RESUMEN
Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade. Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques. Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimensionally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions. Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision. By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.
Asunto(s)
Técnicas de Cocultivo/métodos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Fibroblastos/patología , Humanos , Captura por Microdisección con Láser/métodos , Invasividad Neoplásica , Células del Estroma/patología , Microambiente TumoralRESUMEN
BACKGROUND: In both ulcerative colitis (UC) and Crohn's disease (CD) there is a marked increase in mucosal IgG plasma cells (PC), although their precise role is not well established. In this study we isolated gut PCs from patients with IBD and normal controls and analyzed cytokine production, matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 production, and PC longevity ex vivo. METHODS: Lamina propria mononuclear cells (LPMCs) were isolated from patients with CD (n = 19), UC (n = 27), and normal controls (n = 42). PCs were further selected by immunomagnetic isolation using CD138 microbeads. Cytokine, MMP-3, and TIMP-1 expression was investigated by Taqman polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), Western blotting, and confocal microscopy. PC lifespan in vitro was studied by ELISpot analysis. RESULTS: PCs from both controls and IBD patients contained high levels of transcripts for TGFbeta, whereas they did not contain significant transcripts for IL-4, IL-5, IL-10, IFNgamma, TNF, or IL-12p40. PCs from patients with CD and UC expressed significantly higher levels of MMP-3 protein and transcripts than controls (P < 0.0001). The vast majority of MMP-3-expressing PCs were IgG+ve. In culture, IgA PCs from both IBD patients and controls persisted for only a few days, but IgG PCs from IBD patients persisted for at least 3 weeks. CONCLUSIONS: We have demonstrated that IgG PCs from patients with IBD express large amounts of MMP-3 and that they appear to be long-lived. These results identify a new pathway by which IgG PCs may damage the gut.
Asunto(s)
Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Mucosa Intestinal/inmunología , Metaloproteinasa 3 de la Matriz/metabolismo , Células Plasmáticas/metabolismo , Estudios de Casos y Controles , Citocinas/metabolismo , Humanos , Inmunoglobulina G , Mucosa Intestinal/patología , Células Plasmáticas/inmunología , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
BACKGROUND & AIMS: Dendritic cells (DCs) play a crucial role in immune responses by controlling the extent and type of T-cell response to antigen. Celiac disease is a condition in which T-cell immunity to gluten plays an important pathogenic role, yet information on DCs is scant. We examined mucosal DCs in celiac disease in terms of phenotype, activation/maturation state, cytokine production, and function. METHODS: Mucosal DCs from 48 celiacs and 30 controls were investigated by flow cytometry. In situ distribution of DCs was analyzed by confocal microscopy. Interferon (IFN)-alfa, interleukin (IL)-4, IL-5, IL-12p35, IL-12p40, IL-18, IL-23p19, IL-27, and transforming growth factor-beta transcripts were measured by real-time reverse-transcription polymerase chain reaction in sorted DCs. DC expression of IL-6, IL-12p40, and IL-10 was assessed by intracellular cytokine staining. The effect of IFN-alfa and IL-18 blockade on the gluten-induced IFN-gamma response in celiac biopsy specimens grown ex vivo also was investigated. RESULTS: Mucosal DCs were increased in untreated, but not treated, celiacs. The majority of them were plasmacytoid with higher levels of maturation (CD83) and activation (CD80/CD86) markers. Higher transcripts of Th1 relevant cytokines, such as IFN-alfa, IL-18, and IL-23p19, were produced by celiac DCs, but because IL-12p40 was undetectable, a role for IL-23 is unlikely. Intracellular cytokine staining of celiac DCs showed higher IL-6, but lower IL-10 expression, and confirmed the lack of IL-12p40. Blocking IFN-alfa inhibited IFN-gamma transcripts in ex vivo organ culture of celiac biopsy specimens challenged with gluten. CONCLUSIONS: These data suggest that IFN-alfa-producing DCs contribute to the Th1 response in celiac disease.
