Caracterización de las células inmunes innatas y adaptativas durante una inflamación intestinal gatillada por la ingesta de harina de soya en larvas de pez cebra (Danio rerio)
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2018
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es
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Universidad Andrés Bello
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Resumen
La inflamación intestinal es una característica de varias patologías crónicas tales como las enfermedades inflamatorias del intestino (IBD, del inglés Inflammatory Bowel Disease). En esta condición ocurre una fuerte y permanente infiltración de neutrófilos en el intestino, además la participación de linfocitos con perfil Th1, Th2 y Th17.
A pesar de que diferentes compuestos químicos han sido usados en ratones para generar modelo de estudio de inflamación intestinal, ninguno en particular recapitula el cuadro observado en humanos, además no permiten estudiar el proceso inflamatorio in vivo. Como alternativa a los modelos desarrollados en mamíferos, nosotros proponemos el uso del pez cebra (Danio rerio) como animal modelo. En nuestro laboratorio hemos desarrollado en este pez teleósteo un nuevo modelo de inflamación intestinal, basado en la ingesta de harina de soya. Estudios previos de nuestro laboratorio demostraron que la ingesta de harina de soya por larvas de pez cebra gatilla una significativa migración de neutrófilos hacia el intestino, estrategia que permite detectar inflamación intestinal de forma precoz. En la presente tesis, caracterizamos con mayor profundidad este proceso, analizando la participación de otras células inmunes tanto innatas como adaptativas, como macrófagos, mastocitos y linfocitos. Así mismo, se estandarizaron dos modelos de inflamación; el primero que se caracteriza por ser un intestino que se enfrenta por primera vez a antígenos alimentarios (denominada alimentación breve). El segundo modelo se caracteriza por ser un intestino que ya recibe alimento hace varios días (denominado alimentación prolongada). Los resultados obtenidos mostraron que en la condición control, tanto en el modelo breve como prolongado, se produce un reclutamiento de neutrófilos, macrófagos y linfocitos al intestino, mientras que el número de mastocitos permanece constante. Una situación similar se observa durante un proceso inflamatorio, con la única salvedad de que el número de células reclutadas es significativamente mayor a la condición control. Es importante destacar que por primera vez se observa una respuesta linfocitaria durante un proceso intestinal en larvas de 5 días post fertilización. Anteriormente se había reportado que recién después de 3 semanas de desarrollo la inmunidad adaptativa estaba funcional. Así mismo, pudimos determinar que tanto un proceso inflamatorio breve como prolongado, el perfil de linfocitos Th17 es el predominante y que en condiciones control (de homoestasis) sólo en el modelo prolongado se observa un perfil linfocitario tolerogénico.
Intestinal inflammation is a characteristic of several chronic pathologies such as inflammatory bowel diseases (IBD). In this condition there is a strong and permanent infiltration of neutrophils in the intestine, in addition to the participation of lymphocytes with Th1, Th2 and Th17 profiles. Although different chemical compounds have been used in mice to generate a study model of intestinal inflammation, none of them recapitulates what is observed in humans, neither does allow the study of the inflammatory process in vivo. As an alternative to the models developed in mammals, we propose the use of the zebrafish (Danio rerio) as a model animal. In our laboratory we have developed in this teleost fish a new model of intestinal inflammation, based on the intake of soybean meal. Previous studies from our laboratory showed that the intake of soybean meal by zebrafish larvae triggers a significant migration of neutrophils to the intestine, a strategy that allows detecting intestinal inflammation early. In the present thesis, we characterize this process in greater depth, analyzing the participation of other innate and adaptive immune cells, such as macrophages, mast cells and lymphocytes. Likewise, two models of inflammation were standardized; the first one is characterized as an intestine that faces food antigens for the first time (called brief feeding). The second model is characterized by being an intestine that has been already receiving food for several days ago (called prolonged feeding). The results obtained showed that in the control condition, both in the short and prolonged model, there is a recruitment of neutrophils, macrophages and lymphocytes to the intestine, while the number of mast cells remains constant. A similar situation is observed during an inflammatory process, with the only exception that the number of cells recruited is significantly greater in comparison to the control condition. It is important to note that for the first time a lymphocyte response is observed during an intestinal process in larvae of 5 days after fertilization. Previously, it had been reported that adaptive immunity was functional only after 3 weeks of development. Accordingly, we were able to determine that both a brief and prolonged inflammatory process, the profile of Th17 lymphocytes is the predominant one and that under control conditions (of homoestasis) only in the prolonged model a tolerogenic lymphocyte profile is observed.
Intestinal inflammation is a characteristic of several chronic pathologies such as inflammatory bowel diseases (IBD). In this condition there is a strong and permanent infiltration of neutrophils in the intestine, in addition to the participation of lymphocytes with Th1, Th2 and Th17 profiles. Although different chemical compounds have been used in mice to generate a study model of intestinal inflammation, none of them recapitulates what is observed in humans, neither does allow the study of the inflammatory process in vivo. As an alternative to the models developed in mammals, we propose the use of the zebrafish (Danio rerio) as a model animal. In our laboratory we have developed in this teleost fish a new model of intestinal inflammation, based on the intake of soybean meal. Previous studies from our laboratory showed that the intake of soybean meal by zebrafish larvae triggers a significant migration of neutrophils to the intestine, a strategy that allows detecting intestinal inflammation early. In the present thesis, we characterize this process in greater depth, analyzing the participation of other innate and adaptive immune cells, such as macrophages, mast cells and lymphocytes. Likewise, two models of inflammation were standardized; the first one is characterized as an intestine that faces food antigens for the first time (called brief feeding). The second model is characterized by being an intestine that has been already receiving food for several days ago (called prolonged feeding). The results obtained showed that in the control condition, both in the short and prolonged model, there is a recruitment of neutrophils, macrophages and lymphocytes to the intestine, while the number of mast cells remains constant. A similar situation is observed during an inflammatory process, with the only exception that the number of cells recruited is significantly greater in comparison to the control condition. It is important to note that for the first time a lymphocyte response is observed during an intestinal process in larvae of 5 days after fertilization. Previously, it had been reported that adaptive immunity was functional only after 3 weeks of development. Accordingly, we were able to determine that both a brief and prolonged inflammatory process, the profile of Th17 lymphocytes is the predominant one and that under control conditions (of homoestasis) only in the prolonged model a tolerogenic lymphocyte profile is observed.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Enfermedad Inflamatoria Intestinal, Investigaciones, Peces como Animales de Laboratorio