[en] Autoreactive T lymphocytes crossing the blood-brain barrier (BBB) into the central nervous system (CNS) play a crucial role in the initiation of demyelination and neurodegeneration in multiple sclerosis (MS). Recently, extracellular vesicles (EV) secreted by BBB endothelial cells (BBB-EC) have emerged as a unique form of cell-to-cell communication that contributes to cerebrovascular dysfunction. However, the precise impact of different size-based subpopulations of BBB-EC-derived EV (BBB-EV) on the early stages of MS remains unclear. Therefore, our objective was to investigate the content and function of distinct BBB-EV subpopulations in regulating BBB integrity and their role in T cell transendothelial migration, both in vitro and in vivo. Our study reveals that BBB-ECs release two distinct size based EV populations, namely small EV (sEV; 30-150 nm) and large EV (lEV; 150-300 nm), with a significantly higher secretion of sEV during inflammation. Notably, the expression patterns of cytokines and adhesion markers differ significantly between these BBB-EV subsets, indicating specific functional differences in the regulation of T cell migration. Through in vitro experiments, we demonstrate that lEV, which predominantly reflect their cellular source, play a major role in BBB integrity loss and the enhanced migration of pro-inflammatory Th1 and Th17.1 cells. Conversely, sEV appear to protect BBB function by inducing an anti-inflammatory phenotype in BBB-EC. These findings align with our in vivo data, where the administration of sEV to mice with experimental autoimmune encephalomyelitis (EAE) results in lower disease severity compared to the administration of lEV, which exacerbates disease symptoms. In conclusion, our study highlights the distinct and opposing effects of BBB-EV subpopulations on the BBB, both in vitro and in vivo. These findings underscore the need for further investigation into the diagnostic and therapeutic potential of BBB-EV in the context of MS.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Hosseinkhani, Baharak; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium ; Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology (CCB), VIB, KU Leuven, Leuven, Belgium ; Department of Oncology, Leuven Cancer Institute (LKI), KU Leuven, Leuven, Belgium
Duran, Gayel; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Hoeks, Cindy; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Hermans, Doryssa; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Schepers, Melissa; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium ; Department Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands ; Department of Neuroscience, Biomedical Research Institute, Faculty of Medicine and Life Sciences, Hasselt University, Hasselt, Belgium
Baeten, Paulien; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Poelmans, Joren; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Coenen, Britt; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Bekar, Kubra ; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Pintelon, Isabel; Laboratory of Cell Biology & Histology/Antwerp Centre for Advanced Microscopy (ACAM), University of Antwerp, Universiteitsplein 1, Antwerp, 2610, Belgium
Timmermans, Jean-Pierre; Laboratory of Cell Biology & Histology/Antwerp Centre for Advanced Microscopy (ACAM), University of Antwerp, Universiteitsplein 1, Antwerp, 2610, Belgium
Vanmierlo, Tim; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium ; Department Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands ; Department of Neuroscience, Biomedical Research Institute, Faculty of Medicine and Life Sciences, Hasselt University, Hasselt, Belgium
Michiels, Luc; Bionanotechnology group, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Hellings, Niels; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium
Broux, Bieke; University MS Center, Campus Diepenbeek, Diepenbeek, Belgium. bieke.broux@uhasselt.be ; Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, UHasselt, Diepenbeek, Belgium. bieke.broux@uhasselt.be ; Universiteit Hasselt, Martelarenlaan 42, Hasselt, Belgium. bieke.broux@uhasselt.be
Filippi M, Bar-Or A, Piehl F, Preziosa P, Solari A, Vukusic S, et al. Multiple sclerosis. Nat Reviews Disease Primers. 2018;4(1):43.
Compston A, Coles A. Multiple sclerosis. Lancet. 2008;372(9648):1502–17.
Ajdacic-Gross V, Steinemann N, Horváth G, Rodgers S, Kaufmann M, Xu Y, et al. Onset symptom clusters in multiple sclerosis: characteristics, comorbidities, and risk factors. Front Neurol. 2021;12:693440.
Dendrou CA, Fugger L, Friese MA. Immunopathology of multiple sclerosis. Nat Rev Immunol. 2015;15(9):545–58.
Xiao M, Xiao ZJ, Yang B, Lan Z, Fang F. Blood-brain barrier: more contributor to disruption of Central Nervous System Homeostasis Than victim in neurological disorders. Front Neurosci. 2020;14:764.
Stamatovic SM, Keep RF, Andjelkovic AV. Brain endothelial cell-cell junctions: how to open the blood brain barrier. Curr Neuropharmacol. 2008;6(3):179–92.
Engelhardt B. Development of the blood-brain barrier. Cell Tissue Res. 2003;314(1):119–29.
