Circulating exosomes with unique lipid signature in relapsing remitting multiple sclerosis

Abstract

Exosomes are small, membrane-bound vesicles secreted by most cell types into the extracellular environment. They play a crucial role in intercellular communication by transporting bioactive molecules, including proteins, lipids, and RNAs, thereby influencing the phenotype and potentially the genotype in recipient cells. In recent years, exosomes have gained increasing attention in the study of pathophysiological conditions and numerous diseases, including multiple sclerosis (MS), an autoimmune disorder with myelin sheath and neuroaxonal damage in the central nervous system. In this study, we isolated and purified serum-derived exosomes from patients with relapsing remitting MS (RR-MS) and characterized their lipid profiles using matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS). Lipid analysis was performed in both negative and positive ion modes on intact exosomes, bypassing lipid extraction steps and significantly reducing sample-processing time. The lipid profiles of RR-MS exosomes were compared to those of exosomes isolated from the serum of healthy subjects (HS), and statistical analysis was applied to mass spectra to identify potential lipid biomarkers. The specific phospholipid marker of exosomal membranes, bis(monoacylglycero)phosphate (BMP), was clearly detected in both MALDI lipid profiles, with no significant differences in its content between the two sample groups. However, RR-MS exosomes exhibited significantly lower levels of phosphatidic acid (PA) compared to HS exosomes, despite PA being a key structural component of extracellular vesicles. Notably, comparative analysis revealed an enrichment of several lysophosphatidylcholine (LPC) species in RR-MS exosome membranes, aligning with their known proinflammatory role in MS pathology. Our most significant finding was a markedly lower phosphatidylcholine (PC) to LPC ratio in the pathological group indicating potential alterations in membrane lipid homeostasis. To the best of our knowledge, this study is the first to report a distinct lipid signature in serum-derived exosomes from RR-MS patients using direct MALDI-TOF/MS analysis.

Keywords: MALDI-TOF/MS; autoimmune disorders; extracellular vesicles; lipid biomarkers; serum.

FIGURE 1

Comparison of surface marker profiling of exosomes isolated by two different techniques. (A) Immunoblot analysis of exosomes isolated by the ultracentrifugation protocol (Exo-UC) and the commercial kit (Exo-Kit), probed with anti-CD63 and anti-CD81 antibodies. Albumin contamination (∼69 kDa) is also shown. (B) Immunoblot analysis of two different samples of exosomes isolated from HS sera (HS1, HS2), showing reproducible enrichment of the surface markers TSG101 and CD63, with minimal albumin contamination.

FIGURE 2

Characterization of serum-derived exosomes by DLS. Representative hydrodynamic radius distributions of exosomes measured by intensity distribution (A) and by number (B). Note that the spectra, performed in triplicate (named as record 19, 20, and 21), indicate the presence of vesicular structures at the predicted size of 30–100 nm (A,B).

FIGURE 3

(−) MALDI-TOF/MS lipid profiles of exosomes obtained from HS (upper panel) and RR-MS patients (lower panel). Representative mass spectra obtained in negative ion mode are showed and lipid assignments for main MALDI m/z peaks are reported. The histograms (in the inset) show the differences in intensity between some lipid peaks present in the two series of (−) mass spectra, with p-value < 0.05 (*) set as the threshold to define significant differences. Data are reported as the average value of intensity ± SD. PA, phosphatidic acid; PE, phosphatidylethanolamine; SM, sphingomyelin; BMP, bis (monoacylglycero) phosphate.

FIGURE 4

(+) MALDI-TOF/MS lipid profiles of exosomes obtained from HS (upper panel) and RR-MS patients (lower panel). Representative mass spectra obtained in positive ion mode are showed and lipid assignments for main MALDI m/z peaks are reported. LPC, lysophosphatidylcholine; SM, sphingomyelin; PC, phosphatidylcholine; nd, peak of no interest.

FIGURE 5

Differences in PC and LPC contents (A) and PC/LPC ratio (B) between HS and RR-MS exosomes. The histograms (A) illustrate the differences in both LPC and PC signals detected in the two series of (+) MALDI mass spectra, with p-value < 0.05 set as the threshold to define significant differences. Data are reported as the average value of intensity ± SD (**p < 0.01). The plot (B) illustrates the calculated PC/LPC ratios in exosomes obtained from HS (blue dots) and RR-MS (red dots) (**p < 0.01). LPC, lysophosphatidylcholine; PC, phosphatidylcholine.