Functional roles of extracellular vesicles derived from microglia with diverse activation states (original) (raw)
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Inflammation leads to distinct populations of extracellular vesicles from microglia
Journal of Neuroinflammation, 2018
Background: Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS). Microglia-derived extracellular vesicles (EVs) are suggested to be involved in propagation of inflammatory signals and in the modulation of cell-to-cell communication. However, there is a lack of knowledge on the regulation of EVs and how this in turn facilitates the communication between cells in the brain. Here, we characterized microglial EVs under inflammatory conditions and investigated the effects of inflammation on the EV size, quantity, and protein content. Methods: We have utilized western blot, nanoparticle tracking analysis (NTA), and mass spectrometry to characterize EVs and examine the alterations of secreted EVs from a microglial cell line (BV2) following lipopolysaccharide (LPS) and tumor necrosis factor (TNF) inhibitor (etanercept) treatments, or either alone. The inflammatory responses were measured with multiplex cytokine ELISA and western blot. We also subjected TNF knockout mice to experimental stroke (permanent middle cerebral artery occlusion) and validated the effect of TNF inhibition on EV release. Results: Our analysis of EVs originating from activated BV2 microglia revealed a significant increase in the intravesicular levels of TNF and interleukin (IL)-6. We also observed that the number of EVs released was reduced both in vitro and in vivo when inflammation was inhibited via the TNF pathway. Finally, via mass spectrometry, we identified 49 unique proteins in EVs released from LPS-activated microglia compared to control EVs (58 proteins in EVs released from LPSactivated microglia and 37 from control EVs). According to Gene Ontology (GO) analysis, we found a large increase of proteins related to translation and transcription in EVs from LPS. Importantly, we showed a distinct profile of proteins found in EVs released from LPS treated cells compared to control. Conclusions: We demonstrate altered EV production in BV2 microglial cells and altered cytokine levels and protein composition carried by EVs in response to LPS challenge. Our findings provide new insights into the potential roles of EVs that could be related to the pathogenesis in neuroinflammatory diseases.
Acta Neuropathologica
Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.
Neuroscience, 2010
Brain damage, such as ischemic stroke, enhances proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ). To date, no reliable in vitro systems, which can be used to unravel the potential mechanisms underlying this lesion-induced effect, have been established. Here, we developed an ex vivo method to investigate how the proliferation of NSPCs changes over time after experimental stroke or excitotoxic striatal lesion in the adult rat brain by studying the effects of microglial cells derived from an injured brain on NSPCs. We isolated NSPCs from the SVZ of brains with lesions and analyzed their growth and differentiation when cultured as neurospheres. We found that NSPCs isolated from the brains 1-2 weeks following injury consistently generated more and larger neurospheres than those harvested from naive brains. We attributed these effects to the presence of microglial cells in NSPC cultures that originated from injured brains. We suggest that the effects are due to released factors because we observed increased proliferation of NSPCs isolated from non-injured brains when they were exposed to conditioned medium from cultures containing microglial cells derived from injured brains. Furthermore, we found that NSPCs derived from injured brains were more likely to differentiate into neurons and oligodendrocytes than astrocytes. Our ex vivo system reliably mimics what is observed in vivo following brain injury. It constitutes a powerful tool that could be used to identify factors that promote NSPC proliferation and differentiation in response to injury-induced activation of microglial cells, by using tools such as proteomics and gene array technology.
Isolation of microglia-derived extracellular vesicles: towards miRNA signatures and neuroprotection
Journal of Nanobiotechnology, 2019
The functional preservation of the central nervous system (CNS) is based on the neuronal plasticity and survival. In this context, the neuroinflammatory state plays a key role and involves the microglial cells, the CNS-resident macrophages. In order to better understand the microglial contribution to the neuroprotection, microglia-derived extracellular vesicles (EVs) were isolated and molecularly characterized to be then studied in neurite outgrowth assays. The EVs, mainly composed of exosomes and microparticles, are an important cell-to-cell communication process as they exhibit different types of mediators (proteins, lipids, nucleic acids) to recipient cells. The medicinal leech CNS was initially used as an interesting model of microglia/neuron crosstalk due to their easy collection for primary cultures. After the microglia-derived EV isolation following successive methods, we developed their large-scale and non-targeted proteomic analysis to (i) detect as many EV protein markers ...
