Impact of exercise-induced extracellular vesicles on RAW 264.7 macrophage gene expression

Abstract

The overall process and outcome of muscle regeneration is well established yet communication nuances between different body systems during this process aren’t fully elucidated. One potential communication molecule is the extracellular vesicle (EV) as this molecule can have its regulatory content altered based on different physiological states. If muscles do use EVs as communicators, an ideal target would be the macrophage which nurtures tissue regeneration from start to finish. Since macrophage behavior is tightly regulated throughout regeneration, different macrophage cytokines are released based on the regeneration phase as one marker of their behavior. These cytokines can be inflammatory or non-inflammatory. Human subjects had their blood taken before, immediately after and 24 hours-post exercise and had EVs extracted from each timepoint. These EVs were used to treat macrophages in tissue culture flasks for 6 hours with subsequent RNA extracted for downstream, RT-qPCR analysis. We hypothesized if EVs impacted macrophage behavior, RT-qPCR would detect fold-change in either inflammatory or non-inflammatory cytokines based on the time-points the EVs originated from. Our study established Stx5a as a stable reference gene in murine macrophages for gene normalization and found TGFß and TNF⍺ cytokines were differentially expressed in both young and old human cohorts after EV treatment from various blood collection times.