Publication: The Zebrafish Rag1-Deficient Model Reveals the Complex Roles of Gut Microbiota, Lipid Mediators, and Telomerase in Inflammaging
Authors
Rodríguez Vidal, Juan Francisco
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Escuela Internacional de Doctorado
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García Ayala, Alfonsa ; Cabas Sánchez, Isabel ; Cayuela Fuentes, María Luisa
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Abstract
Introducción
El envejecimiento es un proceso biológico universal caracterizado por el deterioro progresivo de la función fisiológica y mayor riesgo de enfermedades crónicas. Entre sus bases destacan el acortamiento telomérico, la senescencia celular y la inflamación crónica de bajo grado (inflammaging), que forman una red interconectada capaz de impulsar la degeneración tisular. A su vez, la microbiota intestinal, que modula metabolismo e inmunidad, pierde diversidad con la edad y favorece permeabilidad intestinal e inflamación sistémica.
El pez cebra (Danio rerio), y en particular el mutante rag1 —deficiente en linfocitos T/B—, constituye un modelo idóneo para estudiar el eje inmunidad–microbiota–telómeros en el envejecimiento ligado a inflamación, dado su fenotipo inflamatorio, envejecimiento prematuro y su similitud con rasgos humanos de fragilidad.
Objetivos de la tesis
Esta tesis investiga cómo la pérdida de inmunidad adaptativa remodela la microbiota, altera la señalización por eicosanoides y se interconecta con la biología telomérica para acelerar el envejecimiento sistémico. Los objetivos fueron:
Caracterizar las alteraciones intestinales y sistémicas derivadas de la deficiencia de Rag1.
Determinar el papel causal de la microbiota intestinal en el envejecimiento prematuro de los peces mutantes.
Analizar cómo la enzima Lta4h modela la red de eicosanoides inflamatorios en este contexto.
Evaluar cómo el mantenimiento telomérico, y en particular la telomerasa de células inmunitarias, modula el fenotipo rag1-/-.
Metodología
Se emplearon aproximaciones multi-ómicas (transcriptómica, metagenómica, metatranscriptómica, lipidómica) combinadas con manipulación experimental de la microbiota intestinal (trasplantes fecales, tratamiento con antibióticos, suplementación fúngica), modelos genéticos dobles mutantes (rag1-/- lta4h-/- y rag1-/- tert-/-), y restauración dirigida de telomerasa en células hematopoyéticas. Se evaluaron histología, citometría, ensayos funcionales de permeabilidad intestinal, locomoción y supervivencia.
Resultados o conclusiones
Los peces mutantes en rag1 desarrollaron una enteropatía degenerativa con apoptosis epitelial, atrofia vellositaria e infiltrado inmune. A nivel molecular, se activaron vías de senescencia, remodelado de matriz extracelular e inflamación, con pérdida de progenitores intestinales y expansión de enterocitos estresados. Funcionalmente, mostraron mayor permeabilidad intestinal, translocación bacteriana, activación sistémica de NF-κB y deterioro locomotor.
La microbiota de los peces mutantes exhibió disbiosis profunda, con pérdida de Fusobacteriota y Bacteroidota y expansión de Proteobacteria. Esta composición es indispensable para la progresión de la enfermedad: la transferencia de microbiota disbiótica indujo inflamación en hospedadores silvestres, mientras que la depleción bacteriana por antibióticos prolongó la supervivencia y atenuó la inflamación. La micobiota también se alteró, con reducción de levaduras beneficiosas; mientras que su suplementación mejoró la supervivencia, aunque sin revertir el fenotipo.
El epitelio intestinal de los mutantes mostró una activación temprana del metabolismo del ácido araquidónico, con fuerte inducción lta4h. Los dobles mutantes rag1-/- lta4h-/- desviaron el metabolismo lipídico hacia rutas pro-resolutivas, pero presentaron mayor estrés oxidativo y patología agravada, evidenciando el papel dual de Lta4h en la inflamación.