Minagar A, Long A, Ma T, Jackson TH, Kelley RE, Ostanin DV, et al. Interferon (IFN)-beta 1a and IFN-beta 1b block IFN-gamma-induced disintegration of endothelial junction integrity and barrier. Endothelium. 2003;10(6):299–307.
Muller WA. Getting leukocytes to the site of inflammation. Vet Pathol. 2013;50(1):7–22.
Domingues HS, Mues M, Lassmann H, Wekerle H, Krishnamoorthy G. Functional and pathogenic differences of Th1 and Th17 cells in experimental autoimmune encephalomyelitis. PLoS ONE. 2010;5(11):e15531.
Yeh WI, McWilliams IL, Harrington LE. Autoreactive Tbet-positive CD4 T cells develop Independent of classic Th1 cytokine signaling during experimental autoimmune encephalomyelitis. J Immunol. 2011;187(10):4998–5006.
Lee Y, Awasthi A, Yosef N, Quintana FJ, Xiao S, Peters A, et al. Induction and molecular signature of pathogenic TH17 cells. Nat Immunol. 2012;13(10):991–9.
Altan-Bonnet G, Mukherjee R. Cytokine-mediated communication: a quantitative appraisal of immune complexity. Nat Rev Immunol. 2019;19(4):205–17.
Ulivieri C, Baldari CT. Regulation of T cell activation and differentiation by Extracellular vesicles and their pathogenic role in systemic Lupus Erythematosus and multiple sclerosis. Molecules. 2017;22(2).
Tetta C, Ghigo E, Silengo L, Deregibus MC, Camussi G. Extracellular vesicles as an emerging mechanism of cell-to-cell communication. Endocrine. 2013;44(1):11–9.
Théry C. Hosseinkhani B, and, > 300ISEV member. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750.
Xu D, Di K, Fan B, Wu J, Gu X, Sun Y, et al. MicroRNAs in extracellular vesicles: sorting mechanisms, diagnostic value, isolation, and detection technology. Front Bioeng Biotechnol. 2022;10:948959.
Xu K, Liu Q, Wu K, Liu L, Zhao M, Yang H, et al. Extracellular vesicles as potential biomarkers and therapeutic approaches in autoimmune Diseases. J Translational Med. 2020;18(1):432.
Pelissier Vatter FA, Cioffi M, Hanna SJ, Castarede I, Caielli S, Pascual V Extracellular vesicle– and particle-mediated communication shapes innate and adaptive immune responses. J Exp Med. 2021;218(8).
Lucchetti D, Fattorossi A, Sgambato A. Extracellular vesicles in Oncology: Progress and pitfalls in the methods of isolation and analysis. Biotechnol J. 2019;14(1):e1700716.
Morad G, Carman CV, Hagedorn EJ, Perlin JR, Zon LI, Mustafaoglu N, et al. Tumor-derived extracellular vesicles breach the Intact blood–brain barrier via Transcytosis. ACS Nano. 2019;13(12):13853–65.
Balusu S, Van Wonterghem E, De Rycke R, Raemdonck K, Stremersch S, Gevaert K, et al. Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles. EMBO Mol Med. 2016;8(10):1162–83.
Palacio PL, Pleet ML, Reátegui E, Magaña SM. Emerging role of extracellular vesicles in multiple sclerosis: from cellular surrogates to pathogenic mediators and beyond. J Neuroimmunol. 2023;377:578064.
Busatto S, Morad G, Guo P, Moses MA. The role of extracellular vesicles in the physiological and pathological regulation of the blood–brain barrier. FASEB BioAdvances. 2021;3(9):665–75.
Roig-Carles D, Willms E, Fontijn RD, Martinez-Pacheco S, Mäger I, de Vries HE et al. Endothelial-derived extracellular vesicles induce cerebrovascular dysfunction in inflammation. Pharmaceutics. 2021;13(9).
Minagar A, Jy W, Jimenez JJ, Sheremata WA, Mauro LM, Mao WW, et al. Elevated plasma endothelial microparticles in multiple sclerosis. Neurology. 2001;56(10):1319–24.
Raposo G, Stoorvogel W. Extracellular vesicles: Exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373–83.
Dolcetti E, Bruno A, Guadalupi L, Rizzo FR, Musella A, Gentile A, et al. Emerging role of Extracellular vesicles in the pathophysiology of multiple sclerosis. Int J Mol Sci. 2020;21:19.
Marostica G, Gelibter S, Gironi M, Nigro A, Furlan R. Extracellular vesicles in Neuroinflammation. Front Cell Dev Biol. 2020;8:623039.
Wheway J, Latham SL, Combes V, Grau GE. Endothelial microparticles interact with and support the proliferation of T cells. J Immunol. 2014;193(7):3378–87.
