Plasma exosomes are enriched in Hsp70 and modulated by stress and cortisol in rainbow trout

Abstract

Exosomes are endosomally derived vesicles that are secreted from cells and contain a suite of molecules, including proteins and nucleic acids. Recent studies suggest the possibility that exosomes in circulation may be affecting recipient target cell function, but the modes of action are unclear. Here, we tested the hypothesis that exosomes are in circulation in fish plasma and that these vesicles are enriched with heat shock protein 70 (Hsp70). Exosomes were isolated from rainbow trout (Oncorhynchus mykiss) plasma using differential centrifugation, and their presence was confirmed by transmission electron microscopy and the exosomal marker acetylcholinesterase. Plasma exosomes were enriched with Hsp70, and this stress protein was transiently elevated in trout plasma in response to a heat shock in vivo Using trout hepatocytes in primary culture, we tested whether stress levels of cortisol, the principle corticosteroid in teleosts, regulates exosomal Hsp70 content. As expected, a 1-h heat shock (+15°C above ambient) increased Hsp70 expression in hepatocytes, and this led to higher Hsp70 enrichment in exosomes over a 24-h period. However, cortisol treatment significantly reduced the expression of Hsp70 in exosomes released from either unstressed or heat-shocked hepatocytes. This cortisol-mediated suppression was not specific to Hsp70 as beta-actin expression was also reduced in exosomes released from hepatocytes treated with the steroid. Our results suggest that circulating Hsp70 is released from target tissues via exosomes, and their release is modulated by stress and cortisol. Overall, we propose a novel role for extracellular vesicular transport of Hsp70 in the organismal stress response.

Isolation and Characterization of Serum Extracellular Vesicles (EVs) from Atlantic Salmon Infected with Piscirickettsia Salmonis

“…Serum is perhaps the most frequently studied biological fluid in fish and one of the most promising biomarker sources. Studies from Faught et al [ 45 ] show that circulating heat shock protein (Hsp7) is released from target tissues via exosomes, and its release is modulated by stress and cortisol. The authors propose a novel role for EVs transport of Hsp70 in the organismal stress response.…”

The mineralocorticoid receptor is essential for stress axis regulation in zebrafish larvae

“…SDS-PAGE and Western blotting was performed as previously described 46 . Briefly, samples were homogenized in 50 mM Tris buffer (pH 7.5) with a protease inhibitor cocktail (Roche).…”

Integrative Modeling and Visualization of Exosomes

“…We then applied an enrichment factor for proteins known to be enriched in exosomes: six-fold enrichment for HSPA8 (Faught et al 2017), HSP90 at seven times more than HSPA8 (Clayton et al 2005), 124-fold enrichment for tetraspanins CD9 and CD81 and seven-fold for CD63 (Escola et al 1998). ESCRTfamily proteins (PDCD6IP and TSG101) and rab7/rab5 proteins work with tetraspanins for intracellular trafficking and are commonly seen at high levels in proteomics (Tauro et al 2012), so we applied an arbitrary enrichment factor of 20 for them.…”