Caco-2 cells infected with rotavirus release extracellular vesicles that express markers of apoptotic bodies and exosomes

Abstract

Previously, we showed that infecting human intestinal epithelial cells (Caco-2) with rotavirus (RV) increases the release of extracellular vesicles (EVs) with an immunomodulatory function that, upon concentration at 100,000×g, present buoyant densities on a sucrose gradient of between 1.10 to 1.18 g/ml (characteristic of exosomes) and higher than 1.24 g/ml (proposed for apoptotic bodies). The effect of cellular death induced by RV on the composition of these EV is unknown. Here, we evaluated exosome (CD63, Hsc70, and AChE) and apoptotic body (histone H3) markers in EVs isolated by differential centrifugation (4000×g, 10,000×g, and 100,000×g) or filtration/ultracentrifugation (100,000×g) protocols. When we infected cells in the presence of caspase inhibitors, Hsc70 and AChE diminished in EVs obtained at 100,000×g, but not in EVs obtained at 4000×g or 10,000×g. In addition, caspase inhibitors decreased CD63 and AChE in vesicles with low and high buoyant densities. Without caspase inhibitors, RV infection increased exosome markers in all of the EVs obtained by differential centrifugation. However, CD63 preferentially localized in the 100,000×g fraction and H3 only increased in EVs concentrated at 100,000×g and with high buoyant densities on a sucrose gradient. Thus, RV infection increases the release of EVs that, upon concentration at 100,000×g, are composed by exosomes and apoptotic bodies, which can partially be separated using sucrose gradients.

Actin-Dependent Nonlytic Rotavirus Exit and Infectious Virus Morphogenetic Pathway in Nonpolarized Cells

“…We found that a fraction of the rotavirus particles in the cell medium were associated with cellular membranes, in agreement with recent reports that have shown that naked viruses can be released from infected cells within vesicles (22,(40)(41)(42), although in the case of rotavirus the type of association that the viral particles have with membranous structures remains to be determined. In this regard, Barreto et al reported that the structural viral protein VP6 is associated with the exosome marker CDC63 in vitro and in vivo (43), and the same group described that rotavirus infection increases the release of extracellular vesicles (44). While electron microscopy examination of polarized, rotavirus-infected Caco-2 cells did not reveal the presence of extracellular virus within vesicles (15), in this work we found that about 50 to 60% of the extracellular virus was associated with low-density fractions in iodixanol flotation gradients, and the infectivity of the floating virus increased after treatment with Triton X-100, suggesting that a significant fraction of infectious virus is protected by membranous structures.…”

Extracellular vesicles in diagnostics and therapy of the ischaemic heart: Position Paper from the Working Group on Cellular Biology of the Heart of the European Society of Cardiology

“…Eukaryotic EVs include (i) exosomes released by exocytosis of multivesicular bodies (usually 50–150 nm), (ii) Microvesicles (MVs, also called microparticles or ectosomes), vesicles around 0.1–1 μm in diameter shed from the plasma membrane, 10–12 and (iii) apoptotic vesicles released by blebbing of apoptotic cells some of which are > 1 μm (apoptotic bodies), 13 , 14 and (iv) large oncosomes released by migratory tumour cells (> 1 µm) 15 , 16 ( Figure 2 ). However, since current isolation protocols only result in relative enrichment of vesicle subpopulations rather than their complete purification, 17 and that no specific markers are available for the subpopulations, it is preferable to refer to purified vesicles as ‘EVs’ and accurately report the purification method used and characteristics present.…”

Assessing the oncolytic potential of rotavirus on mouse myeloma cell line Sp2/0-Ag14

“…However, rotavirus-infected Sp2/0-Ag14 cells appear to undergo lysis rather than fragmentation into apoptotic bodies. Rotaviruses have been reported to induce apoptosis in Caco-2 cells 53 and oncosis in MA104 cells 54 where virus infection affects cell membrane integrity without inducing DNA fragmentation or formation of apoptotic bodies. On the other hand, rotavirus nonstructural protein NSP1 has been found to suppress apoptotic signaling during the first 6 h post-infection to favor cell survival 55 .…”