Ryoki Kobayashi

Activation of the NLRP3 inflammasome in Porphyromonas gingivalis-accelerated atherosclerosis.

Porphyromonas gingivalis has been shown to accelerate atherosclerotic lesion development in hyperlipidemic animals. Atherosclerosis is a disease characterized by inflammation of the arterial wall. Recent studies have suggested that the NLRP3 inflammasome plays an important role in the development of vascular inflammation and atherosclerosis. Herein, we investigated a possible association between the inflammasome in atherosclerosis and periodontal disease induced by P. gingivalis infection using apolipoprotein E-deficient, spontaneously hyperlipidemic (Apoe(shl)) mice. Oral infection with wild-type (WT) P. gingivalis significantly increased the area of aortic sinus covered with atherosclerotic plaque and alveolar bone loss, compared with KDP136 (gingipain-null mutant) or KDP150 (FimA-deficient mutant) challenge. WT challenge also increased IL-1β, IL-18 and TNF-α production in peritoneal macrophages, and gingival or aortic gene expression of Nod-like receptor family, pyrin domain containing 3 (NLRP3), pro-IL-1β, pro-IL-18 and pro-caspase-1. Porphyromonas gingivalis genomic DNA was detected more in the aorta, gingival tissue, liver and spleen of WT-challenged mice than those in KDP136- or KDP150-challenged mice. We conclude that WT P. gingivalis activates innate immune cells through the NLRP3 inflammasome compared with KDP136 or KDP150. The NLRP3 inflammasome may play a critical role in periodontal disease and atherosclerosis induced by P. gingivalis challenge through sustained inflammation.

Porphyromonas gingivalis infection enhances Th17 responses for development of atherosclerosis

OBJECTIVES Porphyromonas gingivalis has been shown to associate with the development of atherosclerosis. Recent studies indicate that IL-17-producing T helper 17 (Th17) cells have been correlated with the emergence of atherosclerosis. Therefore, we investigated whether the Th17 cell response and expression of Th17-related molecules, in contrast with Th1- and Treg cells, are enhanced by P. gingivalis-challenge in Apolipoprotein E knockout (ApoE KO) mice. DESIGN Five mice were intravenously injected with P. gingivalis three times a week for 3 weeks and killed at 15 weeks of age. The proximal aorta lesion area, flow cytometry analysis and IL-17, IL-10, IFN-γ, and IL-1β levels in splenic cultures, and expression of Th17-related molecules in spleen and hearts were examined. RESULTS P. gingivalis-challenge showed notable accumulation of atherosclerotic plaques by Oil Red O-staining in ApoE KO mice. Intracellular cytokine staining revealed that significantly elevated CD4(+) interleukin (IL)-17A(+) T cells and slightly increased CD4(+) Foxp3(+) T cells was recognized in spleen cells of P. gingivalis-challenged mice compared with those from non-infected mice. P. gingivalis-challenge significantly increased IL-17 and IL-1β production and RORγt expression in splenic cells. Furthermore, the expression of Th17-related genes such as IL-6, TGF-β, RORγt and STAT3 were elevated in splenic cells as well as heart tissue of P. gingivalis-challenged mice. CONCLUSION These results suggest that P. gingivalis infection may enhance pro-inflammatory Th17 cell responses in lesion areas and spleen, thereby accelerating atherosclerosis.

Porphyromonas gingivalis accelerates atherosclerosis in C57BL/6 mice fed a high-fat diet

Objective: Porphyromonas gingivalis has been shown to accelerate atherosclerotic lesion development in atherosclerotic apo E-deficient mice. Here, we investigated whether repeated P. gingivalis injection affected the inflammatory and atherosclerotic responses of C57BL/6 mice fed a high-fat diet (HFD). Materials and methods: Eight-week-old C57BL/6 mice fed either HFD or a regular chow diet (RD) were inoculated intravenously with P. gingivalis or phosphate-buffered saline three times per week for 10 weeks and sacrificed at 19 weeks of age. Atheromatous lesions in the proximal aorta of each animal were analyzed histomorphometrically, and the serum cytokine and C-reactive protein (CRP) levels were determined. Results: Long-term HFD feeding as compared to RD feeding led to a slight increase in atheromatous lesions in the aortic sinus as well as increases in the levels of serum monocyte chemoattractant protein 1. Further, P. gingivalis injection significantly enhanced the formation of atherosclerotic plaque, and increased CRP and inflammatory cytokine levels, in mice fed the HFD, although no further increase in LDL was observed. Conclusion: These results suggest that bacteremia-induced by repeated injection with P. gingivalis accelerates atherosclerosis in normal C57BL/6 mice by initiating inflammation, and is therefore implicated in chronic infection-related pathogenicity.

