RESUMO
Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone-marrow-derived macrophages (BMDM) from wild-type (WT) and Toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855).
Assuntos
Placa Dentária/microbiologia , Imunização , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Doenças Periodontais/imunologia , Doenças Periodontais/microbiologia , Receptor 4 Toll-Like/imunologia , Animais , Biofilmes , Campylobacter rectus/imunologia , Campylobacter rectus/isolamento & purificação , Campylobacter rectus/patogenicidade , Placa Dentária/imunologia , Feminino , Humanos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Knockout , Monócitos , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/imunologia , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD2/deficiência , Doenças Periodontais/fisiopatologia , Porphyromonas/imunologia , Porphyromonas/isolamento & purificação , Porphyromonas/patogenicidade , Porphyromonas endodontalis/imunologia , Porphyromonas endodontalis/isolamento & purificação , Porphyromonas endodontalis/patogenicidade , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/isolamento & purificação , Tannerella forsythia/imunologia , Tannerella forsythia/isolamento & purificação , Tannerella forsythia/patogenicidadeRESUMO
INTRODUCTION: This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-α) throughout the root canal treatment against macrophage cells. METHODS: Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH]2 + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-α and IL-1 beta. RESULTS: Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-α (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)2 + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). CONCLUSIONS: AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.
Assuntos
Interleucina-1beta/imunologia , Ativação de Macrófagos/imunologia , Abscesso Periapical/imunologia , Fator de Necrose Tumoral alfa/imunologia , Anti-Infecciosos Locais/uso terapêutico , Antígenos de Bactérias/imunologia , Hidróxido de Cálcio/uso terapêutico , Linhagem Celular , Clorexidina/uso terapêutico , Cavidade Pulpar/imunologia , Cavidade Pulpar/microbiologia , Endotoxinas/análise , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/imunologia , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/isolamento & purificação , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Peptostreptococcus/imunologia , Peptostreptococcus/isolamento & purificação , Abscesso Periapical/microbiologia , Porphyromonas endodontalis/imunologia , Porphyromonas endodontalis/isolamento & purificação , Prevotella nigrescens/imunologia , Prevotella nigrescens/isolamento & purificação , Irrigantes do Canal Radicular/uso terapêutico , Preparo de Canal Radicular/métodosRESUMO
INTRODUCTION: This study investigated the bacterial community involved in primary endodontic diseases, evaluated its ability to activate the macrophage Toll-like receptor 4 receptor through p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways, and determined the levels of endotoxins and interleukins (interleukin [IL]-6 and -10) produced by endodontic content-stimulated macrophages. METHODS: Samples were taken from 21 root canals by using sterile/apyrogenic paper points. Raw 264.7 macrophages were stimulated with root canal contents. Checkerboard DNA-DNA hybridization was used for bacterial analysis and the limulus amebocyte lysate assay for endotoxin measurement; p38 MAPK and NF-κB activation was determined by Western blot analysis. IL-6 and IL-10 were measured using the enzyme-linked immunosorbent assay. RESULTS: Bacteria and endotoxins were detected in 100% of the samples (21/21). The most frequently observed species were Parvimonas micra (16/21, 76%), Fusobacterium nucleatum ssp. nucleatum (15/21, 71%), and Porphyromonas endodontalis (14/21, 66%). Correlations were found between endotoxins and IL-6 and IL-10 (P < .05); p38 phosphorylation had a peak at 60 minutes, and NF-κB was quickly activated after 10 minutes of stimulation. CONCLUSIONS: It was concluded that the complex bacterial community was shown to be a potent activator of TLR-4 determined by the p38 MAPK and NF-κB signaling pathways, culminating in a high antigenicity against macrophages through the levels of IL-6 and IL-10, all significantly affected by endotoxin levels.