Respecto al mantenimiento telomérico, la deficiencia de telomerasa empeoró el síndrome rag1-/- de forma dosis-dependiente. En contraste, la restauración de telomerasa específicamente en células hematopoyéticas revirtió la disbiosis, redujo la apoptosis intestinal, recuperó la morfología hepática, reprogramó la expresión génica inflamatoria a nivel local y sistémico y extendió la vida media unos 8 meses.
En conjunto, esta tesis demuestra que la pérdida de inmunidad adaptativa desencadena un eje de disbiosis intestinal, inflamación lipídica y disfunción telomérica que precipita el envejecimiento prematuro. Además, identifica la integridad telomérica hematopoyética, la modulación de eicosanoides y la manipulación de la microbiota como nodos clave de intervención para mitigar la inflamación crónica y extender la vida saludable.
Introduction Aging is a universal biological process characterized by the progressive decline of physiological function and an increased risk of chronic diseases. Among its hallmarks are telomere shortening, cellular senescence, and low-grade chronic inflammation (inflammaging), which form an interconnected network that drives tissue degeneration. At the same time, the gut microbiota, which modulates metabolism and immunity, loses diversity with age and promotes intestinal permeability and systemic inflammation. The zebrafish (Danio rerio), particularly the rag1 mutant —deficient in functional T and B lymphocytes—, constitutes an optimal model to study the immunity–microbiota–telomere axis in inflammation-associated aging, given its inflammatory phenotype, premature aging, and similarity to human frailty traits. Thesis objectives This thesis investigates how the loss of adaptive immunity reshapes the microbiota, alters eicosanoid signaling, and intersects with telomere biology to accelerate systemic aging. The objectives were: To characterize intestinal and systemic alterations derived from Rag1 deficiency. To determine the causal role of the gut microbiota in the premature aging of mutant fish. To analyze how the enzyme Lta4h shapes the inflammatory eicosanoid network in this context. To evaluate how telomere maintenance, particularly telomerase in immune cells, modulates the rag1-/- phenotype. Methodology Multi-omic approaches (transcriptomics, metagenomics, metatranscriptomics, lipidomics) were combined with experimental manipulation of the gut microbiota (fecal transplants, antibiotic treatment, fungal supplementation), double mutant models (rag1-/- lta4h-/- and rag1-/- tert-/-), and targeted restoration of telomerase in hematopoietic cells. Histology, flow cytometry, functional assays of intestinal permeability, locomotor activity, and survival were assessed. Results or conclusions rag1 mutant zebrafish developed degenerative enteropathy with epithelial apoptosis, villus atrophy, and immune infiltration. At the molecular level, senescence, extracellular matrix remodeling, and inflammatory pathways were activated, accompanied by loss of intestinal progenitors and expansion of stressed enterocytes. Functionally, mutants showed increased intestinal permeability, bacterial translocation, systemic NF-κB activation, and impaired locomotor activity. The microbiota of mutant fish displayed profound dysbiosis, with loss of Fusobacteriota and Bacteroidota and expansion of Proteobacteria. This composition was indispensable for disease progression: transfer of dysbiotic microbiota induced inflammation in wild-type hosts, while antibiotic-mediated bacterial depletion extended survival and reduced inflammation. The mycobiota was also altered, with a reduction in beneficial yeasts; supplementation improved survival, although without reversing the phenotype. The intestinal epithelium of mutants showed early activation of arachidonic acid metabolism, with strong induction of lta4h. Double mutants (rag1-/- lta4h-/-) redirected lipid metabolism toward pro-resolving pathways but exhibited increased oxidative stress and aggravated pathology, highlighting the dual role of Lta4h in inflammation. Regarding telomere maintenance, telomerase deficiency worsened the rag1-/- syndrome in a dose-dependent manner. In contrast, telomerase restoration specifically in hematopoietic cells reverted dysbiosis, reduced intestinal apoptosis, recovered liver morphology, reprogrammed inflammatory gene expression at both local and systemic levels, and extended median lifespan by about 8 months. Altogether, this thesis demonstrates that the loss of adaptive immunity triggers a triad of gut dysbiosis, lipid-driven inflammation, and telomere dysfunction that accelerates premature aging. Moreover, it identifies hematopoietic telomere integrity, eicosanoid modulation, and microbiota manipulation as key intervention nodes to mitigate chronic inflammation and extend healthy lifespan.
