Extracellular vesicles (EVs) play a major role in cell-to-cell communication in physiological and pathological conditions, and their manipulation may represent a promising therapeutic strategy. Microglia, the parenchymal mononuclear phagocytes of the brain, modulate neighboring cells also through the release of EVs. The production of custom EVs filled with desired molecules, possibly targeted to make their uptake cell specific, and their administration in biological fluids may represent a valid approach for drug delivery. We engineered a murine microglia cell line, BV-2, to release EVs overexpressing the endogenous "eat me" signal Lactadherin (Mfg-e8) on the surface to target phagocytes and containing the anti-inflammatory cytokine IL-4. A single injection of 10 IL-4Mfg-e8 EVs into the cisterna magna modulated established neuroinflammation and significantly reduced clinical signs in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Injected IL-4Mfg-e8 EVs target mainly phagocytes (i.e., macrophages and microglia) surrounding liquoral spaces, and their cargo promote the upregulation of anti-inflammatory markers chitinase 3-like 3 (ym1) and arginase-1 (arg1), significantly reducing tissue damage. Engineered EVs may represent a biological drug delivery tool able to deliver multiple functional molecules simultaneously to treat neuroinflammatory diseases.

BackgroundMyeloid cells, such as macrophages and microglia, play a crucial role in neuroinflammation and have been recently identified as a novel therapeutic target, especially for chronic forms. The general aim would be to change the phenotype of myeloid cells from pro- to anti-inflammatory, favoring their tissue-trophic and regenerative functions. Myeloid cells, however, display a number of functional phenotypes, not immediately identifiable as pro- or anti-inflammatory, and associated to ambiguous markers.MethodsWe employed in vitro assays to study macrophage polarization/differentiation in the presence of classical polarizing stimuli such as IFNγ (pro-inflammatory) and IL4 (anti-inflammatory). We induced neuroinflammation in mice by immunization with a myelin antigen and treated diseased mice with intracisternal delivery of an IL4-expressing lentiviral vector. We analyzed clinical, pathological, and immunological outcomes with a focus on myeloid cells.ResultsWe found that IL6, usually considered a pro-inflammatory cytokine, was released in vitro by macrophages treated with the anti-inflammatory cytokine IL4. We show the existence of macrophages expressing IL6 along with classical anti-inflammatory markers such as CD206 and demonstrate that these cells are immunosuppressive in vitro. In neuroinflamed mice, we show that IL4 delivery in the central nervous system (CNS) is associated with clinical and pathological protection from disease, associated with increased IL6 expression in infiltrating macrophages.ConclusionsIL6 is known to mediate both pro- and anti-inflammatory effects, having two distinct ways to induce cell-signaling: either through the membrane bound receptor (anti-inflammatory) or through trans-signaling (pro-inflammatory). We show here that IL6-expressing macrophages are associated to protection from neuroinflammation, suggesting that IL6 anti-inflammatory properties prevail in the CNS, and calling for a general reconsideration of IL6 in macrophage polarization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0596-5) contains supplementary material, which is available to authorized users.

Since extracellular vesicles (EVs) were discovered in 1983 in sheep reticulocytes samples, they have gradually attracted scientific attention and become a topic of great interest in the life sciences field. EVs are small membrane particles, released by virtually every cell that carries a variety of functional molecules. Their main function is to deliver messages to the surrounding area in both physiological and pathological conditions. Initially, they were thought to be either cell debris, signs of cell death, or unspecific structures. However, accumulating evidence support a theory that EVs are a universal mechanism of communication. Thanks to their biological characteristics and functions, EVs are likely to represent a promising strategy for obtaining pathogen information, identifying therapeutic targets and selecting specific biomarkers for a variety of diseases, such as autoimmune diseases. In this review, we provide a brief overview of recent progress in the study of the biology and functions of EVs. We also discuss their roles in diagnosis and therapy, with particular emphasis on autoimmune diseases.

Autoimmune diseases such as multiple sclerosis (MS) develop because of failed peripheral immune tolerance for a specific self-antigen (Ag). Numerous approaches for Ag-specific suppression of autoimmune neuroinflammation have been proven effective in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. One such approach is intravenous tolerance induction by injecting a myelin Ag used for triggering EAE. However, the translation of this and similar experimental strategies into therapy for MS has been hampered by uncertainty regarding relevant myelin Ags in MS patients. To address this issue, we developed a therapeutic strategy that relies on oligodendrocyte (Ol)–derived extracellular vesicles (Ol-EVs), which naturally contain multiple myelin Ags. Intravenous Ol-EV injection reduced disease pathophysiology in a myelin Ag–dependent manner, both prophylactically and therapeutically, in several EAE models. The treatment was safe and restored immune tolerance by inducing immunosuppressive monocytes and apoptosis of autoreactive CD4+ T cells. Furthermore, we showed that human Ols also released EVs containing most relevant myelin Ags, providing a basis for their use in MS therapy. These findings introduce an approach for suppressing central nervous system (CNS) autoimmunity in a myelin Ag–specific manner, without the need to identify the target Ag.

Microvesicles are a recently described way of cell communication that has been implicated in a number of biological processes, including neuroinflammation. Widely investigated as biomarkers in oncology and neurological disorders, little is known of the role of microvesicles in the pathogenesis of diseases such as multiple sclerosis (MS). Several evidences suggest that pro-inflammatory microglia and infiltrating macrophages release microvesicles that spread inflammatory signals and alter neuronal functions. We review here available information on microvesicles, with a special focus on microglia and macrophage microvesicles, in the pathogenesis of MS, and as potential biomarkers and therapeutic targets.