Characterising the structure-function relationships of apoptotic cell-derived extracellular vesicles in modulation of inflammation

Abstract

Apoptosis and apoptotic cell clearance by phagocytes are essential processes for maintaining tissue homeostasis and resolution of inflammation. Dysfunction in these processes can cause chronic inflammation and autoimmunity. Extracellular vesicles (EVs) mediate intercellular communication and regulate many physiological processes, however, apoptotic cell-derived EVs (ACdEVs) produced by dying immune cells are poorly understood, despite high constitutive levels of immune cell death. This study aimed to address this lack of understanding, in particular the ACdEV proteome, and the role of ACdEV-associated proteins in stimulating macrophage chemoattraction, binding/uptake, and phenotypic changes. Apoptotic cells (UV-induced) released a heterogeneous population of ACdEVs (~50-1000 nm diameter). The THP-1 monocytic cell line was utilised to establish an in vitro model for interactions between macrophages and ACdEVs: chemotaxis, binding/uptake, and phenotype modulation. Using flow cytometry to assess macrophage phenotype, cell surface markers surprisingly suggested a pro-inflammatory response to ACdEVs, and this was supported by analysis of cytokine release. Proteomic analyses revealed the diversity of ACdEV proteomes, and gene ontology identified numerous ACdEV-associated proteins with relevant functions, including calreticulin and various adhesion molecules. Comparing the proteomes of ACdEVs produced during early and late apoptosis showed differences in protein abundance/enrichment. Proteins enriched in early ACdEVs may help to ensure timely clearance of apoptotic cells. This study found that EV function can be differentially affected by the isolation method used. Size exclusion chromatography (SEC) resulted in ACdEVs losing their chemoattractive function, whereas ultracentrifugation (UC) did not. This suggests that surface-associated factors mediate some, but not all ACdEV functions. Functional studies suggest that calreticulin plays a role in recruitment of macrophages, in addition to its previously established role mediating phagocytosis. Data presented here suggest that ACdEVs are complex signalling entities with important roles in apoptotic cell clearance and regulation of inflammation, and much remains to be elucidated regarding mechanisms and molecular players.