Language：English   Publishing type：Research paper (scientific journal)  

Neutrophil elastase (NE) functions as a host defense factor; however, excessive NE activity can potentially destroy human tissues. Although NE activity is positively correlated to gingival crevicular fluid and clinical attachment loss in periodontitis, the underlying mechanisms by which NE aggravates periodontitis remain elusive. In this study, we investigated how NE induces periodontitis severity and whether NE inhibitors were efficacious in periodontitis treatment. In a ligature-induced murine model of periodontitis, neutrophil recruitment, NE activity, and periodontal bone loss were increased in the periodontal tissue. Local administration of an NE inhibitor significantly decreased NE activity in periodontal tissue and attenuated periodontal bone loss. Furthermore, the transcription of proinflammatory cytokines in the gingiva, which was significantly upregulated in the model of periodontitis, was significantly downregulated by NE inhibitor injection. An in vitro study demonstrated that NE cleaved cell adhesion molecules, such as desmoglein 1, occludin, and E-cadherin, and induced exfoliation of the epithelial keratinous layer in three-dimensional human oral epithelial tissue models. The permeability of fluorescein-5-isothiocyanate-dextran or periodontal pathogen was significantly increased by NE treatment in the human gingival epithelial monolayer. These findings suggest that NE induces the disruption of the gingival epithelial barrier and bacterial invasion in periodontal tissues, aggravating periodontitis.

DOI： 10.1038/s41598-022-12358-3

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Periodontitis is one of the most common oral diseases resulting in gingival inflammation and tooth loss. Growing evidence indicates that it results from dysbiosis of the oral microbiome, which interferes with the host immune system, leading to bone destruction. Immune cells activate periodontal ligament cells to express the receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) and promote osteoclast activity. Osteocytes have active roles in periodontitis progression in the bone matrix. Local proteins are involved in bone regeneration through functional immunological plasticity. Here, we discuss the current knowledge of cellular and molecular mechanisms in periodontitis, the roles of local proteins, and promising synthetic compounds generating a periodontal regeneration effect. It is anticipated that this may lead to a better perception of periodontitis pathophysiology.

DOI： 10.3390/ijms23105540

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Language：English   Publishing type：Research paper (scientific journal)  

Streptococcus pneumoniae, the most common cause of community-acquired pneumonia, causes severe invasive infections, including meningitis and bacteremia. The widespread use of macrolides has been reported to increase the prevalence of macrolide-resistant S. pneumoniae (MRSP), thereby leading to treatment failure in patients with pneumococcal pneumonia. However, previous studies have demonstrated that several macrolides and lincosamides have beneficial effects on MRSP infection since they inhibit the production and release of pneumolysin, a pneumococcal pore-forming toxin released during autolysis. In this regard, we previously demonstrated that the mechanisms underlying the inhibition of pneumolysin release by erythromycin involved both the transcriptional downregulation of the gene encoding pneumolysin and the impairment of autolysis in MRSP. Here, using a cell supernatant of the culture, we have shown that clarithromycin inhibits pneumolysin release in MRSP. However, contrary to previous observations in erythromycin-treated MRSP, clarithromycin upregulated the transcription of the pneumococcal autolysis-related lytA gene and enhanced autolysis, leading to the leakage of pneumococcal DNA. On the other hand, compared to erythromycin, clarithromycin significantly downregulated the gene encoding pneumolysin. In a mouse model of MRSP pneumonia, the administration of both clarithromycin and erythromycin significantly decreased the pneumolysin protein level in bronchoalveolar lavage fluid and improved lung injury and arterial oxygen saturation without affecting bacterial load. Collectively, these in vitro and in vivo data reinforce the benefits of macrolides on the clinical outcomes of patients with pneumococcal pneumonia. IMPORTANCE Pneumolysin is a potent intracellular toxin possessing multiple functions that augment pneumococcal virulence. For over 10 years, sub-MICs of macrolides, including clarithromycin, have been recognized to decrease pneumolysin production and release from pneumococcal cells. However, this study indicates that macrolides significantly slowed pneumococcal growth, which may be related to decreased pneumolysin release recorded by previous studies. In this study, we demonstrated that clarithromycin decreases pneumolysin production through downregulation of ply gene transcription, regardless of its inhibitory activity against bacterial growth. Additionally, administration of clarithromycin resulted in the amelioration of lung injury in a mouse model of pneumonia induced by macrolide-resistant pneumococci. Therefore, therapeutic targeting of pneumolysin offers a good strategy to treat pneumococcal pneumonia.

DOI： 10.1128/Spectrum.00318-21

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

Macrolide antibiotics exert anti-inflammatory effects; however, little is known regarding their immunomodulatory mechanisms. In this study, using two distinct mouse models of mucosal inflammatory disease (LPS-induced acute lung injury and ligature-induced periodontitis), we demonstrated that the anti-inflammatory action of erythromycin (ERM) is mediated through upregulation of the secreted homeostatic protein DEL-1. Consistent with the anti-neutrophil recruitment action of endothelial cell-derived DEL-1, ERM inhibited neutrophil infiltration in the lungs and the periodontium in a DEL-1-dependent manner. Whereas ERM (but not other antibiotics such as josamycin and penicillin) protected against lethal pulmonary inflammation and inflammatory periodontal bone loss, these protective effects of ERM were abolished in Del1-deficient mice. By interacting with the growth hormone secretagogue receptor (GHSR) and activating JAK2 in human lung microvascular endothelial cells, ERM induced C/EBPβ-dependent DEL-1 transcription, which was mediated by MAPK p38. Moreover, ERM reversed IL-17-induced inhibition of DEL-1 transcription, in a manner that was not only dependent on JAK2 but also on PI3K/AKT signaling. As DEL-1 levels are severely reduced in inflammatory conditions and with aging, the ability of ERM to upregulate DEL-1 may be a novel approach for the treatment of inflammatory and aging-related diseases.

