Chronic/cyclic neutropenia, leukocyte adhesion deficiency syndrome, Papillon-Lefèvre syndrome, and Chédiak-Higashi syndrome are associated with severe periodontitis, suggesting the importance of neutrophils in the maintenance of periodontal health. Various Toll-like receptor (TLR) ligands are known to stimulate neutrophil function, including FcR-mediated phagocytosis. In the present study, the effect of TLR2 activation on the non-opsonic phagocytosis of oral bacteria and concomitant production of reactive oxygen species (ROS) by human neutrophils was evaluated. Neutrophils isolated from peripheral blood were incubated with Streptococcus sanguinis or Porphyromonas gingivalis in the presence of various concentrations of Pam3CSK4, a synthetic TLR2 ligand, and analyzed for phagocytosis and ROS production by flow cytometry and chemiluminescence, respectively. Pam3CSK4 significantly increased the phagocytosis of both bacterial species in a dose-dependent manner. However, the enhancing effect was greater for S. sanguinis than for P. gingivalis. Pam3CSK4 alone induced ROS production in neutrophils and also increased concomitant ROS production induced by bacteria. Interestingly, incubation with P. gingivalis and Pam3CSK4 decreased the amounts of ROS, as compared to Pam3CSK4 alone, indicating the possibility that P. gingivalis survives within neutrophils. However, neutrophils efficiently killed phagocytosed bacteria of both species despite the absence of Pam3CSK4. Although P. gingivalis is poorly phagocytosed even by the TLR2-activated neutrophils, TLR2 activation of neutrophils may help to reduce the colonization of P. gingivalis by efficiently eliminating S. sanguinis , an early colonizer, in subgingival biofilm.
Bacteria* ; Biofilms ; Colon ; Flow Cytometry ; Humans* ; Leukocyte-Adhesion Deficiency Syndrome ; Ligands ; Luminescence ; Neutropenia ; Neutrophils* ; Periodontitis ; Phagocytosis* ; Porphyromonas gingivalis ; Reactive Oxygen Species* ; Streptococcus ; Toll-Like Receptor 2* ; Toll-Like Receptors*

PURPOSE: Epithelial barrier dysfunction is involved in the pathophysiology of periodontitis and oral lichen planus. Estrogens have been shown to enhance the physical barrier function of intestinal and esophageal epithelia, and we aimed to investigate the effect of estradiol (E2) on the regulation of physical barrier and tight junction (TJ) proteins in human oral epithelial cell monolayers. METHODS: HOK-16B cell monolayers cultured on transwells were treated with E2, an estrogen receptor (ER) antagonist (ICI 182,780), tumor necrosis factor alpha (TNFα), or dexamethasone (Dexa), and the transepithelial electrical resistance (TER) was then measured. Cell proliferation was measured by the cell counting kit (CCK)-8 assay. The levels of TJ proteins and nuclear translocation of nuclear factor (NF)-κB were examined by confocal microscopy. RESULTS: E2 treatment increased the TER and the levels of junctional adhesion molecule (JAM)-A and zonula occludens (ZO)-1 in a dose-dependent manner, without affecting cell proliferation during barrier formation. Treatment of the tight-junctioned cell monolayers with TNFα induced decreases in the TER and the levels of ZO-1 and nuclear translocation of NF-κB. These TNFα-induced changes were inhibited by E2, and this effect was completely reversed by co-treatment with ICI 182,780. Furthermore, E2 and Dexa presented an additive effect on the epithelial barrier function. CONCLUSIONS: E2 reinforces the physical barrier of oral epithelial cells through the nuclear ER-dependent upregulation of TJ proteins. The protective effect of E2 on the TNFα-induced impairment of the epithelial barrier and its additive effect with Dexa suggest its potential use to treat oral inflammatory diseases involving epithelial barrier dysfunction.
Architectural Accessibility ; Cell Count ; Cell Proliferation ; Dexamethasone ; Electric Impedance ; Epithelial Cells ; Estradiol ; Estrogens ; Humans ; Junctional Adhesion Molecule A ; Junctional Adhesion Molecules ; Lichen Planus, Oral ; Microscopy, Confocal ; NF-kappa B ; Periodontitis ; Tight Junctions ; Tumor Necrosis Factor-alpha ; Up-Regulation

