Hypoxia-derived exosomes and RNA-binding protein-enriched fractions modify inflammatory responses in normal microglial cells

Abstract

Introduction: Brain hypoxia triggers neuroinflammatory responses that are partly mediated by microglia and their secreted exosomes. This study investigated whether hypoxia-derived microglial exosomes and RNA-binding protein (RBP)-enriched fractions are associated with alterations in the inflammatory phenotype of healthy microglial cells. Methods: Neonatal Wistar rats were randomly assigned to four groups (n = 5/group): control (C), hypoxia (H), one-week post-hypoxia (HW1), and age-matched one-week control (CW1). Brain tissues and primary microglia were collected to isolate exosomes and RBP-enriched fractions. RBP-enriched fractions were obtained using RNA-conjugated Fe nanoparticles linked by EDC-NHS chemistry and subsequently incorporated into microglial exosomes. Healthy primary microglial cells were treated with exosomes, RBP-enriched fractions, or exosome-loaded RBP-enriched fractions (20 µg/mL; five biological replicates per treatment). Assessments included cell viability, expression of inflammatory and anti-inflammatory genes, innate immune responses, and exosome biodistribution. Results: Microglia-derived exosomes showed preferential accumulation in brain tissue following systemic administration. Treatment by exosomes from the hypoxia group were associated with increased microglial viability and elevated expression of inflammatory markers 24 hours post-treatment. In contrast, exosomes containing hypoxia-derived RBP-enriched fractions were associated with increased ratio of TGF-β/TNF-α and CD206/CD80 expression and reduced expression of CD80, IL-1β, and NOX2 compared with their respective exosome controls. In innate immune assays, RBP-HW1 significantly reduced antiprotease activity, while RBP-H significantly decreased lysozyme and myeloperoxidase activities. Discussion: These findings suggest that the biological effects of microglial exosomes depend on the physiological state of the donor cells, and that hypoxia-derived RBP-enriched fractions are associated with the modulation of inflammatory responses in recipient microglia. However, because the RBP-enriched fractions were not characterized by mass spectrometry or specific immunoblotting, and mechanistic intervention experiments were not conducted, the responsible molecular components and causal pathways remain to be identified.

Keywords: Microglia, hypoxia, inflammation, exosome, RNA binding proteins