Patogénesis de enfermedad celíaca: estudio de los mecanismos de daño en la mucosa intestinal

La enfermedad celíaca (EC) es una enteropatía crónica de base inmune que se desarrolla en individuos genéticamente susceptibles ante la ingesta de un grupo de proteínas derivadas del gluten. Su prevalencia está estimada en un 1-3%. En EC se observa daño a nivel de la mucosa del intestino delgado, caracterizado por atrofia de vellosidades, hiperplasia de criptas y un marcado infiltrado linfocitario, tanto a nivel de la lamina propria como en el compartimento intraepitelial. Estos cambios conducen a la pérdida de la funcionalidad del intestino delgado y a un síndrome de malabsorción asociado a diversos signos clínicos. La EC es un desorden multigénico, cuyo factor de susceptibilidad genética más importante está ubicado en el locus del HLA. Prácticamente el 100% de los pacientes, presenta los alelos del HLA codificantes para las moléculas DQ2 o DQ8. Por fuera de este locus, existen varias regiones que han sido asociadas a EC y que albergan genes con funciones inmunes. El infiltrado de linfocitos T CD4+ (Th1) específicos de péptidos de gluten y restringidos al HLA-DQ2/DQ8, presentes en la lamina propria de la mucosa duodenal, juega un rol relevante al activarse y producir IFNγ, aportando un microambiente altamente proinflamatorio. También es crítico el accionar de los linfocitos intraepiteliales, que se ven aumentados en el curso de la patología, y cuya citotoxicidad sobre enterocitos colaboraría en la instauración de la atrofia de la mucosa. Varios son los aspectos de la inmunopatogenia que aún no fueron descriptos. En este trabajo de Tesis abordaremos el estudio de genes candidatos, los mecanismos de migración y de muerte celular en la mucosa de intestino delgado. Implementando una estrategia de elección de genes candidato posicionales, hemos evidenciado tres genes diferencialmente expresados en el tejido blanco de esta patología: RUNX3, THEMIS y PTPRK, definiéndolos de esta manera como candidatos funcionales. Hemos demostrado además, la participación del eje quimiotáctico CXCR3/CXCL10 en el reclutamiento activo de células mediadoras de daño hacia la mucosa intestinal de pacientes con EC activa, tanto en eventos disparadores de la enfermedad como en el sostenimiento del proceso inflamatorio crónico. Estos resultados sientan precedentes para la evaluación de la determinación sérica de CXCL10 como biomarcador en EC y la inhibición de esta quimoquina como una posible estrategia terapéutica. Finalmente, hemos abordado el análisis de la expresión de moléculas asociadas a estrés de retículo y a vías de muerte celular, pudiendo evidenciar un epitelio intestinal con un marcado estrés de retículo. Nuestras observaciones sugieren que distintas vías de muerte, y no sólo la apoptótica extrínseca como había sido extensamente propuesto, podrían participar activamente en los mecanismos de daño en la mucosa intestinal en EC activa.Facultad de Ciencias Exacta

Transglutaminase 2 expression is enhanced synergistically by interferon-γ and tumour necrosis factor-α in human small intestine

Transglutaminase 2 (TG2) is expressed ubiquitously, has multiple physiological functions and has also been associated with inflammatory diseases, neurodegenerative disorders, autoimmunity and cancer. In particular, TG2 is expressed in small intestine mucosa where it is up-regulated in active coeliac disease (CD). The aim of this work was to investigate the induction of TG2 expression by proinflammatory cytokines [interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-15] and the signalling pathways involved, in human epithelial and monocytic cells and in intestinal tissue from controls and untreated CD patients. Here we report that IFN-γ was the most potent inducer of TG2 expression in the small intestinal mucosa and in four [Caco-2, HT-29, Calu-6 and human acute monocytic leukaemia cell line (THP-1)] of five cell lines tested. The combination of TNF-α and IFN-γ produced a strong synergistic effect. The use of selective inhibitors of signalling pathways revealed that induction of TG2 by IFN-γ was mediated by phosphoinositide 3-kinase (PI3K), while c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were required for TNF-α activation. Quantitative polymerase chain reaction (PCR), flow cytometry and Western blot analysis showed that TG2 expression was blocked completely when stimulation by either TNF-α or IFN-γ was performed in the presence of nuclear factor (NF)-κB inhibitors (sulphasalazine and BAY-117082). TG2 was up-regulated substantially by TNF-α and IFN-γ in intestinal mucosa in untreated CD compared with controls. This study shows that IFN-γ, a dominant cytokine in intestinal mucosa in active CD, is the most potent inducer of TG2, and synergism with TNF-α may contribute to exacerbate the pathogenic mechanism of CD. Selective inhibition of signalling pathways may be of therapeutic benefit. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.Fil: Bayardo, Mariela Paula. 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: Punzi, F.. 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: Bondar, Constanza María. 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: Chopita, Nestor. 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: Chirdo, Fernando Gabriel. 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; Argentin

