Red Blood Cell-Derived Exosomes Deliver Complement C5 to Exacerbate Neuroinflammation and Neuronal Injury after Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) induces neuroinflammation and neuronal damage, partially driven by microglial necroptosis and M1 polarization. Recent evidence implicates red blood cell-derived exosomes (RBC-Exos) in inflammatory pathologies, yet their role in ICH remains unexplored. This study investigates how hemorrhagic RBC-Exos (ICH-Exos) regulate microglial dysfunction and neuropathology through complement signaling. RBC-Exos were isolated and validated using flow cytometry (AnnexinV(+)CD235(+)), transmission electron microscopy, nanoparticle tracking analysis, and western blot (CD63/TSG101). In vitro, hemin-treated microglia were exposed to Normal-Exos or ICH-Exos. Murine ICH models assessed neuropathology via histology (H&E, Nissl staining), adhesion molecule expression (ICAM-1/VCAM-1), and neurobehavioral tests. Proteomics identified exosomal protein cargo, complemented by C5 monoclonal antibody (mAb) blocking experiments to dissect mechanistic pathways. ICH-Exos exacerbated hemin-induced microglial necroptosis, marked by upregulated phosphorylated RIPK1, RIPK3, and MLKL, and amplified M1 polarization (elevated CD86, iNOS, CCL2; suppressed CD163, ARG1). In ICH mice, ICH-Exos aggravated cerebral hemorrhage, neuronal loss, and neurobehavioral deficits while elevating pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Proteomics revealed C5 enrichment in ICH-Exos, correlating with elevated C5a levels in serum and brain tissues. C5 mAb neutralized ICH-Exos effects, rescuing microglial viability, restoring M1/M2 equilibrium, and attenuating necroptosis. In vivo, C5 inhibition reduced hemorrhage volume, restored neuronal Nissl bodies, suppressed ICAM-1/VCAM-1, and mitigated MAPK pathway activation (p-ERK1/2, p-p38). In conclusion, ICH-Exos drive neuroinflammation and neuronal injury post-ICH by promoting C5-dependent microglial necroptosis and M1 polarization. Targeting the C5/C5a axis counteracts these effects, suggesting a novel therapeutic strategy to ameliorate ICH-related neuropathology. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10753-026-02456-z.