Receptor activator of NFkappaB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFalpha, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-gamma, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.
Animals ; Antigen-Presenting Cells/cytology/*drug effects/immunology/*metabolism ; Antigens, CD86/metabolism ; Carrier Proteins/*pharmacology ; Cell Death/drug effects ; Cell Survival/drug effects ; Cells, Cultured ; Cytokines/metabolism ; Flow Cytometry ; Histocompatibility Antigens Class II/metabolism ; Inflammation Mediators ; Interferon Type II/pharmacology ; Lipopolysaccharides/pharmacology ; Macrophages/cytology/*drug effects/immunology/*metabolism ; Membrane Glycoproteins/*pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Nitric Oxide Synthase Type II/metabolism ; Phagocytosis/drug effects ; Research Support, Non-U.S. Gov't ; T-Lymphocytes/immunology/metabolism ; Up-Regulation/drug effects/genetics

Molecular mimicry is an attractive mechanism for triggering autoimmunity. In this review, we explore the potential role of evolutionary conserved bacterial proteins in the production of autoantibodies with focus on granulomatosis with polyangiitis (GPA) and rheumatoid arthritis (RA). Seven autoantigens characterized in GPA and RA were BLASTed against a bacterial protein database. Of the seven autoantigens, proteinase 3, type II collagen, binding immunoglobulin protein, glucose-6-phosphate isomerase, alpha-enolase, and heterogeneous nuclear ribonuclear protein have well-conserved bacterial orthologs. Importantly, those bacterial orthologs are also found in human-associated bacteria. The wide distribution of the highly conserved stress proteins or enzymes among the members of the normal flora and common infectious microorganisms raises a new question on how cross-reactive autoantibodies are not produced during the immune response to these bacteria in most healthy people. Understanding the mechanisms that deselect auto-reactive B cell clones during the germinal center reaction to homologous foreign antigens may provide a novel strategy to treat autoimmune diseases.
Arthritis, Rheumatoid ; Autoantibodies ; Autoantigens ; Autoimmune Diseases* ; Autoimmunity ; Bacteria ; Bacterial Infections* ; Bacterial Proteins ; Clone Cells ; Collagen Type II ; Germinal Center ; Glucose-6-Phosphate Isomerase ; Heat-Shock Proteins ; Immunoglobulins ; Molecular Mimicry* ; Myeloblastin ; Phosphopyruvate Hydratase

Recurrent aphthous stomatitis (RAS) is a common oral mucosal disorder for which no curative treatment is available. We previously reported that decreased Streptococcus salivarius and increased Acinetobacter johnsonii on the oral mucosa are associated with RAS risk. The purpose of this study was to identify antibiotics that selectively inhibit A. johnsonii but minimally inhibit oral mucosal commensals. S. salivarius KCTC 5512, S. salivarius KCTC 3960, A. johnsonii KCTC 12405, Rothia mucilaginosa KCTC 19862, and Veillonella dispar KCOM 1864 were subjected to antibiotic susceptibility test using amoxicillin, cefotaxime, gentamicin, clindamycin, and metronidazole in liquid culture. The minimal inhibitory concentration (MIC) was defined as the concentration that inhibits 90% of growth. Only gentamicin presented a higher MIC for A. johnsonii than MICs for S. salivarius and several oral mucosal commensals. Interestingly, the growth of S. salivarius increased 10~200% in the presence of sub-MIC concentrations of gentamicin, which was independent of development of resistance to gentamicin. In conclusion, gentamicin may be useful to restore RAS-associated imbalance in oral microbiota by selectively inhibiting the growth of A. johnsonii but enhancing the growth of S. salivarius.
Acinetobacter ; Amoxicillin ; Anti-Bacterial Agents* ; Cefotaxime ; Clindamycin ; Gentamicins ; Mass Screening* ; Metronidazole ; Microbiota ; Mouth Mucosa ; Stomatitis, Aphthous* ; Streptococcus ; Veillonella

