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In the past decade, cell-to-cell communication mediated by exosomes has attracted growing attention from biomedical scientists and physicians, leading to several recent publications in top-tier journals. Exosomes are generally defined as secreted membrane vesicles, or extracellular vesicles (EVs), corresponding to the intraluminal vesicles of late endosomal compartments, which are secreted upon fusion of multi-vesicular endosomes with the cell's plasma membrane. Cells, however, were shown to release other types of EVs, for instance, by direct budding off their plasma membrane. Some of these EVs share with exosomes major biophysical and biochemical characteristics, such as size, density and membrane orientation, which impose difficulties in their efficient separation. Despite frequent claims in the literature, whether exosomes really display more important patho/physiological functions, or are endowed with higher potential as diagnostic or therapeutic tools than other EVs, is not yet convincingly demonstrated. In this opinion article, we describe reasons for this lack of precision knowledge in the current stage of the EV field, we review recently described approaches to overcome these caveats, and we propose ways to improve our knowledge on the respective functions of distinct EVs, which will be crucial for future development of well-designed EV-based clinical applications. This article is part of the discussion meeting issue ‘Extracellular vesicles and the tumour microenvironment’.

The twenty-first century has witnessed major developments in the field of extracellular vesicle (EV) research, including significant steps towards defining standard criteria for the separation and detection of EVs. The recent recognition that EVs have the potential to function as biomarkers or as therapeutic tools has attracted even greater attention to their study. With this progress in mind, an updated comprehensive overview of the roles of EVs in the immune system is timely. This Review summarizes the roles of EVs in basic processes of innate and adaptive immunity, including inflammation, antigen presentation, and the development and activation of B cells and T cells. It also highlights key progress related to deciphering the roles of EVs in antimicrobial defence and in allergic, autoimmune and antitumour immune responses. It ends with a focus on the relevance of EVs to immunotherapy and vaccination, drawing attention to ongoing or recently completed clinical trials that aim to harness the therapeutic potential of EVs.Extracellular vesicles (EVs) are increasingly recognized as having ubiquitous roles in the immune system. This Review focuses on the progress made in the field in the past 5 years, including the roles of EVs in innate and adaptive immunity and their potential use in diagnosis and therapy.

Circulating extracellular vesicles, which are produced by normal and diseased tissues and released into body fluids, can be markers of disease processes and perhaps even tools to deliver new therapies.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.This Review describes the state of the art in imaging extracellular vesicles in animals to study their release, biodistribution and uptake, and covers labeling strategies, microscopy methods and discoveries made in model organisms.

Beyond the consolidated role in degrading and recycling cellular waste, the autophagic- and endo-lysosomal systems play a crucial role in extracellular release pathways. Lysosomal exocytosis is a process leading to the secretion of lysosomal content upon lysosome fusion with plasma membrane and is an important mechanism of cellular clearance, necessary to maintain cell fitness. Exosomes are a class of extracellular vesicles originating from the inward budding of the membrane of late endosomes, which may not fuse with lysosomes but be released extracellularly upon exocytosis. In addition to garbage disposal tools, they are now considered a cell-to-cell communication mechanism. Autophagy is a cellular process leading to sequestration of cytosolic cargoes for their degradation within lysosomes. However, the autophagic machinery is also involved in unconventional protein secretion and autophagy-dependent secretion, which are fundamental mechanisms for toxic protein disposal, immune signalling and pathogen surveillance. These cellular processes underline the crosstalk between the autophagic and the endosomal system and indicate an intersection between degradative and secretory functions. Further, they suggest that the molecular mechanisms underlying fusion, either with lysosomes or plasma membrane, are key determinants to maintain cell homeostasis upon stressing stimuli. When they fail, the accumulation of undigested substrates leads to pathological consequences, as indicated by the involvement of autophagic and lysosomal alteration in human diseases, namely lysosomal storage disorders, age-related neurodegenerative diseases and cancer. In this paper, we reviewed the current knowledge on the functional role of extracellular release pathways involving lysosomes and the autophagic- and endo-lysosomal systems, evaluating their implication in health and disease.

Acute respiratory distress syndrome (ARDS) remains a major cause of respiratory failure in critically ill patients. Mesenchymal stromal cells (MSCs) are a promising candidate for a cell-based therapy. However, the mechanisms of MSCs' effects in ARDS are not well understood. In this study, we focused on the paracrine effect of MSCs on macrophage polarization and the role of extracellular vesicle (EV)-mediated mitochondrial transfer. To determine the effects of human MSCs on macrophage function in the ARDS environment and to elucidate the mechanisms of these effects. Human monocyte-derived macrophages (MDMs) were studied in noncontact coculture with human MSCs when stimulated with LPS or bronchoalveolar lavage fluid (BALF) from patients with ARDS. Murine alveolar macrophages (AMs) were cultured ex vivo with/without human MSC-derived EVs before adoptive transfer to LPS-injured mice. MSCs suppressed cytokine production, increased M2 macrophage marker expression, and augmented phagocytic capacity of human MDMs stimulated with LPS or ARDS BALF. These effects were partially mediated by CD44-expressing EVs. Adoptive transfer of AMs pretreated with MSC-derived EVs reduced inflammation and lung injury in LPS-injured mice. Inhibition of oxidative phosphorylation in MDMs prevented the modulatory effects of MSCs. Generating dysfunctional mitochondria in MSCs using rhodamine 6G pretreatment also abrogated these effects. In the ARDS environment, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype through EV-mediated mitochondrial transfer. MSC-induced changes in macrophage phenotype critically depend on enhancement of macrophage oxidative phosphorylation. AMs treated with MSC-derived EVs ameliorate lung injury in vivo.

