RESUMO
Oncostatin M (OSM) is a pleiotropic cytokine elevated in a number of inflammatory conditions including periodontal disease. OSM is produced by a variety of immune cells and has diverse functionality such as regulation of metabolic processes, cell differentiation, and the inflammatory response to bacterial pathogens. The oral cavity is under constant immune surveillance including complementary neutrophil and macrophage populations, due to a persistent symbiotic bacterial presence. Periodontal disease is characterized by a dysbiotic bacterial community, with an abundance of Treponema denticola. Despite strong associations with severe periodontal disease, the source and mechanism of the release of OSM have not been defined in the oral cavity. We show that OSM protein is elevated in the gingival epithelium and immune cell infiltrate during periodontal disease. Furthermore, salivary and oral neutrophil OSM is elevated in correlation with the presence of T. denticola. In an air pouch infection model, T. denticola stimulated higher levels of OSM than the oral pathogen Porphorymonas gingivalis, despite differential recruitment of innate immune cells suggesting T. denticola has distinct properties to elevate OSM levels. OSM release and transcription were increased in isolated human blood, oral neutrophils, or macrophages exposed to T. denticola in vitro as measured by ELISA, qPCR, and microscopy. Using transcription, translation, and actin polymerization inhibition, we found that T. denticola stimulates both OSM release through degranulation and de novo synthesis in neutrophils and also OSM release and synthesis in macrophages. Differential induction of OSM by T. denticola may promote clinical periodontal disease.
Assuntos
Macrófagos/imunologia , Neutrófilos/imunologia , Oncostatina M/imunologia , Treponema denticola/imunologia , Infecções por Treponema/imunologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Treponema (T.) denticola is one of the key etiological agents in the development of periodontitis. The major outer sheath protein (Msp) of T. denticola has been shown to mediate pathogenesis and to facilitate adhesion of T. denticola to mucosal surfaces. This study aimed to find short polypeptides in the amino acid sequence of Msp which may be immunogenic and might elicit protective antisera against T. denticola. The complete msp sequence was divided into six fragments and the corresponding genes were cloned and expressed. Antisera against the polypeptides were raised in rabbits and fragment 3 (F3), hereinafter called PerioVax3 was the most potent fragment of the Msp in terms of yielding high titer antiserum. An adhesion assay was done to examine the inhibitory effects of antisera on the attachment of T. denticola to human gingival fibroblasts (HGFs) and human fibronectin. Antiserum against PerioVax3 significantly inhibited attachment of T. denticola to the substratum. Also, antiserum against PerioVax3 inhibited detachment of HGFs upon T. denticola exposure. To begin examining the clinical relevance of this work, blood samples from 12 sever periodontitis patients were collected and the sera were used in western blotting against the recombinant polypeptides. Periodontitis patient antisera exclusively detected PerioVax3 in western blotting. The data suggest that PerioVax3 carries epitopes that may trigger humoral immunity against T. denticola, which may protect against its adhesion functions. The complexity of periodontitis suggests that PerioVax3 may be considered for testing as a component of an experimental multivalent periodontal vaccine in further preclinical and clinical studies.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Epitopos/imunologia , Periodontite/imunologia , Treponema denticola/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/farmacologia , Antígenos de Bactérias/sangue , Antígenos de Bactérias/genética , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/imunologia , Proteínas da Membrana Bacteriana Externa/sangue , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/genética , Linhagem Celular , Clonagem Molecular , Modelos Animais de Doenças , Fibroblastos , Fibronectinas , Humanos , Masculino , Periodontite/sangue , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Treponema denticola/genética , Vacinas , Fatores de Virulência/imunologiaRESUMO
Autoantibodies against alpha-enolase (ENO1) are often detected in various infectious and autoimmune diseases. Anti-ENO1 antibody titers were reported to be associated with the severity of periodontitis in patients with rheumatoid arthritis. Because the enolase of the periodontal pathogen Treponema denticola (TdEno) has the highest homology with ENO1 among the enolases of human-associated bacteria, we hypothesized that anti-ENO1 autoantibodies produced during the immune response to TdEno may contribute to the progression of periodontitis and tested it in human and mouse systems. In human subjects with healthy periodontium or chronic periodontitis, a strong positive correlation between the levels of anti-TdEno and anti-ENO1 antibodies was observed. In addition, the purified anti-TdEno antibodies recognized ENO1 as well as TdEno in a dot blot, confirming the cross-reactivity between TdEno and ENO1. However, anti-ENO1 antibody titers were not associated with the severity of periodontitis. To further investigate the role of TdEno in the production of anti-ENO1 antibodies and the progression of periodontitis, mice received an oral gavage of P. gingivalis alone, subcutaneous immunization with TdEno alone, or both P. gingivalis oral gavage and TdEno immunization. Immunization with TdEno induced not only anti-TdEno but also anti-mouse Eno1 (mEno1) antibodies and increased the expression of TNFα in the gingival tissues. However, alveolar bone loss was not increased by TdEno immunization. In conclusion, autoreactive anti-ENO1/mEno1 antibodies that are produced as byproducts during the antibody response to TdEno play a minimal role in the progression of periodontitis in the absence of rheumatoid arthritis.
