AQP4-IgG-Induced Astrocyte-Derived Small Extracellular Vesicles Carrying Mitochondrial DNA Regulate the TLR9/MyD88/NF-κB Pathway to Drive Microglial Activation and Neuromyelitis Optica

Neuromyelitis optica spectrum disorder (NMOSD) is a rare but serious inflammatory demyelinating disease. A key characteristic of NMOSD is the presence of a pathogenic autoantibody in serum called aquaporin-4 immunoglobulin G (AQP4-IgG). This study investigates the mechanism of astrocyte-derived small extracellular vesicles (EVs) carrying mitochondrial DNA (mtDNA) to promote AQP4-IgG-induced microglial activation in neuromyelitis optica (NMO) via the Toll-like Receptor 9 (TLR9)/myeloid differentiation primary response 88 (MyD88)/nuclear factor-kappa B (NF-κB) pathway. Serum IgG was isolated from NMOSD patients (AQP4-IgG) and healthy controls (Con-IgG). Astrocytes were treated with AQP4-IgG or Con-IgG. EVs were isolated via ultracentrifugation, characterized, and examined for internalization. Microglia were exposed to EVs, and mtDNA levels were assessed. An NMO mouse model was established, with neurological damage, mouse behaviors, tissue damage, and microglial characterization evaluated using modified neurological severity score, open-field test, rotarod test, luxol fast blue staining, and flow cytometry. Inflammatory cytokines, TLR9, MyD88, p65, IκBα, p-p65, and p-IκBα in BV2 cells and spinal cord tissues were analyzed via ELISA, RT-qPCR, and western blot. AQP4-IgG-induced astrocyte-derived EVs increased Iba1-high-expressing and CD86/tumor necrosis factor-α-high-expressing cells, reduced CD206/transforming growth factor-β-high-expressing cells, and boosted inflammatory responses. AQP4-IgG-induced EVs carried mtDNA to activate microglia via the TLR9/MyD88/NF-κB pathway. TLR9/MyD88/NF-κB pathway inhibition reversed AQP4-IgG-induced EVs' promotion on microglial activation. In vivo, AQP4-IgG-induced EVs-mtDNA exacerbated microglial activation and NMO through the TLR9/MyD88/NF-κB pathway. AQP4-IgG-induced EVs carried mtDNA to upregulate TLR9, further activating the MyD88/NF-κB pathway, thereby promoting microglial activation and transition toward pro-inflammatory gene-high-expressing cells to drive NMO progression.

Aquaporin-4 immunoglobulin g; Astrocyte-Derived small extracellular vesicles; Microglia; Mitochondrial DNA; Neuroinflammation; Neuromyelitis optica; Toll-like receptor 9/myeloid differentiation primary response 88/Nuclear factor-kappa b.