Key Points Extracellular vesicle (EV) research in Gram-positive bacteria, mycobacteria and fungi was neglected until recently, owing to the presumption that vesicles could not traverse the thick cell walls found in these organisms. EVs are now understood to be produced by all types of microorganism, including those with thick cell walls, and are biologically active. EVs from bacteria, mycobacteria and fungi contain virulence factors, such as toxins, that are involved in pathogenesis and elicit strong host immune responses. For example, Cryptococcus neoformans EVs carry the capsular polysaccharide glucuronoxylomannan, which is an important virulence factor. Interaction of EVs with the host is specific to the microorganism from which the EVs were produced and is based on the lipid content and cargo of the EVs. Research into EVs produced by microorganisms with thick cell walls is a very young field. By learning how these microorganisms use EVs, we hope that researchers will gain insight into pathogenesis, therapeutics and vaccines. How extracellular vesicles traverse the thick cell walls of Gram-positive bacteria, mycobacteria and fungi has perplexed researchers. In this Review, Prados-Rosales and colleagues consider possible solutions to this conundrum and describe the diverse functions of the extracellular vesicles produced by these organisms. Extracellular vesicles (EVs) are produced by all domains of life. In Gram-negative bacteria, EVs are produced by the pinching off of the outer membrane; however, how EVs escape the thick cell walls of Gram-positive bacteria, mycobacteria and fungi is still unknown. Nonetheless, EVs have been described in a variety of cell-walled organisms, including Staphylococcus aureus , Mycobacterium tuberculosis and Cryptococcus neoformans . These EVs contain varied cargo, including nucleic acids, toxins, lipoproteins and enzymes, and have important roles in microbial physiology and pathogenesis. In this Review, we describe the current status of vesiculogenesis research in thick-walled microorganisms and discuss the cargo and functions associated with EVs in these species.

Key Points The transfer of extracellular vesicles (EVs) from one cell to another is thought to be an important mechanism for cell–cell communication, and EVs that are produced by virus-infected cells may modulate uninfected cells. Viruses have specific receptors and therefore usually have a more restricted cellular tropism. By contrast, the uptake of EVs is almost universal and can occur through several cellular endocytic mechanisms in addition to direct fusion, a property that enables systemic delivery of their content. The differences between viral and EV receptor usage can be used to separate and purify EVs. This enables the identification of specific EV-mediated effects and enables compounds that potentially inhibit the delivery and function of extracellular vesicles to be tested. Viruses that establish chronic and persistent infections in a host probably use EVs to enhance the establishment and maintenance of infection. The EVs that are produced from virus-infected cells (and may therefore differ in content) may also restrict virus infection and enable continued host viability and persistent viral infection. The incorporation of virions into EVs could prevent the recognition of viral proteins by the immune system and facilitate spread in the host. The release of membrane-bound vesicles from cells is being increasingly recognized as a mechanism of intercellular communication. In this Review, Raab-Traub and Dittmer discuss the roles that extracellular vesicles have during virus infection. The release of membrane-bound vesicles from cells is being increasingly recognized as a mechanism of intercellular communication. Extracellular vesicles (EVs) or exosomes are produced by virus-infected cells and are thought to be involved in intercellular communication between infected and uninfected cells. Viruses, in particular oncogenic viruses and viruses that establish chronic infections, have been shown to modulate the production and content of EVs. Viral microRNAs, proteins and even entire virions can be incorporated into EVs, which can affect the immune recognition of viruses or modulate neighbouring cells. In this Review, we discuss the roles that EVs have during viral infection to either promote or restrict viral replication in target cells. We will also discuss our current understanding of the molecular mechanisms that underlie these roles, the potential consequences for the infected host and possible future diagnostic applications.

Cells from all kingdoms of life produce extracellular vesicles (EVs). Their cargo is protected from the environment by the surrounding lipid bilayer. EVs from many organisms have been shown to function in cell-cell communication, relaying signals that impact metazoan development, microbial quorum sensing, and pathogenic host-microbe interactions. Here, we have investigated the production and functional activities of EVs in a surface-associated microbial community or biofilm of the fungal pathogen Candida albicans. Crowded communities like biofilms are a context in which EVs are likely to function. Biofilms are noteworthy because they are encased in an extracellular polymeric matrix and because biofilm cells exhibit extreme tolerance to antimicrobial compounds. We found that biofilm EVs are distinct from those produced by free-living planktonic cells and display strong parallels in composition to biofilm matrix material. The functions of biofilm EVs were delineated with a panel of mutants defective in orthologs of endosomal sorting complexes required for transport (ESCRT) subunits, which are required for normal EV production in diverse eukaryotes. Most ESCRT-defective mutations caused reduced biofilm EV production, reduced matrix polysaccharide levels, and greatly increased sensitivity to the antifungal drug fluconazole. Matrix accumulation and drug hypersensitivity of ESCRT mutants were reversed by addition of wild-type (WT) biofilm EVs. Vesicle complementation showed that biofilm EV function derives from specific cargo proteins. Our studies indicate that C. albicans biofilm EVs have a pivotal role in matrix production and biofilm drug resistance. Biofilm matrix synthesis is a community enterprise; prior studies of mixed cell biofilms have demonstrated extracellular complementation. Therefore, EVs function not only in cell-cell communication but also in the sharing of microbial community resources.

Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation. This protocol describes how to isolate up to six different subpopulations of extracellular vesicles (EVs) from tissues. The procedure includes detailed instructions for EV characterization using electron microscopy, RNA and protein analysis.