Abstract
Objective To identify whether factors toxic to oligodendrocytes (OLs), released by B cells from patients with MS, are found in extracellular microvesicles enriched in exosomes.
Methods Conditioned medium (Sup) was obtained from cultures of blood B cells of patients with MS and normal controls (NCs). Exosome-enriched (Ex-En) fractions were prepared by solvent precipitation from Sup containing bovine serum and from serum-free Sup by ultracentrifugation (UC) or immunoprecipitation (IP) with antibodies to CD9. Ex-En fractions were diluted 1:4 with OL culture medium and screened for toxic effects on cultured rat OLs as measured by trypan blue uptake. Proteomic analysis was performed on Sup fractions.
Results MS B cell–derived Ex-En fractions prepared from Sup by solvent extraction, UC, or IP induced OL death, whereas corresponding Ex-En fractions from NC showed little toxicity. Proteomic analysis of Sup demonstrated enrichment of proteins characteristic of exosomes from both NC and MS B-cell Sup. Ontology enrichment analysis suggested differences in the types and cargo of exosomes from MS Sup compared with NC, with proteins related to cell surface, extracellular plasma membrane, and gliogenesis enriched in MS.
Conclusions Much of the in vitro toxicity of Sup from B cells of patients with relapsing-remitting MS is found in Ex-En fractions, as confirmed by 3 methods. Proteomic analysis of B-cell Sup indicates multiple differences between MS and NC.
Glossary
- AMBIC=
- ammonium bicarbonate;
- DMEM=
- Dulbecco's modified Eagle medium;
- DTT=
- dithiothreitol;
- Ex-En=
- exosome enriched;
- FDR=
- false discovery rate;
- GM=
- gray matter;
- IAA=
- iodoacetamide;
- Ig=
- immunoglobulin;
- IP=
- immunoprecipitation;
- NC=
- normal control;
- OL=
- oligodendrocyte;
- PIANO=
- Platform for Integrated Analysis of Omics data;
- PPMS=
- primary progressive MS;
- RRMS=
- relapsing-remitting MS;
- Sup=
- conditioned medium from B-cell cultures;
- UC=
- ultracentrifugation;
- WM=
- white matter
Footnotes
Funding information and disclosures are provided at the end of the article. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NN.
↵* These authors contributed equally to the manuscript.
The Article Processing Charge was funded by the authors.
- Received October 22, 2018.
- Accepted in final form January 16, 2019.
- Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
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