Formation of a protein corona on the surface of extracellular vesicles in blood plasma.

In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research.

An implanted device enables in vivo monitoring of extracellular vesicle-mediated spread of pro-inflammatory mast cell response in mice.

Mast cells have been shown to release extracellular vesicles (EVs) in vitro. However, EV-mediated mast cell communication in vivo remains unexplored. Primary mast cells from GFP-transgenic and wild type mice, were grown in the presence or absence of lipopolysaccharide (LPS), and the secreted EVs were separated from the conditioned media. Mast cell-derived EVs were next cultured with LPS-naïve mast cells, and the induction of TNF-α expression was monitored. In addition, primary mast cells were seeded in diffusion chambers that were implanted into the peritoneal cavities of mice. Diffusion chambers enabled the release of GFP+ mast cell-derived EVs in vivo into the peritoneal cavity. Peritoneal lavage cells were assessed for the uptake of GFP+ EVs and for TNF-α production. In vitro, LPS-stimulated mast cell-derived EVs were efficiently taken up by non-stimulated mast cells, and induced TNF-α expression in a TLR4, JNK and P38 MAPK dependent manner. In vivo, using implanted diffusion chambers, we confirmed the release and transmission of mast cell-derived EVs to other mast cells with subsequent induction of TNF-α expression. These data show an EV-mediated spreading of pro-inflammatory response between mast cells, and provide the first in vivo evidence for the biological role of mast cell-derived EVs.

Mast cell secretome: Soluble and vesicular components.

Mast cells are multifunctional master cells implicated in both innate and adaptive immune responses. Their role has been best characterized in allergy and anaphylaxis; however, emerging evidences support their contribution to a wide variety of human diseases. Mast cells, being capable of both degranulation and subsequent recovery, have recently attracted substantial attention as also being rich sources of secreted extracellular vesicles (including exosomes and microvesicles). Along with secreted de novo synthesized soluble molecules and secreted preformed granules, the membrane-enclosed extracellular vesicles represent a previously unexplored part of the mast cell secretome. In this review article we summarize available data regarding the different soluble molecules and membrane-enclosed structures secreted by mast cells. Furthermore, we provide an overview of the release mechanisms including degranulation, piecemeal degranulation, transgranulation, and secretion of different types of extracellular vesicles. Finally, we aim to give a summary of the known biological functions associated with the different mast cell-derived secretion products. The increasingly recognized complexity of mast cell secretome may provide important novel clues to processes by which mast cells contribute to the development of different pathologies and are capable of orchestrating immune responses both in health and disease.