Structural and Functional Characterisation of Apoptotic Cell-Derived Extracellular Vesicles

Abstract

Persistent and uncontrolled inflammation promotes the development of numerous chronic pathologies. During efferocytosis, apoptotic cells (AC) are recognized and engulfed by professional phagocytes. This phagocytic event is guided by the release of “find-me” signals creating a chemotactic gradient that aids the recruitment of macrophages. AC also release extracellular vesicles (ACdEVs) in the environment, and although this subset of EVs has not yet been extensively characterized, evidence highlights their role as key mediators of efferocytosis. This study aims to understand the complex mechanisms of the resolution of inflammation mediated by ACdEVs. AC (UV induced) were shown to continuously release highly heterogeneous EVs, as observed by their various structures/shapes (Cryo-TEM), and polydisperse size distribution (resistive pulse sensing). ACdEVs were shown to have a similar membrane fluidity as their parent cells as measured by laurdan fluorescence. Interestingly, comparing early and late ACdEVs revealed differences in lipid composition, which may reflect different biogenesis pathways. ACdEVs samples depleted of soluble factors lose the ability to recruit macrophages whereas their uptake remained unchanged. Depleting the membrane of cholesterol resulted in an increased membrane rigidity and surface charges which was found to promote the ACdEVs uptake. Early and late apoptosis timepoints were defined by the kinetic study of UV-induced apoptosis as 6h and 18h, respectively. Early ACdEVs were more efficiently engulfed but less chemo attractive than the late ACdEVs, which may result in different immunomodulatory functions. The characterization of EV subtypes in different contexts is extremely challenging. Here, an alternative passive microfluidic method was shown to continuously separate EVs (above 500 nm) based on their size, while retaining soluble factors. Altogether, these data suggest that ACdEVs are complex mediators of efferocytosis and also highlights the importance of eat-me and find-me signals in ACdEV’s functions. Lipidomics should support a new and fundamental way of discriminating between different EV subtypes and, indeed, the lipid composition of the membrane is likely vital in tuning the activity of these subtypes.