Funciones de las células T reguladoras en el control inflamatorio e integridad de la barrera intestina en la cirrosis

Antecedentes: Las infecciones bacterianas suponen una complicación frecuente en la cirrosis avanzada siendo la más recurrente la peritonitis bacteriana espontánea (PBE). El mecanismo patogénico que explica su desarrollo es la traslocación bacteriana (TB), que se produce por una disbiosis de la microbiota intestinal, un incremento en la permeabilidad intestinal a antígenos bacterianos y una alteración en la respuesta inmune del huésped. La inflamación inducida por la TB de bacterias o de sus productos durante la cirrosis contribuye a su progresión y facilita el desarrollo de complicaciones. El uso de estrategias enfocadas a disminuir los episodios de TB es deseable en estos pacientes. El tratamiento con quinolonas poco absorbibles, norfloxacino (Nflx), como medida profiláctica para reducir la carga entérica de bacterias ha demostrado reducir la incidencia de la PBE, pero se ha relacionado con el desarrollo de resistencias. El estudio de los mecanismos por los que el Nflx ejerce un efecto inmunomodulador puede ayudar a la comprensión de la interacción entre la microbiota y su huésped. En nuestro grupo hemos descrito la implicación de la interleucina (IL)-10 en este efecto. Las células T reguladoras (Treg) son una población de células T CD4+ productoras de IL-10, que resulta clave en la inducción de homeostasis intestinal al restringir las respuestas proinflamatorias. Objetivos: Identificar si la población Treg está involucrada en el mecanismo por el que el Nflx compensa el ambiente inflamatorio durante la cirrosis, así como evaluar el efecto que ejerce la homeostasis intestinal inducida por esta población celular en la prevención de episodios de TB durante la cirrosis experimental. Métodos: En la tesis doctoral se han realizado experimentos utilizando muestras de pacientes cirróticos y ratones con cirrosis inducida por administración de CCl4 por vía intragástrica durante 12 semanas. De esta forma, se incluyeron pacientes ingresados de forma consecutiva con cirrosis y líquido ascítico (LA) que presentasen: PBE, LA no infectado y en tratamiento con Nflx como profilaxis secundaria de PBE (grupo DIS). Las células Treg de sangre periférica de pacientes se definieron mediante citometría de flujo como células CD4+CD25+FoxP3+. Se evaluó la señalización coestimuladora de células dendríticas (CDs) humanas y se midieron los niveles séricos de Nflx e IL-10. Para conocer la contribución específica de las Treg, los protocolos de daño hepático fueron realizados sobre ratones wild type (WT) y Recombination activating gene (Rag)-1 knockout (Rag1-/-). Así, se realizaron experimentos de transferencia de células T CD4+ vírgenes y Treg procedentes de ratones WT no cirróticos a ratones inmunodeficientes Rag1-/- con cirrosis. Subgrupos de animales fueron tratados con Nflx, así como estimulados con Escherichia coli (E. coli) por vía oral. En estos animales se evaluó: 1) la señalización coestimuladora en CDs, 2) los niveles sistémicos de interferón (IFN)-γ, IL-2 e IL-10, 3) la TB de ADN bacteriano (ADNbact), lipopolisacárido (LPS) y dextrano, 4) los niveles de expresión génica y proteica de proteínas tight-junction (TJ) en muestras de colon, 5) el perfil Th y producción génica de IL-10 de linfocitos intestinales en respuesta a E. coli y 6) los niveles de ácidos grasos de cadena corta (AGCCs) antes y después de la administración de E. coli. Resultados: En el primer estudio se incluyeron 84 pacientes. El porcentaje de Treg aumentó significativamente en pacientes del grupo DIS en comparación con pacientes con PBE o con LA no infectado. Se observó una correlación positiva entre Treg y los niveles séricos de Nflx e IL-10. Las CDs de pacientes con DIS mostraron una disminución significativa en la expresión de CD80 y CD86 en comparación con pacientes con PBE y con LA no infectado y se correlacionó con los niveles de Nflx. La modulación de la señalización coestimuladora por Nflx no se detectó en ratones Rag1-/- ni en ratones Rag1-/- reconstituidos con células T vírgenes. Sin embargo, la administración de células T vírgenes y Treg se asoció con una expresión significativamente disminuida de CD80 y CD86 en presencia de Nflx. Por último, el efecto inmunomodulador del Nflx sobre la reducción de IL-2 e IFN-γ y sobre el aumento de IL-10 se logró significativamente sólo cuando se restauró la población Treg en ratones Rag1-/-. En el segundo estudio se observó que los ratones Rag1-/- cotransferidos con células T vírgenes y Treg mostraron una reducción significativa en la permeabilidad intestinal a la TB de antígenos bacterianos y se recuperó la expresión de las proteínas TJ en el colon de ratones Rag1-/-. La administración de células T vírgenes y Treg en ratones Rag1-/- restringió la diferenciación proinflamatoria Th1/Th17 de los linfocitos intestinales en respuesta a E. coli. La concentración de los principales AGCCs resultó en una reducción significativa en ratones Rag1-/- después de la administración oral de E. coli, pero permaneció inalterada en ratones Rag1-/- cotransferidos con células T vírgenes y Treg. De la misma forma, la expresión de los receptores de AGCCs se redujo por el tratamiento con E. coli pero sus niveles fueron restablecidos en ratones reconstituidos con linfocitos T vírgenes y Treg en ratones Rag1-/-. Conclusiones: Estos resultados muestran la implicación de las Treg en el mecanismo por el cual el Nflx ejerce efectos inmunomoduladores más allá de su efecto bactericida en la cirrosis. Además, la mejora en la homeostasis intestinal inducida por este tipo celular al restringir la permeabilidad intestinal, limitar la diferenciación inflamatoria local de los linfocitos intestinales y disminuir la pérdida de AGCCs refuerzan el valor de esta población celular en la prevención de la TB en la cirrosis