Sublingual Vaccine with GroEL Attenuates Atherosclerosis

Autoimmune responses to heat-shock protein 60 (HSP60) contribute to the progression of atherosclerosis, whereas immunization with HSP60 may induce atheroprotective responses. We assessed the capacity of an atheroprotective vaccine that targeted a recombinant HSP60 from Porphyromonas gingivalis (rGroEL) to induce a protective mucosal immune response. Female apolipoprotein E-deficient spontaneously hyperlipidemic (Apoeshl) mice received sublingual delivery of rGroEL prior to P. gingivalis 381 injection. The animals were euthanized 16 weeks later. Sublingual immunization with rGroEL induced significant rGroEL-specific serum IgG responses. Antigen-specific cells isolated from spleen produced significantly high levels of IL-10 and IFN-γ after antigen re-stimulation in vitro. Flow cytometric analysis indicated that the frequencies of both IL-10+ and IFN-γ+ CD4+ Foxp3+ cells increased significantly in submandibular glands (SMG). Furthermore, sublingual immunization with rGroEL significantly reduced atherosclerosis lesion formation in the aortic sinus and decreased serum CRP, MCP-1, and ox-LDL levels. These findings suggest that sublingual immunization with rGroEL is associated with the increase of IFNγ+ or IL-10+ Foxp3+ cells in SMG and a systemic humoral response, which could be an effective strategy for the prevention of naturally occurring or P. gingivalis-accelerated atherosclerosis.

Oral administration of Lactobacillus gasseri SBT2055 is effective in preventing Porphyromonas gingivalis-accelerated periodontal disease.

Probiotics have been used to treat gastrointestinal disorders. However, the effect of orally intubated probiotics on oral disease remains unclear. We assessed the potential of oral administration of Lactobacillus gasseri SBT2055 (LG2055) for Porphyromonas gingivalis infection. LG2055 treatment significantly reduced alveolar bone loss, detachment and disorganization of the periodontal ligament, and bacterial colonization by subsequent P. gingivalis challenge. Furthermore, the expression and secretion of TNF-α and IL-6 in gingival tissue was significantly decreased in LG2055-administered mice after bacterial infection. Conversely, mouse β-defensin-14 (mBD-14) mRNA and its peptide products were significantly increased in distant mucosal components as well as the intestinal tract to which LG2055 was introduced. Moreover, IL-1β and TNF-α production from THP-1 monocytes stimulated with P. gingivalis antigen was significantly reduced by the addition of human β-defensin-3. These results suggest that gastrically administered LG2055 can enhance immunoregulation followed by periodontitis prevention in oral mucosa via the gut immune system; i.e., the possibility of homing in innate immunity.

Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva

The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.

Regulation of the NLRP3 inflammasome in Porphyromonas gingivalis-accelerated periodontal disease.

ObjectivePorphyromonas gingivalis is involved in the pathogenesis of chronic inflammatory periodontal disease. Recent studies have suggested that the NLRP3 inflammasome plays an important role in the development of chronic inflammation. We investigated a possible association between the inflammasome in gingival inflammation and bone loss induced by P. gingivalis infection using NLRP3-deficient mice.MethodsWild-type and NLRP3-deficient mice were injected orally with P. gingivalis. We assessed alveolar bone loss, expression of pro-interleukin (IL)-1β, pro-IL-18, receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) in gingival tissue, as well as IL-1β, IL-18, and IL-6 production and caspase-1 activity in peritoneal macrophages.ResultsPorphyromonas gingivalis challenge significantly increased alveolar bone loss; gingival gene expression of pro-IL-1β, pro-IL-18, and RANKL; production of IL-1β, IL-18, and IL-6; and caspase-1 activity in peritoneal macrophages of wild-type mice, but did not affect NLRP3-deficient mice. Meanwhile, OPG mRNA expression in gingival tissue and peritoneal IL-6 production were significantly higher in NLRP3-knockout mice.ConclusionsPorphyromonas gingivalis activated innate immune cells via the NLRP3 inflammasome. These results suggest that the NLRP3 inflammasome, followed by a response from the IL-1 family, is critical in periodontal disease induced by wild-type P. gingivalis challenge via sustained inflammation.

