RESUMEN
Genome-wide association studies have identified risk loci linked to inflammatory bowel disease (IBD)1-a complex chronic inflammatory disorder of the gastrointestinal tract. The increasing prevalence of IBD in industrialized countries and the augmented disease risk observed in migrants who move into areas of higher disease prevalence suggest that environmental factors are also important determinants of IBD susceptibility and severity2. However, the identification of environmental factors relevant to IBD and the mechanisms by which they influence disease has been hampered by the lack of platforms for their systematic investigation. Here we describe an integrated systems approach, combining publicly available databases, zebrafish chemical screens, machine learning and mouse preclinical models to identify environmental factors that control intestinal inflammation. This approach established that the herbicide propyzamide increases inflammation in the small and large intestine. Moreover, we show that an AHR-NF-κB-C/EBPß signalling axis operates in T cells and dendritic cells to promote intestinal inflammation, and is targeted by propyzamide. In conclusion, we developed a pipeline for the identification of environmental factors and mechanisms of pathogenesis in IBD and, potentially, other inflammatory diseases.
Asunto(s)
Ambiente , Herbicidas , Inflamación , Enfermedades Inflamatorias del Intestino , Intestinos , Animales , Ratones , Inflamación/inducido químicamente , Inflamación/etiología , Inflamación/inmunología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Pez Cebra , Aprendizaje Automático , Bases de Datos Factuales , Modelos Animales de Enfermedad , Intestinos/efectos de los fármacos , Intestinos/inmunología , Intestinos/metabolismo , Intestinos/patología , FN-kappa B , Proteína beta Potenciadora de Unión a CCAAT , Receptores de Hidrocarburo de Aril , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Herbicidas/efectos adversosRESUMEN
Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.
Asunto(s)
Adenosina Trifosfato/metabolismo , Apirasa/metabolismo , Enfermedades Inflamatorias del Intestino/terapia , Probióticos/uso terapéutico , Receptores Purinérgicos P2Y2/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Apirasa/genética , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Disbiosis/prevención & control , Femenino , Fibrosis/prevención & control , Microbioma Gastrointestinal/fisiología , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/patología , Humanos , Enfermedades Inflamatorias del Intestino/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Purinérgicos P2Y2/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets.
Asunto(s)
Astrocitos/fisiología , Comunicación Celular , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Microglía/fisiología , Esclerosis Múltiple/fisiopatología , Análisis de la Célula Individual , Animales , Antígenos CD/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Sistema Nervioso Central/fisiopatología , Encefalomielitis Autoinmune Experimental/patología , Efrina-B3/metabolismo , Herpesvirus Suido 1/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Esclerosis Múltiple/patología , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , RNA-Seq , Especies Reactivas de Oxígeno/metabolismo , Receptor EphB3/antagonistas & inhibidores , Receptor EphB3/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforinas/metabolismo , Transducción de Señal , Linfocitos T/fisiología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Metabolism has been shown to control peripheral immunity, but little is known about its role in central nervous system (CNS) inflammation. Through a combination of proteomic, metabolomic, transcriptomic, and perturbation studies, we found that sphingolipid metabolism in astrocytes triggers the interaction of the C2 domain in cytosolic phospholipase A2 (cPLA2) with the CARD domain in mitochondrial antiviral signaling protein (MAVS), boosting NF-κB-driven transcriptional programs that promote CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis. cPLA2 recruitment to MAVS also disrupts MAVS-hexokinase 2 (HK2) interactions, decreasing HK enzymatic activity and the production of lactate involved in the metabolic support of neurons. Miglustat, a drug used to treat Gaucher and Niemann-Pick disease, suppresses astrocyte pathogenic activities and ameliorates EAE. Collectively, these findings define a novel immunometabolic mechanism that drives pro-inflammatory astrocyte activities, outlines a new role for MAVS in CNS inflammation, and identifies candidate targets for therapeutic intervention.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Fosfolipasas A2 Secretoras/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , 1-Desoxinojirimicina/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Hexoquinasa/metabolismo , Humanos , Ácido Láctico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Fosfolipasas A2 Secretoras/genéticaRESUMEN
Genome-wide studies have identified genetic variants linked to neurologic diseases. Environmental factors also play important roles, but no methods are available for their comprehensive investigation. We developed an approach that combines genomic data, screens in a novel zebrafish model, computational modeling, perturbation studies, and multiple sclerosis (MS) patient samples to evaluate the effects of environmental exposure on CNS inflammation. We found that the herbicide linuron amplifies astrocyte pro-inflammatory activities by activating signaling via sigma receptor 1, inositol-requiring enzyme-1α (IRE1α), and X-box binding protein 1 (XBP1). Indeed, astrocyte-specific shRNA- and CRISPR/Cas9-driven gene inactivation combined with RNA-seq, ATAC-seq, ChIP-seq, and study of patient samples suggest that IRE1α-XBP1 signaling promotes CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, MS. In summary, these studies define environmental mechanisms that control astrocyte pathogenic activities and establish a multidisciplinary approach for the systematic investigation of the effects of environmental exposure in neurologic disorders.