Intraluminal administration of poly I:C causes an enteropathy that is exacerbated by administration of oral dietary antigen

Systemic administration of polyinosinic:polycytidylic acid (poly I:C), mimics virally-induced activation of TLR3 signalling causing acute small intestine damage, but whether and how mucosal administration of poly I:C causes enteropathy is less clear. Our aim was to investigate the inflammatory pathways elicited after intraluminal administration of poly I:C and determine acute and delayed consequences of this locally induced immune activation. Intraluminal poly I:C induced rapid mucosal immune activation in C57BL/6 mice involving IFNβ and the CXCL10/CXCR3 axis, that may drive inflammation towards a Th1 profile. Intraluminal poly I:C also caused enteropathy and gut dysfunction in gliadin-sensitive NOD-DQ8 mice, and this was prolonged by concomitant oral administration of gliadin. Our results indicate that small intestine pathology can be induced in mice by intraluminal administration of poly I:C and that this is exacerbated by subsequent oral delivery of a relevant dietary antigen.Laboratorio de Investigaciones del Sistema Inmun

Role of CXCR3/CXCL10 axis in immune cell recruitment into the small intestine in celiac disease

Lymphocytic infiltration in the lamina propria (LP), which is primarily composed of CD4+ Th1 cells and plasma cells, and increased numbers of intraepithelial lymphocytes (IELs), is a characteristic finding in active celiac disease (CD). Signals for this selective cell recruitment have not been fully established. CXCR3 and its ligands, particularly CXCL10, have been suggested to be one of the most relevant pathways in the attraction of cells into inflamed tissues. In addition, CXCR3 is characteristically expressed by Th1 cells. The aim of this work was to investigate the participation of the chemokine CXCL10/CXCR3 axis in CD pathogenesis. A higher concentration of CXCL10 was found in the serum of untreated CD patients. The mRNA levels of CXCL10 and CXCL11 but not CXCL9 were significantly higher in duodenal biopsies from untreated CD patients compared with non-CD controls or treated patients. The results demonstrate that CXCL10 is abundantly produced in untreated CD and reduced in treated patients, and the expression of CXCL10 was found to be correlated with the IFNγ levels in the tissue. Plasma cells and enterocytes were identified as CXCL10-producing cells. Moreover, the CXCL10 expression in intestinal tissues was upregulated by poly I:C and IL-15. IELs, LP T lymphocytes, and plasma cells, which infiltrate the intestinal mucosa in untreated CD, express CXCR3. The CXCR3/CXCL10 signalling axis is overactivated in the small intestinal mucosa in untreated patients, and this finding explains the specific recruitment of the major cell populations that infiltrate the epithelium and the LP in CD.Facultad de Ciencias Exacta

Unraveling the mystery of Gaucher bone density pathophysiology

Gaucher disease (GD) is caused by pathogenic mutations in GBA1, the gene that encodes the lysosomal enzyme β-glucocerebrosidase. Despite the existence of a variety of specific treatments for GD, they cannot completely reverse bone complications. Many studies have evidenced the impairment in bone tissue of GD, and molecular mechanisms of bone density alterations in GD are being studied during the last years and different reports emphasized its efforts trying to unravel why and how bone tissue is affected. The cause of skeletal density affection in GD is a matter of debates between research groups. and there are two opposing hypotheses trying to explain reduced bone mineral density in GD: increased bone resorption versus impaired bone formation. In this review, we discuss the diverse mechanisms of bone alterations implicated in GD revealed until the present, along with a presentation of normal bone physiology and its regulation. With this information in mind, we discuss effectiveness of specific therapies, introduce possible adjunctive therapies and present a novel model for GD-associated bone density pathogenesis. Under the exposed evidence, we may conclude that both sides of the balance of remodeling process are altered. In GD the observed osteopenia/osteoporosis may be the result of contribution of both reduced bone formation and increased bone resorption.Fil: Rozenfeld, Paula Adriana. 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: Crivaro, Andrea Natalia. 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: Ormazabal, Maximiliano Emanuel. 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: Mucci, Juan Marcos. 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: Bondar, Constanza María. 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: Delpino, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentin

Efficacy of pentosan polysulfate in in vitro models of lysosomal storage disorders: Fabry and Gaucher Disease