Assuntos
Anticorpos Antibacterianos/sangue , Autoanticorpos/sangue , Biomarcadores Tumorais/imunologia , Proteínas de Ligação a DNA/imunologia , Fosfopiruvato Hidratase/imunologia , Treponema denticola/enzimologia , Treponema denticola/imunologia , Proteínas Supressoras de Tumor/imunologia , Adulto , Idoso , Animais , Artrite Reumatoide , Periodontite Crônica/microbiologia , Progressão da Doença , Feminino , Gengiva/imunologia , Gengiva/microbiologia , Humanos , Imunização , Masculino , Camundongos , Pessoa de Meia-Idade , Porphyromonas gingivalis , Fator de Necrose Tumoral alfa/genéticaRESUMO
Treponema denticola is an indigenous oral spirochete that inhabits the gingival sulcus or periodontal pocket. Increased numbers of oral treponemes within this environment are associated with localized periodontal inflammation, and they are also part of an anaerobic polymicrobial consortium responsible for endodontic infections. Previous studies have indicated that T. denticola stimulates the innate immune system through Toll-like receptor 2 (TLR2); however, the pathogen-associated molecular patterns (PAMPs) responsible for T. denticola activation of the innate immune system are currently not well defined. In this study, we investigated the role played by T. denticola periplasmic flagella (PF), unique motility organelles of spirochetes, in stimulating an innate immune response. Wild-type T. denticola stimulated the production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-10, and IL-12 by monocytes from human peripheral blood mononuclear cells, while its isogenic nonmotile mutant lacking PF resulted in significantly diminished cytokine stimulation. In addition, highly purified PF were able to dose dependently stimulate cytokine TNF-α, IL-1ß, IL-6, IL-10, and IL-12 production in human monocytes. Wild-type T. denticola and the purified PF triggered activation of NF-κB through TLR2, as determined using a variety of TLR-transfected human embryonic 293 cell lines, while the PF-deficient mutants lacked the ability to stimulate, and the complemented PF-positive T. denticola strain restored the activation. These findings suggest that T. denticola stimulates the innate immune system in a TLR2-dependent fashion and that PF are a key bacterial component involved in this process.
Assuntos
Flagelos/imunologia , Imunidade Inata/imunologia , Receptor 2 Toll-Like/imunologia , Treponema denticola/imunologia , Células Cultivadas , Gengiva/imunologia , Gengiva/microbiologia , Células HEK293 , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Interleucina-10 , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Monócitos/imunologia , Monócitos/microbiologia , NF-kappa B/imunologia , Moléculas com Motivos Associados a Patógenos/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Treponema denticola is an oral spirochete strongly associated with severe periodontal disease. A prominent virulence factor, the major outer sheath protein (Msp), disorients neutrophil chemotaxis by altering the cellular phosphoinositide balance, leading to impairment of downstream chemotactic events including actin rearrangement, Rac1 activation, and Akt activation in response to chemoattractant stimulation. The specific regions of Msp responsible for interactions with neutrophils remain unknown. In this study, we investigated the inhibitory effect of truncated Msp regions on neutrophil chemotaxis and associated signaling pathways. Murine neutrophils were treated with recombinant protein truncations followed by assessment of chemotaxis and associated signal pathway activation. Chemotaxis assays indicate sequences within the C-terminal region; particularly the first 130 amino acids, have the strongest inhibitory effect on neutrophil chemotaxis. Neutrophils incubated with the C-terminal region protein also demonstrated the greatest inhibition of Rac1 activation, increased phosphoinositide phosphatase activity, and decreased Akt activation; orchestrating impairment of chemotaxis. Furthermore, incubation with antibodies specific to only the C-terminal region blocked the Msp-induced inhibition of chemotaxis and denaturing the protein restored Rac1 activation. Msp from the strain OTK, with numerous amino acid substitutions throughout the polypeptide, including the C-terminal region compared with strain 35405, showed increased ability to impair neutrophil chemotaxis. Collectively, these results indicate that the C-terminal region of Msp is the most potent region to modulate neutrophil chemotactic signaling and that specific sequences and structures are likely to be required. Knowledge of how spirochetes dampen the neutrophil response is limited and Msp may represent a novel therapeutic target for periodontal disease.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Neutrófilos/fisiologia , Porinas/química , Porinas/metabolismo , Treponema denticola/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Interações Hospedeiro-Patógeno , Camundongos , Neuropeptídeos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Porinas/genética , Porinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Treponema denticola/efeitos dos fármacos , Treponema denticola/imunologia , Fatores de Virulência , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin ß6(-/-) mouse model to study the causality. We investigated the ability of a polymicrobial consortium of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum to colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infected Itgß6(-/-) mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques in Itgß6(-/-) mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescence in situ hybridization demonstrated active invasion of the aortic adventitial layer by P. gingivalis. Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the mechanism of the host aortic inflammatory response induced by polymicrobial infection with well-characterized periodontal pathogens.
