Glucocorticoid receptor intestinal epithelial knockout mice show attenuated colonic inflammatory response but unaffected permeability in early experimental sepsis

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

A Standardized Extract of Lentinula edodes Cultured Mycelium Inhibits Pseudomonas aeruginosa Infectivity Mechanisms

This work was supported by grants from FEDER project of Junta de Andalucia, Spain (30B572F301), Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds (SAF2017-88457-R and AGL2017-85270-R), and by Junta de Andalucia (CTS235 and CTS164). MT-G was supported by the University of the Ministry of Education (Spain). CIBERehd is funded by Instituto de Salud Carlos III.The priority pathogen list of the World Health Organization classified Pseudomonas aeruginosa as the second top critical pathogen. Hence, the development of novel antibacterial strategies to tackle this bacterium is highly necessary. Herein we explore the potential antibacterial effect of a standardized extract of cultured mycelium of Lentinula edodes (AHCCR ) on P. aeruginosa. AHCCR was found to inhibit the growth rate and biofilm formation of strain PAO1. No change in swarming was observed, but AHCCR hampered swimming and twitching motility. In accordance, a decreased expression of metabolism, growth, and biofilm formation genes was shown. AHCCR also diminished the levels of exotoxin A and bacteria inside IEC18 cells and the secretion of IL-6, IL-10 and TNF by infected macrophages. This effect was related to a reduced phosphorylation of MAPKs and to bacteria internalization. Taken together, our data suggest that AHCCR has a potential role to prevent P. aeruginosa infections and may lead to the development of new therapies.FEDER project of Junta de Andalucia, Spain 30B572F301Ministry of Economy and CompetitivityEuropean Commission SAF2017-88457-R AGL2017-85270-RUniversity of the Ministry of Education (Spain)Junta de Andalucia CTS235 CTS164Instituto de Salud Carlos III European Commissio

Papel de la enzima fosfatasa alcalina no específica de tejido (TNAP) en el epitelio intestinal en la inflamación

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

Deficiency in Tissue Non-Specific Alkaline Phosphatase Leads to Steatohepatitis in Mice Fed a High Fat Diet Similar to That Produced by a Methionine and Choline Deficient Diet

Funding: This research was funded by the Ministry of Economy and Competitivity of Spain, partly with Fondo Europeo de Desarrollo Regional FEDER funds [BFU2014-57736-P, AGL2014-58883-R, SAF2017-88457-R, AGL2017-85270-R] and by Junta de Andalucía [CTS235, CTS164]. MTG, RGB and CHC were supported by fellowships from the Ministry of Education. CIBERehd is funded by Instituto de Salud Carlos III. Institutional Review Board Statement: The study was conducted according to the guidelines of the Guide for the Care and Use of Laboratory Animals, and approved by the Animal Welfare Committee of the University of Granada (registry number: CEEA 01/03/2017–029). Informed Consent Statement: Not applicable for studies not involving humans. Acknowledgments: We gratefully acknowledge the assistance of Mercedes González and the rest of the group.The liver expresses tissue-nonspecific alkaline phosphatase (TNAP), which may participate in the defense against bacterial components, in cell regulation as part of the purinome or in bile secretion, among other roles. We aimed to study the role of TNAP in the development of hepatosteatosis. TNAP+/− haplodeficient and wild type (WT) mice were fed a control diet (containing 10% fat w/w) or the same diet deficient in methionine and choline (MCD diet). The MCD diet induced substantial weight loss together with hepatic steatosis and increased alanine aminotransferase (ALT) plasma levels, but no differences in IL-6, TNF, insulin or resistin. There were no substantial differences between TNAP+/− and WT mice fed the MCD diet. In turn, TNAP+/− mice receiving the control diet presented hepatic steatosis with alterations in metabolic parameters very similar to those induced by the MCD diet. Nevertheless, no weight loss, increased ALT plasma levels or hypoglycemia were observed. These mice also presented increased levels of liver TNF and systemic resistin and glucagon compared to WT mice. The phenotype of TNAP+/− mice fed a standard diet was normal. In conclusion, TNAP haplodeficiency induces steatosis comparable to that produced by a MCD diet when fed a control diet.Ministry of Economy and Competitivity of SpainEuropean Commission BFU2014-57736-P AGL2014-58883-R SAF2017-88457-R AGL2017-85270-RJunta de Andalucia CTS235 CTS164Ministry of EducationInstituto de Salud Carlos III European Commissio

Papel biológico de genes ubicados en una isla genómica en Pseudomonas aeruginosa

Pseudomonas aeruginosa es un patógeno humano oportunista Gram negativo omnipresente que no se considera parte de la microbiota intestinal comensal humana. Sin embargo, el agotamiento de la microbiota intestinal (disbiosis) después del tratamiento con antibióticos facilita la colonización del tracto intestinal por P. aeruginosa resistente a múltiples fármacos. Una posible alternativa a los tratamientos convencionales se basa en el uso de alimentos funcionales con actividad prebiótica. El efecto bifidogénico de fructooligosacáridos (FOS) está bien establecido, se ha demostrado que promueve el crecimiento de bacterias intestinales beneficiosas específicas como las bifidobacterias. Estudios previos del patógeno oportunista nosocomial Pseudomonas aeruginosa PAO1 han demostrado que los FOS reducen el crecimiento y la formación de biopelículas, debido a una disminución en la motilidad y la secreción de exotoxina. Sin embargo, la base transcripcional de estas alteraciones fenotípicas sigue sin estar clara. Para abordar esta pregunta, se realizó un análisis de secuencia de ARN (RNAseq) que nos permitió detectar la presencia de una isla genómica formada por 15 genes que fueron reprimidos en presencia de FOS. Anteriormente, se demostró mediante el análisis funcional de mutantes isogénicos, que los genes PA0643, PA0644 y PA0646, ubicados en esta isla génica, codifican proteínas involucradas en el crecimiento, la formación de biopelículas, la motilidad. En este contexto, este trabajo plasmo la implicación de estos genes en la modulación de la respuesta inflamatoria