Asunto(s)
Astrocitos/metabolismo , Sistema Nervioso Central/metabolismo , Animales , Sistema Nervioso Central/inmunología , Biología Computacional/métodos , Encefalomielitis Autoinmune Experimental/inmunología , Endorribonucleasas/metabolismo , Ambiente , Exposición a Riesgos Ambientales/efectos adversos , Genoma , Genómica , Humanos , Inflamación/metabolismo , Linurona/efectos adversos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores sigma/efectos de los fármacos , Receptores sigma/metabolismo , Transducción de Señal , Proteína 1 de Unión a la X-Box/metabolismo , Pez CebraRESUMEN
Microglia and astrocytes modulate inflammation and neurodegeneration in the central nervous system (CNS)1-3. Microglia modulate pro-inflammatory and neurotoxic activities in astrocytes, but the mechanisms involved are not completely understood4,5. Here we report that TGFα and VEGF-B produced by microglia regulate the pathogenic activities of astrocytes in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Microglia-derived TGFα acts via the ErbB1 receptor in astrocytes to limit their pathogenic activities and EAE development. Conversely, microglial VEGF-B triggers FLT-1 signalling in astrocytes and worsens EAE. VEGF-B and TGFα also participate in the microglial control of human astrocytes. Furthermore, expression of TGFα and VEGF-B in CD14+ cells correlates with the multiple sclerosis lesion stage. Finally, metabolites of dietary tryptophan produced by the commensal flora control microglial activation and TGFα and VEGF-B production, modulating the transcriptional program of astrocytes and CNS inflammation through a mechanism mediated by the aryl hydrocarbon receptor. In summary, we identified positive and negative regulators that mediate the microglial control of astrocytes. Moreover, these findings define a pathway through which microbial metabolites limit pathogenic activities of microglia and astrocytes, and suppress CNS inflammation. This pathway may guide new therapies for multiple sclerosis and other neurological disorders.
Asunto(s)
Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/microbiología , Microglía/metabolismo , Animales , Astrocitos/patología , Células Cultivadas , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/microbiología , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/prevención & control , Receptores ErbB/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Inflamación/prevención & control , Receptores de Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/patología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Receptores de Hidrocarburo de Aril/metabolismo , Simbiosis , Factor de Crecimiento Transformador alfa/biosíntesis , Factor de Crecimiento Transformador alfa/metabolismo , Triptófano/deficiencia , Triptófano/metabolismo , Factor B de Crecimiento Endotelial Vascular/biosíntesis , Factor B de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with important functions in the immune response and cancer. AHR agonists are provided by the environment, the commensal flora and the metabolism. Considering AHR physiological functions, AHR agonists may have important effects on health and disease. Thus, the quantification of AHR agonists in biological samples is of scientific and clinical relevance. We compared different reporter systems for the detection of AHR agonists in serum samples of Multiple Sclerosis (MS) patients, and assessed the influence of transfection methods and cell lines in a reporter-based in vitro assay. While the use of stable or transient reporters did not influence the measurement of AHR agonistic activity, the species of the cell lines used in these reporter assays had important effects on the reporter readings. These observations suggest that cell-specific factors influence AHR activation and signaling. Thus, based on the reported species selectivity of AHR ligands and the cell species-of-origin effects that we describe in this manuscript, the use of human cell lines is encouraged for the analysis of AHR agonistic activity in human samples. These findings may be relevant for the analysis of AHR agonists in human samples in the context of inflammatory and neoplastic disorders.