Assuntos
Túnica Adventícia/patologia , Antígenos de Neoplasias/imunologia , Aorta/patologia , Aterosclerose/complicações , Integrinas/imunologia , Periodontite/complicações , Placa Aterosclerótica/complicações , Túnica Adventícia/imunologia , Túnica Adventícia/microbiologia , Animais , Antígenos de Neoplasias/genética , Aorta/imunologia , Aorta/microbiologia , Aterosclerose/imunologia , Aterosclerose/microbiologia , Aterosclerose/patologia , Bacteroidetes/crescimento & desenvolvimento , Bacteroidetes/imunologia , Bacteroidetes/patogenicidade , Reabsorção Óssea , Modelos Animais de Doenças , Fusobacterium nucleatum/crescimento & desenvolvimento , Fusobacterium nucleatum/imunologia , Fusobacterium nucleatum/patogenicidade , Expressão Gênica , Gengiva/imunologia , Gengiva/microbiologia , Gengiva/patologia , Hibridização in Situ Fluorescente , Inflamassomos , Integrinas/deficiência , Integrinas/genética , Lipoproteínas LDL/genética , Lipoproteínas LDL/imunologia , Camundongos , Camundongos Knockout , Consórcios Microbianos , Periodontite/imunologia , Periodontite/microbiologia , Periodontite/patologia , Periodonto/imunologia , Periodonto/microbiologia , Periodonto/patologia , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/microbiologia , Placa Aterosclerótica/patologia , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidade , Treponema denticola/crescimento & desenvolvimento , Treponema denticola/imunologia , Treponema denticola/patogenicidadeRESUMO
BACKGROUND: Periodontitis is a bacteria-induced inflammatory disease mainly affecting periodontal tissues, leading to periodontal inflammation, bone breakdown, and loss of the tooth. The main obstacle for treating periodontitis effectively is the difficulty in finding a target that can inhibit bone loss and inflammation simultaneously. Recent studies showed that cathepsin K (CTSK) might have functions in the immune system besides its role in osteoclasts. Thus, targeting CTSK would have a potential therapeutic effect in both the bone system and the immune system during the progression of periodontitis. METHODS: In the current study, a small molecular inhibitor (odanacatib [ODN]) is explored to inhibit the function of CTSK in a bacteria-induced periodontitis mouse model. RESULTS: The application of ODN decreased the number of osteoclasts, macrophages, and T cells, as well as the expression of Toll-like receptors (TLRs) in the periodontitis lesion area. Furthermore, lack of CTSK inhibited the expression of TLR4, TLR5, and TLR9 and their downstream cytokine signaling in the gingival epithelial cells in periodontitis lesions, demonstrating that the innate immune response was inhibited in periodontitis. CONCLUSION: The present results show that inhibition of CTSK can prevent bone loss and the immune response during the progression of periodontitis, indicating that CTSK is a promising target for treating inflammatory diseases such as periodontitis by affecting both osteoclasts and the immune system.
Assuntos
Perda do Osso Alveolar/prevenção & controle , Anti-Inflamatórios/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Catepsina K/antagonistas & inibidores , Periodontite/prevenção & controle , Perda do Osso Alveolar/microbiologia , Animais , Infecções por Bacteroidaceae/imunologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Feminino , Gengiva/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/imunologia , Imunidade Inata/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Osteoclastos/efeitos dos fármacos , Periodontite/microbiologia , Porphyromonas gingivalis/imunologia , Linfócitos T/efeitos dos fármacos , Tannerella forsythia/imunologia , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 5 Toll-Like/antagonistas & inibidores , Receptor Toll-Like 9/antagonistas & inibidores , Treponema denticola/imunologia , Infecções por Treponema/imunologiaRESUMO
Using rheumatoid arthritis (RA) and periodontitis mouse models, we demonstrate that RA and periodontitis share many pathological features, such as deregulated cytokine production, increased immune-cell infiltration, increased expression of Toll-like receptors (TLRs), and enhanced osteoclast activity and bone erosion. We reveal that genetic deletion of cathepsin K (Ctsk) caused a radical reduction in inflammation and bone erosion within RA joint capsules and periodontal lesions, a drastic decrease in immune-cell infiltration, and a significant reduction in osteoclasts, macrophages, dendritic and T-cells. Deficiency of Ctsk greatly decreased the expression of TLR-4, 5, and 9 and their downstream cytokines in periodontal gingival epithelial lesions and synovial RA lesions. Hence, Ctsk may be targeted to treat RA and periodontitis simultaneously due to its shared osteoimmune role.
