Extracellular Vesicles Derived from M1-Activated Microglia Induce Pro-Retinopathic Functional Changes in Microvascular Cells

DESIGN. Experimental study aimed at understanding the interactions between microglia and vascular cells in the diabetic retina. PURPOSE. Microglial cells are the main responsible for the modulation of the immune response to inflammatory stimuli inside the diabetic retina. Most studies addressing microglial potential to modulate diabetic retinopathy use rodent cells. An immortalized human microglial line, tested to verify its susceptibility to inflammation, is now available and represents a potential tool to investigate the pathophysiology of the inflammatory component of diabetic retinopathy in species-specific models. Extracellular vesicles (EVs) carry several molecules (miRNAs, mRNAs, proteins) and are known to exert a paracrine function in the transmission of signals between neighbouring tissues. Our aim was to characterize EVs released by microglial cells in inflammatory conditions and investigate their effects on the vascular components of the retina. METHODS. Human microglial cells were exposed for 24 h to an inflammatory cocktail (20 ng/mL hIL − 1β + 10 ng/ml hTNFα + 50 ng/mL hINFγ), able to induce M1 pro-inflammatory polarization. 50 µmL/L thiamine were also added, to test the anti-inflammatory potential of this vitamin. EVs were isolated from the supernatants and characterized by Nanosight and Western blotting. EV content in miRNAs and mRNAs was investigated. Human retinal endothelial cells (HRECs) and pericytes (HRPs) were exposed for 24 h to microglial-derived EVs and their response in terms of proliferation, apoptosis, migration, and ROS production assessed. RESULTS. All EVs isolated from the supernatants of microglial cells showed characteristic EV markers (Alix, CD63, CD81). Pro-inflammatory miR-21 and miR-155, as well as CCL-2 and MMP-2 expression, were increased in M1 microglia-derived EVs, and normalized by the addition of thiamine to microglia cultures. M1-derived EVs increased apoptosis, migration, and ROS production in both HRPs and HRECs, and HRP proliferation. Addition of thiamine normalized all these parameters. CONCLUSIONS. EVs derived from M1-activated microglia can stimulate functional changes in microvascular cells and induce features of retinopathy. Thiamine exerts an anti-inflammatory protective effect, when added to M1 microglial cultures.