Asunto(s)
Enfermedad Celíaca/metabolismo , Células Dendríticas/metabolismo , Glútenes/inmunología , Inmunidad Celular , Inmunidad Mucosa , Interferón-alfa/metabolismo , Mucosa Intestinal/metabolismo , Células TH1/metabolismo , Anticuerpos , Antígenos CD/análisis , Estudios de Casos y Controles , Enfermedad Celíaca/dietoterapia , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Diferenciación Celular , Separación Celular , Células Cultivadas , Ciclooxigenasa 2/análisis , Células Dendríticas/inmunología , Dieta con Restricción de Proteínas , Citometría de Flujo , Gliadina/inmunología , Humanos , Interferón-alfa/genética , Interferón-alfa/inmunología , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Mucosa Intestinal/enzimología , Mucosa Intestinal/inmunología , Microscopía Confocal , Fragmentos de Péptidos/inmunología , Fenotipo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1/inmunología , Técnicas de Cultivo de Tejidos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhagic Escherichia coli. We have previously utilized bioluminescence imaging (BLI) to determine the in vivo colonization dynamics of C. rodentium. However, due to the oxygen requirement of the bioluminescence system and the colonic localization of C. rodentium, in vivo localization studies were performed using harvested organs. Here, we report the detection of bioluminescent C. rodentium and commensal E. coli during colonization of the gastrointestinal tract in intact living animals. Bioluminescence was dependent on intact blood circulation, suggesting that the colonic environment is not anaerobic but nanaerobic. In addition, BLI revealed that C. rodentium colonizes the rectum, a site previously unreported for this pathogen.
Asunto(s)
Citrobacter rodentium/aislamiento & purificación , Tracto Gastrointestinal/microbiología , Mediciones Luminiscentes/métodos , Recto/microbiología , Animales , Escherichia coli/aislamiento & purificación , RatonesRESUMEN
Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. Matrix metalloproteinases (MMPs) have been shown to mediate matrix remodeling and cell migration during tissue injury and repair in the intestine. We have previously shown enhanced pathology in infected TNFRp55-/-, IL-12p40-/-, and IFN-gamma-/- mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues. We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3-/- mice. In MMP3-/- mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice. Colonic tissues from MMP3-/- mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12. However, MMP3-/- mice showed delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal lamina propria. CSFE-labeled mesenteric lymph node CD4+ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon of MMP3-/- mice than into those of WT mice. These studies show that mucosal remodeling can occur in the absence of MMP3, but that MMP3 plays a role in the migration of CD4+ T lymphocytes to the intestinal mucosa.
Asunto(s)
Citrobacter rodentium , Infecciones por Enterobacteriaceae/inmunología , Enfermedades Intestinales/inmunología , Metaloproteinasa 3 de la Matriz/fisiología , Animales , Antígenos CD/fisiología , Linfocitos T CD4-Positivos/fisiología , Movimiento Celular , Colon/enzimología , Colon/patología , Células Dendríticas/fisiología , Infecciones por Enterobacteriaceae/patología , Femenino , Interferón gamma/fisiología , Interleucina-12/fisiología , Subunidad p40 de la Interleucina-12 , Enfermedades Intestinales/patología , Metaloproteinasa 3 de la Matriz/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidades de Proteína/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis TumoralRESUMEN
The indigenous bacterial microflora colonize the gut at birth and remain there throughout life. Approximately 10(14) bacteria are present in the ileum and colon and they are clearly immunogenic. The evidence is strong that the vast majority of IgA plasma cells in normal human gut are responding to the antigens of the flora, and although the flora is also responsible for producing the large numbers of T cells which are present in the gut of healthy individuals, the types of T cell response which the flora elicits are less well understood. A major challenge for the immune system is to distinguish between the antigens of the flora and the antigens of pathogens. There is also a growing realization that the normal flora can also influence gene expression in antigen-presenting cells in the gut and so set the context in which T cells respond to food antigen and vaccines.