Assuntos
Artrite Reumatoide/metabolismo , Reabsorção Óssea/etiologia , Catepsina K/metabolismo , Imunidade Inata , Osteocondrite/etiologia , Osteoclastos/imunologia , Periodontite/metabolismo , Animais , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Bacteroidetes/crescimento & desenvolvimento , Bacteroidetes/imunologia , Catepsina K/genética , Cruzamentos Genéticos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Feminino , Cápsula Articular/imunologia , Cápsula Articular/metabolismo , Cápsula Articular/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Periodontite/imunologia , Periodontite/microbiologia , Periodontite/fisiopatologia , Periodonto/imunologia , Periodonto/metabolismo , Periodonto/microbiologia , Periodonto/patologia , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Treponema denticola/crescimento & desenvolvimento , Treponema denticola/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Treponema denticola, a gram-negative and anaerobic spirochete, is associated with advancing severity of chronic periodontitis. In this study, we confirmed that two major antigenic proteins were Msp and TmpC, and examined their physiological and pathological roles using gene-deletion mutants. Msp formed a large complex that localized to the outer membrane, while TmpC existed as a monomer and largely localized to the inner membrane. However, TmpC was also detected in the outer membrane fraction, but its cell-surface exposure was not detected. Msp defects increased cell-surface hydrophobicity and secretion of TNF-α from macrophage-like cells, whereas TmpC defects decreased autoagglutination and chymotrypsin-like protease activities. Both mutants adhered to gingival epithelial cells similarly to the wild-type and showed slightly decreased motility. In addition, in Msp-defective mutants, the TDE1072 protein, which is a major membrane protein, was abolished; therefore, phenotypic changes in the mutant can be, at least in part, attributed to the loss of the TDE1072 protein. Thus, the major antigenic proteins, Msp and TmpC, have significant and diverse impacts on the characteristics of T. denticola, especially cell surface properties.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Células Epiteliais/microbiologia , Gengiva/microbiologia , Mutação/genética , Porinas/genética , Treponema denticola/genética , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Imunofluorescência , Gengiva/citologia , Gengiva/imunologia , Gengiva/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Porinas/imunologia , Porinas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Treponema denticola/crescimento & desenvolvimento , Treponema denticola/imunologia , Treponema denticola/metabolismoRESUMO
The purpose of this study was to investigate the mechanism(s) of interleukin (IL)-8 suppression by Treponema denticola, one of the major periodontal pathogens, in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with wild-type (WT), dentilisin-deficient (K1) or flagellin-deficient (flgE) T. denticola in the presence or absence of 2% human serum for 24 h. The levels of IL-8 expression were measured with real-time reverse transcription PCR and ELISA. In the absence of human serum, the WT and flgE, but not K1, substantially reduced not only the levels of IL-8 protein but also of IL-8 mRNA. Such downregulation of IL-8 mRNA was independent of bacterial invasion. Degradation of cytokine mixture by the WT, K1 and flgE revealed dentilisin-dependent preferential degradation of tumor necrosis factor (TNF)-α, an IL-8-inducing cytokine. WT and flgE significantly decreased the levels of TNFα secreted by HOK-16B cells, suggesting modulation of IL-8 through dentilisin-mediated degradation of TNFα. The addition of human serum to the culture potentiated the suppressive effect of T. denticola, resulting in substantial reductions of IL-8 and TNFα levels, even by K1. The serum-dependent effects of T. denticola were attributed to its ability to suppress the accumulation of intracellular reactive-oxygen species (ROS), a group of ubiquitous signaling molecules. Pretreatment with an antioxidant suppressed TNFα-induced IL-8 expression, confirming the role of ROS in TNFα signaling. Collectively, T. denticola targeted a key inflammatory cytokine and its signaling molecule to modulate the host innate immune response, which provides a new insight into modulation of host immunity by a periodontal pathogen.
