Increased STAT3 Activation in Periodontitis Drives Inflammatory Bone Loss.

Periodontitis is one of the most prevalent human inflammatory diseases. It is characterized by periodontal tissue destruction, progressively driven by the host response. In this regard, cytokines associated with tissue destruction, such as interleukin (IL)-6 and IL-23, use a common signaling pathway mediated by STAT3. This transcription factor is also needed for IL-17A production, a key mediator in periodontitis pathogenesis. Although several studies have reported increased activation of STAT3 in experimental periodontitis, a detailed characterization of STAT3 activation in human gingival tissues and its involvement in alveolar bone loss has yet to be explored. Using a cross-sectional study design, we detected increased proportions of pSTAT3-positive cells during periodontitis compared with health, particularly in epithelial cells and T cells. Other cell types of hematopoietic and nonhematopoietic origin also display STAT3 activation in gingival tissues. We detected increased STAT3 phosphorylation and expression of STAT3-related genes during experimental periodontitis. Next, we evaluated the role of STAT3 in alveolar bone destruction using a mouse model of STAT3 loss of function (mut-Stat3 mice). Compared with controls, mut-Stat3 mice had reduced alveolar bone loss following ligature-induced periodontitis. We also evaluated pharmacologic inhibition of STAT3 in ligature-induced periodontitis. Like mut-Stat3 mice, mice treated with STAT3 small-molecule inhibitor had reduced bone loss compared with controls. Our results demonstrate that STAT3 activation is increased in epithelial and T cells during periodontitis and indicate a pathogenic role of STAT3 in inflammatory alveolar bone loss.

Establishment and Stability of the Murine Oral Microbiome.

Commensal microbiomes exert critical functions at barrier sites. In particular, establishment of the commensal microbiome after birth dictates immune functionality and tissue homeostasis at mucosal surfaces. To investigate the establishment and stability of the oral mucosal microbiome in mice, we evaluated oral microbiome communities shortly after birth, through adulthood, and up to 1 y of life in a controlled manner, using sequential oral samples from the same mice over time. We further evaluated transmissibility of oral microbiomes from parents and during cohousing experiments and evaluated susceptibility to oral inflammatory disease in mice harboring distinct microbiomes. Our work reveals basic principles in the establishment and stability of a health-associated oral microbiome after birth and provides insights that may be important for host-microbiome experimentation in animal models.

Presencia de marcadores de las vías de activación de los macrófagos en periodontitis crónica / Presence of markers of activation pathways of macrophages in chronic periodontitis

La periodontitis crónica es una patología infecciosa, causada por un complejo de especies bacterianas, que afecta principalmente los tejidos de inserción de los dientes. La respuesta inmune-inflamatoria producida se caracteriza por la presencia de un infiltrado inflamatorio, en el cual los macrófagos representan entre 5 al 30 por ciento. Es sabido que los macrófagos se activan mediante dos vías: Clásica y Alterna, caracterizadas por la presencia de marcadores indirectos: IFN-y e IL-6 para la vía clásica e IL-4 para la vía alterna, ampliamente abordados. Recientemente, se ha descrito a la subunidad A del factor XIII de la coagulación (FXIII-A) como un buen marcador de la vía alterna. El objetivo de este estudio consiste en determinar la presencia de IFN-y, IL-6, FXIII-A e IL-4 como marcadores de las vías de activación de los macrófagos, en pacientes con periodontitis crónica. Para tal efecto, se realizó inmunohistoquímica y Western-Blot para los cuatro marcadores junto a CD-68, marcador de macrófagos, en 18 biopsias de tejido periodontal sano y 18 con periodontitis crónica. Se detectó la presencia de IFN-y, IL-6, IL-4 y FXIII-A junto a CD68+, en todas las muestras de pacientes sanos y con periodontitis. Los resultados obtenidos sugieren que al estar presente IFN-y, IL-6, IL-4 y FXIII-A, los macrófagos se activarían a través de ambas vías, lo cual, produciría una respuesta tanto proinflamatoria (Th1) como antinflamatoria (Th2). Son necesarios más estudios para determinar si existe una vía preferencial de activación.

