Pro-inflammatory small extracellular vesicles: neuroinflammation propagation and target spinal cord cells in explant cultures

Within the vast landscape of intercellular messengers, extracellular vesicles, namely membrane-enclosed nanosized-particles released by virtually all cells, are increasingly tied to central nervous system transcellular communication, yet their roles in propagating neuro-inflammation, in kindling astrocytic reactivity and neuronal dysfunctions, as well as the relationship among vesicle source cells and the recipient ones, remain largely unclear. To dissect extracellular vesicles’ dynamics and their still-elusive impact on neuron-glia interactions, we collect and separate vesicles from cultured ex-vivo organ spinal explants immuno-challenged by a cytokines’ cocktail. Inflammation-released vesicles are isolated either by ultracentrifugation or by size exclusion chromatography, identified by ultramicroscopy and immunoblot, and transferred to naïve spinal explants, devoid of inflammatory features. We report that spinal extracellular vesicles, only when released during cytokine treatment, act as pro-inflammatory messengers and trigger a distinct temporal pattern of tissue reactivity by spreading spinal reactivity to naïve astrocytes, neurons and microglia in recipient slices. We combine live calcium imaging, electrophysiology, confocal microscopy and Luminex assay to depict changes in astrocytic calcium events, neuronal synaptic activity and microglia morphology, as well as the emergence of pro-inflammatory cytokines and chemokines in recipient spinal organ slices. By delivering cre-packing extracellular vesicles to cre-reporter Ai9 organ explant slices, we further indicate that, even when cre-packing is selectively transduced only by neurons, astrocytes are pro-inflammatory vesicles’ preferential cell recipients.