Assuntos
Regulação da Expressão Gênica/imunologia , Gengiva/imunologia , Interleucina-8/imunologia , Queratinócitos/imunologia , Treponema denticola/imunologia , Infecções por Treponema/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Quimotripsina/genética , Quimotripsina/imunologia , Quimotripsina/metabolismo , Gengiva/metabolismo , Gengiva/microbiologia , Gengiva/patologia , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Interleucina-8/biossíntese , Interleucina-8/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Peptídeo Hidrolases , Proteólise , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Treponema denticola/genética , Treponema denticola/metabolismo , Infecções por Treponema/genética , Infecções por Treponema/metabolismo , Infecções por Treponema/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Smoking has been reported to increase the risk of periodontal disease by disrupting the balance of immune responses and tissue repair processes; however, this risk varies among smokers. Cotinine levels in saliva are routinely used to measure the level of smoking, and reflect the quantity of nicotine, and other smoking-related xenobiotics that challenge host systems. This study delineated characteristics of inflammatory mediators in saliva and serum antibody responses to both periodontal pathogens and commensal bacteria in smokers as they related to cotinine levels. MATERIALS AND METHODS: This case-control study (n = 279) examined salivary inflammatory mediator responses [interleukin (IL)-1ß, IL-10, prostaglandin E2, myeloperoxidase and plasminogen activator inhibitor-1], and serum IgG antibody responses to three periodontal pathogens (Aggregatibacter actinomyce-temcomitans, Porphyromonas gingivalis, Treponema denticola) and five commensal oral microorganisms (Veillonella parvula, Streptococcus sanguis, Prevotella loescheii, Actinomyces naeslundii, Capnocytophaga ochracea). RESULTS: The patients were stratified into health (n = 30), gingivitis (n = 55) and periodontitis (n = 184); cotinine levels correlated with reported smoking habits in health, less so with gingivitis, and were not correlated in periodontitis. Of the inflammatory mediators/acute phase proteins, only IL-1ß levels were positively associated (p < 0.001) with the pack years and cotinine levels. As might be predicted, patients with periodontitis smoked more (p < 0.001) and had higher levels of cotinine. IL-1ß and antibody to A. actinomycetemcomitans, P. gingivalis and T. denticola were significantly higher in the patients with periodontitis than either patients with gingivitis or who were healthy. CONCLUSIONS: Generally, antibody to the pathogens and commensals was lower with decreased cotinine levels. Smoking exacerbated differences in both inflammatory mediators and three antibody in periodontal disease compared to healthy subjects.
Assuntos
Cotinina/análise , Mediadores da Inflamação/análise , Saliva/química , Fumar/metabolismo , Actinomyces/imunologia , Adulto , Idoso , Aggregatibacter actinomycetemcomitans/imunologia , Anticorpos Antibacterianos/sangue , Capnocytophaga/imunologia , Estudos de Casos e Controles , Dinoprostona/análise , Feminino , Gengivite/metabolismo , Gengivite/microbiologia , Humanos , Imunoglobulina G/sangue , Interleucina-10/análise , Interleucina-1beta/análise , Masculino , Pessoa de Meia-Idade , Periodontite/metabolismo , Periodontite/microbiologia , Peroxidase/análise , Inibidor 1 de Ativador de Plasminogênio/análise , Porphyromonas gingivalis/imunologia , Prevotella/imunologia , Saliva/microbiologia , Fumar/imunologia , Streptococcus sanguis/imunologia , Treponema denticola/imunologia , Veillonella/imunologia , Adulto JovemRESUMO
Periodontitis is a polymicrobial disease that arises from the dysbiosis of the plaque biofilm. To study polymicrobial interactions with gingival epithelial cells, the oral commensal Fusobacterium nucleatum and the periodontal pathogen Treponema denticola were chosen due to their opposing effects on the expression of human beta-defensins (HBDs) and interleukin (IL)-8 in gingival epithelial cells. Immortalized gingival epithelial HOK-16B cells were infected with either F. nucleatum or T. denticola alone or together, and the expression of HBDs and IL-8 was investigated. Coinfection with F. nucleatum and T. denticola neutralized the stimulatory and suppressive effects on the expression of HBD-2 and -3, but the suppressive effect of T. denticola on IL-8 expression remained. In CHO/CD14/TLR2 reporter cells, T. denticola attenuated F. nucleatum-induced activation of TLR2, a receptor that mediates HBD induction. Although F. nucleatum facilitated the invasion of T. denticola into host cells, T. denticola interfered with the fusion of internalized F. nucleatum with lysosomes, which may avert TLR9-dependent IL-8 induction. Furthermore, T. denticola suppressed the F. nucleatum-stimulated accumulation of intracellular reactive oxygen species (ROS), a group of essential signaling molecules for the TLR2 and TLR9 pathways. The elimination of ROS using N-acetyl cysteine completely blocked the inductions of HBD-3 and IL-8 and significantly reduced HBD-2 induction by F. nucleatum, confirming the importance of ROS in the host response. In sum, T. denticola incapacitates the F. nucleatum-induced expression of HBDs and IL-8 in gingival epithelial cells by interrupting endo-lysosomal maturation and ROS-dependent TLR activation. These results may provide new insights into polymicrobial interactions in the gingival sulcus.