Periodontitis is a chronic infectious disease caused by a bacterial species complex, which affects mainly the insertion tissues of the teeth. The immune-inflammatory response produced is characterized by an inflammatory infiltrate in which macrophages represent between 5 to 30 percent. It is known and has been widely discussed that macrophages are activated in two ways: Classical and Alterna, characterized by the presence of indirect markers: IFN-y and IL-6 for the classical pathway and IL-4 for the alternative pathway. Recently the subunit A of the clotting factor XIII (FXIII-A) has been described as a good marker of the alternative pathway. The objective of this study is to determine the presence of IFN-y, IL-6, IL-4 and FXIII-A as markers of the macrophage activation pathways in patients with chronic periodontitis. To this end, we performed immunohistochemistry and Western blot for the four markers with CD68 macrophage marker, in 18 healthy periodontal tissue biopsies and 18 with chronic periodontitis. We detected the presence of IFN-y, IL-6, IL-4 and FXIII-A with CD68 +, in all samples of healthy patients and periodontitis. The results suggest that when present, IFN-y, IL-6, IL-4 and FXIII-A, activate macrophages through both routes, which would produce a proinflammatory response (Th1) as antiinflammatory (Th2). Further studies are necessary to determine whether there is a preferential pathway activation.

Host-pathogen interactions in progressive chronic periodontitis.

Periodontitis is an infection characterized by the occurrence of supporting tissue destruction with an episodic nature. Disease progression is often determined by the loss of attachment level or alveolar bone, and sequential probing of periodontal attachment remains the most commonly utilized method to diagnose progressive destruction of the periodontium. The tolerance method has been the most extensive clinical method used in recent years to determine site-specific attachment level changes. There is abundant evidence that major tissue destruction in periodontal lesions results from the recruitment of immune cells. Considerable effort has been made to study the host cell and mediator profiles involved in the pathogenesis of chronic periodontitis, but the definition of active sites, where current periodontal breakdown occurs, and consecutive characterization of the mediators involved are still among the main concerns. In the present review, we summarize periodontopathic bacteria and host factors, including infiltrating cell populations, cytokines, and host matrix metalloproteinases, associated with under-going episodic attachment loss that could partly explain the mechanisms involved in destruction of the supporting tissues of the tooth.

Presencia de linfocitos T reguladores en periodontitis crónica / Presence of T regulatory cells in chronic periodontitis

La enfermedad periodontal requiere de un hospedero susceptible para su desarrollo y progresión. Dentro de las características del hospedero se encuentra la respuesta T reguladora, que otorga tolerancia frente a antígenos propios, participa durante las enfermedades infecciosas limitando el daño tisular, sin disminuir la respuesta antibacteriana. El presente estudio tiene por objetivo determinar la presencia, reclutamiento y función de Tregs en pacientes con periodontitis crónica. En 10 biopsias de tejido periodontal sano y con periodontits crónica se realizó inmunohistoquímica para marcadores (CD4, CD25, Foxp3), quimioquinas (CCL17, CCL22) y citoquinas (TGF-B, IL-10) de Tregs. Además de Western-Blot para detectar las citoquinas. Los resultados obtenidos sugieren una posible asociación entre células Tregs y la infección periodontal, ya que se confirma su reclutamiento y presencia. Sin embargo, son necesarios más estudios del posible desbalance con su contraparte pro-inflamatoria Th17, que expliquen en parte la compleja etiopatogenia de la enfermedad periodontal.

Periodontal disease requires a susceptible host to initiation, development and progression. T regulatory response is one of these inmunoregulatory characteristics of the susceptible host, which provide tolerance, tissular protection during infection without impairing the control of periodontopathogens. The aim of this study is to determinate the presence, homing and function of T regulatory cells (Tregs) in patients with chronic periodontitis. Ten biopsies were taken from pockets, the presence of Tregs markers (CD4, CD25, Foxp3), chemokines (CCL17, CCL22) and cytokines (TGF-p, IL-10) were determinate by immunohistochemistry. Cytokines also were detected with Western-Blot. Our results suggest a possible association between Tregs and periodontal infection, confirming homing and presence of Tregs. However, further studies are required to determine the possible imbalance with pro-inflammatory part Th17, that might explain the complex etiopathogenesis of periodontal disease.

Associations between matrix metalloproteinase-8 and -14 and myeloperoxidase in gingival crevicular fluid from subjects with progressive chronic periodontitis: a longitudinal study.

