Activated Retinal Microglia Secrete Exosomes Containing Galectin‐3

Abstract

PurposeNumerous reports support a potential role for activated microglial cells in the mechanisms of degenerative retinal and brain diseases. Although the exact mechanism remains unknown, our study and those of others describe the transport and release of cytotoxic substances by activated microglia during the initial stages of neuronal cell death. Among these substances, galectin‐3 has emerged as an important player in inflammatory conditions and neurodegenerative diseases by mediating and/or modulating microglial activation. Galectin‐3, a member of an evolutionary conserved family of carbohydrate‐binding lectins, lacks a signal peptide and is most likely secreted via a non‐classical exocytic pathway involving extracellular vesicles (exosomes and microparticles).MethodsTo investigate the role of galectin‐3‐derived retinal microglia in degenerative retinal diseases, activated microglia were isolated from 4–5 week old mice retinas with a targeted mutation of the c‐mer proto‐oncogene tyrosine kinase gene (Mertk−/−) and characterized for expression of microglia specific markers. Extracellular vesicles were purified from 48h conditioned media of cultured microglia and characterized for expression of galectin‐3 and Alix, an exosome marker, using immunocytochemistry and Western blots.ResultsRetinal microglia from Mertk−/− mice displayed an activated phenotype characterized by expression of CX3Cr1, CD11b, and Griffonia simplicifolia isolectin B4 but not of the Müller glial marker glial fibrillary acidic protein (GFAP) nor the vascular endothelial cell marker PECAM‐1. Intense galectin‐3 immunofluorescence was observed throughout the cell body and processes of microglial cells; producing a ~30 kDa galectin‐3 reactive band in the cytosolic component in Western blots. Abundant amounts of extracellular vesicles (~100–200 nm in size) secreted by the cells in 48h conditioned medium displayed a ~30 kDa galectin‐3‐reactive band and an Alix‐reactive band, consistent with exosomes, in Western blots.ConclusionsOur study shows that activated microglia isolated from early Mertk−/− mice with retinal degeneration expressed high levels of galectin‐3. Moreover, galectin‐3 co‐localized with Alix‐expressing exosomes secreted by cultured microglia. The increased production and release of exosomal galectin‐3 by activated retinal microglia support a potential role for galectin‐3 and microglial cells in the mechanisms of degenerative retinal diseases.