Human milk-derived extracellular vesicles promote the heat shock response in polarized microglia

Proteotoxic stress induces microglia polarization and attenuates cytoprotective, pro-survival cellular cascades. Milk-derived extracellular vesicles (MEVs) are lipid-coated nanovesicles that combat pro-inflammation in peripheral cells and tissues; however, the cytoprotective potential of MEVs remains unknown in brain macrophages. We investigated whether MEVs reduce neuroinflammation in human microglia by activating the heat shock response (HSR). The HSR triggers the upregulation of molecular chaperones (heat shock proteins; HSPs) to restore proteostasis by refolding or degrading misfolded aggregates. MEVs were isolated from unpasteurized human donor milk. Human microglia clone 3 (HMC3) cells were primed with 10 ng/mL IFN-γ to induce polarization, and a subset of cells were supplemented with 200 µg of MEVs. The abundance of HSF1 and candidate HSPs (Hsp70, Hsp90, Hsp40, Hsp27) were analyzed via RT-qPCR and western immunoblotting at 6h, 12h, and 24h post-MEV supplementation. We found that MEV supplementation promoted the HSR in polarized microglia, compared to homeostatic cells. Furthermore, MEVs increased the duration of the HSR in response to pro-inflammatory stress, exerting robust and continued pro-survival benefits.