Introduction Aging is a universal biological process characterized by the progressive decline of physiological function and an increased risk of chronic diseases. Among its hallmarks are telomere shortening, cellular senescence, and low-grade chronic inflammation (inflammaging), which form an interconnected network that drives tissue degeneration. At the same time, the gut microbiota, which modulates metabolism and immunity, loses diversity with age and promotes intestinal permeability and systemic inflammation. The zebrafish (Danio rerio), particularly the rag1 mutant —deficient in functional T and B lymphocytes—, constitutes an optimal model to study the immunity–microbiota–telomere axis in inflammation-associated aging, given its inflammatory phenotype, premature aging, and similarity to human frailty traits. Thesis objectives This thesis investigates how the loss of adaptive immunity reshapes the microbiota, alters eicosanoid signaling, and intersects with telomere biology to accelerate systemic aging. The objectives were: To characterize intestinal and systemic alterations derived from Rag1 deficiency. To determine the causal role of the gut microbiota in the premature aging of mutant fish. To analyze how the enzyme Lta4h shapes the inflammatory eicosanoid network in this context. To evaluate how telomere maintenance, particularly telomerase in immune cells, modulates the rag1-/- phenotype. Methodology Multi-omic approaches (transcriptomics, metagenomics, metatranscriptomics, lipidomics) were combined with experimental manipulation of the gut microbiota (fecal transplants, antibiotic treatment, fungal supplementation), double mutant models (rag1-/- lta4h-/- and rag1-/- tert-/-), and targeted restoration of telomerase in hematopoietic cells. Histology, flow cytometry, functional assays of intestinal permeability, locomotor activity, and survival were assessed. Results or conclusions rag1 mutant zebrafish developed degenerative enteropathy with epithelial apoptosis, villus atrophy, and immune infiltration. At the molecular level, senescence, extracellular matrix remodeling, and inflammatory pathways were activated, accompanied by loss of intestinal progenitors and expansion of stressed enterocytes. Functionally, mutants showed increased intestinal permeability, bacterial translocation, systemic NF-κB activation, and impaired locomotor activity. The microbiota of mutant fish displayed profound dysbiosis, with loss of Fusobacteriota and Bacteroidota and expansion of Proteobacteria. This composition was indispensable for disease progression: transfer of dysbiotic microbiota induced inflammation in wild-type hosts, while antibiotic-mediated bacterial depletion extended survival and reduced inflammation. The mycobiota was also altered, with a reduction in beneficial yeasts; supplementation improved survival, although without reversing the phenotype. The intestinal epithelium of mutants showed early activation of arachidonic acid metabolism, with strong induction of lta4h. Double mutants (rag1-/- lta4h-/-) redirected lipid metabolism toward pro-resolving pathways but exhibited increased oxidative stress and aggravated pathology, highlighting the dual role of Lta4h in inflammation. Regarding telomere maintenance, telomerase deficiency worsened the rag1-/- syndrome in a dose-dependent manner. In contrast, telomerase restoration specifically in hematopoietic cells reverted dysbiosis, reduced intestinal apoptosis, recovered liver morphology, reprogrammed inflammatory gene expression at both local and systemic levels, and extended median lifespan by about 8 months. Altogether, this thesis demonstrates that the loss of adaptive immunity triggers a triad of gut dysbiosis, lipid-driven inflammation, and telomere dysfunction that accelerates premature aging. Moreover, it identifies hematopoietic telomere integrity, eicosanoid modulation, and microbiota manipulation as key intervention nodes to mitigate chronic inflammation and extend healthy lifespan.
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24-ene-2027
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