Apoptosis is a form of programmed cell death that occurs throughout life as part of normal development as well as pathologic processes including chronic inflammation and infection. Although the death of a cell is often considered as the only biological outcome of a cell committed to apoptosis, it is becoming increasingly clear that the dying cell can actively communicate with other cells via soluble factors as well as membrane‐bound extracellular vesicles (EVs) to regulate processes including cell clearance, immunity and tissue repair. Compared to EVs generated from viable cells such as exosomes and microvesicles, apoptotic cell‐derived EVs (ApoEVs) are less well defined and the basic criteria for ApoEV characterization have not been established in the field. In this study, we will examine the current understanding of ApoEVs, in particular, the ApoEV subtype called apoptotic bodies (ApoBDs). We described that a subset of ApoBDs can be larger than 5 μm and smaller than 1 μm based on flow cytometry and live time‐lapse microscopy analysis, respectively. We also described that a subset of ApoBDs can expose a relatively low level of phosphatidylserine on its surface based on annexin A5 staining. Furthermore, we characterized the presence of caspase‐cleaved proteins (in particular plasma membrane‐associated or cytoplasmic proteins) in samples enriched in ApoBDs. Lastly, using a combination of biochemical‐, live imaging‐ and flow cytometry‐based approaches, we characterized the progressive lysis of ApoBDs. Taken together, these results extended our understanding of ApoBDs.

Extracellular vesicles (EVs) are an important class of membrane-bound structures that have been widely investigated for their roles in intercellular communication in the contexts of tumor progression, vascular function, immunity and regenerative medicine. Much of the current knowledge on the functions of EVs pertains to those derived from viable cells (e.g. exosomes and microvesicles) or apoptotic cells (e.g. apoptotic bodies) whilst the generation of EVs from dying cells under non-apoptotic conditions remains poorly characterized. Herein, the release of EVs from THP-1 monocytes under conditions of primary necrosis, secondary necrosis and pyroptosis, was investigated. A comprehensive analysis of THP-1-derived EVs revealed that cells undergoing lytic forms of cell death generated a high number of EVs compared with viable or apoptotic cells in vitro. Differential centrifugation via 16,000 g and 100,000 g revealed that dying THP-1 cells release both medium and small EVs, respectively, consistent with the known characteristics of microvesicles and/or exosomes. In addition, large EVs isolated via 2000 g centrifugation were also present in all samples. These findings suggest that lytic cell death under both sterile and non-sterile inflammatory conditions induces monocytes to generate EVs, which could potentially act as mediators of cell-to-cell communication.

Communication between dying cells and their environment is a critical process that promotes tissue homeostasis during normal cellular turnover, whilst during disease settings, it can contribute to inflammation through the release of intracellular factors. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound cell-derived structures that can engage in intercellular communication via the trafficking of bioactive molecules between cells and tissues. In addition to the well-described functions of EVs derived from living cells, the ability of dying cells to release EVs capable of mediating functions on target cells or tissues is also of significant interest. In particular, during inflammatory settings such as acute tissue injury, infection and autoimmunity, the EV-mediated transfer of proinflammatory cargo from dying cells is an important process that can elicit profound proinflammatory effects in recipient cells and tissues. Furthermore, the biogenesis of EVs via unique cell-death-associated pathways has also been recently described, highlighting an emerging niche in EV biology. This review outlines the mechanisms and functions of dying-cell-derived EVs and their ability to drive inflammation during various modes of cell death, whilst reflecting on the challenges and knowledge gaps in investigating this subgenre of extracellular vesicles research.