Exosomes derived from N2 neutrophils reprogram the immune microenvironment to promote spinal cord injury repair

Introduction Spinal cord injury induces irreversible neurological impairments via secondary pathological cascades dominated by excessive inflammation, oxidative stress, and neuronal apoptosis. As the first-responder immune cells following spinal cord trauma, neutrophils can acquire an N2-like reparative phenotype, yet direct neutrophil transplantation is hampered by poor cellular survival. Exosomes, nanoscale cell-secreted vesicles with low immunogenicity and favourable biostability, constitute promising cell-free candidates for regenerative therapy. Nevertheless, the therapeutic potential of exosomes derived from N2-polarized neutrophils remains poorly defined for spinal cord injury repair. Methods Murine bone-marrow-derived neutrophils were polarized toward the N2 phenotype using interleukin-6 (IL-6) and granulocyte-colony-stimulating factor (G-CSF), with non-polarized neutrophils set as the control group. Exosomes were purified by ultracentrifugation and comprehensively characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. In vitro functional assays were performed to evaluate macrophage phenotypic reprogramming, neuronal axonal regeneration, and neuronal apoptosis. A modified Allen's weight-drop mouse spinal cord injury model was adopted for in vivo intervention. Motor functional recovery was evaluated using the Basso Mouse Scale and gait analysis, while neural tissue repair was assessed via immunofluorescence staining. Results Exosomes derived from N2-polarized neutrophils (N2-exos) exhibited canonical exosomal hallmarks. In vitro, these exosomes drove lipopolysaccharide-primed macrophages to switch to an anti-inflammatory reparative phenotype, alleviated oxidative-stress-mediated axonal injury, and suppressed neuronal apoptosis. In vivo administration of N2-exos markedly enhanced hind-limb motor performance and gait coordination, and facilitated axonal regeneration across the injury epicenter. Conclusions N2-exos remodel the post-traumatic immune microenvironment and confer combined neuroprotective and axon regenerative effects. This cell-free modality represents a promising therapeutic candidate for regenerative interventions against spinal cord injury.