyses, a majority of B- and T-cells in the diseased gingival tissue expressed RANKL, whereas few lymphocytes expressed RANKL in healthy gingival tissue. Expression of RANKL by monocytes/macrophages, or by other cell types, was not as prominent as B- or T-cells in the diseased gingival tissues. The lymphocytes isolated from patient’s gingival tissue induced in vitro osteoclast differentiation in a RANKL-dependent manner, indicating that RANKL expressed by the periodontal lymphocytes appeared to be functionally active. According to examination using gingival tissue homogenates, the concentration of RANKL and IgG antibodies to some of the oral commensal bacteria showed a positive correlation, thus indicating that immune recognition (i.e., breakdown of tolerance) to oral commensal bacteria is increased in the bone resorpive lesion of periodontal disease.
A mouse model to investigate the commensal host-bacterial relationship

In order to elucidate the commensal host-bacterial relationship in the oral cavity, we have recently established a mouse model of periodontal disease utilizing commensal bacteria in laboratory mice. It is the mouse oral commensal bacterium, Pasteurella pneumotropica (Pp) that allows us to study the oral mucosal tolerance in the context of periodontal disease. Very importantly, Pp is phylogenically related to a pathogen of localized aggressive periodontitis (LAP), Actinobacillus actimomycetemcomitans (Aa).

In our model, oral colonization of Pasteurella pneumotropica (Pp) establishes a host-commensal relationship in the oral cavity of BALB/c mice. Mucosal T cell tolerance is then elicited to orally colonized Pp, which, while characterized by salivary IgA responses to 27 kD protein antigen (OmpA) of Pp, produces little, or no, Th1-, Th2- or IgG-responses to this organism. Systemic immunization with Aa (not by oral inoculation) broke the T cell tolerance to Pp in conjunction with the induction of serum IgG antibody to Pp OmpA. It appeared that the strong antigenicity of Aa Omp29 elicited a cross reactive immune response to Pp OmpA, consequently breaking down the tolerance to Pp. The breakdown of tolerance also resulted in periodontal bone resorption in a RANKL-dependent manner, as well as reduction of FOXP+ T cells in the tissue. Adoptive transfer of FOXP+/RANKA-/ CD4+ Treg cells reduced the expression of RANKL in the gingival tissue of animals receiving systemic immunization with Aa, and, as a result, abrogated the periodontal bone resorption. These results suggest that systemic challenge with cross-reactive antigen can disturb T cell tolerance to oral commensal bacteria and can, therefore, result in RANKLmediated periodontal bone resorption. FOXP+ Treg cells appear to play a key role in the maintenance of such T cell tolerance to oral commensal bacteria.
Future directions

Using this mouse model of periodontal disease as well as previously established rat models, our investigations are directed toward the development of therapeutic approaches to ameliorate periodontal disease. To this end, several different approaches are currently being tested: (1) RNAi-based gene therapy to silence the key signaling molecule that causes pathogenic bio-reactions; (2) Auto-regulatory lymphocyte transplantation therapy; and (3) CAM (complementary and alternative medicine) therapy using natural products that promote the oral commensal host-bacterial relationship.

Selected Publications

Kim DM, Koszeghy KL, Badovinac RL, Kawai T, Hosokawa I, Howell TH, Karimbux NY. 2007. The effect of aspirin on gingival crevicular fluid levels of inflammatory and anti-inflammatory mediators in patients with gingivitis. J Periodontol. 78(8):1620-1626

Ernst CW, Lee JE, Nakanishi T, Karimbux NY, Rezende TM, Stashenko P, Seki M, Taubman MA, Kawai T. 2007. Diminished forkhead box P3/CD25 double-postive T regulatory cells are associated with the increased nuclear factor-kappaB ligand (RANKL+) T cells in bone resorption lesion of peridontal disease. Clin Exp Immunol. 148(2):271-80.

Kawai T, Paster BJ, Komatsuzawa H, Ernst CW, Goncalves RB, Sasaki H, Ouhara K, Stashenko PP, Sugai M, Taubman MA. 2007. Cross-reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent periodontal bone resorption in a mouse model. Oral Microbiol Immunol. 22(3):208-15.

Bartlett JD, Ball RL, Kawai T, Tye FE, Tsuchiya M, Simmer JP. 2006. Origin, splicing, and expression of rodent amelogenin exon 8. J. Dent. Res. 85(10) :894-899.

