Hye-Kyoung Jun

Antibacterial and lipopolysaccharide (LPS)-neutralising activity of human cationic antimicrobial peptides against periodontopathogens.

In this study, we investigated the antibacterial activity of eight antimicrobial peptides (AMPs), comprising four human beta-defensins (HBDs), three human neutrophil defensins (HNPs) and the cathelicidin LL-37, against two representative periodontopathogens, Prevotella intermedia and Tannerella forsythia. The neutralising effect of these AMPs on expression of interleukin (IL)-1beta, IL-8 and intercellular adhesion molecule 1 (ICAM-1) induced by lipopolysaccharide (LPS) from P. intermedia and T. forsythia was also tested in THP-1 cells and human gingival fibroblasts. Prevotella intermedia was susceptible to HBD-3 and LL-37 but was resistant to HBD-1, HBD-2, HBD-4, HNP-1, HNP-2 and HNP-3 at concentrations up to 10microM. However, all of the AMPs except HNP-2 at 5microM significantly inhibited the expression of IL-1beta, IL-8 and ICAM-1 induced by P. intermedia LPS. Tannerella forsythia showed marked susceptibility to the AMPs tested in the following order: LL-37, HBD-3, HBD-2, HBD-1, HNP-1 and HBD-4. All of the AMPs except HNP-3 had significant neutralising effects on T. forsythia LPS activity. The AMPs showing LPS-neutralising activity inhibited LPS binding to the cells. These results suggest that AMPs may be considered as preventive and therapeutic agents against mixed bacterial infections such as periodontitis by eliminating the pathogens themselves as well as reducing the activity of LPS.

Integrin α5β1 Activates the NLRP3 Inflammasome by Direct Interaction with a Bacterial Surface Protein

Integrins are cell-surface heterodimeric glycoproteins composed of alpha and beta subunits that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. In this study, we report a specific role of integrin α5β1 in NLRP3 inflammasome activation in macrophages stimulated by Td92, a surface protein of the periodontopathogen, Treponema denticola. The direct interaction of Td92 with the cell membrane integrin α5β1 resulted in ATP release and K(+) efflux, which are the main events in NLRP3 activation. This interaction was arginine-glycine-aspartate (RGD)-independent, and Td92 internalization was not required for the activity. An integrin α5β1 antibody and oxATP, an ATP receptor antagonist, inhibited NLRP3 expression, caspase-1 activation, interleukin-1β (IL-1β) secretion, and proIL-1β synthesis, all of which were regulated by NF-κB activation. Therefore, our data has identified the integrin α5β1 as a principal cell membrane receptor for both NLRP3 inflammasome activation and IL-1β transcription by a bacterial protein, which could exaggerate inflammation, a characteristic of periodontitis.

Fusobacterium nucleatum GroEL induces risk factors of atherosclerosis in human microvascular endothelial cells and ApoE(-/-) mice.

Infection and inflammation are risk factors in the initiation and progression of atherosclerosis. Periodontitis is one of the most prevalent chronic inflammations of the oral cavity, and has been reported to be associated with systemic disease. In this study, we evaluated whether the heat-shock protein GroEL of Fusobacterium nucleatum, one of the most prevalent bacteria in periodontitis, induces factors that predispose to atherosclerosis in human microvascular endothelial cells (HMEC-1) and apolipoprotein E-deficient (ApoE(-/-)) mice. GroEL induced the expression of chemokines such as monocyte chemoattractant protein-1 and interleukin-8 as well as cell adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin. GroEL induced the activity of tissue factor and reduced the activity of the tissue factor pathway inhibitor. Foam cell formation was induced by GroEL. GroEL-injected ApoE(-/-) mice showed significant atherosclerotic lesion progression compared with control mice. Serum levels of risk factors for atherosclerosis such as interleukin-6, C-reactive protein, and low-density lipoprotein were increased in GroEL-injected ApoE(-/-) mice compared with control mice, whereas serum levels of high-density lipoprotein were decreased. We could detect significantly higher levels of anti-F. nucleatum GroEL antibody in serum and F. nucleatum DNA in gingival crevicular fluid from patients with periodontitis than in that from healthy subjects. Our results indicate that the host response to the GroEL of periodontal pathogens like F. nucleatum may be a mechanism involved in atherosclerosis, supporting the association of periodontitis and systemic infection.

Highly Conserved Surface Proteins of Oral Spirochetes as Adhesins and Potent Inducers of Proinflammatory and Osteoclastogenic Factors

