RESUMEN
Mesenchymal stem cell (MSC) transplantation is an effective periodontal regenerative therapy. MSCs are multipotent, have self-renewal ability, and can differentiate into periodontal cells. However, senescence is inevitable for MSCs. In vitro, cell senescence can be induced by long-term culture with/without cell passage. However, the regulatory mechanism of MSC senescence remains unclear. Undifferentiated MSC-specific transcription factors can regulate MSC function. Herein, we identified the regulatory transcription factors involved in MSC senescence and elucidated their mechanisms of action. We cultured human MSCs (hMSCs) with repetitive cell passages to induce cell senescence and evaluated the mRNA and protein expression of cell senescence-related genes. Additionally, we silenced the cell senescence-induced transcription factors, GATA binding protein 6 (GATA6) and SRY-box 11 (SOX11), and investigated senescence-related signaling pathways. With repeated passages, the number of senescent cells increased, while the cell proliferation capacity decreased; GATA6 mRNA expression was upregulated and that of SOX11 was downregulated. Repetitive cell passages decreased Wnt and bone morphogenetic protein (BMP) signaling pathway-related gene expression. Silencing of GATA6 and SOX11 regulated Wnt and BMP signaling pathway-related genes and affected cell senescence-related genes; moreover, SOX11 silencing regulated GATA6 expression. Hence, we identified them as pair of regulatory transcription factors for cell senescence in hMSCs via the Wnt and BMP signaling pathways.
Asunto(s)
Senescencia Celular/genética , Células de la Médula Ósea/citología , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Factor de Transcripción GATA6/antagonistas & inhibidores , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Transcripción SOXC/antagonistas & inhibidores , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismo , Transducción de Señal/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
RNA-binding proteins (RBPs) regulate mRNA stability by binding to the 3'-untranslated region (UTR) region of mRNA. Human antigen-R (HuR), one of the RBPs, is involved in the progression of diseases, such as rheumatoid arthritis, diabetes mellitus and some inflammatory diseases. Interleukin (IL)-6 is a major inflammatory cytokine regulated by HuR binding to mRNA. Periodontal disease (PD) is also an inflammatory disease caused by elevations in IL-6 following an infection by periodontopathogenic bacteria. The involvement of HuR in the progression of PD was assessed using in-vitro and in-vivo experiments. Immunohistochemistry of inflamed periodontal tissue showed strong staining of HuR in the epithelium and connective tissue. HuR mRNA and protein level was increased following stimulation with Porphyromonas gingivalis (Pg), one of the periodontopathogenic bacteria, lipopolysacchride (LPS)-derived from Pg (PgLPS) and tumour necrosis factor (TNF)-α in OBA-9, an immortalized human gingival epithelial cell. The luciferase activity of 3'-UTR of IL-6 mRNA was increased by TNF-α, Pg and PgLPS in OBA-9. Luciferase activity was also increased in HuR-over-expressing OBA-9 following a bacterial stimulation. Down-regulation of HuR by siRNA resulted in a decrease in mRNA expression and production of IL-6. In contrast, the over-expression of HuR increased IL-6 mRNA expression and production in OBA-9. The HuR inhibitor, quercetin, suppressed Pg-induced HuR mRNA expression and IL-6 production in OBA-9. An oral inoculation with quercetin also inhibited bone resorption in ligature-induced periodontitis model mice as a result of down-regulation of IL-6. These results show that HuR modulates inflammatory responses by regulating IL-6.
Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Encía/patología , Interleucina-6/genética , Periodontitis/patología , Regiones no Traducidas 3'/genética , Adulto , Anciano , Animales , Resorción Ósea/tratamiento farmacológico , Línea Celular , Proteína 1 Similar a ELAV/genética , Células Epiteliales/metabolismo , Femenino , Encía/citología , Humanos , Lipopolisacáridos/inmunología , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Periodontitis/tratamiento farmacológico , Porphyromonas gingivalis/inmunología , Porphyromonas gingivalis/patogenicidad , Quercetina/farmacología , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Factor de Necrosis Tumoral alfaRESUMEN
AIM: To report a case of reparative bone-like tissue formation in the tooth of a patient with systemic sclerosis. SUMMARY: A 58-year-old Japanese female patient with systemic sclerosis was referred because of tooth fracture. Cone beam computerized tomography (CBCT) revealed multiple root resorption and the unclear transition from alveolar bone to root profile. A sample from a fractured tooth was analysed histologically. Haematoxylin and eosin-stained sections revealed the irregular replacement of pulp and dentine by bone-like tissue. Calcinosis was noted in various parts of the body and a histological analysis identified it as dystrophic calcification on sclerosed fibrous connective tissue. Bite force and the occlusal area were markedly weaker than the means for female of the same age. KEY LEARNING POINTS: CBCT may be more useful than dental radiography for diagnosing multiple root resorption in systemic sclerosis patients. When systemic sclerosis patients have calcinosis, their root status must be examined carefully. When root resorption is present in systemic sclerosis patients, reparative bone-like tissue formation in teeth needs to be taken into account prior to the initiation of dental treatment.
Asunto(s)
Resorción Radicular/etiología , Esclerodermia Sistémica/complicaciones , Fracturas de los Dientes/etiología , Calcinosis/etiología , Tomografía Computarizada de Haz Cónico , Femenino , Humanos , Persona de Mediana Edad , Osteogénesis , Radiografía Dental , Resorción Radicular/diagnóstico por imagen , Resorción Radicular/patología , Fracturas de los Dientes/diagnóstico por imagenRESUMEN
Transplantation of mesenchymal stem cells (MSCs), which possess self-renewing properties and multipotency, into a periodontal defect is thought to be a useful option for periodontal tissue regeneration. However, developing more reliable and predictable implantation techniques is still needed. Recently, we generated clumps of an MSC/extracellular matrix (ECM) complex (C-MSC), which consisted of cells and self-produced ECM. C-MSCs can regulate their cellular functions in vitro and can be grafted into a defect site, without any artificial scaffold, to induce bone regeneration. Accordingly, this study aimed to evaluate the effect of C-MSC transplantation on periodontal tissue regeneration in beagle dogs. Seven beagle dogs were employed to generate a premolar class III furcation defect model. MSCs isolated from dog ilium were seeded at a density of 7.0 × 104 cells/well into 24-well plates and cultured in growth medium supplemented with 50 µg/mL ascorbic acid for 4 d. To obtain C-MSCs, confluent cells were scratched using a micropipette tip and were then torn off as a cellular sheet. The sheet was rolled up to make round clumps of cells. C-MSCs were maintained in growth medium or osteoinductive medium (OIM) for 5 or 10 d. The biological properties of C-MSCs were evaluated in vitro, and their periodontal tissue regenerative activity was tested by using a dog class III furcation defect model. Immunofluorescence analysis revealed that type I collagen fabricated the form of C-MSCs. OIM markedly elevated calcium deposition in C-MSCs at day 10, suggesting its osteogenic differentiation capacity. Both C-MSCs and C-MSCs cultured with OIM transplantation without an artificial scaffold into the dog furcation defect induced periodontal tissue regeneration successfully compared with no graft, whereas osteogenic-differentiated C-MSCs led to rapid alveolar bone regeneration. These findings suggested that the use of C-MSCs refined by self-produced ECM may represent a novel predictable periodontal tissue regenerative therapy.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Matriz Extracelular/metabolismo , Regeneración Tisular Guiada Periodontal/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Enfermedades Periodontales/terapia , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Perros , Ilion/citología , Células Madre Mesenquimatosas/citología , Microtomografía por Rayos XRESUMEN
Epidemiological studies have linked periodontitis to rheumatoid arthritis (RA). Porphyromonas gingivalis (Pg) was reported recently to produce citrullinated protein (CP) and increase anti-cyclic CP antibody (ACPA), both of which have been identified as causative factors of RA. In the present study, we determined the effects of Pg infection on the exacerbation of RA in a mouse model. RA model mice (SKG mice) were established by an intraperitoneal (i.p.) injection of laminarin (LA). Mice were divided into six groups, Ctrl (PBS injection), LA (LA injection), Pg/LA (Pg + LA injection), Pg (Pg injection), Ec/LA (Escherichia coli and LA injection) and Ec (E. coli injection). In order to evaluate RA, joint swelling by the arthritis score, bone morphology by microcomputed tomography (microCT), haematoxylin and eosin staining, ACPA, matrix metalloproteinase-3 (MMP-3) and cytokine level in serum by enzyme-linked immunosorbent assay were determined. Osteoclast differentiation from bone marrow mononuclear cells (BMCs) was examined to clarify the underlying mechanisms of RA. The presence of Pg and CP in joint tissue was also investigated. The arthritis score was threefold higher in the Pg/LA group than in the LA group. Severe bone destruction was observed in joint tissue of the Pg/LA group. A microCT analysis of the Pg/LA group revealed a decrease in bone density. ACPA, MMP-3, interleukin (IL)-2, IL-6, CXCL1 and macrophage inflammatory protein (MIP)-1α levels from the Pg/LA group were the highest. The osteoclastogenesis of BMCs was enhanced in the Pg/LA group. Furthermore, large amounts of Pg components and CP were detected in the Pg/LA group. In conclusion, Pg infection has the potential to exacerbate RA.