Assuntos
Coinfecção/imunologia , Células Epiteliais/imunologia , Fusobacterium nucleatum/imunologia , Periodontite/imunologia , Treponema denticola/imunologia , Animais , Células CHO , Cricetulus , Endossomos/metabolismo , Gengiva/patologia , Interações Hospedeiro-Patógeno , Humanos , Fusão de Membrana , Microbiota , Espécies Reativas de Oxigênio/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Transgenes/genética , beta-Defensinas/metabolismoRESUMO
AIM(S): To explore the associations between the presence of periodontal pathogens and the expression of toll-like receptors (TLR-2 and TLR-4) in the placental tissue of patients with hypertensive disorders compared to the placentas of healthy normotensive patients. MATERIAL AND METHODS: A case-control study was performed. From a cohort composed of 126 pregnant women, 33 normotensive healthy pregnant women were randomly selected, and 25 cases of patients with hypertensive disorders of pregnancy, including gestational hypertension and pre-eclampsia, were selected. Placental biopsy was obtained after aseptic placental collection at the time of delivery. All of the samples were processed and analysed for the detection of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Treponema denticola and Tannerella forsythia using the polymerase chain reaction (PCR) technique. Determination of the expressions of TLR-2 and TLR-4 was performed in samples of total purified protein isolated from placental tissues and analysed by ELISA. The data were assessed using descriptive statistics. The associations among variables were estimated through multiple logistic regression models and the Mann-Whitney test to evaluate the differences between the two groups. RESULTS: A significant increase was observed in the expression of TLR-2 in the placentas of patients with hypertensive disorders (p = 0.04). Additionally, the multiple logistic regression models demonstrated an association between the presence of T. denticola and P. gingivalis in placental tissues and hypertensive disorders (OR: 9.39, p = 0.001, CI 95% 2.39-36.88 and OR: 7.59, p = 0.019, CI 95% 1.39-41.51, respectively). CONCLUSIONS: In the present study, pregnant women with periodontal disease presented an association in the placental tissue between the presence of T. denticola and P. gingivalis and hypertensive disorders. Additionally, increased expression of TLR-2 was observed. However, further studies are required to determine the specific roles of periodontal pathogens and TLRs in the placental tissue of patients with pregnancy-related hypertensive disorders.
Assuntos
Hipertensão Induzida pela Gravidez/microbiologia , Placenta/imunologia , Porphyromonas gingivalis/isolamento & purificação , Receptor 2 Toll-Like/análise , Treponema denticola/isolamento & purificação , Adulto , Aggregatibacter actinomycetemcomitans/imunologia , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Bacteroides/imunologia , Bacteroides/isolamento & purificação , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Fusobacterium nucleatum/imunologia , Fusobacterium nucleatum/isolamento & purificação , Gengivite/imunologia , Gengivite/microbiologia , Humanos , Hipertensão Induzida pela Gravidez/imunologia , Perda da Inserção Periodontal/imunologia , Perda da Inserção Periodontal/microbiologia , Índice Periodontal , Bolsa Periodontal/imunologia , Bolsa Periodontal/microbiologia , Periodontite/imunologia , Periodontite/microbiologia , Placenta/microbiologia , Porphyromonas gingivalis/imunologia , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/microbiologia , Gravidez , Receptor 4 Toll-Like/análise , Treponema denticola/imunologiaRESUMO
PURPOSE: Periodontal disease is considered to be a risk factor for threatened preterm labor (TPL) and preterm birth (PB), but pathogenic mechanisms have not yet been elucidated. We hypothesized that infection with periodontopathic bacteria may enhance thrombosis through molecular mimicry with TLRVYK peptides on beta-2 glycoprotein I, a target molecule in anti-phospholipid syndrome. This study aimed to examine the effects of periodontitis on TPL and PB. METHODS: Ninety-five pregnant women (47 TPL and 48 healthy subjects) participated. Periodontal clinical parameters and periodontopathic bacteria were examined. Molecular mimicry between TLRVYK peptides and homologous peptides on the periodontopathic bacteria was examined by enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal antibodies specific for the respective peptides (SIRVYK on Aggregatibacter actinomycetemcomitans, TLRIYT on Porphyromonus gingivalis, and TLALYK on Treponema denticola). Serum high-sensitivity C-reactive protein, anti-TLRVYK and anti-SIRVYK IgG antibodies were measured using ELISA. RESULTS: Among the rabbit antibodies specific for the bacterial homologous peptides, only anti-SIRVYK IgG antibody reacted with TLRVYK peptides. Multivariable analysis showed that anti-SIRVYK IgG antibody was significantly associated with diagnosis of TPL. Of 95 births, 14 (14.7 %) delivered preterm. The preterm birth rate was higher in the anti-SIRVYK IgG antibody >median group than in the ≤median group. Of the 47 TPL subjects 13 had PB, and ordinal logistic regression analysis revealed that past smoking, presence of P. gingivalis and anti-SIRVYK IgG antibody were significantly correlated with PB. CONCLUSIONS: Infection with P. gingivalis and the antibody response to SIRVYK might be associated with TPL and PB.
Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina G/sangue , Mimetismo Molecular , Oligopeptídeos/imunologia , Periodontite/imunologia , Nascimento Prematuro/imunologia , Adulto , Aggregatibacter actinomycetemcomitans/imunologia , Peso ao Nascer , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Intervalos de Confiança , Reações Cruzadas , Estudos Transversais , Feminino , Idade Gestacional , Humanos , Trabalho de Parto Prematuro/imunologia , Trabalho de Parto Prematuro/microbiologia , Periodontite/complicações , Periodontite/microbiologia , Porphyromonas gingivalis/imunologia , Gravidez , Nascimento Prematuro/microbiologia , Nascimento a Termo/imunologia , Treponema denticola/imunologia , Adulto JovemRESUMO
Membrane-anchored complement regulatory proteins (CRPs), including CD46, CD55, and CD59, protect host cells from complement attack. In the present study, we investigated whether periodontopathogen lipopolysaccharide and proinflammatory cytokines modulate CRP gene/protein expression in human oral epithelial cells. The lipopolysaccharide of Treponema denticola and Tannerella forsythia were the most potent for increasing the gene expression of CD55 and CD59, and to a lesser extent CD46, after a 48-h stimulation. An lipopolysaccharide-induced upregulation of epithelial cell-surface CRP was also demonstrated. The stimulation of epithelial cells with lipopolysaccharide was associated with interleukin-6 (IL-6) and IL-8 secretion. Although these two cytokines had no effect on CD46 and CD55 gene expression in epithelial cells, IL-1ß and tumor necrosis factor-α induced a significant upregulation. The cell-surface expression of CRP was also increased by the stimulation of epithelial cells with cytokines. The CD46, CD55, and CD59 gene/protein expression was upregulated by periodontopathogen lipopolysaccharide and proinflammatory cytokines. It can be hypothesized that, when faced with bacterial challenges and inflammatory conditions associated with active periodontal sites, oral epithelial cells may respond by increasing CRP gene/protein expression to avoid cell lysis by the complement system, which is activated during periodontitis.
Assuntos
Antígenos CD/genética , Citocinas/farmacologia , Lipopolissacarídeos/farmacologia , Mucosa Bucal/efeitos dos fármacos , Antígenos CD/biossíntese , Bacteroidetes/imunologia , Antígenos CD55/biossíntese , Antígenos CD55/genética , Antígenos CD59/biossíntese , Antígenos CD59/genética , Linhagem Celular Transformada , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Expressão Gênica/efeitos dos fármacos , Humanos , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Microscopia de Fluorescência , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , Pasteurellaceae/imunologia , Porphyromonas gingivalis/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Treponema denticola/imunologiaRESUMO
Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are periodontal pathogens associated with the etiology of adult periodontitis as polymicrobial infections. Recent studies demonstrated that oral infection with P. gingivalis induces both periodontal disease and atherosclerosis in hyperlipidemic and proatherogenic ApoE(-/-) mice. In this study, we explored the expression of microRNAs (miRNAs) in maxillas (periodontium) and spleens isolated from ApoE(-/-) mice infected with P. gingivalis, T. denticola, and T. forsythia as a polymicrobial infection. miRNA expression levels, including miRNA miR-146a, and associated mRNA expression levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) were measured in the maxillas and spleens from mice infected with periodontal pathogens and compared to those in the maxillas and spleens from sham-infected controls. Furthermore, in response to these periodontal pathogens (as mono- and polymicrobial heat-killed and live bacteria), human THP-1 monocytes demonstrated similar miRNA expression patterns, including that of miR-146a, in vitro. Strikingly, miR-146a had a negative correlation with TNF-α secretion in vitro, reducing levels of the adaptor kinases IL-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6). Thus, our studies revealed a persistent association of miR-146a expression with these periodontal pathogens, suggesting that miR-146a may directly or indirectly modulate or alter the chronic periodontal pathology induced by these microorganisms.
Assuntos
Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , MicroRNAs/biossíntese , Periodontite/genética , Periodontite/microbiologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/imunologia , Bacteroides/imunologia , Western Blotting , Modelos Animais de Doenças , Infecções por Bactérias Gram-Negativas/imunologia , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/imunologia , Monócitos/imunologia , Monócitos/metabolismo , Periodontite/imunologia , Porphyromonas gingivalis/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia , Baço/metabolismo , Treponema denticola/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND AND OBJECTIVE: Treponema denticola is a micro-organism that is involved in the pathogenesis of periodontitis. Major surface protein complex (MSPc), which is expressed on the envelope of this treponeme, plays a key role in the interaction between T. denticola and gingival cells. The peptidoglycan extracted from T. denticola induces the production of a large variety of inflammatory mediators by macrophage-like cells, suggesting that individual components of T. denticola cells induce the inflammatory response during periodontal disease. This study was designed to demonstrate that MSPc of T. denticola stimulates release of proinflammatory mediators in primary human monocytes. MATERIAL AND METHODS: Primary human monocytes were separated from the blood of healthy donors and incubated for up to 24 h with varying concentrations of MSPc. The production of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and matrix metalloproteinase 9 (MMP-9) was measured at different time points with commercially available enzyme-linked immunosorbent assays. RESULTS: T. denticola MSPc induced the synthesis of TNF-alpha, IL-1 beta, IL-6 and MMP-9 in a dose- and time-dependent manner. Similar patterns of TNF-alpha, IL-1 beta and IL-6 release were observed when cells were stimulated with 100 and 1000 ng/mL of MSPc. The production of MMP-9 was significant only when cells were treated with 1000 ng/mL of MSPc. CONCLUSION: These results indicate that T. denticola MSPc, at concentrations ranging from 100 ng/mL to 1.0 microg/mL, activates a proinflammatory response in primary human monocytes.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Interleucina-1beta/análise , Interleucina-6/análise , Metaloproteinase 9 da Matriz/análise , Proteínas de Membrana/imunologia , Monócitos/imunologia , Treponema denticola/imunologia , Fator de Necrose Tumoral alfa/análise , Células Cultivadas , Humanos , Mediadores da Inflamação/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Monócitos/enzimologia , Fatores de TempoRESUMO
Treponema denticola has been identified as an important cause of periodontal disease and hypothesized to be involved in extra-oral infections. The objective of this study was to investigate the role of T. denticola cell length and motility during mouse peritoneal macrophages in vitro uptake. Macrophages, incubated under aerobic and anaerobic conditions, produced a similar amount of TNF-alpha when stimulated with Escherichia coli LPS. The uptake of FlgE- and CfpA-deficient mutants of T. denticola was significantly increased compared with the wild-type strain, due to cell size or lack of motility. Opsonization with specific antibodies considerably improved the treponemes' uptake. These results suggest that macrophages, in addition to other phagocytes, could play an important role in the control of T. denticola infection, and that the raising of specific antibodies could improve the efficacy of the immune response toward T. denticola, either at an oral site or during dissemination.