Weksler B, Romero IA, Couraud PO. The hCMEC/D3 cell line as a model of the human blood brain barrier. Fluids Barriers CNS. 2013;10(1):16.
Hermans D, Houben E, Baeten P, Slaets H, Janssens K, Hoeks C, et al. Oncostatin M triggers Brain Inflammation by compromising blood-brain barrier integrity. Acta Neuropathol. 2022;144(2):259–81.
Mateescu B, Kowal EJ, van Balkom BW, Bartel S, Bhattacharyya SN, Buzás EI, et al. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper. J Extracell Vesicles. 2017;6(1):1286095.
Słomka A, Urban SK, Lukacs-Kornek V, Żekanowska E, Kornek M. Large extracellular vesicles: have we found the Holy Grail of inflammation? Front Immunol. 2018;9:2723.
Kuypers S, Smisdom N, Pintelon I, Timmermans J-P, Ameloot M, Michiels L, et al. Unsupervised machine learning-based clustering of Nanosized fluorescent extracellular vesicles. Small. 2021;17(5):2006786.
Sork H, Corso G, Krjutskov K, Johansson HJ, Nordin JZ, Wiklander OPB, et al. Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome. Sci Rep. 2018;8(1):10813.
Hosseini-Beheshti E, Pham S, Adomat H, Li N, Tomlinson Guns ES. Exosomes as biomarker enriched microvesicles: characterization of exosomal proteins derived from a panel of prostate cell lines with distinct AR phenotypes. Mol Cell Proteomics. 2012;11(10):863–85.
Broux B, Stinissen P, Hellings N. Which Immune Cells Matter? The Immunopathogenesis of Multiple Sclerosis. 2013;33(4):283–306.
Blonda M, Amoruso A, Martino T, Avolio C. New insights into Immune Cell-Derived Extracellular vesicles in multiple sclerosis. Front Neurol. 2018;9:604.
Gardiner C, Di Vizio D, Sahoo S, Théry C, Witwer KW, Wauben M, et al. Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J Extracell Vesicles. 2016;5:32945.
Coursey TG, Bohat R, Barbosa FL, Pflugfelder SC, de Paiva CS. Desiccating stress–Induced Chemokine expression in the epithelium is dependent on Upregulation of NKG2D/RAE-1 and Release of IFN-γ in experimental Dry Eye. J Immunol. 2014;193(10):5264–72.
Murphy AC, Lalor SJ, Lynch MA, Mills KH. Infiltration of Th1 and Th17 cells and activation of microglia in the CNS during the course of experimental autoimmune encephalomyelitis. Brain Behav Immun. 2010;24(4):641–51.
van Langelaar J, van der Vuurst RM, Janssen M, Wierenga-Wolf AF, Spilt IM, Siepman TA, et al. T helper 17.1 cells associate with multiple sclerosis Disease activity: perspectives for early intervention. Brain. 2018;141(5):1334–49.
Janoschka C, Lindner M, Koppers N, Starost L, Liebmann M, Eschborn M, et al. Enhanced pathogenicity of Th17 cells due to natalizumab treatment: implications for MS Disease rebound. Proc Natl Acad Sci U S A. 2023;120(1):e2209944120.
Croxford JL, Feldmann M, Chernajovsky Y, Baker D. Different therapeutic outcomes in experimental allergic encephalomyelitis dependent upon the mode of delivery of IL-10: a comparison of the effects of protein, adenoviral or retroviral IL-10 delivery into the central nervous system. J Immunol. 2001;166(6):4124–30.
McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T, et al. TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol. 2007;8(12):1390–7.
Driedonks T, Jiang L, Carlson B, Han Z, Liu G, Queen SE, et al. Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina. J Extracell Biology. 2022;1(10):e59.
Sabat R, Grütz G, Warszawska K, Kirsch S, Witte E, Wolk K, et al. Biology of interleukin-10. Cytokine Growth Factor Rev. 2010;21(5):331–44.
Musette P, Benveniste O, Lim A, Bequet D, Kourilsky P, Dormont D, et al. The pattern of production of cytokine mRNAs is markedly altered at the onset of multiple sclerosis. Res Immunol. 1996;147(7):435–41.
Salmaggi A, Dufour A, Eoli M, Corsini E, La Mantia L, Massa G, et al. Low serum interleukin-10 levels in multiple sclerosis: further evidence for decreased systemic immunosuppression? J Neurol. 1996;243(1):13–7.
Özenci K, Huang X, Kivisäkk F, et al. Multiple sclerosis: levels of Interleukin-10-Secreting blood mononuclear cells are low in untreated patients but augmented during Interferon-β-1b treatment. Scand J Immunol. 1999;49(5):554–61.
