RESUMEN
Extracellular vesicles (EVs) play a major role in cell-to-cell communication in physiological and pathological conditions, and their manipulation may represent a promising therapeutic strategy. Microglia, the parenchymal mononuclear phagocytes of the brain, modulate neighboring cells also through the release of EVs. The production of custom EVs filled with desired molecules, possibly targeted to make their uptake cell specific, and their administration in biological fluids may represent a valid approach for drug delivery. We engineered a murine microglia cell line, BV-2, to release EVs overexpressing the endogenous "eat me" signal Lactadherin (Mfg-e8) on the surface to target phagocytes and containing the anti-inflammatory cytokine IL-4. A single injection of 107 IL-4+Mfg-e8+ EVs into the cisterna magna modulated established neuroinflammation and significantly reduced clinical signs in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Injected IL-4+Mfg-e8+ EVs target mainly phagocytes (i.e., macrophages and microglia) surrounding liquoral spaces, and their cargo promote the upregulation of anti-inflammatory markers chitinase 3-like 3 (ym1) and arginase-1 (arg1), significantly reducing tissue damage. Engineered EVs may represent a biological drug delivery tool able to deliver multiple functional molecules simultaneously to treat neuroinflammatory diseases.
Asunto(s)
Vesículas Extracelulares/metabolismo , Interleucina-4/metabolismo , Esclerosis Múltiple/metabolismo , Animales , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/ultraestructura , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Vesículas Extracelulares/ultraestructura , Femenino , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV-neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.
Asunto(s)
Vesículas Extracelulares/inmunología , Inflamación/metabolismo , MicroARNs/metabolismo , Neuroglía/inmunología , Neuronas/inmunología , Sinapsis/inmunología , Animales , Células Cultivadas , Líquido Cefalorraquídeo/metabolismo , Técnicas de Cocultivo , Vesículas Extracelulares/patología , Femenino , Hipocampo/inmunología , Hipocampo/patología , Humanos , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Neuroglía/patología , Plasticidad Neuronal/fisiología , Neuronas/patología , Cultivo Primario de Células , Ratas Sprague-Dawley , Sinapsis/patologíaRESUMEN
Microvesicles (MVs) are membrane particles of 200-500 nm released by all cell types constitutively. MVs of myeloid origin are found increased in the cerebrospinal fluid (CSF) of patients suffering from neuroinflammatory disorders, although the factors triggering their production have never been defined. Here, we report that both pro- and anti-inflammatory cytokines, specifically interferon-γ and interleukin-4, are equally able to stimulate the production of MVs from microglia cells and monocytes. Additionally, we found this process to be independent from the best characterized molecular pathway so far described for membrane shedding, which is centered on the purinergic receptor P2X7, whose activation by high concentrations of extracellular ATP (exATP) results in membrane blebbing operated by the secreted enzyme acid sphingomyelinase (ASMase). Moreover, a potent inhibitor of ASMase, injected in a mouse model of multiple sclerosis, failed to reduce the number of MVs in their CSF. This suggests that cytokines, rather than exATP, may exert a long-term control of MV production in the context of chronic inflammation, where both pro- and anti-inflammatory factors play coordinated roles.
Asunto(s)
Micropartículas Derivadas de Células/inmunología , Citocinas/inmunología , Esclerosis Múltiple/inmunología , Células Mieloides/inmunología , Transducción de Señal/inmunología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/inmunología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Línea Celular , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Imipramina/farmacología , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/metabolismo , Células Mieloides/metabolismo , Receptores Purinérgicos P2X7/inmunología , Receptores Purinérgicos P2X7/metabolismo , Retinoides/farmacología , Transducción de Señal/efectos de los fármacos , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/inmunología , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Muscular dystrophies are severe disorders due to mutations in structural genes, and are characterized by skeletal muscle wasting, compromised patient mobility, and respiratory functions. Although previous works suggested enhancing regeneration and muscle mass as therapeutic strategies, these led to no long-term benefits in humans. Mice lacking the transcription factor Nfix have delayed regeneration and a shift toward an oxidative fiber type. Here, we show that ablating or silencing the transcription factor Nfix ameliorates pathology in several forms of muscular dystrophy. Silencing Nfix in postnatal dystrophic mice, when the first signs of the disease already occurred, rescues the pathology and, conversely, Nfix overexpression in dystrophic muscles increases regeneration and markedly exacerbates the pathology. We therefore offer a proof of principle for a novel therapeutic approach for muscular dystrophies based on delaying muscle regeneration.