Paneth Cells Regulate Lymphangiogenesis under Control of Microbial Signals during Experimental Portal Hypertension

Intestinal microbiota can modulate portal hypertension through the regulation of the intestinal vasculature. We have recently demonstrated that bacterial antigens activate Paneth cells (PCs) to secrete products that regulate angiogenesis and portal hypertension. In the present work we hypothesized that Paneth cells regulate the development of lymphatic vessels under the control of intestinal microbiota during experimental portal hypertension. We used a mouse model of inducible PCs depletion (Math1Lox/LoxVilCreERT2) and performed partial portal vein ligation (PPVL) to induce portal hypertension. After 14 days, we performed mRNA sequencing and evaluated the expression of specific lymphangiogenic genes in small intestinal tissue. Intestinal and mesenteric lymphatic vessels proliferation was assessed by immunohistochemistry. Intestinal organoids with or without PCs were exposed to pathogen-associated molecular patterns, and conditioned media (CM) was used to stimulate human lymphatic endothelial cells (LECs). The lymphangiogenic activity of stimulated LECs was assessed by tube formation and wound healing assays. Secretome analysis of CM was performed using label-free proteomics quantification methods. Intestinal immune cell infiltration was evaluated by immunohistochemistry. We observed that the intestinal gene expression pattern was altered by the absence of PCs only in portal hypertensive mice. We found a decreased expression of specific lymphangiogenic genes in the absence of PCs during portal hypertension, resulting in a reduced proliferation of intestinal and mesenteric lymphatic vessels as compared to controls. In vitro analyses demonstrated that lymphatic tube formation and endothelial wound healing responses were reduced significantly in LECs treated with CM from organoids without PCs. Secretome analyses of CM revealed that PCs secrete proteins that are involved in lipid metabolism, cell growth and proliferation. Additionally, intestinal macrophages infiltrated the ileal mucosa and submucosa of mice with and without Paneth cells in response to portal hypertension. Our results suggest that intestinal microbiota signals stimulate Paneth cells to secrete factors that modulate the intestinal and mesenteric lymphatic vessels network during experimental portal hypertension

Paneth Cells Regulate Lymphangiogenesis under Control of Microbial Signals during Experimental Portal Hypertension.

