Method Article

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

DOI:

10.3791/60118

⸱

June 2nd, 2020

 ,  ,  ,  , 
1U1192-Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse (PRISM), Univ. Lille, INSERM

In This Article

Summary

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The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

Abstract

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The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

Introduction

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Many neuropathologies are related to the neuro-inflammatory state which is a complex mechanism that is increasingly considered, but still poorly understood because the immune processes are diverse and depend upon the cell environment. Indeed, the CNS disorders do not systematically involve the same activation signals and immune cell populations and thus the pro- or anti-inflammatory responses are difficult to evaluate as causes or consequences of pathologies. The brain resident macrophages called “microglia” appear to be at the interface between the nervous and immune systems1. Microglia have a myeloid origin and are derived from th....

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Protocol

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1. Primary Culture of Microglia/Macrophages

  1. Primary culture of microglia
    1. Culture commercial rat primary microglia (2 x 106 cells) (see the Table of Materials) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% exosome-free serum, 100 U/mL of penicillin, 100 µg/mL streptomycin, and 9.0 g/L glucose at 37 °C and 5% CO2.
    2. Collect the conditioned medium after a 48 h culture and proceed to the isolation of EVs.
  2. Primary culture of macrophages
    1. Culture commercial rat primary macrophages (1 x 106 ce....

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Results

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One of the main challenges to attributing biologicals effects to extracellular vesicles (EVs) is the ability to isolate the EVs from the whole culture medium. In this report, we present a method using ultracentrifugation (UC) and size-exclusion chromatography (SEC) which is coupled to the large-scale analysis of protein signatures to validate EV markers and identify bioactive compounds. The macrophage- or microglia-derived EVs were isolated from the conditioned medium after a 24 h or 48 h culture respectively (