Assuntos
Necrose da Polpa Dentária/microbiologia , Mediadores da Inflamação/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Adolescente , Adulto , Idoso , Antígenos de Bactérias/imunologia , Linhagem Celular , Cavidade Pulpar/microbiologia , Necrose da Polpa Dentária/imunologia , Endotoxinas/análise , Infecções por Fusobacterium/imunologia , Fusobacterium nucleatum/imunologia , Fusobacterium nucleatum/isolamento & purificação , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Humanos , Interleucina-10/análise , Interleucina-6/análise , Pessoa de Meia-Idade , NF-kappa B/imunologia , Peptostreptococcus/imunologia , Peptostreptococcus/isolamento & purificação , Porphyromonas endodontalis/imunologia , Porphyromonas endodontalis/isolamento & purificação , Fatores de Tempo , Receptor 4 Toll-Like/imunologia , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/análiseRESUMO
INTRODUCTION: Root canal contents are potent stimuli for proinflammatory cytokines involved in apical periodontitis. This study investigated target gram-negative bacterial species and endotoxins in primary endodontic infection with apical periodontitis, determined their antigenicity against macrophages through the levels of PGE(2), and evaluated their relationship with clinical findings. METHODS: Samples were taken from 21 root canals with primary infection and apical periodontitis by using paper points. Polymerase chain reaction (16S rDNA) was used for bacterial detection and limulus amebocyte lysate assay for endotoxin measurement. Levels of prostaglandin E2 (PGE(2)) were measured by enzyme-linked immunosorbent assay (Duoset Kit; R&D, Minneapolis, MN). RESULTS: Prevotella nigrescens (13/21), Fusobacterium nucleatum (6/21), and Porphyromonas endodontalis (6/21) were the most frequently observed species. A positive association was found between F. nucleatum and P. endodontalis (P < .05). A correlation was found between the number of gram-negative bacterial species and the levels of endotoxins, such as PGE(2) (P < .05). Higher levels of endotoxin were detected in teeth with exudation, whereas elevated levels of PGE(2) were found in teeth with tenderness to percussion and pain on palpation. CONCLUSIONS: Our findings imply an additive effect between the number of gram-negative bacterial species involved in endodontic infection regarding the induction of proinflammatory cytokine by macrophage cells. Moreover, teeth with clinical symptomatology were related to higher levels of endotoxins and PGE(2) secretion.
Assuntos
Antígenos de Bactérias/imunologia , Necrose da Polpa Dentária/microbiologia , Dinoprostona/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Macrófagos/imunologia , Adolescente , Adulto , Idoso , Técnicas de Cultura de Células , Sobrevivência Celular/fisiologia , Citocinas/análise , Citocinas/imunologia , Necrose da Polpa Dentária/imunologia , Dinoprostona/análise , Endotoxinas/análise , Endotoxinas/imunologia , Exsudatos e Transudatos/química , Infecções por Fusobacterium/imunologia , Fusobacterium nucleatum/imunologia , Humanos , Mediadores da Inflamação/imunologia , Pessoa de Meia-Idade , Periodontite Periapical/imunologia , Periodontite Periapical/microbiologia , Porphyromonas endodontalis/imunologia , Porphyromonas gingivalis/imunologia , Prevotella intermedia/imunologia , Prevotella nigrescens/imunologia , RNA Bacteriano/análise , Adulto JovemRESUMO
Caries-induced pulpitis is typically accompanied by an increase in dental pulp microvascular density. However, the mechanisms by which dental pulp cells recognize lipopolysaccharides (LPSs) remain unclear. We hypothesized that Porphyromonas endodontalis and Escherichia coli LPSs induce vascular endothelial growth factor (VEGF) expression in dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF) through mitogen-activated protein kinase (MAPK) signaling. ELISA, semi-quantitative RT-PCR, immunofluorescence, and Western blots were used. Here, we observed that LPSs induced VEGF expression in DPSC and HDPF cells, and both cell types express Toll-like receptor 4 (TLR- 4). Notably, LPS-induced VEGF is associated with phosphorylation of protein kinase C (PKC zeta) and extracellular signal-regulator kinase (ERK1/2) and is dependent upon MAPK activation. Analysis of these data, collectively, unveils a signaling pathway responsible for synthesis of VEGF by pulp cells and suggests a novel therapeutic target for the management of vascular responses in teeth with pulpitis.