Intestinal microbiota can modulate portal hypertension through the regulation of the intestinal vasculature. We have recently demonstrated that bacterial antigens activate Paneth cells (PCs) to secrete products that regulate angiogenesis and portal hypertension. In the present work we hypothesized that Paneth cells regulate the development of lymphatic vessels under the control of intestinal microbiota during experimental portal hypertension. We used a mouse model of inducible PCs depletion (Math1Lox/LoxVilCreERT2) and performed partial portal vein ligation (PPVL) to induce portal hypertension. After 14 days, we performed mRNA sequencing and evaluated the expression of specific lymphangiogenic genes in small intestinal tissue. Intestinal and mesenteric lymphatic vessels proliferation was assessed by immunohistochemistry. Intestinal organoids with or without PCs were exposed to pathogen-associated molecular patterns, and conditioned media (CM) was used to stimulate human lymphatic endothelial cells (LECs). The lymphangiogenic activity of stimulated LECs was assessed by tube formation and wound healing assays. Secretome analysis of CM was performed using label-free proteomics quantification methods. Intestinal immune cell infiltration was evaluated by immunohistochemistry. We observed that the intestinal gene expression pattern was altered by the absence of PCs only in portal hypertensive mice. We found a decreased expression of specific lymphangiogenic genes in the absence of PCs during portal hypertension, resulting in a reduced proliferation of intestinal and mesenteric lymphatic vessels as compared to controls. In vitro analyses demonstrated that lymphatic tube formation and endothelial wound healing responses were reduced significantly in LECs treated with CM from organoids without PCs. Secretome analyses of CM revealed that PCs secrete proteins that are involved in lipid metabolism, cell growth and proliferation. Additionally, intestinal macrophages infiltrated the ileal mucosa and submucosa of mice with and without Paneth cells in response to portal hypertension. Our results suggest that intestinal microbiota signals stimulate Paneth cells to secrete factors that modulate the intestinal and mesenteric lymphatic vessels network during experimental portal hypertension

Improved hemodynamic and liver function in portal hypertensive cirrhotic rats after administration of B. pseudocatenulatum CECT 7765

Purpose: Evaluating whether changes in gut microbiota induced by a bifidobacterial strain may have an effect on the hepatic vascular function in portal hypertensive cirrhotic rats.Methods: Bile duct ligation (BDL) was performed in rats. A subgroup of animals received B. pseudocatenulatum CECT7765 (109 cfu/daily ig.) for 1 week prior to laparotomy. Hemodynamic, biochemical and inflammatory markers were evaluated. Ileal microbiota composition was identified. Statistical analysis was performed.Results: Sham-operated (n = 6), BDL (n = 6) and BDL treated with bifidobacteria (n = 8) rats were included. B. pseudocatenulatum CECT7765 significantly decreased proteobacteria (p = 0.001) and increased Bacteroidetes (p = 0.001) relative abundance. The bifidobacteria decreased the Firmicutes/Bacteroidetes ratio in the BDL model (p = 0.03). BDL with bifidobacteria vs BDL rats showed: significantly reduced portal vein area, portal flow, congestion index, alkaline phosphatase and total bilirubin, significantly increased serum cytokines and nitric oxide levels, gene expression levels of bile acids receptor FXR and endothelial nitric oxide synthase. Quantitative changes in the Clostridiales and Bacteroidales orders were independently associated with variations in portal vein area and portal flow, while changes in the Proteobacteria phylum were independently associated with congestion. Variations in all liver function markers significantly correlated with total OTUs mainly in the Firmicutes, but only changes in the Clostridiales were independently associated with alkaline phosphatase in the ANCOVA analysis.Conclusion: Hemodynamic alterations and liver dysfunction induced by BDL in rats are partially restored after oral administration of B. pseudocatenulatum CECT7765. Results provide a proof-of-concept for the beneficial effect of this bifidobacterial strain in reducing complications derived from portal hypertension in cirrhosis

Bacterial antigen translocation and age as BMI-independent contributing factors on systemic inflammation in NAFLD patients