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Discussion

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The central nervous system (CNS) is a complex tissue in which cell-to-cell communication regulates normal neuronal functions necessary for homeostasis30. EVs are now widely studied and described as important molecular cargos for cell-to-cell communication31. They specifically deliver a cocktail of mediators to recipient cells thereby affecting their functions in healthy and pathological conditions32. Recent studies indicate that EVs play a crucial ro.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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The presented work was supported by the Ministère de L’Education Nationale, de L’Enseignement Supérieur et de la Recherche and INSERM. We gratefully acknowledge the BICeL- Campus Scientific City Facility for access to instruments and technical advices. We gratefully acknowledge Jean-Pascal Gimeno, Soulaimane Aboulouard and Isabelle Fournier for the Mass spectrometry assistance. We gratefully acknowledge Tanina Arab, Christelle van Camp, Francoise le Marrec-Croq, Jacopo Vizioli and Pierre-Eric Sautière for their strong contribution to the scientific and technical developments.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
12% Mini-PROTEAN TGX Precast Protein GelsBio-rad4561045EDU 
AcetonitrileFisher ChemicalsA955-1 
Amicon 50 kDa centrifugal filterMerckUFC505024 
Ammonium bicarbonateSigma-Aldrich9830 
HSP90 α/β antibody (RRID: AB_675659)Santa-cruzsc-13119 
B27 Plus supplementGibcoA3582801 
BenchMixer V2 Vortex MixerBenchmark ScientificBV1003 
Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford)Bio-Rad5000006 
C18 ZipTipsMerck MilliporeZTC18S096 
C6 rat glioma cellATCCATCC CCL-107 
Canonical tubesSarstedt62.554.002 
CentrifugeEppendorf5804000010 
CO2 IncubatorThermoFisher  
Confocal microscope LSM880Carl ZeissLSM880 
Cover glassMarienfeld111580 
Culture Dish (60 mm)Sarstedt82.1473 
DithiothreitolSigma-Aldrich43819 
DMEMGibco41966029 
EASY-nLC 1000 Liquid ChromatographThermoFisher  
Electron microscope JEM-2100JEOL  
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acidSigma-Aldrich03777-10G 
Ethylenediaminetetraacetic acidSigma-AldrichED-100G 
Exo-FBSOzymeEXO-FBS-50A-1Exosome depleted FBS
ExoCarta database (top 100 proteins of Evs)  http://www.exocarta.org/
Fetal Bovine SerumGibco16140071 
Fetal Horse SerumBiowestS0960-500 
Filtropur S 0.2Sarstedt83.1826.001 
Fisherbrand Q500 Sonicator with ProbeFisherbrand12893543 
FlexAnalysisBrucker  
Fluorescence mounting mediumAgilentS3023 
Formic AcidSigma-Aldrich695076 
Formvar-carbon coated copper gridsAgar scientific LtdAGS162-3 
GlucoseSigma-AldrichG8769 
GlutaraldehydeSigma-Aldrich340855 
Hoechst 33342Euromedex17535-AAT 
IdoacetamideSigma-AldrichI1149 
InstantBlue Coomassie Protein StainExpedeonISB1L 
Invert light microscope CKX53Olympus  
L-glutamineGibco25030-024 
LabTek II 8 wells Nunc154534 
Laemmli 2xBio-Rad1610737 
LamininCorning354232 
MaxQuant software (proteins identification software)  https://maxquant.net/maxquant/
MBT Polish stellBrucker8268711 
MEM 10xGibco21090-022 
MethylcelluloseSigma-AldrichM6385-100G 
MiliQ waterMerck Millipore  
MilkRegilaitREGILAIT300 
Mini PROTEAN Vertical Electrophoresis CellBio-Rad1658000FC 
MonoP FPLC columnGE Healthcare no longer available
Nanosight NS300Malvern PanalyticalNS300 
NanoSight NTA software v3.2Malvern Panalytical  
NanoSight syringe pumpMalvern Panalytical  
NeurobasalGibco21103-049 
Nitrocellulose membraneGE Healthcare10600007 
Nonidet P-40Fluka56741 
Nunc multidish 24 wellsThermoFisher82.1473 
ParaformaldehydeElectro microscopy Science15713 
PC-12 cell lineATCCATCC CRL-1721 
Penicillin-StreptomycinGibco15140-122 
Peptide calibration mixLaserBio LabsC101 
Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)Jackson ImmunoResearch115-035-003 
Perseus software (Processing of identified proteins)  https://maxquant.net/perseus/
Phalloidin-tetramethylrhodamine conjugateSanta-cruzsc-362065 
Phenylmethanesulfonyl fluorideSigma-Aldrich78830 
Phosphate Buffer SalineInvitrogen14190094no calcium, no magnesium
pluriStrainer M/ 60 µmpluriSelect43-50060 
Poly-D-lysineSigma-AldrichP6407 
Polycarbonate centrifuge tubesBeckman Coulter355651 
Protease InhibitorSigma-AldrichS8830-20TAB 
PureColCell Systems5005 
Q-Exactive mass spectrometerThermoFisher  
rapifleX mass spectrometerBrucker  
Rat cortical neuronsCell ApplicationsR882N-20Cell origin : Derived from cerebral cortices of day 18 embryonic Sprague Dawley rat brains
Rat Macrophage & Microglia Culture MediumCell ApplicationsR620K-100Cell orgin : Normal healthy Rat bone marrow
Rat primary macrophagesCell ApplicationsR8818-10a 
Rat primary microgliaLonzaRG535 
Sepharose CL-2BGE Healthcare17014001 
Sequencing Grade Modified TrypsinPromegaV5111 
SlideDustsher100204 
Sodium ChlorideScharlauSO0227 
Sodium Dodecyl SulfateSigma-AldrichL3771 
Sodium FluorideSigma-AldrichS7920-100G 
Sodium hydroxideScharlabSO0420005P 
Sodium pyrophosphateSigma-AldrichS6422-100G 
SpeedVac Vacuum ConcentratorThermoFisher  
String software (functional protein association networks)  https://string-db.org/
SuperSignal West Dura extended Duration SubstrateThermoFisher34075 
Syringe 1.0 mLTerumo8SS01H1 
Trans-Blot SD Semi-Dry Transfer cellBio-Rad1703940 
Trifluoroacetic acidSigma-AldrichT6508 
TrisInterchimUP031657 
Tris-GlycineEuromedexEU0550 
Tween 20Sigma-AldrichP2287 
UltracentrifugeBeckman CoulterA95765 
Ultracentrifuge Rotor 70.1 TiBeckman Coulter342184 
Uranyl acetateAgar Scientific LtdAGR1260A 
Whatman filter paperSigma-AldrichWHA10347510 
α-Cyano-4-hydroxycinnamic acidSigma-AldrichC2020-25G 

References

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  1. Thion, M. S., Ginhoux, F., Garel, S. Microglia and early brain development: An intimate journey. Science. 362 (6411), 185-189 (2018).
  2. Ginhoux, F., et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 330<....

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Tags

Extracellular VesiclesImmune CellsUltracentrifugationSize Exclusion ChromatographyNanoparticle Tracking AnalysisWestern BlotProteomics AnalysisNeurite OutgrowthGlioma Cell InvasionMicroglia derived EVs

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