Exosome-enriched fractions from MS B cells induce oligodendrocyte death

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.

Figure 1. Toxic factors are enriched in Ex-En fractions prepared from MS B-cell Sup by water exclusion precipitation

B-cell Sup from 5 MS and 5 NC were centrifuged at 2,000g for 30 minutes to remove cell debris; medium alone was processed in parallel as control. The supernatants were removed for isolation of Ex-En fractions; the pellets were suspended and tested for OL toxicity. The supernatants were mixed with isolation reagent. Samples were incubated at 4^° overnight and then centrifuged at 10,000g for 1 hour. The exosome-enriched precipitates, designated Ex-En, were tested for OL toxicity. The background value for NC Ex-En and MS Ex-En toxicity is the value for the toxicity of the medium Ex-En fraction (19%); subtraction of that value gives a range of −2% to 13% for NC Ex-En and a range of 37%–43% for MS Ex-En. Values represent averages, n = 2 ± range; *p < 0.0001 by analysis of variance with the Tukey post-test. Ex-En = exosome enriched; NC = normal control; OL = oligodendrocyte.

Figure 2. Toxic factors are enriched in exosome-enriched fractions prepared from MS B-cell Sup by ultracentrifugation

(A) B cells were cultured in serum-free X-VIVO 10 defined medium. Five MS Sup showed greater toxicity to OL than 5 NC Sup. These results were similar to those obtained previously from B cells cultured in medium containing bovine serum (Lisak et al. 2017). (B) Medium alone, 3 NC, and 4 MS Sup were centrifuged at 62,500g for 2 hours (see Methods); the pellets, enriched in extracellular vesicles, were suspended in DMEM, diluted 1:4 with OL medium, and tested for OL toxicity. The pellets from MS samples retained toxicity compared with the NC samples. The background value for NC UC and MS UC toxicity is the value for the toxicity of the medium UC fraction (16%); subtraction of that value gives a range of −5% to 16% for NC UC and a range of 29%–40% for MS UC. Values represent averages, n = 2 ± range; *p < 0.0001 by analysis of variance with the Tukey post-test. NC = normal control; OL = oligodendrocyte; UC = ultracentrifugation.

Figure 3. Toxic factors are enriched in exosome-enriched fractions prepared from MS B-cell Sup by immunoprecipitation with CD9 antibody

The >300-kDa fractions prepared from medium alone, 3 NC, and 3 MS Sup were incubated with antibody to the exosomal marker CD9 conjugated to Protein A Sepharose. In the MS samples, 2 were RRMS and 1 was PPMS (MS36). Immunoprecipitates were tested for OL toxicity. Although the IP medium sample alone and IP NC fractions showed some OL toxicity (A), after subtraction of the medium background, MS IP fractions from each of the 3 patients with MS were 3–4-fold more toxic to OL than NC IP fractions (B). Values represent averages, n = 2 ± range; *p < 0.002 by analysis of variance with the Tukey post-test. IP = immunoprecipitation; NC = normal control; OL = oligodendrocyte; PPMS = primary progressive MS; RRMS = relapsing-remitting MS.

Figure 4. Venn diagram for B-cell Sup proteins compared with proteins from exosome-enriched fractions from a human B-cell line

(A) Venn diagram. (B) The chart shows examples of representative proteins and groups of proteins from the 219 proteins common to both the B-cell Sup proteins and the exosome-enriched fractions from the human B-cell line, including both common exosomal markers and B cell–enriched proteins (*).