Gaucher and Fabry diseases are the most prevalent sphingolipidoses. Chronic inflammation is activated in those disorders, which could play a role in pathogenesis. Significant degrees of amelioration occur in patients upon introduction of specific therapies; however, restoration to complete health status is not always achieved. The idea of an adjunctive therapy that targets inflammation may be a suitable option for patients. PPS is a mixture of semisynthetic sulfated polyanions that have been shown to have anti-inflammatory effects in mucopolysaccharidosis type I and II patients and animal models of type I, IIIA and VI. We hypothesized PPS could be a useful adjunctive therapy to inflammation for Gaucher and Fabry diseases. The objective of this work is to analyze the in vitro effect of PPS on inflammatory cytokines in cellular models of Gaucher and Fabry diseases, and to study its effect in Gaucher disease associated in vitro bone alterations. Cultures of peripheral blood mononuclear cells from Fabry and Gaucher patients were exposed to PPS. The secretion of proinflammatory cytokines was significantly reduced. Peripheral blood cells exposed to PPS from Gaucher patients revealed a reduced tendency to differentiate to osteoclasts. Osteoblasts and osteocytes cell lines were incubated with an inhibitor of glucocerebrosidase, and conditioned media was harvested in order to analyze if those cells secrete factors that induce osteoclastogenesis. Conditioned media from this cell cultures exposed to PPS produced lower numbers of osteoclasts. We could demonstrate PPS is an effective molecule to reduce the production of proinflammatory cytokines in in vitro models of Fabry and Gaucher diseases. Moreover, it was effective at ameliorating bone alterations of in vitro models of Gaucher disease. These results serve as preclinical supportive data to start clinical trials in human patients to analyze the effect of PPS as a potential adjunctive therapy for Fabry and Gaucher diseases.Fil: Crivaro, Andrea Natalia. 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: Mucci, Juan Marcos. 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: Bondar, Constanza María. 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: Ormazabal, Maximiliano Emanuel. 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: Ceci, Romina. 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: Simonaro, Calogera. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Rozenfeld, Paula Adriana. 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; Argentin

Osteocyte alterations induce osteoclastogenesis in an in vitro model of gaucher disease

Gaucher disease (GD) is caused by mutations in the glucosylceramidase β (GBA 1) gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to the accumulation of the glycolipid glucocerebroside in the lysosomes of cells, mainly in the monocyte/macrophage lineage. Its mildest form is Type I GD, characterized by non-neuronopathic involvement. Bone compromise is the most disabling aspect of the Gaucher disease. However, the pathophysiological aspects of skeletal alterations are not yet fully understood. The bone tissue homeostasis is maintained by a balance between resorption of old bone by osteoclasts and new bone formation by osteoblasts. A central player in this balance is the osteocyte as it controls both processes. We studied the involvement of osteocytes in an in vitro chemical model of Gaucher disease. The osteocyte cell line MLO-Y4 was exposed to conduritol-β-epoxide (CBE), an inhibitor of GCase, for a period of 7, 14 and 21 days. Conditioned media from CBE-treated osteocytes was found to induce osteoclast differentiation. GCase inhibition caused alterations in Cx43 expression and distribution pattern and an increase in osteocyte apoptosis. Osteoclast differentiation involved osteocyte apoptotic bodies, receptor activator of nuclear factor κ-B ligand (RANKL) and soluble factors. Thus, our results indicate that osteocytes may have a role to play in the bone pathophysiology of GD.Instituto de Estudios Inmunológicos y Fisiopatológico

Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Estudios Inmunológicos y Fisiopatológico

Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Estudios Inmunológicos y Fisiopatológico

In vitro osteoclastogenesis from Gaucher patients' cells correlates with bone mineral density but not with Chitotriosidase

Gaucher disease (GD) is caused by mutations on the gene encoding for the lysosomal enzyme glucocerebrosidase. Type I GD (GD1) patients present anemia, hepatosplenomegaly and bone alterations. In spite of treatment, bone alterations in GD patients persist, including poor bone mineral density (BMD). Mechanisms leading to bone damage are not completely understood, but previous reports suggest that osteoclasts are involved. Chitotriosidase (CHIT) is the most reliable biomarker used in the follow up of patients, although its correlation with bone status is unknown. The aim of this work was to study the pro-osteoclastogenic potential in patients and to evaluate its correlation with CHIT activity levels and clinical parameters. PBMCs from treated patients and healthy controls were cultured in the presence of M-CSF, and mature osteoclasts were counted. BMD, blood CHIT activity and serum levels of CTX, BAP, and cytokines were evaluated in patients. We found that blood CHIT activity and osteoclast differentiation were significantly increased in patients, but no correlation between them was observed. Interestingly, osteoclast numbers but not CHIT, presented a negative correlation with BMD expressed as Z-score. CTX, BAP and serum cytokines involved in bone remodeling were found altered in GD1 patients. These results show for the first time a correlation between osteoclast differentiation and BMD in GD1 patients, supporting the involvement of osteoclasts in the bone pathology of GD1. Our results also suggest that an altered immune response may play an important role in bone damage.Instituto de Estudios Inmunológicos y FisiopatológicosLaboratorio de Investigación en Osteopatías y Metabolismo Minera