Assuntos
Macrófagos Peritoneais/imunologia , Fagocitose/imunologia , Treponema denticola/imunologia , Aerobiose , Anaerobiose , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Células Cultivadas , Proteínas do Citoesqueleto/genética , Escherichia coli , Técnica Indireta de Fluorescência para Anticorpo , Imunoglobulina E/genética , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Proteínas de Membrana/imunologia , Camundongos , Mutação/genética , Treponema denticola/genética , Fator de Necrose Tumoral alfa/análiseRESUMO
We previously reported that Treponema denticola, a periodontal pathogen, suppressed the expression of human beta-defensins (HBDs) and IL-8 in human gingival epithelial cells. To clarify the receptor(s) involved in the suppression of HBD-2, immortalized gingival epithelial (HOK-16B) cells were infected with live or heat-killed T. denticola for 24 h, and the expression of HBD-2 was examined by real-time RT-PCR. Live T. denticola, but not heat-killed bacteria, suppressed the expression of HBD-2 about 40%. Time courses of suppression revealed that T. denticola suppressed HBD-2 expression only at late time points, which was accompanied with the suppression of TNFalpha production. Neutralization of TNFalpha with an antibody abrogated the suppressive effect of T. denticola on HBD-2. Accordingly, heat-killed T. denticola did not suppress TNFalpha production. Knock-down of toll-like receptor (TLR) 2 via RNA interference reversed the suppressive effect of T. denticola on the expression of HBD-3, but not on the production of TNFalpha. Collectively, T. denticola suppresses the expression of HBD-2 in gingival epithelial cells by inhibiting the TLR2 axis and TNFalpha production, which may contribute to the pathogenesis of periodontitis by T. denticola.
Assuntos
Células Epiteliais/metabolismo , Receptor 2 Toll-Like/genética , Treponema denticola/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/genética , beta-Defensinas/genética , Vacinas Bacterianas/imunologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/microbiologia , Expressão Gênica , Gengiva/citologia , Interações Hospedeiro-Patógeno , Humanos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Receptor 2 Toll-Like/metabolismo , Treponema denticola/imunologia , Treponema denticola/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , beta-Defensinas/metabolismoRESUMO
We reported previously that Treponema denticola, one of the periodontal pathogens, suppresses the expression of human beta-defensins (HBDs) in human gingival epithelial cells. To identify the mechanisms involved in this suppression, immortalized and normal human gingival epithelial cells were infected with live or heat-killed T. denticola for 24 h, and then the expression of HBDs was examined by real-time RT-PCR. Live T. denticola suppressed the expression of HBD-3 substantially and also suppressed the expression of HBD-1 and HBD-2. However, heat-killed bacteria did not produce a suppressive effect but instead slightly upregulated the levels of HBD-2 and HBD-3. In contrast to live T. denticola, which reduced the activation of mitogen-activated protein kinase (MAPK) and NF-kappaB within an hour of infection, heat-killed bacteria did not show any inhibitory effect on the MAPK and NF-kappaB signaling pathways. Knockdown of Toll-like receptor 2 (TLR2) via RNA interference abolished the suppressive effect of T. denticola on the expression of HBD-3. Heat-killed T. denticola but not live bacteria could activate TLR2 in CHO/CD14/TLR2 reporter cells, suggesting that T. denticola contains a heat-labile inhibitor(s) of TLR2 in addition to ligands recognized by TLR2. Indeed, live T. denticola was able to inhibit TLR2 activation by Pam(3)CSK. In conclusion, T. denticola suppressed the expression of HBD-3 by inhibiting the TLR2 axis in gingival epithelial cells. These results may provide new insight into the pathogenesis of periodontitis caused by T. denticola.