Exosomes: unconventional players in spinal cord neuroinflammation

Neuroinflammation is a common underlying event in all central nervous system diseases, essentially based on altered communication patterns between resident cells (neurons and glia), which finally leads to detrimental functional alteration and potentially irreversible cell damage. Extracellular vesicles (EVs) are a recently discovered intercellular delivery system secreted by cells for specific intercellular communication. They are composed by a lipid bilayer containing a wide range of molecular signals like cytokines, microRNA, receptors and enzymes able to modify the phenotype of the recipient cells, integrating coordinated physiological response to environmental stimuli. Given their pivotal role in conveying key regulatory molecules, EVs are now intensively studied as novel fundamental players in many pathological processes, including neuroinflammation. Here, we studied the dysfunctional glial cells behaviour induced by EVs in the context of spinal cord neuroinflammation. Particularly, we focused on dissecting the role of exosomes, the smaller class of EVs originated by the endolysosomal secretory pathway, in promoting a proinflammatory phenotype in astrocytes and microglia, in organotypic spinal cord slices. We first realized an efficient protocol to isolate small extracellular vesicle (sEVs), primarily constituted by exosomes, to our knowledge for the first time, from spinal slices after LPS-induced neuroinflammation. We then used such vesicles (from here on referred to as exosomes or EXO) isolated from the inflammatory microenvironment to treat naïve slices and characterize their impact on healthy spinal tissue. Our results showed that upon inflammatory stimulation with LPS, EXOs secretion significantly increase compared to control condition. Interestingly, EXOs isolated from LPS treated slices were able to induce microglia activation and proliferation comparably to LPS. Furthermore, live calcium imaging recordings using GCaMP6f expressed only in GFAP+ cells showed that inflammatory EXOs can disrupt astrocytic physiological calcium dynamics increasing the frequency of Ca2+ events. In order to obtain novel mechanistic cues about the ability of inflammatory EXOs in modulating glial calcium dynamics we focused on connexin 43 (Cx43) hemichannels. Cx43 is the main component of astrocytic gap junctions and uncoupled hemichannels, key macromolecular structures in the regulation of intra- and intercellular Ca2+ signalling. Interestingly, inflammatory EXOs treatment was able to promote Cx43 hemichannel opening in GFAP+ cells, as revealed by Lucifer Yellow (LY) permeability assay. Our previous experiments showed that gap27, a Cx43 inhibitor peptide, was able to restore physiological calcium signals in spinal slices challenged with diverse danger stimuli, including LPS. Interestingly, inflammatory EXOs preincubated with gap27, didn’t affect astrocytic calcium dynamics suggesting a pivotal role for exosomal Cx43 hemichannels in transfer danger signals to the recipient cells. All these evidences suggest that exosomes play an active role in mediating and spreading neuroinflammatory events, modulating glial cells reactivity through a Cx43-mediated mechanism.