Nichols FC, Riep B, Mun J, Morton MD, Kawai T, Dewhirst FE, Smith MB. 2006. Structures and biological activities of novel phosphatidylethanolamine lipids of Porphyromonas gingivalis. J. Lipid Res. 47(4):844–853.

Ouhara K, Komatsuzawa H, Shiba H, Uchida Y, Kawai T, Sayama K, Hashimoto K, Taubman MA, Kurihara H, Sugai M. 2006. Actinobacillus actinomycemcomitans OMP-1 00 triggers innate immunity, ß-deficient and CAP18 (LL37) production, through the fibronectin-integrin pathway in human gingival epithelial cells. Infect. Immun. 74(9):5211–5220.

Hosokawa I, Hosokawa Y, Komatsuzawa H, Goncalves RB, Karimbux NY, Napimoga MH, Seki M, Ouhara K, Sugai M, Taubman MA, Kawai T. 2006 Innate immune peptide LL-37 displays distinct expression pattern from beta-defensins in inflamed gingival tissue. Clin. Exp. Immun. In press.

Kawai T, Matsuyama T, Hosokawa Y, Makihira S, Seki M, Karimbux NY, Goncalves RB, Valverde P, Dibart S, Li YP, Miranda LA, Ernst CWO, Izumi Y, Taubman MA. 2006. B and T lymphocytes are the primary sources of RANKL in the bone resorptive lesion of periodontal disease. Am. J. Pathol. 1 69(3):987–998.

Kawai T. Hypermucoviscosity: An Extremely Sticky Phenotype of Klebsiella pneumoniae Associated with Emerging Destructive Tissue Abscess Syndrome (Editorial Comment), 2006. Clinical Infect. Dis., 42(10):1359–1361.

Han X, Kawai T, Eastcott JW, Taubman MA. 2006. Bacterial-responsive B lymphocytes induce periodontal bone resorption. J. Immunol. 176(1):625–631.

Taubman MA, Valverde P, Han X, Kawai T. 2005. Immune response: The key to bone resorption in periodontal disease. J. Periodontol. 76(1 1-s):2033–2041.

Valverde P, Kawai T, Taubman MA. 2005. Potassium channel-blockers as therapeutic agents to interfere with periodontal bone resorption. J. Dent. Res. 84(1) :488–499.

Matsuyama T, Kawai T, Izumi Y, Taubman MA. 2005. Expression of major histocompatibility complex class II and CD80 by gingival epithelial cells induces activation of CD4+ T cells in response to bacterial challenge. Infect. Immun. 73(2):1044–1 051.

Sasaki H, Okamatsu Y, Kawai T, Kent R, Taubman M, Stashenko P. 2004. The interleukin-10 knockout mouse is highly susceptible to Porphyromonas gingivalis-induced alveolar bone loss. J. Periodontal Res. 39(6):432–441.

Valverde P, Kawai T, Taubman MA. 2004. Selective blockade of voltage-gated potassium channels reduces inflammatory bone resorption in experimental periodontal disease. J. Bone Miner. Res. 19(1) :155–1 64.

Asakawa R, Komatsuzawa H, Kawai T, Yamada S, Goncalves RB, Izumi S, Fujiwara T, Nakano Y, Suzuki N, Uchida Y, Ouhara K, Shiba H, Taubman MA, Kurihara H, Sugai M. 2003. Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans. Mol. Microbiol. 50(4) :1125–1139.

Komatsuzawa H, Asakawa R, Kawai T, Ochiai K, Fujiwara T, Taubman MA, Ohara M, Kurihara H, Sugai M. 2002. Identification of six major outer membrane proteins from Actinobacillus ctinomycetemcomitans. Gene 288(1– 2) :195–201.

Taubman MA, Kawai T. 2001. Involvement of T lymphocytes in periodontal disease and indirect and direct induction of bone resorption. Crit. Rev. Oral Biol. Med. 12(2) :125–1 35.

Takeichi O, Haber J, Kawai T, Smith DJ, Moro I, Taubman MA. 2000. Cytokine profiles of cells from gingival tissue with pathological pocketing. J. Dent. Res. 79(8):1548–1 555.

Kawai T, Seki M, Watanabe H, Eastcott JW, Smith DJ, Taubman MA. 2000. Th1 transmigration anergy; a new concept of endothelial cell—T cell regulatory interaction. Int. Immunol. 12:937–948.