Rol de galectina-1 en la fisiopatología de las Enfermedades Inflamatorias Intestinales

Las Enfermedades Inflamatorias Intestinales (EII) son un conjunto de patologías crónicas multifactoriales en las cuales se produce una respuesta inmune aberrante contra componentes de la microbiota intestinal. Dentro de las EII, la colitis ulcerosa (CU) y la enfermedad de Crohn (EC) son las dos formas más estudiadas. Si bien existen diferencias en la presentación clínica de estas patologías, en ambos casos los linfocitos T (LT) de la mucosa sobre-expresan citoquinas pro-inflamatorias del perfil Th1. La cronicidad de este proceso desencadena complicaciones tales como la formación de abscesos, fístulas, estenosis e inclusive la necesidad de resección quirúrgica de diversos segmentos del intestino debido a lo extenso del daño. Galectina-1 (Gal-1) es una lectina endógena con propiedades anti-infamatorias ampliamente documentadas. Entre los distintos efectos descriptos para esta proteína podríamos mencionar el control de la sobrevida de los LT Th1 y la modulación en la producción de citoquinas. En el presente trabajo de tesis se propuso estudiar la expresión de Gal-1 en la mucosa colónica humana y murina en situaciones fisiológicas y patológicas; analizar la correlación entre la producción de esta lectina y la actividad inflamatoria del tejido y su capacidad de modular la sobrevida de los LT y de las células epiteliales intestinales (CEI) en dichos entornos. Pudimos demostrar que el análisis conjunto de la expresión intestinal de diferentes galectinas permite distinguir entre pacientes con EII activa y pacientes control, EII inactiva o en remisión. Además, concluimos que este análisis es una herramienta eficiente para determinar el grado de inflamación de dicha mucosa y distinguirla de otras patologías inflamatorias intestinales agudas y crónicas. Por lo tanto, podría constituir un marcador de la historia de la patología en el paciente. En cuanto a su rol en la fisiología intestinal, en homeostasis Gal-1 ejerce un efecto pro-apoptótico sobre los LT de la lámina propia colónica, efecto que permite controlar la activación celular frente al constante desafío antigénico por parte de la microbiota. Además, comprobamos que la respuesta fisiológica del intestino frente a un estímulo inflamatorio consiste en inducir un aumento en los niveles de expresión de esta lectina. Sin embargo, este mecanismo compensatorio resulta inefectivo dado que los LT de la lámina propia alteran su maquinaria de glicosilacióin, inhibiendo la expresión de los ligandos específicos para Gal-1. Asimismo, las proteasas presentes en el entorno inflamatorio tisular degradan a esta proteína. Ambos efectos en la lámina propia favorecen la persistencia del foco inflamatorio y la no resolución del daño tisular. Por otra parte, la inflamación sostenida incrementa el porcentaje de CEI que expresan los ligandos para esta lectina. Este fenómeno intensifica el efecto pro-apoptótico que Gal-1 ejerce sobre dichas células. Este mecanismo contribuye a la perdida de la integridad epitelial, la alteración de la permeabilidad intestinal y propicia la traslocación de los antígenos del lumen a la lámina propia. Por lo tanto, en condiciones de homeostasis, Gal-1 contribuiría a la normal fisiología del intestino. Sin embargo, en procesos inflamatorios crónicos, dicho rol se vería alterado, hecho que propiciaría la continuidad de la enfermedad. Una mejor comprensión de los mecanismos que gobiernan la expresión de los ligandos específicos de esta lectina, y el rol que ésta ejerce sobre otras células del sistema inmune, permitirá plantear nuevas terapias o procedimientos para restablecer el glicofenotipo de los LT y de las CEI y, de esta manera, controlar la inflamación intestinalFacultad de Ciencias Exacta

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine

Abstract: Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals-1-) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals-1- mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.Facultad de Ciencias Exacta