Background & Aims Low‐grade systemic inflammation is a crucial landmark in NAFLD favouring disease progression and comorbidities. We evaluated the input of circulating bacterial antigens on systemic markers of inflammation in NAFLD patients. Patients & Methods Multicenter cross‐sectional study including consecutive patients with biopsy‐proven NAFLD. Demographic, metabolic and fibrosis‐related variables were collected. Circulating bacterial antigens were quantified in blood. Toll‐like receptor SNPs were genotyped. Serum cytokine levels were evaluated. Peripheral blood mononuclear cell response to bacterial antigens was evaluated in vitro. Results Three hundred and fifteen patients from five Spanish hospitals were distributed by BMI. At least, one bacterial antigenic type was found in 66 patients with BMI 30 (77.3%) (P = .014). HOMA‐IR was significantly higher in the presence of circulating antigens among patients with BMI < 30. NASH and significant fibrosis in non‐obese patients were more frequent in the presence of at least two circulating antigenic types. Allelic frequencies of TLR variants were similar to controls and did not affect clinical or laboratory parameters. Pro‐inflammatory cytokines were significantly increased in patients with bacterial antigens, regardless of BMI. TLR gene and protein expression levels were significantly increased in PBMCs from patients with bacterial antigens. Antigen concentrations independently influenced TNF‐α and IL‐6, in both BMI subgroups of patients. Age independently influenced TNF‐α and IL‐6 in non‐obese patients, and TNF‐α in obese patients. Conclusion Serum circulating bacterial antigens as well as age were BMI‐independent factors related to increased systemic inflammation in NAFLD and provides insight on the multifaceted sources of inflammation in these patients.This work has been funded by grants PI16/0967 and PI17/0535 from Instituto de Salud Carlos III, Madrid, Spain, and PROMETEO 2016/001 from Generalitat Valenciana, Valencia, Spain. IGH was recipient of a Young Investigator Grant by CIBERehd, Instituto de Salud Carlos III, Madrid, Spain

Circulating levels of butyrate are inversely related to portal hypertension, endotoxemia, and systemic inflammation in patients with cirrhosis

Short-chain fatty acids (SCFAs) are gut microbiota-derived products that participate in maintaining the gut barrier integrity and host's immune response. We hypothesize that reduced SCFA levels are associated with systemic inflammation, endotoxemia, and more severe hemodynamic alterations in cirrhosis. Patients with cirrhosis referred for a hepatic venous pressure gradient (HVPG) measurement (n = 62) or a transjugular intrahepatic portosystemic shunt placement (n = 12) were included. SCFAs were measured in portal (when available), hepatic, and peripheral blood samples by GC-MS. Serum endotoxins, proinflammatory cytokines, and NO levels were quantified. SCFA levels were significantly higher in portal vs. hepatic and peripheral blood. There were inverse relationships between SCFAs and the severity of disease. SCFAs (mainly butyric acid) inversely correlated with the model for end-stage liver disease score and were further reduced in patients with history of ascites, hepatic encephalopathy, and spontaneous bacterial peritonitis. There was an inverse relationship between butyric acid and HVPG values. SCFAs were directly related with systemic vascular resistance and inversely with cardiac index. Butyric acid inversely correlated with inflammatory markers and serum endotoxin. A global reduction in the blood levels of SCFA in patients with cirrhosis is associated with a more advanced liver disease, suggesting its contribution to disease progression.-Juanola, O., Ferrusquía-Acosta, J., García-Villalba, R., Zapater, P., Magaz, M., Marín, A., Olivas, P., Baiges, A., Bellot, P., Turon, F., Hernández-Gea, V., González-Navajas, J. M., Tomás-Barberán, F. A., García-Pagán, J. C., Francés, R. Circulating levels of butyrate are inversely related to portal hypertension, endotoxemia, and systemic inflammation in patients with cirrhosis

Aula de innovación educativa

Resumen basado en el de la publicaciónTítulo, resumen, palabras clave en español e inglesSe descrbe cómo en el marco del proyecto Erasmus+ Think Global, la escuela de educación infantil y primaria Pembroke Dock Community School de Gales ha contribuido al desarrollo de la competencia global y al logro de los objetivos de desarrollo sostenible por medio del proyecto La vida submarina, que promueve la adopción de medidas para el bienestar colectivo y el desarrollo sostenible, la participación, la igualdad de género y las interacciones interculturales.Biblioteca del Ministerio de Educación y Formación Profesional; Calle San Agustín, 5; 28014 Madrid; Tel. +34917748000; [email protected]

Non-alcoholic fatty liver disease: metabolic, genetic, epigenetic and environmental risk factors