Asunto(s)
Artritis Reumatoide/etiología , Artritis Reumatoide/patología , Infecciones por Bacteroidaceae/complicaciones , Infecciones por Bacteroidaceae/microbiología , Porphyromonas gingivalis , Animales , Articulación del Tobillo/diagnóstico por imagen , Articulación del Tobillo/patología , Artritis Reumatoide/diagnóstico por imagen , Biomarcadores , Diferenciación Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Expresión Génica , Ratones , Osteoclastos/citología , Osteoclastos/metabolismo , Microtomografía por Rayos XRESUMEN
OBJECTIVE: A large number of individuals have halitosis. The total amount of volatile sulfur compounds, which are the main cause of halitosis, has been correlated with periodontitis following bacterial infection. In this study, Porphyromonas gingivalis (Pg), a major periodontopathogenic bacterium, was isolated from patients with halitosis by the amplification of 16S rRNA, and the ability of isolated Pg to produce methyl mercaptan (CH3 SH) was determined to clarify the relationship between halitosis and Pg infection. MATERIALS AND METHODS: CH3 SH concentrations were measured in patients using Oral Chroma. The production of CH3 SH by Pg standard and clinical strains was also measured in vitro. Real-time PCR was performed to compare the expression of mgl mRNA (which encoded l-methionine-a-deamino-g-mercaptomethane-lyase) among the Pg strains. The production of CH3 SH and the expression of mgl mRNA were also determined to assess the effects of oriental medicine. RESULTS: The production of CH3 SH and the expression of mgl mRNA strongly correlated with each other in the presence of l-methionine. The expression of mgl mRNA by Pg W83 was strongly inhibited by magnoliaceae. CONCLUSION: The production of CH3 SH was correlated with the expression of mgl. Furthermore, the oriental medicine, magnoliaceae, may represent a potential treatment for halitosis.
Asunto(s)
Halitosis/microbiología , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , ARN Mensajero/biosíntesis , Compuestos de Sulfhidrilo/metabolismo , Adulto , Anciano , Antiinfecciosos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/microbiología , Medicamentos Herbarios Chinos/farmacología , Femenino , Humanos , Magnoliaceae , Masculino , Metionina/metabolismo , Metionina/farmacología , Persona de Mediana Edad , Periodontitis/metabolismo , Periodontitis/microbiología , Porphyromonas gingivalis/aislamiento & purificación , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adulto JovenRESUMEN
AIM: To examine the in vitro effects of LL37 on the expression of vascular endothelial growth factor (VEGF) in human pulp cells and to identify the intracellular signalling pathway involved. METHODOLOGY: Pulp cells at passage 6 were treated with 10 µg mL(-1) synthesized LL37, and an inhibition assay was performed with MAPK or NF-κB inhibitors to determine the possible signalling pathway. VEGF mRNA, VEGF protein and phosphorylated ERK1/2 levels were determined by real-time PCR, ELISA and Western blot, respectively. Data were analysed using t-tests. RESULTS: LL37 significantly increased both the mRNA and protein levels of VEGF in pulp cells (P < 0.01). However, pre-treatment with an ERK kinase inhibitor suppressed these increases. Furthermore, the inhibitor blocked LL37-induced ERK1/2 phosphorylation. CONCLUSIONS: LL37 activated the ERK pathway to boost VEGF secretion from human pulp cells. Because of this angiogenic effect and its reported induction of pulp cell migration and antimicrobial activity against cariogenic bacteria, LL37 may be applicable as a pulp capping material.