Galactomannan as a potential modulator of intestinal ischemia–reperfusion injury

Background: Galactomannan (GAL), a polysaccharide present on the cell wall of several fungi, has shown an ability to modulate inflammatory responses through the dectin-1 receptor in human macrophages. However, studies evaluating the modulatory properties of this polysaccharide in in vivo inflammatory scenarios are scarce. We hypothesized that GAL pretreatment would modulate local and remote damage related to intestinal reperfusion after an ischemic insult. Materials and methods: Adult male Balb/c mice were subjected to intestinal ischemia–reperfusion injury by reversible occlusion of the superior mesenteric artery, consisting of 45 min of ischemia followed by 3 or 24 h of reperfusion. Intragastric GAL (70 mg/kg) was administered 12 h before ischemia, and saline solution was used in the control animals. Jejunum, lung, and blood samples were taken for the analysis of histology, gene expression, plasma cytokine levels, and nitrosative stress. Results: Intestinal and lung histologic alterations were attenuated by GAL pretreatment, showing significant differences compared with nontreated animals. Interleukin 1β, monocyte chemoattractant protein 1, and IL-6 messenger RNA expression were considerably downregulated in the small intestine of the GAL group. In addition, GAL treatment significantly prevented plasma interleukin 6 and monocyte chemoattractant protein 1 upregulation and diminished nitrate and nitrite levels after 3 h of intestinal reperfusion. Conclusions: GAL pretreatment constitutes a novel and promising therapy to reduce local and remote damage triggered by intestinal ischemia–reperfusion injury. Further in vivo and in vitro studies to understand GAL's modulatory effects are warranted.Fil: Stringa, Pablo. Hospital Universitario la Paz; EspañaFil: Toledano, Victor. Hospital Universitario la Paz; EspañaFil: Papa Gobbi, Rodrigo. Hospital Universitario la Paz; EspañaFil: Arreola, Miguel. Hospital Universitario la Paz; EspañaFil: Largo, Carlota. Hospital Universitario la Paz; EspañaFil: Machuca, Mariana. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Aguirre, Luis A.. Hospital Universitario la Paz; EspañaFil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: López Collazo, Eduardo. Hospital Universitario la Paz; EspañaFil: Hernández Oliveros, Francisco. Hospital Universitario la Paz; Españ

A galectin-specific signature in the gut delineates Crohn’s disease and ulcerative colitis from other human inflammatory intestinal disorders

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions of the gastrointestinal tract including Crohn’s disease (CD) and ulcerative colitis (UC). Galectins, defined by shared consensus amino acid sequence and affinity for b-galactosides, are critical modulators of the inflammatory response. However, the relevance of the galectin network in the pathogenesis of human IBD has not yet been explored. Here, we analyzed the expression of relevant members of the galectin family in intestinal biopsies, and identified their contribution as novel mucosal markers in IBD. Colonic biopsies were obtained from 59 IBD patients (22 CD and 37 UC), 9 patients with gut rejection after transplantation, 8 adult celiac patients, and 32 non-IBD donors. Galectin mRNA expression was analyzed by RT-PCR and qPCR using specific primers for individual galectins. A linear discriminant analysis (LDA) was used to analyze galectin expression in individual intestinal samples. Expression of common mucosalassociated galectins (Gal-1, 23, 24, 29) is dysregulated in inflamed tissues of IBD patients compared with noninflamed IBD or control samples. LDA discriminated between different inflammation grades in active IBD and showed that remission IBD samples were clusterized with control samples. Galectin profiling could not distinguish CD and UC. Furthermore, inflamed IBD was discriminated from inflamed tissue of rejected gut in transplanted patients and duodenum of celiac patients, which could not be distinguished from control duodenum samples. The integrative analysis of galectins discriminated IBD from other intestinal inflammatory conditions and could be used as potential mucosal biomarker.Instituto de Estudios Inmunológicos y Fisiopatológico

Experimental Assessment of Intestinal Damage in Controlled Donation After Circulatory Death for Visceral Transplantation