Th17‐cells in atopic dermatitis stimulate orthodontic root resorption

OBJECTIVE The aim of this study was to investigate how atopic dermatitis (AD) contributes to root resorption during orthodontic tooth movement. MATERIALS AND METHODS Atopic dermatitis model mice and wild-type mice were subjected to an excessive orthodontic force (OF) to induce movement of the upper first molars. The expression levels of the tartrate-resistant acid phosphatase (TRAP), IL-17, IL-6, and RANKL proteins were determined in the periodontal ligament (PDL) by an immunohistochemical analysis. Furthermore, the effects of the compression force on co-cultures of CD4(+) cells from AD patients or healthy individuals and human PDL cells were investigated with regard to the levels of secretion and mRNA expression of IL-17, IL-6, RANKL, and osteoprotegerin. RESULTS The immunoreactivities for TRAP, IL-17, IL-6, and RANKL in the AD group were found to be significantly increased. The double immunofluorescence analysis for IL-17/CD4 detected immunoreaction. The secretion of IL-17, IL-6, and RANKL, and the mRNA levels of IL-6 and RANKL in the AD patients were increased compared with those in healthy individuals. CONCLUSION Th17 cells may therefore be associated with the deterioration of root resorption of AD mice, and may explain why AD patients are more susceptible to root resorption than healthy individuals when an excessive OF is applied.

Sublingual vaccination with fusion protein consisting of the functional domain of hemagglutinin A of Porphyromonas gingivalis and Escherichia coli maltose‐binding protein elicits protective immunity in the oral cavity

This study demonstrated that sublingual immunization with a fusion protein, 25k-hagA-MBP, which consists of a 25-kDa antigenic region of hemagglutinin A purified from Porphyromonas gingivalis fused to maltose-binding protein (MBP) originating from Escherichia coli as an adjuvant, elicited protective immune responses. Immunization with 25k-hagA-MBP induced high levels of antigen-specific serum IgG and IgA, as well as salivary IgA. High level titers of serum IgG and IgA were also induced for almost 1 year. In an IgG subclass analysis, sublingual immunization with 25k-hagA-MBP induced both IgG1 and IgG2b antibody responses. Additionally, numerous antigen-specific IgA antibody-forming cells were detected from the salivary gland 7 days after the final immunization. Mononuclear cells isolated from submandibular lymph nodes (SMLs) showed significant levels of proliferation upon restimulation with 25k-hagA-MBP. An analysis of cytokine responses showed that antigen-specific mononuclear cells isolated from SMLs produced significantly high levels of IL-4, IFN-γ, and TGF-β. These results indicate that sublingual immunization with 25k-hagA-MBP induces efficient protective immunity against P. gingivalis infection in the oral cavity via Th1-type and Th2-type cytokine production.

Pseudopropionibacterium rubrum sp. nov., a novel red-pigmented species isolated from human gingival sulcus: Novel Pseudopropionibacterium

In this study, sStrain SK‐1T, a novel gram‐positive, pleomorphic, rod‐shaped, non‐spore forming, non‐motile organism, designated SK‐1T, was isolated from human gingival sulcus and found to produce acetic acid, propionic acid, lactic acid, and succinic acid as end products of glucose fermentation. Strain SK‐1T is most closely related to Pseudopropionibacterium (Propionibacterium) propionicum with sequence homologies of the 16S rRNA and RNA polymerase β subunit (rpoB) genes of 96.6% and 93.1%, respectively. The genomic DNA G + C content of the isolate was 61.8 mol%. On the basis of the sequence data of the 16S rRNA and housekeeping (rpoB) genes, a novel taxon is here proposed, Pseudopropionibacterium rubrum sp. nov. (type strain SK‐1T = JCM 31317T = DSM 100122T). The 16S rRNA and rpoB gene sequences of strain SK‐1T have been deposited in the DDBJ under the accession numbers LC002971 and LC102236, respectively.