Abstract
BackgroundActivated 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.MethodsWe 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.ResultsOur 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 LPS-activated 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.ConclusionsWe 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.
Highlights
Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS)
Proinflammatory responses from LPS-stimulated BV2 microglial cells First, we examined the activation of BV2 cells after 12 h culture in the presence of 1 μg/ml LPS followed by deprivation of serum for 12 h to elicit a strong inflammatory reaction
The inflammatory enzyme inducible nitric oxide synthase (iNOS) is expressed by activated microglia [22] and as expected, the level of iNOS was significantly increased in cells upon LPS stimulation (Fig. 1a)
Summary
Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS). Microglia are considered the main innate-immune cells of the central nervous system (CNS). They continuously survey their microenvironment and have the ability to interact with neurons to regulate their activity [1]. Microglia continuously survey their surroundings with highly dynamic processes [2] and become activated in response to injury, infection or neurodegenerative processes [3]. Microglia are highly dynamic cells with the ability to transform their morphology from ramified to amoeboid and alter their phenotypes corresponding to diverse conditions. M1-microglia are proinflammatory, secreting inflammatory cytokines, chemokines, and nitric oxide (NO), which is believed to result in neuronal dysfunction and
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