There is an urgent need to address the shortage of potential multivisceral grafts in order to reduce the average time in waiting list. Since donation after circulatory death (DCD) has been successfully employed for other solid organs, a thorough evaluation of the use of intestinal grafts from DCD is warranted. Here, we have generated a model of Maastricht III DCD in rodents, focusing on the viability of intestinal and multivisceral grafts at five (DCD5) and twenty (DCD20) minutes of cardiac arrest compared to living and brain death donors. DCD groups exhibited time-dependent damage. DCD20 generated substantial intestinal mucosal injury and decreased number of Goblet cells whereas grafts from DCD5 closely resemble those of brain death and living donors groups in terms intestinal morphology, expression of tight junction proteins and number of Paneth and Globet cells. Upon transplantation, intestines from DCD5 showed increased ischemia/reperfusion damage compared to living donor grafts, however mucosal integrity was recovered 48 h after transplantation. No differences in terms of graft rejection, gene expression and absorptive function between DCD5 and living donor were observed at 7 post-transplant days. Collectively, our results highlight DCD as a possible strategy to increase multivisceral donation and transplantation procedures.Instituto de Estudios Inmunológicos y FisiopatológicosConsejo Nacional de Investigaciones Científicas y Técnica

Novel coronavirus (SARS-CoV-2) infection in a patient with multivisceral transplant

Coronavirus disease 2019 (COVID-19) pandemic has become one of the most challenging episodes in the history of modern public health, with particular emphasis in high-risk population. However, the evidence regarding their response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent responsible for COVID-19 is scant. Herein, we present the clinical and therapeutic course of a SARS-CoV-2 infection in a patient with multivisceral transplant and a recent tuberculosis infection.Instituto de Estudios Inmunológicos y Fisiopatológico

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine

Abstract: Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals-1-) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals-1- mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.Facultad de Ciencias Exacta

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model

Background. Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function.Methods. ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed. Results. The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR. Conclusions. Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.Facultad de Ciencias MédicasInstituto de Estudios Inmunológicos y FisiopatológicosLaboratorio y Programa de Trasplante de Organos, Tejidos y CélulasInstituto de Investigaciones Bioquímicas de La Plat

Galactomannan as a Potential Modulator of Intestinal Ischemia-Reperfusion Injury

Background: Galactomannan (GAL), a polysaccharide present on the cell wall of several fungi, has shown an ability to modulate inflammatory responses through the dectin-1 receptor in human macrophages. However, studies evaluating the modulatory properties of this polysaccharide in in vivo inflammatory scenarios are scarce. We hypothesized that GAL pretreatment would modulate local and remote damage related to intestinal reperfusion after an ischemic insult. Materials and methods: Adult male Balb/c mice were subjected to intestinal ischemia–reperfusion injury by reversible occlusion of the superior mesenteric artery, consisting of 45 min of ischemia followed by 3 or 24 h of reperfusion. Intragastric GAL (70 mg/kg) was administered 12 h before ischemia, and saline solution was used in the control animals. Jejunum, lung, and blood samples were taken for the analysis of histology, gene expression, plasma cytokine levels, and nitrosative stress. Results: Intestinal and lung histologic alterations were attenuated by GAL pretreatment, showing significant differences compared with nontreated animals. Interleukin 1β, monocyte chemoattractant protein 1, and IL-6 messenger RNA expression were considerably downregulated in the small intestine of the GAL group. In addition, GAL treatment significantly prevented plasma interleukin 6 and monocyte chemoattractant protein 1 upregulation and diminished nitrate and nitrite levels after 3 h of intestinal reperfusion. Conclusions: GAL pretreatment constitutes a novel and promising therapy to reduce local and remote damage triggered by intestinal ischemia–reperfusion injury. Further in vivo and in vitro studies to understand GAL's modulatory effects are warranted.Facultad de Ciencias VeterinariasInstituto de Estudios Inmunológicos y Fisiopatológico

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model

Background. Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function.Methods. ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed. Results. The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR. Conclusions. Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.Facultad de Ciencias MédicasInstituto de Estudios Inmunológicos y FisiopatológicosLaboratorio y Programa de Trasplante de Organos, Tejidos y CélulasInstituto de Investigaciones Bioquímicas de La Plat