Regulación de la dinámica mitocondrial en la Sinapsis Inmunológica de Linfocitos B y sus implicancias funcionales
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2024
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Los linfocitos B son células del sistema inmune adaptativo capaces de reconocer antígenos procedentes de células presentadoras de antígeno, como macrófagos, y extraerlos, procesarlos y presentarlos en moléculas de histocompatibilidad de clase II a los linfocitos T. Este proceso ocurre mediante la formación de la sinapsis inmunológica (SI). Cuando el receptor de células B (BCR) reconoce el antígeno desencadena la activación de los linfocitos B, iniciando vías de señalización que conducen a la reorganización de microtúbulos y actina hacia la SI. Además, el posicionamiento del centro organizador de microtúbulos (MTOC) establece una polaridad celular que facilita el reclutamiento de organelos, tales como: los lisosomas y las mitocondrias.En los linfocitos B, las mitocondrias desempeñan un papel metabólico que influye en procesos como la diferenciación hacia células plasmáticas o el cambio de clase de inmunoglobulina. Sin embargo, su papel en el establecimiento de la SI sigue siendo un tema poco explorado. En este trabajo, evaluamos la organización de las mitocondrias durante la formación de la SI y el impacto de la pérdida de función de Drp1, una GTPasa involucrada en la fisión mitocondrial, en el establecimiento de la SI y las funciones de los linfocitos B, particularmente su capacidad para extraer y presentar antígenos. Para ello, se activaron linfocitos B con beads recubiertas con antígenos específicos para el BCR o con coverslips que imitan la SI, lo que permitió la visualización y cuantificación de los componentes intracelulares y la evaluación de la extracción antigénica. La dinámica mitocondrial se interrumpió utilizando 50 µM de mdivi-1 antes de la activación en cada experimento. La extracción de antígenos se midió evaluando la cantidad remanente de antígeno en las beads incubadas con linfocitos B en diferentes condiciones, mientras que la presentación de antígenos se evaluó mediante un ensayo de co-cultivo con un hibridoma de linfocitos T que reconoce un antígeno específico.Nuestros resultados indican que Drp1 es reclutado a la SI y fosforilado en su forma activa en respuesta a la activación del BCR. Este proceso ocurre simultáneamente con el desplazamiento de mitocondrias y lisosomas hacia la SI. Además, la interrupción de la dinámica mitocondrial afectó el reclutamiento del MTOC y las mitocondrias a la SI, acompañado de un aumento de α-tubulina acetilada alrededor del MTOC y una reducción en el spreading celular de los linfocitos B. Nuestros hallazgos también revelan una disminución de la fusión lisosomal en la SI cuando la dinámica mitocondrial está alterada, lo que lleva a una menor extracción y presentación de antígenos a los linfocitos T.En conjunto, nuestros resultados sugieren que la fisión mitocondrial mediada por Drp1 es esencial para el establecimiento de la SI y la función de los linfocitos B. El reclutamiento mitocondrial a la SI permite la función lisosomal, que es crítica para el desarrollo de la SI. Este estudio proporciona nuevas perspectivas sobre la modulación de las respuestas inmunes a través de las mitocondrias, potencialmente relevante para enfermedades inflamatorias o autoinmunes relacionadas con disfunciones mitocondriales.
