Characterization of miRNAs in Extracellular Vesicles Released From Atlantic Salmon Monocyte-Like and Macrophage-Like Cells

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.

Why Does Piscirickettsia salmonis Break the Immunological Paradigm in Farmed Salmon? Biological Context to Understand the Relative Control of Piscirickettsiosis

“…( 113 ) observed a change in the morphology, phagocytic ability, and miRNA profile of Atlantic salmon head-kidney leukocytes (HKLs) in vitro , showing that the cells differentiate from “monocyte-like” (Day 1) to “macrophage-like” (Day 5). At the same time, the abundance of some miRNAs in EVs was significantly different from the abundance of miRNAs in HKLs, suggesting that these miRNAs are involved in the immune response and/or macrophage activation ( 114 ). Smith et al.…”

Transcriptome Profiling of Atlantic Salmon Adherent Head Kidney Leukocytes Reveals That Macrophages Are Selectively Enriched During Culture

“…HKLs were isolated as previously described (32,36). Atlantic salmon were euthanized with an overdose of MS222 (0.4 g/L, Syndel Laboratories, Vancouver, BC, Canada).…”

Exosomal miRNAs in the plasma of Cynoglossus semilaevis infected with Vibrio harveyi: Pleiotropic regulators and potential biomarkers involved in inflammatory and immune responses

“…There were 19 miRNAs differently enriched in the EVs released from the “monocyte-like” head kidney leukocytes (HKLs) in comparison with the “macrophage-like” HKLs in Atlantic salmon. Among them, ssa-miR-146a-5p, ssa-miR-155-5p, and ssa-miR-731-5p were significantly upregulated ( P <0.001) in the “macrophage-like” HKLs, suggesting their important roles in HKL differentiation ( 32 ). A total of 10 viral proteins were identified in the exosomes derived from the kidney cells of the grass carp infected by the grass carp reovirus (GCRV), suggesting that exosomes may play important roles during GCRV infection ( 33 ).…”