Periodontitis is a chronic inflammation of periodontal tissue caused by subgingival plaque-associated bacteria. Periodontitis has long been understood to be the result of an excessive host response to plaque bacteria. In addition, periodontal pathogens have been regarded as the causative agents that induce a hyperinflammatory response from the host. In this brief review, host-microbe interaction of nonperiodontopathic versus periodontopathic bacteria with innate immune components encountered in the gingival sulcus will be described. In particular, we will describe the susceptibility of these microbes to antimicrobial peptides (AMPs) and phagocytosis by neutrophils, the induction of tissue-destructive mediators from neutrophils, the induction of AMPs and interleukin (IL)-8 from gingival epithelial cells, and the pattern recognition receptors that mediate the regulation of AMPs and IL-8 in gingival epithelial cells. This review indicates that true periodontal pathogens are poor activators/suppressors of a host immune response, and they evade host defense mechanisms.
Bacteria ; Defense Mechanisms ; Epithelial Cells ; Host-Pathogen Interactions ; Immune Evasion ; Immunity, Innate ; Inflammation ; Interleukin-8 ; Interleukins ; Neutrophils ; Peptides ; Periodontitis ; Phagocytosis ; Receptors, Pattern Recognition

Purpose: Culture-based methods for microbiological diagnosis and antibiotic susceptibility tests have limitations in the management of orofacial infections. We aimed to profile pus microbiota and identify antibiotic resistance genes (ARGs) using a culture-independent approach. Materials and Methods: Genomic DNA samples extracted from the pus specimens of two patients with orofacial abscesses were subjected to shotgun sequencing on the NovaSeq system. Taxonomic profiling and prediction of ARGs were performed directly from the metagenomic raw reads.Result: Taxonomic profiling revealed obligate anaerobic polymicrobial communities associated with infections of odontogenic origins: the microbial community of Patient 1 consisted of one predominant species (Prevotella oris 74.6%) with 27 minor species, while the sample from Patient 2 contained 3 abundant species (Porphyromonas endodontalis 33.0%; P. oris 31.6%; and Prevotella koreensis 13.4%) with five minor species. A total of 150 and 136 putative ARGs were predicted in the metagenome of each pus sample. The coverage of most predicted ARGs was less than 10%, and only the CfxA2 gene identified in Patient 1 was covered 100%. ARG analysis of the seven assembled genome/ metagenome datasets of P. oris revealed that strain C735 carried the CfxA2 gene. Conclusion A metagenomics-based approach is useful to profile predominantly anaerobic polymicrobial communities but needs further verification for reliable ARG detection.