BACKGROUND: Matrix metalloproteinase (MMP)-8 is a central mediator in chronic periodontitis. MMP-8 can be activated by the cooperative action of other MMPs such as MMP-14, reactive oxygen species, and microbial proteases. The aim of this study is to associate the levels, molecular forms, isoenzyme distribution, and degree of activation of MMP-8 and -14, myeloperoxidase (MPO), and tissue inhibitor of MMP (TIMP)-1 in gingival crevicular fluid (GCF) from patients with progressive periodontitis at baseline and after periodontal therapy. METHODS: In this longitudinal study, GCF samples from active (n = 25) and inactive (n = 25) sites of subjects with periodontitis were screened at baseline for GCF levels of MMP-8 by immunofluorometric assay, of MMP-14 by specific activity assay, and of MPO and TIMP-1 by enzyme-linked immunosorbent assay. MMP-8 and MPO were also measured after periodontal treatment. Molecular forms were determined by immuno-Western blot analyses and subjected to densitometric scanning and statistical analyses. RESULTS: High MMP-8 and MPO levels and a strong MPO/MMP-8 positive correlation were found in active and inactive sites at baseline. After treatment, decreases in MPO and MMP-8 were seen, except for active sites in which MMP-8 differences were not significant (P <0.05). CONCLUSIONS: We present initial data that show that GCF levels and associations between MPO and MMP-8 are related to progression episodes and treatment responses in patients with chronic periodontitis. Our results suggest an interaction between the MPO oxidative pathway and MMP-8 activation, and this cascade might be useful as a potential biomarker for treatment outcomes.

Monocyte chemotactic protein-3: possible involvement in apical periodontitis chemotaxis.

AIM: To study the expression of monocyte chemotactic protein-3 (MCP-3, also known as chemokine CCL-7) in tissue from apical lesions (AL) and to associate MCP-3 expression with symptomatic or asymptomatic apical periodontitis. METHODOLOGY: To determine the expression of MCP-3 in AL, biopsies obtained during tooth extraction procedures were fixed, subjected to routine processing and diagnosed as apical granuloma (AG) (n = 7) or radicular cyst (RC) (n = 5). As controls, apical periodontal ligament (PDL) specimens from healthy premolars extracted for orthodontics reasons were included (n = 7). All specimens were immunostained for MCP-3 and examined under a light microscope. In addition, homogenates from AL (n = 14) and healthy PDL samples (n = 7) were studied through immunowestern blot. Finally, periapical exudates samples were collected from root canals of teeth having diagnosis of symptomatic (n = 14) and asymptomatic apical periodontitis (n = 14) during routine endodontic treatments and analysed by immunowestern blot and densitometry. RESULTS: MCP-3 was detected in AG and RC and localized mainly to inflammatory leucocytes, whereas no expression was observed in healthy PDLs. MCP-3 was also detected in periapical exudate, and its levels were significantly higher in symptomatic than in asymptomatic apical periodontitis. CONCLUSIONS: MCP-3 was expressed in AL and its levels associated with clinical symptoms. MCP-3 might play a role in disease pathogenesis, possibly by stimulating mononuclear chemotaxis.

Papel de los linfocitos T CD4+ en la destrucción ósea observada durante la periodontitis crónica / Role of CD4+ T cells in bone destruction during chronic periodontitis

Propósito: La periodontitis crónica es una enfermedad de naturaleza inflamatoria y etiología infecciosa, caracterizada por la destrucción del aparato de inserción del diente, cemento radicular, ligamento periodontal y hueso alveolar, y que causa la pérdida de los dientes. Durante su desarrollo se establece un denso infiltrado inflamatorio celular, constituido principalmente por linfocitos T, con la capacidad de secretar una serie de citoquinas que participan en los eventos patogénicos de la enfermedad, regulando la inflamación de los tejidos periodontales y la destrucción del hueso alveolar. En la artritis reumatoide, la citoquina recientemente identificada RANKL (Ligando del receptor activador del factor nuclear κB) es expresada por los linfocitos T CD4+, participando directamente en los procesos de estimulación de la diferenciación osteoclástica y en la activación de osteoclastos maduros, y por lo tanto, en la destrucción ósea articular. El objetivo del presente estudio es determinar si mayores niveles de RANKL se encuentran asociados a la periodontitis crónica y si estos son sintetizados por los linfocitos T CD4+ reclutados en los sitios con periodontitis crónica. Material y métodos: En 33 pacientes mayores de 35 años de edad afectados de periodontitis crónica y 20 individuos controles sanos, se determinaron los niveles de mensajero de ácido desoxiribonucleico (mARN) de RANKL en biopsias de encía mediante transcriptasa reversa-reacción en cadena de la polimerasa (RT-PCR) en tiempo real. A partir de las mismas biopsias, células gingivales totales fueron aisladas para inmunotipificar y cuantificar los leucocitos infiltrantes gingivales e identificar las células responsables de la expresión de RANKL mediante doble tinción por citometría de flujo. Resultados: Los pacientes con periodontitis mostraron mayores niveles de mARN de RANKL en comparación a individuos sanos, observándose que la expresión de RANKL se incrementó en 238,3 veces con relación a los sujetos controles. Los individuos con periodontitis mostraron mayores porcentajes de linfocitos T CD4+ y CD8+. Además, una asociación entre RANKL y los linfocitos T CD4+ fue determinada mediante doble tinción por citometría de flujo. Conclusión: Estos datos demuestran que mayores niveles de RANKL se encuentran asociados a la periodontitis y que estos mayores niveles se pueden explicar en parte a la actividad de los linfocitos T CD4+ en el sitio de la infección. La determinación de la asociación entre la periodontitis crónica y la síntesis de RANKL constituye un interesante mecanismo molecular que contribuye a explicar en parte la destrucción tisular asociada y permite proyectar posibles nuevas estrategias inmunoterapéuticas que ayudarían a controlar la pérdida de tejido característica de la enfermedad periodontal (AU)

