Differences in Intercellular Communication During Clinical Relapse and Gadolinium-Enhanced MRI in Patients With Relapsing Remitting Multiple Sclerosis: A Study of the Composition of Extracellular Vesicles in Cerebrospinal Fluid.

Fabiana Geraci,Paolo Ragonese,Giovanni Savettieri,Sabrina Realmuto,Giuseppe Salemi,Maria Magdalena Barreca,Emanuele Aliotta,Maria Antonietta Mazzola,Giulia Vazzoler,Gabriella Sconzo

doi:10.3389/fncel.2018.00418

Abstract

This study was designed based on the hypothesis that changes in both the levels and surface marker expression of extracellular vesicles (EVs) isolated from the cerebrospinal fluid (CSF) may be associated with the clinical form, disease activity, and severity of multiple sclerosis (MS). The analyzes were performed on subjects affected by MS or other neurological disorders. EVs, which were isolated by ultracentrifugation of CSF samples, were characterized by flow cytometry. A panel of fluorescent antibodies was used to identify the EV origin: CD4, CCR3, CCR5, CD19, and CD200, as well as isolectin IB4. The Mann–Whitney U-test and Kruskal–Wallis test were used for statistical analyzes. EVs isolated from the CSF were more abundant in patients with progressive MS and in those with a clinically isolated syndrome than in all the other groups examined. Furthermore, an important change in the number of EVs and in their surface marker expression occurred during active phases of MS [i.e., clinical relapses and the presence of enhancing lesions on magnetic resonance imaging (MRI)]. In particular, the number of CSF-EVs increased in patients affected by MS during clinical relapse; this finding was associated with a decrease in the number of CD19+/CD200+ (naïve B cells) EVs. These markers are expressed by immature and naïve B lymphocytes, and to the best of our knowledge, this double staining has never been associated with MS, but their reduction has been observed in patients with another type of Th1 cell-mediated autoimmune disease. In contrast, the presence of lesions in the brain and spine on gadolinium-enhanced MRI was associated with an increase in the numbers of CCR3+/CCR5+ (subset of CD8 memory T cells), CD4+/CCR3+ (Th2 cells), and CD4+/CCR5+ (Th1 cells) CSF-EVs. Two points are worth emphasizing: (i) the data obtained in this study confirm that CSF-EVs represent a potentially promising tool to identify biomarkers specific for different phases of MS; and (ii) Considering the role of EVs in intercellular communication, our results provide some insights that improve our understanding of the relationships among some of the cell types that are mainly involved in MS pathogenesis (e.g., lymphocytes, glia, and neurons).

Highlights

Extracellular vesicles (EVs) are small double lipid membrane vesicles that are released by all cell types
RMS, relapsing multiple sclerosis; pMS, progressive multiple sclerosis; clinically isolated demyelinating syndrome (CIS), clinically isolated syndrome; other inflammatory neurological disorders (OINDs), other inflammatory neurological disorder; other non-inflammatory neurological disorders (ONINDs), other noninflammatory neurological disorder; EDSS, expanded disability status scale; GAD, gadolinium; MRI, magnetic resonance imaging; NR, not reported; NA, not applicable. ◦The values are presented as medians; minima-maxima. ◦◦The two subjects with OIND were affected by myelitis. ◦◦◦Four of the subjects with ONIND were diagnosed with a chronic cerebrovascular disease, four with normal pressure hydrocephalus, and one each with acute ischaemic stroke, benign intracranial hypertension, spastic paraparesis, parkinsonism, diabetic polyneuropathy, brain tumor outcomes, unspecified consciousness disorder, and conversion disorder
CSF-EVs, cerebrospinal fluid extracellular vesicles; CIS, clinically isolated syndrome; rMS, relapsing multiple sclerosis; pMS, progressive multiple sclerosis; OIND, other inflammatory neurological disorder; ONIND, other non-inflammatory neurological disorder; EDSS, expanded disability status scale; GAD, gadolinium; MRI, magnetic resonance imaging; NR, not reported; NA, not applicable. ◦The values are presented as medians; minima-maxima. ◦◦The two subjects with OIND were affected by myelitis. ◦◦◦Four of the subjects with ONIND were diagnosed with normal pressure hydrocephalus, four with chronic cerebrovascular disorders, and one each with acute ischaemic stroke, spastic tetraparesis, benign intracranial hypertension, parkinsonism, diabetic polyneuropathy, and conversion disorder

Summary

Introduction

Extracellular vesicles (EVs) are small double lipid membrane vesicles that are released by all cell types. These specialized structures were recently shown to be involved in intercellular communication via both autocrine and paracrine signaling. EVs contain a variety of cell surface receptors, signaling proteins, lipids, and nucleic acid, depending on the donor cell type. The functions of EVs rely on the type of the parental cells. A selective enrichment of specific molecules has been observed, allowing EVs to have different properties and roles from their parental cells (Del Conde et al, 2005; Li et al, 2013). Several types of interactions between EVs and target cells have been identified. The interplay may be mediated by ligand/receptor binding, or may be direct, followed by fusion or endocytosis (Turturici et al, 2014)

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