Browsing by Author "Ceacero-Heras, Diego"

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    Memoria de congreso
    Glucocorticoid receptor intestinal epithelial knockout mice show attenuated colonic inflammatory response but unaffected permeability in early experimental sepsis
    (2023-03-04) Ceacero-Heras, Diego; Tena-Garitaonaindia, Mireia; Seguí-Pérez, Alba; Ruiz-Henares, G.; Córdova, Samir; Martínez-Augustin, Olga; Sánchez de Medina, Fermín
    Introduction: Sepsis is defined as an organic dysfunction that threatens the life of patients due to an abnormally regulated response to infection [1]. The initial phase of sepsis is dominated by an increased production of proinflammatory cytokines, which leads to augmented capillary permeability, extravasation, hypercoagulability and myelopoiesis. One of the main sources of infection in sepsis is believed to be the intestinal microbiota via traslocation through the mucosa to the bloodstream. Systemic inflammation weakens intestinal barrier function (IBF) in animal models, resulting in increased bacterial traslocation [2]. Even if the management of sepsis has advanced in the last decades, mortality is still high and there are blanks in terms of pathological systems and long-term consequences. Thus, the search for effective treatments is clearly justified. Glucocorticoids (GC) are part of the drugs used in sepsis, but they have only shown a moderate therapeutic effect. This fact may be caused by harmful effects of GCs on IBF, whose compromise may limit GC clinical benefit by facilitating luminal translocation of microorganisms. Besides, GC treatment impairs epithelial healing in experimental colitis in mice [3]. Previous results of our research group have shown that mice with induced deletion of the GC receptor (GR) in intestinal epithelial cells (i.e. NR3C1ΔIEC mice) are protected against dextran sulphate sodium (DSS)-induced colitis [4]. In turn, gene deletion results in a short lived inflammatory response in the colon [5]. Objective: Understanding the role of the intestinal epithelial GR and its involvement in IBF regulation in experimental sepsis, with the ultimate goal of improving the management of sepsis with GCs. Matherial and methods: The cecal ligation and puncture (CLP) model of sepsis was applied to WT C57BL/6J and NR3C1ΔIEC mice. Ceacum-exposed mice were used as control (Sham). Mice were sacrificed 24 hours after surgery. Four hours before sacrifice, mice were administered 4 kD FITC-dextran, a fluorescent marker of permeability. Colon, jejunum, adrenes, kidney and liver RT-qPCRs were performed as well as determination of plasma FITC-dextran and corticosterone plasma levels. Results: After 24 h, CLP mice exhibited elevated corticosterone plasma levels with hypoglycemia and splenomegaly. Intestinal barrier function was weakened, as indicated by increased FITC-dextran plasma levels. A modest increase in inflammatory markers (S100a8, Cxcl1) was noted in the colon and jejunum. The expression of Tjp1, involved in barrier function, was downregulated in CLP mice. Similarly, the colonic expression of Cyp11a1 and Lrh1, involved in local steroidogenesis, was lower in CLP mice, regardless of genotype. Markers of inflammation were also augmented in the lung and kidney. CLP mice exhibited hypercorticosteronemia, which was associated to increased Cyp11a1 in the adrenes. Of note, both parameters were less pronounced in KO mice. The latter also exhibited dampened inflammatory response in the colon but not the jejunum. FITC-dextran plasma levels were similarly increased in WT and KO mice. Conclusions: In the early stages of the CLP model of sepsis the colon and jejunum are inflamed, and epithelial deletion of the glucocorticoid receptor appears to modulate inflammation in the former, with no change in barrier function. Further studies will characterize the microbiota composition and phenotype in later stages and in the response to glucocorticoid treatment.
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    Memoria de congreso
    Papel de la enzima fosfatasa alcalina no específica de tejido (TNAP) en el epitelio intestinal en la inflamación
    (2023-03-04) Tena Garitaonaindia, Mireia; Cordova, Samir; Ceacero-Heras, Diego; Seguí, Alba; Sánchez de Medina, Fermín; Martínez Augustin, Olga
    Introducción: la fosfatasa alcalina (AP) es una familia de enzimas que ha sido relacionada con la protección frente a inflamación intestinal. Se ha descrito que una de sus isoformas, la fosfatasa alcalina intestinal (IAP), es capaz de desfosforilar diferentes antígenos bacterianos, de tal forma que la enzima regula el crecimiento de la microbiota e impide el paso de antígenos activos. En cuanto a la isoforma TNAP, se ha observado que su expresión se encuentra incrementada en la colitis experimental, no solo debido a la infiltración de células del sistema inmunológico, sino también por el incremento de expresión de esta enzima en las células del epitelio intestinal. Objetivo: conocer el papel de la TNAP en la inflamación intestinal. Métodos y resultados: se ha generado un modelo de ratón con deleción condicional inducible del gen que codifica TNAP (Alpl) en el epitelio intestinal (ratones AlplIEC-/-). El silenciamiento específico de TNAP en IECs en inflamación por DSS (7 días) supuso una pérdida mayor de peso en los ratones, sin observarse diferencias en el índice de actividad de la enfermedad (DAI). A nivel histológico se observó un mayor nivel de infiltración en la submucosa en el colon de los ratones sin TNAP. Los ratones AlplIEC-/- presentaron una expresión reducida de marcadores inflamatorios en el colon, como S100a8, Il6 y Tnf. Por el contrario, la deficiencia en TNAP en el epitelio intestinal supuso un aumento en la expresión de la fosfatasa alcalina intestinal global (Akp6) en el colon, sugiriendo que podría existir algún mecanismo de compensación. Además, la ausencia de TNAP en el epitelio intestinal provocó un aumento de expresión de genes relacionados en el mantenimiento de la función de barreara, como Muc4, Tjp1 y Tff3. Conclusión: los ratones AlplIEC-/- presentan un fenotipo mixto, con mayor infiltración y daño histológico pero menor expresión de marcadores inflamatorios en la colitis por DSS. Perspectivas futuras: se realizarán estudios de transcriptómica para conocer el efecto de la TNAP presente en el intestino sobre la función de barrera intestinal y sobre la microbiota.