Defining the surface proteome of apoptotic cell-derived extracellular vesicles

Abstract

The release of extracellular vesicles is essential for intercellular communication and transport of molecular cargo. During apoptosis, cells release a significant quantity of apoptotic cell-derived extracellular vesicles (ACdEVs), which play a critical role in regulating inflammation by attracting immune cells for apoptotic cell clearance and enhancing their pro-resolving functions, thus promoting tissue homeostasis. Despite their abundance, the molecular mechanisms governing ACdEV interactions with recipient immune cells remain poorly understood. This project aimed to elucidate the surface proteome of ACdEVs to identify key surface molecules involved in immune cell engagement, with only CX3CL1 and ICAM-3 identified to date. ACdEVs from various immune cell sources and apoptotic timepoints revealed fundamental differences in physical and molecular properties, highlighting their heterogeneity. Data mining of in-house mass spectrometry proteomic ACdEV datasets led to the selection of adhesion proteins and known immunomodulators CD31, CD47, CD49d, and CD166, for investigation. Notably, these proteins were lost from the apoptotic cell surface and detected on ACdEVs. The interactions of ACdEVs with macrophages, key regulators of apoptotic cell clearance, were also examined to assess their influence on macrophage migration, binding, internalisation, and phenotype. Strikingly, ACdEVs were chemoattractive to macrophages, but this property was abolished by size-exclusion chromatography, suggesting disruption of a functional chemoattractive ACdEV corona. Antibody blockade enabled the functional assessment of individual proteins at the ACdEV surface. Together, these data indicated a ‘don't eat me’ function exerted by CD47 and ‘eat me’ functions exerted by CD31 and CD49d, highlighting their roles in regulating ACdEV surface interactions. Furthermore, the four adhesion proteins did not appear to modulate macrophage phenotype. This research, therefore, identifies surface proteins involved in the interactions between ACdEVs and recipient macrophages that do not affect phenotypic changes, thereby enhancing our understanding of the molecular mechanisms that facilitate ACdEV interactions with recipient cells and their role in immunomodulation.