ABSTRACT Oral spirochetes include enormously heterogeneous Treponema species, and some have been implicated in the etiology of periodontitis. In this study, we characterized highly conserved surface proteins in four representative oral spirochetes (Treponema denticola, T. lecithinolyticum, T. maltophilum, and T. socranskii subsp. socranskii) that are homologs of T. pallidum Tp92, with opsonophagocytic potential and protective capacity against syphilis. Tp92 homologs of oral spirochetes had predicted signal peptides (20 to 31 amino acids) and molecular masses of 88 to 92 kDa for mature proteins. They showed amino acid sequence identities of 37.9 to 49.3% and similarities of 54.5 to 66.9% to Tp92. The sequence identities and similarities of Tp92 homologs of oral treponemes to one another were 41.6 to 71.6% and 59.9 to 85.6%, respectively. The tp92 gene homologs were successfully expressed in Escherichia coli, and the recombinant proteins were capable of binding to KB cells, an epithelial cell line, and inhibited the binding of the whole bacteria to the cells. Antiserum (the immunoglobulin G fraction) raised against a recombinant form of the T. denticola Tp92 homolog cross-reacted with homologs from three other species of treponemes. The Tp92 homologs stimulated various factors involved in inflammation and osteoclastogenesis, like interleukin-1β (IL-1β), tumor necrosis factor alpha, IL-6, prostaglandin E2, and matrix metalloproteinase 9, in host cells like monocytes and fibroblasts. Our results demonstrate that Tp92 homologs of oral spirochetes are highly conserved and may play an important role in cell attachment, inflammation, and tissue destruction. The coexistence of various Treponema species in a single periodontal pocket and, therefore, the accumulation of multiple Tp92 homologs may amplify the pathological effect in periodontitis.

Differential effect of autoinducer 2 of Fusobacterium nucleatum on oral streptococci

Autoinducer 2 (AI-2) is a quorum sensing molecule and plays an important role in dental biofilm formation, mediating interspecies communication and virulence expression of oral bacteria. Fusobacterium nucleatum connects early colonizing commensals and late colonizing periodontopathogens. F. nucleatum AI-2 and quorum sensing inhibitors (QSIs) can manipulate dental biofilm formation. In this study, we evaluated the effect of F. nucleatum AI-2 and QSIs on biofilm formation of Streptococcus gordonii and Streptococcus oralis, which are initial colonizers in dental biofilm. F. nucleatum AI-2 significantly enhanced biofilm growth of S. gordonii and attachment of F. nucleatum to preformed S. gordonii biofilms. By contrast, F. nucleatum AI-2 reduced biofilm growth of S. oralis and attachment of F. nucleatum to preformed S. oralis biofilms. The QSIs, (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone and d-ribose, reversed the stimulatory and inhibitory effects of AI-2 on S. gordonii and S. oralis, respectively. In addition, co-culture using a two-compartment system showed that secreted molecules of F. nucleatum had the same effect on biofilm growth of the streptococci as AI-2. Our results demonstrate that early colonizing bacteria can influence the accretion of F. nucleatum, a secondary colonizer, which ultimately influences the binding of periodontopathogens.

Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia induce cell death and release of endogenous danger signals

OBJECTIVE The aim of this study was to analyze whether periodontopathogens induced inflammatory cell death and the release of diverse endogenous danger molecules in THP-1-derived macrophages. METHODS The macrophages were treated with Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia. Activation of caspase-1 and caspase-4 was detected by Western blotting. Cell death of bacteria-stimulated macrophages was examined using a lactate dehydrogenase (LDH) assay and propidium iodide (PI)/annexin V (AV) staining. Levels of endogenous danger signals, including adenosine triphosphate (ATP), uric acid, heat shock protein 60 (HSP60), high-mobility group box protein 1 (HMGB1), and fibronectin in the culture supernatants were determined using an ATP bioluminescence assay kit, a uric acid assay kit, and Western blotting, respectively. RESULTS T. denticola, P. gingivalis, and T. forsythia induced activation of caspase-1 and caspase-4. The LDH assay and PI/AV staining showed that all three pathogens induced pyroptotic cell death. All three bacteria induced release of ATP, which is an important ligand for inflammasome activation; the increase in ATP ultimately leads to caspase-1 activation. T. denticola induced release of HSP60 and fibronectin, while T. forsythia induced release of HMGB1 in addition to HSP60 and fibronectin. None of the endogenous molecules except for fibronectin were detected in P. gingivalis-infected cells, possibly due to degradation of these factors by the proteolytic activity of the bacteria. Interestingly, P. gingivalis induced uric acid release. CONCLUSION Inflammatory cell death and endogenous danger molecules released from cells infected with periodontopathogens may play critical roles in the pathogenesis and progression of periodontitis by augmenting immune and inflammatory responses.

Enterococcus faecalis activates caspase-1 leading to increased interleukin-1 beta secretion in macrophages.

INTRODUCTION Recent studies of inflammasome activation have focused on the pathogenesis of diverse inflammatory and autoimmune diseases. Inflammasome activation results in caspase-1 activation, which is required for processing of prointerleukin (IL)-1 beta to its secreted form as well as a proinflammatory cell death (ie, pyroptosis). The purpose of this study was to analyze whether Enterococcus faecalis associated with endodontic infection induces inflammasome activation. METHODS THP-1 macrophages were treated with E. faecalis in the presence or absence of caspase-1 inhibitors. Caspase-1 activation, pro-IL-1 beta expression, and IL-1 beta secretion were detected by immunoblotting, real-time reverse-transcription polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. Cell death was measured by lactate dehydrogenase release and propidium iodide staining. Adenosine triphosphate (ATP) release was measured by an ATP bioluminescence assay kit. RESULTS E. faecalis induced caspase-1 activation and pro-IL-1 beta expression, which resulted in IL-1 beta secretion in macrophages. E. faecalis significantly induced ATP release, which is a mechanism of Nod-like receptor family protein 3 (NLRP3) inflammasome activation, whereas oxATP treatment inhibited E. faecalis-induced caspase-1 activation. E. faecalis significantly increased lactate dehydrogenase release and propidium iodide uptake, which are characteristics of pyroptosis. CONCLUSIONS Our results show that E. faecalis may contribute to the progression of pulpal inflammation by stimulating excessive secretion of IL-1 beta and cell death.