Asunto(s)
Antibacterianos/farmacología , Catelicidinas/farmacología , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos , Diente Premolar , Western Blotting , Técnicas de Cultivo de Célula , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
BACKGROUND AND OBJECTIVE: Periodontitis is an infectious disease caused by an interaction between the host and periodontopathogenic bacteria. Regulating the immune response in human gingival epithelial cells (HGEC) may contribute to the prevention of periodontitis. Irsogladine maleate (IM) has previously been shown to regulate inflammation and the cell-cell junctional barrier in HGEC. In addition to these functions, control of bacterial recognition is important for preventing inflammation in periodontal tissue. Innate immunity in gingival epithelium is the first line of defense and plays a crucial role against bacterial challenge. Therefore, the effect of IM on regulating toll-like receptor 2 (TLR2), which is part of the innate immunity, was determined in this study. MATERIAL AND METHODS: OBA-9, an immortalized human gingival epithelial cell line, and primary cultured HGEC were used in this study. Real-time PCR and western blotting were performed in OBA-9 or HGEC stimulated with whole cells of Porphyromonas gingivalis or with lipopolysaccharide (LPS) derived from P. gingivalis (PgLPS) in the presence or absence of IM to determine expression of TLR2 mRNA and production of TLR2 protein. Small interfering RNA (siRNA) against TLR2 was transfected into OBA-9 to clarify the association between the induction of TLR2 and interleukin-8 (IL-8) production. RESULTS: The addition of IM into P. gingivalis or PgLPS-induced OBA-9 suppressed IL-8 production (p < 0.01). The addition of IM also abolished the induction of TLR2 by P. gingivalis or PgLPS in OBA-9 and primary cultured HGEC (p < 0.01). The suppressive effect of IM on the induction of TLR2 was also confirmed by immunohistostaining. Stimulation with peptidoglycan, a specific ligand for TLR2, suppressed the expression of toll-like receptor 4 (TLR4) mRNA in the presence of IM (p < 0.01). However, LPS derived from Escherichia coli, a ligand for TLR4, did not induce the expression of TLR2 mRNA. The PgLPS-induced expression of TLR4 mRNA was abolished by IM. Knockdown of TLR2 by siRNA transfection resulted in a weaker response of induction of IL8 mRNA in P. gingivalis or PgLPS-stimulated OBA-9. CONCLUSION: These results suggest that IM suppresses the induction of IL-8 production by regulating increased levels of TLR2.
Asunto(s)
Encía/efectos de los fármacos , Inmunosupresores/farmacología , Interleucina-8/efectos de los fármacos , Porphyromonas gingivalis/inmunología , Receptor Toll-Like 2/efectos de los fármacos , Triazinas/farmacología , Línea Celular , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Encía/citología , Humanos , Inmunidad Innata/efectos de los fármacos , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , ARN Interferente Pequeño/genética , Receptor Toll-Like 2/genéticaRESUMEN
Apoptosis is thought to contribute to the progression of periodontitis. It has been suggested that the apoptosis of epithelial cells may contribute to the loss of epithelial barrier function. Smad2, a downstream signaling molecule of TGF-ß receptors (TGF-ßRs), is critically involved in apoptosis in several cell types. However, the relationship between smad2 and bacteria-induced apoptosis has not yet been elucidated. It is possible that the regulation of apoptosis induced by periodontopathic bacteria may lead to novel preventive therapies for periodontitis. Therefore, in the present study, we investigated the involvement of smad2 phosphorylation in apoptosis of human gingival epithelial cells induced by Aggregatibacter actinomycetemcomitans (Aa). Aa apparently induced the phosphorylation of smad2 in primary human gingival epithelial cells (HGECs) or the human gingival epithelial cell line, OBA9 cells. In addition, Aa induced phosphorylation of the serine residue of the TGF-ß type I receptor (TGF-ßRI) in OBA9 cells. SB431542 (a TGF-ßRI inhibitor) and siRNA transfection for TGF-ßRI, which reduced both TGF-ßRI mRNA and protein levels, markedly attenuated the Aa-induced phosphorylation of smad2. Furthermore, the disruption of TGF-ßRI signaling cascade by SB431542 and siRNA transfection for TGF-ßRI abrogated the activation of cleaved caspase-3 expression and repressed apoptosis in OBA9 cells treated with Aa. Thus, Aa induced apoptosis in gingival epithelial cells by activating the TGF-ßRI-smad2-caspase-3 signaling pathway. The results of the present study may suggest that the periodontopathic bacteria, Aa, activates the TGF-ßR/smad2 signaling pathway in human gingival epithelial cells and induces apoptosis in epithelial cells, which may lead to new therapeutic strategies that modulate the initiation of periodontitis.
Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Apoptosis/fisiología , Inserción Epitelial/microbiología , Proteína Smad2/fisiología , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Caspasa 3/efectos de los fármacos , Inhibidores de Caspasas/farmacología , Línea Celular , Células Cultivadas , Dioxoles/farmacología , Inserción Epitelial/patología , Células Epiteliales/microbiología , Células Epiteliales/fisiología , Silenciador del Gen , Humanos , Periodontitis/microbiología , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
BACKGROUND AND OBJECTIVE: LL37, originally found in the innate immune system, is a robust antimicrobial peptide. LL37 exhibits multiple bio-functions in various cell types, such as migration, cytokine production, apoptosis, and angiogenesis besides its antimicrobial activity Periodontal ligament (PL) cells play a pivotal role in periodontal tissue regeneration. Based on these findings, we hypothesized that LL37 can regulate PL cell function to promote regeneration of periodontal tissue. To prove this hypothesis, we investigated the effect of LL37 on the potent angiogenic inducer vascular endothelial growth factor (VEGF) expression in cultures of human PL (HPL) cells because neovascularization is indispensable for the progress of tissue regeneration. Moreover, we investigated the signaling cascade associated with LL37-induced VEGF expression. MATERIAL AND METHOD: HPL cells were treated with synthesized LL37 in the presence or absence of PD98059, a MEK-ERK inhibitor, or PDTC, an NF-κB inhibitor. VEGF expression levels were assessed by real-time polymerase chain reaction analysis and an enzyme-linked immunoassay. Phosphorylation levels of ERK1/2 or NF-κB p65 were determined by Western blotting. RESULTS: LL37 upregulated VEGF-A expression at the mRNA and protein levels in HPL cells, while VEGF-B mRNA expression was not affected. Both ERK and NF-κB inhibitors clearly abrogated the increase in VEGF-A levels induced by LL37 in HPL cells. Importantly, LL37 increased phosphorylated levels of ERK1/2 and NF-κB p65 in HPL cells. CONCLUSION: LL37 induces VEGF-A production in HPL cells via ERK and NF-κB signaling cascades, which may result in angiogenesis, thereby contributing to periodontal regeneration.