PURPOSE: We previously reported that human serum significantly reduces the invasion of various oral bacterial species into gingival epithelial cells in vitro. The aims of the present study were to characterize the serum component(s) responsible for the inhibition of bacterial invasion of epithelial cells and to examine their effect on periodontitis induced in mice. METHODS: Immortalized human gingival epithelial (HOK-16B) cells were infected with various 5- (and 6-) carboxy-fluorescein diacetate succinimidyl ester-labeled oral bacteria, including Fusobacterium nucleatum, Provetella intermedia, Porphyromonas gingivalis, and Treponiema denticola, in the absence or presence of three major serum components (human serum albumin [HSA], pooled human IgG [phIgG] and alpha1-antitrypsin). Bacterial adhesion and invasion were determined by flow cytometry. The levels of intracellular reactive oxygen species (ROS) and activation of small GTPases were examined. Experimental periodontitis was induced by oral inoculation of P. gingivalis and T. denticola in Balb/c mice. RESULTS: HSA and phIgG, but not alpha1-antitrypsin, efficiently inhibited the invasion of various oral bacterial species into HOK-16B cells. HSA but not phIgG decreased the adhesion of F. nucleatum onto host cells and the levels of intracellular ROS in HOK-16B cells. N-acetylcysteine (NAC), a ROS scavenger, decreased both the levels of intracellular ROS and invasion of F. nucleatum into HOK-16B cells, confirming the role of ROS in bacterial invasion. Infection with F. nucleatum activated Rac1, a regulator of actin cytoskeleton dynamics. Not only HSA and NAC but also phIgG decreased the F. nucleatum-induced activation of Rac1. Furthermore, both HSA plus phIgG and NAC significantly reduced the alveolar bone loss in the experimental periodontitis induced by P. gingivalis and T. denticola in mice. CONCLUSIONS: NAC and the serum components HSA and phIgG, which inhibit bacterial invasion of oral epithelial cells in vitro, can successfully prevent experimental periodontitis.
Acetylcysteine* ; Actin Cytoskeleton ; Albumins ; Alveolar Bone Loss ; Animals ; Bacteria ; Bacterial Adhesion ; Epithelial Cells* ; Flow Cytometry ; Fusobacterium nucleatum ; Humans ; Immunoglobulin G ; Mice* ; Monomeric GTP-Binding Proteins ; Periodontitis* ; Porphyromonas gingivalis ; Reactive Oxygen Species ; Serum Albumin

The pathophysiology of glandular dysfunction in Sjögren's syndrome (SS) has not been fully elucidated. Previously, we reported the presence of autoantibodies to AQP-5 in patients with SS, which was associated with a low resting salivary flow. The purpose of this study was to investigate the presence of anti-AQP1 autoantibodies. To detect anti-AQP1 autoantibodies, cell-based indirect immunofluorescence assay was developed using MDCK cells that overexpressed human AQP1. By screening 112 SS and 52 control sera, anti-AQP1 autoantibodies were detected in 27.7% of the SS but in none of the control sera. Interestingly, the sera that were positive for anti-AQP1 autoantibodies also contained anti-AQP5 autoantibodies in the previous study. Different from anti-AQP5 autoantibodies, the presence of anti-AQP1 autoantibodies was not associated with the salivary flow rate. Although anti-AQP1 autoantibodies are not useful as a diagnostic marker, the presence of autoantibodies to AQP1 may be an obstacle to AQP1 gene therapy for SS.
Aquaporin 1 ; Autoantibodies* ; Fluorescent Antibody Technique ; Fluorescent Antibody Technique, Indirect ; Genetic Therapy ; Humans ; Madin Darby Canine Kidney Cells ; Mass Screening

Although neutrophils function in both defense and tissue destruction, their defensive roles have rarely been studied in association with periodontitis. We hypothesized that peripheral neutrophils are pre-activated in vivo in periodontitis and that hyperactive neutrophils would show enhanced phagocytic ability as well as an increased production of reactive oxygen species (ROS). Peripheral blood neutrophils from patients with aggressive periodontitis and age/gender-matched healthy subjects (10 pairs) were isolated. The levels of CD11b and CD64 expression on the neutrophils and the level of plasma endotoxin were determined by flow cytometry and a limulus amebocyte lysate test, respectively. In addition, neutrophils were subjected to a flow cytometric phagocytosis assay and luminol-enhanced chemiluminescence for non-opsonized Fusobacterium nucleatum in parallel. The neutrophilsfrom most patients expressed increased levels of both CD11b and CD64. In addition, the plasma from these patients tended to contain a higher level of endotoxin than the healthy controls. In contrast, no differences were found between the two groups with regard to phagocytosis or ROS generation by F. nucleatum. The ability to phagocytose F. nucleatum was found to positively correlate with the ability to produce ROS. In conclusion, peripheral neutrophils from patients with aggressive periodontitis are hyperactive but not hyperreactive to F. nucleatum.
Aggressive Periodontitis ; Flow Cytometry ; Fusobacterium nucleatum ; Horseshoe Crabs ; Humans ; Luminescence ; Neutrophils ; Periodontitis ; Phagocytosis ; Phenotype ; Plasma ; Reactive Oxygen Species