B lymphocytes are cells of the adaptive immune system capable of recognizing antigens from antigen-presenting cells, such as macrophages, extracting, processing, and presenting them on class II histocompatibility molecules to T lymphocytes. This process occurs through the formation of the immunological synapse (IS). Recognition of antigens by the B-cell receptor (BCR), triggers the activation of B cells, initiating signaling pathways that lead to microtubule, and actin reorganization toward the IS. Additionally, the positioning of the microtubule-organizing center (MTOC) establishes cellular polarity that facilitates the recruitment of organelles such as lysosomes and mitochondria. In B lymphocytes, mitochondria play a metabolic role that influences processes involving the differentiation into plasma cells or immunoglobulin class switching. However, their role in the establishment of an IS remains poorly explored. In this study, we evaluated mitochondrial organization during IS formation and the impact of Drp1 loss of function—a GTPase involved in mitochondrial fission—on IS establishment and B cell functions, particularly their ability to extract and present antigens. To achieve this, B lymphocytes were activated with antigen-coated beads specific to the BCR or coverslips mimicking the IS, enabling visualization and quantification of intracellular components and assessment of antigen extraction. Mitochondrial dynamic was disrupted using 50 µM mdivi-1 prior to activation in each experiment. Antigen extraction was measured by evaluating the remaining amount of antigens on beads incubated with B cells under different conditions, while antigen presentation was assessed through a co-culture assay with a T-cell hybridoma recognizing a specific antigen. Our results indicate that Drp1 is recruited to the IS and phosphorylated into its activeform in response to BCR activation. This process occurs concomitantly with the displacementof mitochondria and lysosomes towards the IS. Furthermore, disruption of mitochondrialdynamic impaired MTOC and mitochondrial recruitment to the IS, accompanied by increasedacetylated α-tubulin around the MTOC and reduced B cell spreading. Our findings also revealdecreased lysosomal fusion at the IS when mitochondrial dynamics are attenuated, leadingto reduced antigen extraction and presentation to T lymphocytes Overall, our results suggest that mitochondrial fission mediated by Drp1 is essentialfor IS establishment and B cell function. Mitochondrial recruitment to the IS enables lysosomalfunction critical for IS development. This study offers new insights into modulating immuneresponses through mitochondria, potentially relevant to inflammatory or autoimmunediseases linked to mitochondrial dysfunction.
B lymphocytes are cells of the adaptive immune system capable of recognizing antigens from antigen-presenting cells, such as macrophages, extracting, processing, and presenting them on class II histocompatibility molecules to T lymphocytes. This process occurs through the formation of the immunological synapse (IS). Recognition of antigens by the B-cell receptor (BCR), triggers the activation of B cells, initiating signaling pathways that lead to microtubule, and actin reorganization toward the IS. Additionally, the positioning of the microtubule-organizing center (MTOC) establishes cellular polarity that facilitates the recruitment of organelles such as lysosomes and mitochondria. In B lymphocytes, mitochondria play a metabolic role that influences processes involving the differentiation into plasma cells or immunoglobulin class switching. However, their role in the establishment of an IS remains poorly explored. In this study, we evaluated mitochondrial organization during IS formation and the impact of Drp1 loss of function—a GTPase involved in mitochondrial fission—on IS establishment and B cell functions, particularly their ability to extract and present antigens. To achieve this, B lymphocytes were activated with antigen-coated beads specific to the BCR or coverslips mimicking the IS, enabling visualization and quantification of intracellular components and assessment of antigen extraction. Mitochondrial dynamic was disrupted using 50 µM mdivi-1 prior to activation in each experiment. Antigen extraction was measured by evaluating the remaining amount of antigens on beads incubated with B cells under different conditions, while antigen presentation was assessed through a co-culture assay with a T-cell hybridoma recognizing a specific antigen. Our results indicate that Drp1 is recruited to the IS and phosphorylated into its activeform in response to BCR activation. This process occurs concomitantly with the displacementof mitochondria and lysosomes towards the IS. Furthermore, disruption of mitochondrialdynamic impaired MTOC and mitochondrial recruitment to the IS, accompanied by increasedacetylated α-tubulin around the MTOC and reduced B cell spreading. Our findings also revealdecreased lysosomal fusion at the IS when mitochondrial dynamics are attenuated, leadingto reduced antigen extraction and presentation to T lymphocytes Overall, our results suggest that mitochondrial fission mediated by Drp1 is essentialfor IS establishment and B cell function. Mitochondrial recruitment to the IS enables lysosomalfunction critical for IS development. This study offers new insights into modulating immuneresponses through mitochondria, potentially relevant to inflammatory or autoimmunediseases linked to mitochondrial dysfunction.
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Tesis (Doctor en Ciencias con mención en Biología Celular y Molecular)--Pontificia Universidad Católica de Chile, 2024