Activated Microglia-Derived Extracellular Vesicles Elicit a Pro-Inflammatory Astrocytic Response via Cargo-Dependent Mechanisms

Neuroinflammation plays a dual role in brain health supporting defense and repair, but causes neurotoxicity when persistent. Microglia and astrocytes coordinate these responses through cytokine signaling and extracellular vesicles (EVs), though their vesicle-mediated communication remains unclear. This study investigated whether EVs from activated microglia (ABEVs) influence astrocyte polarization and inflammatory signaling. BV-2 microglial cells were activated with lipopolysaccharide (LPS), and microvesicle (ABMVs) and exosome (ABEXs) EVs were isolated via sequential ultracentrifugation. Primary mouse astrocytes were treated with LPS, ABMVs, or ABEXs, and expression of reactive astrocyte markers (<i>C3</i>, <i>Serpina3n</i>, <i>S100a10</i>, <i>Sphk1</i>) and inflammatory mediators (<i>Lcn2</i>, <i>Il-1β</i>, <i>Ccl2</i>, <i>Ccl5</i>, <i>Cxcl10</i>) was quantified, and EV protein cargo was analyzed by mass spectrometry and proteomics. LPS-treated astrocytes exhibited increased <i>C3</i> and <i>Serpina3n</i> and decreased <i>S100a10</i>, consistent with reactive polarization. ABEXs mimicked this effect, significantly inducing <i>C3</i>, <i>Serpina3n</i>, and <i>Sphk1</i>, whereas ABMVs had a weaker influence. ABEXs also upregulated <i>Lcn2</i> and <i>Il-1β</i>, partially reproducing microglial inflammatory effects. Proteomic profiling revealed marked cargo differences: ABEXs exhibited 16 upregulated proteins linked to NOD-like receptor signaling compared to non-activated BEXs, and 165 proteins associated with ribosome biogenesis and spliceosome pathways compared to ABMVs, indicating subtype-specific signaling potential. Collectively, our findings demonstrate that microglia-derived EVs modulate astrocytic polarization and cytokine profiles in a cargo-dependent manner, emphasizing their importance in interglial communication and revealing novel targets for neuroinflammatory modulation.