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Abstract
Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily “monocyte-like” at Day 1 to primarily “macrophage-like” at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.
Highlights
Extracellular vesicles (EVs) are cell-derived, lipid bilayerenclosed particles that are secreted from many, if not all, cell types, including immune cells [1,2,3]
Due to the limiting number of head kidney leukocytes (HKLs) isolated per fish, and the low amount of RNA available in extracellular vesicles (EVs), a total of 16 Atlantic salmon were used in this study: five individuals for RNA-seq, five individuals for reverse transcriptase (RT)-qPCR and nanoparticle tracking analysis (NTA), three individuals for Western blot, and three individuals for transmission electron microscopy (TEM)
We recognize that detection of additional protein markers is suggested in the MISEV2018 guidelines; the wide testing of available antibodies for the canonical EV markers that cross-react with Atlantic salmon proteins is beyond our present capacity
Summary
Extracellular vesicles (EVs) are cell-derived, lipid bilayerenclosed particles that are secreted from many, if not all, cell types, including immune cells [1,2,3]. Three categories of EVs have been described: exosomes (30–100 nm in diameter), which are formed when multivesicular bodies fuse with the plasma membrane to release intraluminal vesicles; microvesicles (100– 1000 nm in diameter), which are formed from direct budding of the plasma membrane; and apoptotic bodies (>1 mM in diameter), which are formed from the blebbing membrane of an apoptotic cell [4, 5]. For the purpose of this study, the term EV will refer to exosomes and microvesicles since, due to our isolation methods, large apoptotic bodies are unlikely to represent a major contribution to the observed results. EVs share some common characteristics, which enable their identification from cells and other particles. While EVs have been widely studied in mammals, there are only a few studies that examine EVs in teleost fish, which will be discussed below [7,8,9,10,11,12]
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