Hyperlipidemia abolishes, but immune balancing by DNase-I restores neuroprotection by MSC-derived extracellular vesicles

Background: Owing to their potent immunomodulatory properties, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (EVs) have emerged as promising neuroprotective treatments for ischemic stroke. Preclinical studies using MSC-EVs have mainly been performed in young, otherwise healthy rodents. Stroke patients frequently carry vascular risk factors and comorbidities. We herein investigated whether MSC-EVs retain neuroprotective activity in hyperlipidemic mice on cholesterol-rich Western diet. Methods: Male C57BL/6J mice were exposed to regular normal diet or Western diet for 6 weeks. At the age of 9-10 weeks, mice were exposed to transient intraluminal middle cerebral artery occlusion (MCAO). Vehicle or MSC-EVs (2x10^6 or 6x10^6 cell equivalents) were intravenously administered immediately after reperfusion, and vehicle or rosuvastatin (5 mg/kg/day) were intraperitoneally applied starting immediately after or seven days before MCAO. Neurological deficits, ischemic injury, and immune responses were evaluated up to 72 hours post-ischemia. To investigate the hyperlipidemia-associated immune dysregulation, mice received DNase-I before or immediately after MCAO. In defined subgroups, monocytes/ macrophages or neutrophils were additionally depleted by clodronate liposomes or anti-Ly6G antibodies, respectively. Results: In contrast to normolipidemic control mice, MSC-EVs failed to induce post-ischemic neuroprotection in hyperlipidemic mice. Neither MSC-EV dose escalation nor rosuvastatin co-treatment restored the therapeutic efficacy of MSC-EVs. Hyperlipidemia induced systemic innate immune dysregulation characterized by reduced monocyte/ macrophage activation, increased neutrophil activation, and elevated circulating cell-free DNA. DNase-I treatment before, but not after MCAO reversed these immune abnormalities and restored neuroprotection by MSC-EVs, decreasing neurological deficits, infarct volume and brain edema. Depletion of either monocytes/ macrophages or neutrophils abolished the neuroprotective effects of MSC-EVs in DNase-I-pretreated hyperlipidemic mice. Conclusions: Immune dysregulation abolishes MSC-EV-induced neuroprotection after ischemic stroke in hyperlipidemic mice. DNase-I priming restores MSC-EV responsiveness through mechanisms critically involving monocyte/ macrophage and neutrophil rebalancing. Our data highlight the host immune status as determinant of EV therapeutic efficacy.