Estudio de internalización y mecanismos de endocitosis de las vesículas de membrana externa de Prevotella copri en macrófago
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Fecha
2024
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Facultad/escuela
Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
La Artritis Reumatoide (AR) es una enfermedad autoinmune por etapas en donde la inflamación
crónica generada en tejido sinovial puede causar deformidad en distintas articulaciones como manos,
rodillas, muñecas, pies, entre otros. Se describe como enfermedad autoinmune debido a la presencia
de autoanticuerpos como el factor reumatoide (FR) y anticuerpos anti-peptidos citrulinadas (ACPA).
Hay distintos factores genéticos y ambientales que pueden influenciar en el padecimiento de AR
como la dieta occidental, fumar, la sobreabundancia o disminución de algún microorganismo en la
microbiota intestinal de la persona.
Distintos estudios han encontrado directa relación entre la disbiosis en la microbiota gastrointestinal
y la inflamación dada en el tejido sinovial de las articulaciones tanto en pacientes como en modelos
murinos para AR. La bacteria anaerobia Gram negativo Prevotella copri se encuentra normalmente
en la microbiota de las personas, pero en situaciones de AR se encuentra en mayor abundancia. Se ha
reportado que el péptido de P. copri denominado Pc-p27 es presentado por la molécula MHC-II de
las células presentadoras de antígenos, como los macrófagos, desencadenando respuesta de células T
y la producción de anticuerpos en pacientes con AR. Por otra parte, se encuentran las vesículas de
membrana externa (OMV) que desempeñan un papel fundamental en la interacción bacteria-huésped
gracias a que presentan estructuras de la bacteria parental como LPS, proteínas de membrana,
peptidoglucanos, etc.
El principal objetivo que tiene este proyecto es evaluar el mecanismo de endocitosis utilizado por los
macrófagos para internalizar las OMVs de Prevotella copri. Se realizó el cultivo anaerobio de P.
copri para la obtención de sus OMVs y posterior incubación en macrófagos primarios derivados de
PBMC adquiriendo imágenes mediante microscopía confocal. También se realizó estimulación con
OMVs en células RAW264.7 para realizar cinéticas de internalización e inhibición de vías de
endocitosis mediante inhibidores farmacológicos.
Se obtuvieron imágenes con señal fluorescente de las OMVs-DiO en macrófagos primarios, cinética
internalización de las OMVs durante 2 horas, internalización de OMVs-DiO dependiente de
concentración, además de sugerirse que la vía de internalización de las vesículas fue la endocitosis
mediada por clatrinas.
La relevancia de este proyecto fue comprender el mecanismo por el cual células presentadoras de
antígenos como los macrófagos pueden internalizar las OMVs pertenecientes a una bacteria
relacionada actualmente con el desarrollo de la artritis reumatoide.
Rheumatoid arthritis (RA) is a staged autoimmune disease where chronic inflammation generated in synovial tissue can cause deformity in different joints such as hands, knees, wrists, feet, among others. It is described as an autoimmune disease due to the presence of autoantibodies such as rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA). There are different genetic and environmental factors that can influence the development of RA such as western diet, smoking, overabundance or decrease of some microorganism in the intestinal microbiota of the person. Several studies have found a direct relationship between dysbiosis in the gastrointestinal microbiota and inflammation in the synovial tissue of joints in both patients and murine models of RA. The Gram-negative anaerobic bacterium Prevotella copri is normally found in the microbiota of humans, but in RA situations it is found in greater abundance. It has been reported that the P. copri peptide called Pc-p27 is presented by the MHC-II molecule of antigen-presenting cells, such as macrophages, triggering T-cell responses and antibody production in patients with RA. On the other hand, outer membrane vesicles (OMVs) play a fundamental role in the bacteria-host interaction because they present structures of the parental bacteria such as LPS, membrane proteins, peptidoglycans. The main objective of this project is to evaluate the endocytosis mechanism used by macrophages to internalize Prevotella copri OMVs. Anaerobic culture of P. copri was performed to obtain its OMVs and subsequent incubation in primary PBMC-derived macrophages, acquiring images by confocal microscopy. Stimulation with OMVs was also performed in RAW264.7 cells to perform internalization kinetics and inhibition of endocytosis pathways by means of pharmacological inhibitors. Fluorescent signal imaging of OMVs-DiO in primary macrophages, 2-hour OMV internalization kinetics, a concentration-dependent increased internalization of OMVs-DiO, and the suggestion that the vesicle internalization pathway was clathrin-mediated endocytosis were obtained. The relevance of this project was to understand the mechanism by which antigen-presenting cells such as macrophages can internalize OMVs belonging to a bacterium currently linked to the development of rheumatoid arthritis.
Rheumatoid arthritis (RA) is a staged autoimmune disease where chronic inflammation generated in synovial tissue can cause deformity in different joints such as hands, knees, wrists, feet, among others. It is described as an autoimmune disease due to the presence of autoantibodies such as rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA). There are different genetic and environmental factors that can influence the development of RA such as western diet, smoking, overabundance or decrease of some microorganism in the intestinal microbiota of the person. Several studies have found a direct relationship between dysbiosis in the gastrointestinal microbiota and inflammation in the synovial tissue of joints in both patients and murine models of RA. The Gram-negative anaerobic bacterium Prevotella copri is normally found in the microbiota of humans, but in RA situations it is found in greater abundance. It has been reported that the P. copri peptide called Pc-p27 is presented by the MHC-II molecule of antigen-presenting cells, such as macrophages, triggering T-cell responses and antibody production in patients with RA. On the other hand, outer membrane vesicles (OMVs) play a fundamental role in the bacteria-host interaction because they present structures of the parental bacteria such as LPS, membrane proteins, peptidoglycans. The main objective of this project is to evaluate the endocytosis mechanism used by macrophages to internalize Prevotella copri OMVs. Anaerobic culture of P. copri was performed to obtain its OMVs and subsequent incubation in primary PBMC-derived macrophages, acquiring images by confocal microscopy. Stimulation with OMVs was also performed in RAW264.7 cells to perform internalization kinetics and inhibition of endocytosis pathways by means of pharmacological inhibitors. Fluorescent signal imaging of OMVs-DiO in primary macrophages, 2-hour OMV internalization kinetics, a concentration-dependent increased internalization of OMVs-DiO, and the suggestion that the vesicle internalization pathway was clathrin-mediated endocytosis were obtained. The relevance of this project was to understand the mechanism by which antigen-presenting cells such as macrophages can internalize OMVs belonging to a bacterium currently linked to the development of rheumatoid arthritis.
Notas
Memoria (Ingeniera en Biotecnología)
Palabras clave
Artritis Reumatoide, Bacterias Anaeróbicas, Macrófagos