Glia, 2008
The contribution of microglia to the modulation of neurogenesis under pathological conditions is unclear. Both proand anti-neurogenic effects have been reported, likely reflecting the complexity of microglial activation process. We previously demonstrated that prolonged (72 hr) in vitro exposure to lipopolysaccharide (LPS) endows microglia with a potentially neuroprotective phenotype, here referred as to ''chronic''. In the present study we further characterized the chronic phenotype and investigated whether it might differently regulate the properties of embryonic and adult neural precursor cells (NPC) with respect to the ''acute'' phenotype acquired following a single (24 hr) LPS stimulation. We show that the LPS-dependent induction of the proinflammatory cytokines interleukin (IL)-1a, IL-1b, IL-6, and tumor necrosis factor (TNF)-a was strongly reduced after chronic stimulation of microglia, as compared with acute stimulation. Conversely, the synthesis of the anti-inflammatory cytokine IL-10 and the immunomodulatory prostaglandin E 2 (PGE 2 ) was still elevated or further increased, after chronic LPS exposure, as revealed by real time PCR and ELISA techniques. Acutely activated microglia, or their conditioned medium, reduced NPC survival, prevented neuronal differentiation and strongly increased glial differentiation, likely through the release of proinflammatory cytokines, whereas chronically activated microglia were permissive to neuronal differentiation and cell survival, and still supported glial differentiation. Our data suggest that, in a chronically altered environment, persistently activated microglia can display protective functions that favor rather than hinder brain repair processes. V V C
Extracellular vesicles can act as a potent immunomodulators of human microglial cells
Journal of Tissue Engineering and Regenerative Medicine, 2019
Functional impairments of microglia have been recently associated with several neurological conditions. Therefore modulation of anti-inflammatory and phagocytic properties of microglial cells could represent a novel therapeutic approach. In the present study we investigated the effects of extracellular vesicles (EVs) derived from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs) on the inflammatory response and functional properties of immortalized human microglial cells. NFκB reporter assays demonstrated that EVs suppressed LPS-induced activation of NFκB signalling pathway in human microglial cells. The effect was similar to that obtained with anti-TLR4 blocking antibody. We also show, that EVs differentially affected phagocytic activity of unpolarized (M0) and polarized (M1 and M2) microglial cells. EVs induced significant upregulation of phagocytic activity in M0 cells (by 39 %), slight decrease in M1 cells and moderate increase (by 21 %) in M2 cells. The Seahorse XF Glycolysis stress test revealed that EVs induced an immediate and sustained increase of glycolytic activity in M0, M1 and M2 cells. Interestingly, EVs acted in an inverse dose-dependent manner. These findings indicate that EVs can induce glycolytic reprogramming of unpolarized and polarized human microglial cells. In conclusion, our pilot study demonstrates that EVs derived from SHEDs can act as a potent immunomodulators of human microglial cells. These findings could be potentially exploited for the development of new therapeutic strategies targeting neuroinflammatory microglia.
Microglia: dismantling and rebuilding circuits after acute neurological injury
Metabolic Brain Disease, 2014
The brain is comprised of neurons and its support system including astrocytes, glial cells and microglia, thereby forming neurovascular units. Neurons require support from glial cells to establish and maintain functional circuits, but microglia are often overlooked. Microglia function as the immune cell of the central nervous system, acting to monitor the microenvironment for changes in signaling, pathogens and injury. More recently, other functional roles for microglia within the healthy brain have been identified, including regulating synapse formation, elimination and function. This review aims to highlight and discuss these alternate microglial roles in the healthy and in contrast, diseased brain with a focus on two acute neurological diseases, traumatic brain injury and epilepsy. In these conditions, microglial roles in synaptic stripping and stabilization as part of neuronal:glial interactions may position them as mediators of the transition between injury-induced circuit dismantling and subsequent reorganization. Increased understanding of microglia roles could identify therapeutic targets to mitigate the consequences of neurological disease. Microglia: Amoeboid to ramified and back again Microglial morphology has long been interpreted to follow function, with surface antigens changing dependent on the stimulus for activation and the role required to play in the brain. This simplistic view is now being challenged, with data collected over the previous decades indicating alternate roles for microglia, particularly in the uninjured brain. During development of the brain, microglial precursors undergo three developmental milestones toward becoming fully integrated microglia. Microglia proliferate and migrate to populate different central nervous system (CNS) regions, and then differentiate from an amoeboidlike form into their ramified morphology. Within the non-pathological brain, ramified microglia constantly survey the microenvironment by movement of their fine processes, sampling the surface of cells and interstitial fluid in their immediate vicinity [1, 2] and
Microglia: first responders in the central nervous system
Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie, 2013
Microglia has emerged not only as an essential inflammatory cell but also as a major player in the development of the adult brain. Microglia phagocytize extra-numerical synapses during postnatal development, maintain and strengthen the remaining subset of synapses, remodel synaptic circuits and clearing apoptotic newborn neurons. Thereby, microglia plays a crucial role for the establishment, plasticity and function of adult neural circuits. In addition to the key role in normal brain function, any imbalance in microglia activity has been associated with neurodegenerative diseases. Microglial cells respond rapidly to smallest pathological changes, this being a vital aspect in many tissue scaring and the local confinement of focal lesions. It is assumed that the high motility of microglial cells represents an important requirement to fulfill the numerous functions. In this review will highlight the role of microglial motility in the healthy and the injured brain, and discuss how impai...
The Journal of Neuroscience, 2021
Excessive inflammation within the CNS is injurious, but an immune response is also required for regeneration. Macrophages and microglia adopt different properties depending on their microenvironment, and exposure to IL4 and IL13 has been used to elicit repair. Unexpectedly, while LPS-exposed macrophages and microglia killed neural cells in culture, the addition of LPS to IL4/IL13-treated macrophages and microglia profoundly elevated IL10, repair metabolites, heparin binding epidermal growth factor trophic factor, antioxidants, and matrix-remodeling proteases. In C57BL/6 female mice, the generation of M(LPS/IL4/IL13) macrophages required TLR4 and MyD88 signaling, downstream activation of phosphatidylinositol-3 kinase/mTOR and MAP kinases, and convergence on phospho-CREB, STAT6, and NFE2. Following mouse spinal cord demyelination, local LPS/IL4/IL13 deposition markedly increased lesional phagocytic macrophages/microglia, lactate and heparin binding epidermal growth factor, matrix remo...