Asunto(s)
Antibacterianos/farmacología , Catelicidinas/farmacología , Ligamento Periodontal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Células Cultivadas , Relación Dosis-Respuesta a Droga , Flavonoides/farmacología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/análisis , FN-kappa B/análisis , FN-kappa B/antagonistas & inhibidores , Neovascularización Fisiológica/efectos de los fármacos , Ligamento Periodontal/citología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Pirrolidinas/farmacología , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tiocarbamatos/farmacología , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , eIF-2 Quinasa/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
BACKGROUND AND OBJECTIVE: As epithelial cells function as a mechanical barrier, the permeability of the gingival epithelial cell layer indicates a defensive capability against invasion by periodontal pathogens. We have reported the expression of claudin-1 and E-cadherin, key regulators of permeability, in the gingival junctional epithelium. Irsogladine maleate (IM) is a medication for gastric ulcers and also regulates Aggregatibacter actinomycetemcomitans-stimuated chemokine secretion and E-cadherin expression in gingival epithelium. In this study, we have further investigated the effects of IM on the barrier functions of gingival epithelial cells under inflammatory conditions. MATERIAL AND METHODS: We examined the permeability, and the expression of claudin-1 and E-cadherin, in human gingival epithelial cells (HGECs) stimulated with tumor necrosis factor (TNF)-α, with or without IM. RESULTS: TNF-α increased the permeability of HGECs, and IM abolished the increase. TNF-α reduced the expression of E-cadherin in HGECs, and IM reversed the reduction. In addition, immunofluorescence staining showed that TNF-α disrupted claudin-1 expression in HGECs, and IM reversed this effect. CONCLUSION: The results suggest that IM reverses the TNF-α-induced disruption of the gingival epithelial barrier by regulating E-cadherin and claudin-1.
Asunto(s)
Encía/efectos de los fármacos , Triazinas/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Western Blotting , Cadherinas/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Claudina-1 , Impedancia Eléctrica , Inserción Epitelial/citología , Inserción Epitelial/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Femenino , Fluoresceína , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Encía/citología , Encía/inmunología , Humanos , Masculino , Proteínas de la Membrana/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Uniones Estrechas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto JovenRESUMEN
INTRODUCTION: The present study examined whether induction of an adaptive immune response to orally colonizing non-pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. METHODS: BALB/c mice harboring P. pneumotropica (P. pneumotropica(+) mice) in the oral cavity or control P. pneumotropica-free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T-cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor-kappaB ligand (RANKL) -positive T cells in gingival tissue. RESULTS: Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29-kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross-reactivity with P. pneumotropica OmpA compared to results in the control non-immunized mice. The A. actinomycetemcomitans-immunized P. pneumotropica(+) mice developed remarkable periodontal bone loss in a RANKL-dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin-Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double-color confocal microscopy demonstrated that the frequency of RANKL(+) T cells in the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice was remarkably elevated compared to control mice. CONCLUSION: The induction of an adaptive immune response to orally colonizing non-pathogenic P. pneumotropica results in RANKL-dependent periodontal bone loss in mice.
Asunto(s)
Aggregatibacter actinomycetemcomitans/inmunología , Pérdida de Hueso Alveolar/inmunología , Pérdida de Hueso Alveolar/microbiología , Pasteurella pneumotropica/inmunología , Ligando RANK/inmunología , Animales , Antígenos Bacterianos , Proteínas de la Membrana Bacteriana Externa/inmunología , Reacciones Cruzadas , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Osteoprotegerina/farmacología , Pasteurella pneumotropica/patogenicidad , Ligando RANK/antagonistas & inhibidores , Linfocitos T/inmunologíaRESUMEN
Anti-microbial peptides produced from mucosal epithelium appear to play pivotal roles in the host innate immune defence system in the oral cavity. In particular, human beta-defensins (hBDs) and the cathelicidin-type anti-microbial peptide, LL-37, were reported to kill periodontal disease-associated bacteria. In contrast to well-studied hBDs, little is known about the expression profiles of LL-37 in gingival tissue. In this study, the anti-microbial peptides expressed in gingival tissue were analysed using immunohistochemistry and enxyme-linked immunosorbent assay (ELISA). Immunohistochemistry revealed that neutrophils expressed only LL-37, but not hBD-2 or hBD-3, and that such expression was prominent in the inflammatory lesions when compared to healthy gingivae which showed very few or no LL-37 expressing neutrophils. Gingival epithelial cells (GEC), however, expressed all three examined anti-microbial peptides, irrespective of the presence or absence of inflammation. Moreover, as determined by ELISA, the concentration of LL-37 in the gingival tissue homogenates determined was correlated positively with the depth of the gingival crevice. Stimulation with periodontal bacteria in vitro induced both hBD-2 and LL-37 expressions by GEC, whereas peripheral blood neutrophils produced only LL-37 production, but not hBD-2, in response to the bacterial stimulation. These findings suggest that LL-37 displays distinct expression patterns from those of hBDs in gingival tissue.