DOI： 10.1172/jci.insight.136706

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DOI： 10.1111/1348-0421.12688

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Language：English   Publishing type：Research paper (scientific journal)  

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.

DOI： 10.1111/1348-0421.12672

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVE: Food-derived peptides have been reported to exhibit antibacterial activity against periodontal pathogenic bacteria. However, no effect has been shown on inflammation and bone resorption in periodontal pathology. The overall objective of the current study was to investigate how rice peptides influence biological defense mechanisms against periodontitis-induced inflammatory bone loss, and identify their novel functions as a potential anti-inflammatory drug. DESIGN: The expression of inflammatory and osteoclast-related molecules was examined in mouse macrophage-derived RAW 264.7 cell cultures using qPCR. Subsequently, the effect of these peptides on inflammatory bone loss in mouse periodontitis was examined using a mouse model of tooth ligation. Briefly, periodontal bone loss was induced for 7 days in mice by ligating the maxillary second molar and leaving the contralateral tooth un-ligated (baseline control). The mice were microinjected daily with the peptide in the gingiva until the day before euthanization. One week after the ligation, TRAP-positive multinucleated cells (MNCs) were enumerated from five random coronal sections of the ligated sites in each mouse. RESULTS: Rice peptides REP9 and REP11 significantly inhibited transcription activity of inflammatory and osteoclast-related molecules. Local treatment with the rice peptides, in mice subjected to ligature-induced periodontitis, inhibited inflammatory bone loss, explaining the decreased numbers of osteoclasts in bone tissue sections. CONCLUSION: Therefore, these data suggested that the rice peptides possess a protective effect against periodontitis.

DOI： 10.1016/j.archoralbio.2018.11.021

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Language：English   Publishing type：Research paper (scientific journal)  

Vaccination is an effective strategy to prevent pneumococcal diseases. Currently, licensed vaccines include the pneumococcal polysaccharide vaccine (PPSV) and the pneumococcal conjugate vaccine (PCV), which target some of the most common of the 94 serotypes of S. pneumoniae based on their capsular composition. However, it has been reported that PPSV is not effective in children aged less than 2 years old and PCV induces serotype replacement, which means that the pneumococcal population has changed following widespread introduction of these vaccines, and the non-vaccine serotypes have increased in being the cause of invasive pneumococcal disease. Therefore, it is important that there is development of novel pneumococcal vaccines to either replace or complement current polysaccharide-based vaccines. Our previous study suggested that S. pneumoniae releases elongation factor Tu (EF-Tu) through autolysis followed by the induction of proinflammatory cytokines in macrophages via toll-like receptor 4, that may contribute to the development of pneumococcal diseases. In this study, we investigated the expression of EF-Tu in various S. pneumoniae strains and whether EF-Tu could be an antigen candidate for serotype-independent vaccine against pneumococcal infection. Western blotting and flow cytometry analysis revealed that EF-Tu is a common factor expressed on the surface of all pneumococcal strains tested, as well as intracellularly. In addition, we demonstrate that immunization with recombinant (r) EF-Tu induced the production of inflammatory cytokines and the IgG1 and IgG2a antibodies in mice, and increased the CD4+ T-cells proportion in splenocytes. We also reveal that anti-EF-Tu serum increased the phagocytic activity of mouse peritoneal macrophages against S. pneumoniae infection, independent of their serotypes. Finally, our results indicate that mice immunized with rEF-Tu were significantly and non-specifically protected against lethal challenges with S. pneumoniae serotypes (2 and 15A). Therefore, pneumococcal EF-Tu could be an antigen candidate for the serotype-independent vaccine against pneumococcal infection.

DOI： 10.1016/j.vaccine.2018.11.015

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Language：English   Publishing type：Research paper (scientific journal)  

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

DOI： 10.1016/j.cellimm.2018.01.006

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Language：English  

Excessive activation of neutrophils results in the release of neutrophil elastase (NE), which leads to lung injury in severe pneumonia. Previously, we demonstrated a novel immune subversion mechanism involving microbial exploitation of this NE ability, which eventually promotes disruption of the pulmonary epithelial barrier. In the present study, we investigated the effect of NE on host innate immune response. THP-1-derived macrophages were stimulated with heat-killed Streptococcus pneumoniae or lipopolysaccharide in the presence or absence of NE followed by analysis of toll-like receptor (TLR) and cytokine expression. Additionally, the biological significance of NE was confirmed in an in vivo mouse intratracheal infection model. NE downregulated the gene transcription of multiple cytokines in THP-1-derived macrophages through the cleavage of TLRs and myeloid differentiation factor 2. Additionally, NE cleaved inflammatory cytokines and chemokines. In a mouse model of intratracheal pneumococcal challenge, administration of an NE inhibitor significantly increased proinflammatory cytokine levels in bronchoalveolar lavage fluid, enhanced bacterial clearance, and improved survival rates. Our work indicates that NE subverts the innate immune response and that inhibition of this enzyme may constitute a novel therapeutic option for the treatment of pneumococcal pneumonia.

DOI： 10.3389/fimmu.2018.00732

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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