Background: Chronic periodontitis is an infectious disease, characterized by alveolar bone destruction and teeth loss. Receptor activator of nuclear factor kB- ligand (RANKL) is an osteoclastogenic cytokine, central regulatory factor in the osteoclasts life-span and physiological and pathological bone resorption. Gingival T cells synthesize RANKL contributing to molecular local unbalance that entail to the alveolar bone resorption seen in periodontitis. Our study was aimed at associating the levels of RANKL with the CD4+ T cell activity present in gingival tissues of chronic periodontitis patients. Methods: Gingival biopsies were obtained from 33 chronic periodontitis patients and 20 healthy controls. Specimens were either formalin fixed and paraffin embedded for real-time reverse transcription polymerase chain reaction (RT-PCR) and histological analysis, or tissue digestion processed for cell culture and flow cytometry analysis. RANKL mRNA and protein levels were determined by quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) in gingival cells culture supernatants. Gingival leukocytes were quantified by flow cytometry. RANKL and CD4 immunoreactivity was analyzed by flow cytometry and confocal microscopy. Results: RANKL mRNA levels were higher in periodontitis than in healthy subjects and spontaneous and LPSand PHA-stimulated RANKL synthesis were higher also in patients than controls. CD4+ T lymphocytes were the predominant infiltrate cell subset present in gingival tissues of periodontitis patients. Furthermore, an association between RANKL and CD4+ T cells was determined by double-staining flow cytometry and confocal microscopy. Conclusion: Taken together, these data demonstrate that gingival CD4+ T cells are the main cells responsible for the higher levels of RANKL observed in chronic periodontitis human patients (AU)

RANKL in human periapical granuloma: possible involvement in periapical bone destruction.

OBJECTIVES: The cytokine receptor activator of nuclear factor kappaB-ligand (RANKL) has been involved in both the physiological and pathological regulation of osteoclast life span and bone metabolism. Periapical granuloma is a periradicular lesion characterized by periapical bone destruction. The aims of this study were to associate the RANKL mRNA levels to periapical granulomas using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) technique and to determine the specific cell involved in RANKL synthesis. METHODS: In eight periapical granuloma and eight periodontal ligament samples from periodontally healthy volunteers, RANKL mRNA was detected by real-time RT-PCR. Expression of RANKL on infiltrate leukocytes was further investigated by flow cytometry in six periapical granulomas. RESULTS: Receptor activator of nuclear factor kappaB-ligand mRNA levels were higher in periapical granulomas than in healthy periodontal ligament as its RANKL mRNA cycle threshold (Ct) and DeltaCt were significantly lower than that of controls (33.07 +/- 1.24 vs 36.96 +/- 1.69 and 11.58 +/- 3.02 vs 15.60 +/- 3.31, respectively). A 16.2-fold (2.0-131.6) higher RANKL gene expression was detected in the granulomas compared with the control tissues. We determined by flow cytometry that lymphocytes were the predominant leukocyte cells (53.31%), and monocytes and dendritic cells were the main RANKL synthesizers in granuloma lesions. CONCLUSIONS: These data indicate that monocytes synthesized RANKL in periapical granulomas and suggest that RANKL is involved in bone loss associated with periapical lesions.