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of chronic liver disease in the Western world, probably due to the growing prevalence of obesity, metabolic diseases, and exposure to some environmental agents. In certain patients, simple hepatic steatosis can progress to non-alcoholic steatohepatitis (NASH), which can sometimes lead to liver cirrhosis and its complications including hepatocellular carcinoma. Understanding the mechanisms that cause the progression of NAFLD to NASH is crucial to be able to control the advancement of the disease. The main hypothesis considers that it is due to multiple factors that act together on genetically predisposed subjects to suffer from NAFLD including insulin resistance, nutritional factors, gut microbiota, and genetic and epigenetic factors. In this article, we will discuss the epidemiology of NAFLD, and we overview several topics that influence the development of the disease from simple steatosis to liver cirrhosis and its possible complications

Peripheral blood regulatory T cells and disease activity, quality of life, and outcomes in children with juvenile idiopathic arthritis

Abstract Objectives To measure regulatory T cell (Treg) levels in the peripheral blood of children with juvenile idiopathic arthritis (JIA) and analyse the association of this measure with disease activity, quality of life, adjustment of treatment, and hospitalisation. Methods We conducted a two-phase study (cross-sectional and prospective), including consecutive children with a JIA diagnosis according to ILAR criteria. Our independent variables were Tregs, Th1, Th2, and cytokines in peripheral blood, and our dependent variables in the cross-sectional phase were arthritis category, JIA activity, and patient-reported outcomes. To test associations, we used Spearman’s correlation coefficient and the Mann-Whitney U test. In the prospective phase, we explored the probability of treatment adjustment and hospitalisation for JIA during follow-up according to Tregs levels at baseline, using Cox proportional regression. Results Our sample included 87 participants (median age 11 years, 63.2% girls). Tregs were not associated with most variables of interest. However, we found that higher Tregs concentration was associated with lower erythrocyte sedimentation rate (ESR) and better subjective disease status and course, while higher IL-10 and TGF-β levels were associated with lower ESR, less pain, and better subjective disease status We found no association between Tregs and treatment adjustments or hospitalisation. Conclusions Higher baseline Treg levels in the peripheral blood of children with JIA may be associated with reduced disease activity and better quality of life, though were not informative on the inflammatory progression on the follow-up

Gut bacterial DNA translocation is an independent risk factor of flare at short term in patients with crohn's disease

OBJECTIVES We aimed at evaluating bacterial DNA (bactDNA) presence in blood of Crohn's disease (CD) patients in remission as an independent risk factor of flare at 6 months. METHODS This is a prospective, multicenter study on CD patients with Crohn's disease activity index (CDAI)150 in the following 6 months. BactDNA in blood, the nucleotide-binding oligomerization domain containing 2 (NOD2) genotype, and serum cytokine levels were determined at baseline. RESULTS A total of 288 patients were included. BactDNA was detected in 98 patients (34.0%). A variant-NOD2 genotype was identified in 114 patients (39.6%). Forty patients (14%) relapsed during follow-up. Multivariate survival analysis identified bactDNA as an independent risk factor of flare (hazard ratio (HR) 8.75 (4.02-19.06) 95% confidence interval (CI)). Hospitalization, surgery, switch of treatment, initiation and escalation of anti-tumor necrosis factor (TNF) therapy, steroids initiation, and increased fecal calprotectin levels at 6 months were associated with bactDNA at baseline. A logistic regression analysis showed bactDNA as an independent and significant predictive factor of hospitalization (odds ratio (OR) 11.9 (3.4-42.3); P<0.001), steroids startup (OR 8.5 (2.7-27.1); P<0.001), and switch of treatment (OR 3.5 (1.6-7.7); P=0.002) at 6 months. No relationship was observed between bactDNA and mucosal lesions in patients with colonoscopy at admission. Serum pro-inflammatory cytokines were significantly increased in patients with bactDNA or a variant-NOD2 genotype. The combination of both factors induced decreased anti-TNF-α levels and a higher percentage of patients on intensified anti-TNF therapy. CONCLUSIONS BactDNA is an independent risk factor of relapse at 6 months in CD patients. BactDNA is also independently associated with an increased risk of hospitalization, switch of treatment, and steroids initiation