Contradictory roles of Porphyromonas gingivalis gingipains in caspase-1 activation

Porphyromonas gingivalis utilizes its major proteases, Arg gingipains (RgpA and RgpB) and Lys gingipain (Kgp), for dysregulation of host immune systems. The aim of this study was to investigate the roles of gingipains in caspase‐1 activation and its sequelae in P. gingivalis‐infected macrophages. Infection with P. gingivalis at low multiplicity of infections (MOIs), but not at high MOIs, resulted in low levels of interleukin‐1β and lactate dehydrogenase without detectable active caspase‐1 in the culture supernatants. The proteins released from caspase‐1‐activated cells were rapidly degraded by gingipains. However, P. gingivalis with gingipains induced higher intracellular caspase‐1 activity in the infected cells than the gingipain‐null mutant, which was associated with ATP release from the infected cells. In addition, growing the gingipain‐null mutant with gingipains enhanced caspase‐1 activation by the mutant. In contrast, inhibition of the protease activity of Kgp or Rgps increased the caspase‐1‐activating potential of wild‐type P. gingivalis, indicating an inhibitory effect of the collaborative action of Kgp and Rgps. These results illuminate the contradictory roles of gingipains in the manipulation of host defence systems by P. gingivalis, as they act by both stimulating and inhibiting innate immune responses.

The Major Outer Membrane Protein of a Periodontopathogen Induces IFN-β and IFN-Stimulated Genes in Monocytes via Lipid Raft and TANK-Binding Kinase 1/IFN Regulatory Factor-3

Surface molecules of pathogens play an important role in stimulating host immune responses. Elucidation of the signaling pathways activated by critical surface molecules in host cells provides insight into the molecular pathogenesis resulting from bacteria-host interactions. MspTL is the most abundant outer membrane protein of Treponema lecithinolyticum, which is associated with periodontitis, and induces expression of a variety of proinflammatory factors. Although bacteria and bacterial components like LPS and flagellin are known to induce IFN-β, induction by bacterial surface proteins has not been reported. In the present study, we investigated MspTL-mediated activation of signaling pathways stimulating up-regulation of IFN-β and IFN-stimulated genes in a human monocytic cell line, THP-1 cells, and primary cultured human gingival fibroblasts. MspTL treatment of the cells induced IFN-β and the IFN-stimulated genes IFN-γ-inducible protein-10 (IP-10) and RANTES. A neutralizing anti-IFN-β Ab significantly reduced the expression of IP-10 and RANTES, as well as STAT-1 activation, which was also induced by MspTL. Experiments using specific small interfering RNA showed that MspTL activated TANK-binding kinase 1 (TBK1), but not inducible IκB kinase (IKKi). MspTL also induced dimerization of IFN regulatory factor-3 (IRF-3) and translocation into the nucleus. The lipid rapid-disrupting agents methyl-β-cyclodextrin, nystatin, and filipin inhibited the MspTL internalization and cellular responses, demonstrating that lipid raft activation was a prerequisite for MspTL cellular signaling. Our results demonstrate that MspTL, the major outer protein of T. lecithinolyticum, induced IFN-β expression and subsequent up-regulation of IP-10 and RANTES via TBK1/IRF-3/STAT-1 signaling secondary to lipid raft activation.

Caspase-4 activation by a bacterial surface protein is mediated by cathepsin G in human gingival fibroblasts

Caspase-4 is an inflammatory caspase; however, its mechanism of activation is poorly understood. In this study, we demonstrate that Td92, a surface protein of the periodontal pathogen Treponema denticola and a homolog of the Treponema pallidum surface protein Tp92, activates caspase-4 and induces pyroptosis in primary cultured human gingival fibroblasts (HGFs) via cathepsin G activation. Cathepsin G inhibition or siRNA knockdown of cathepsin G inhibited Td92-induced caspase-4 activation and cell death. Td92-induced cell death was significantly inhibited by siRNA knockdown of gasdermin D. Td92 treatment resulted in the binding of cathepsin G to caspase-4 and the coaggregation of these two molecules. In addition, Td92 induced IL-1α expression and secretion, and this was inhibited by caspase-4 knockdown. Cytochalasin D did not block Td92-induced caspase-4 activation, suggesting that Td92 internalization is not required for caspase-4 activation. Our results demonstrate that cathepsin G is directly engaged in caspase-4 activation by a bacterial ligand, which is responsible for cell death and IL-1α secretion in HGFs.