Extracellular vesicles released from endothelial cells of the blood-brain barrier mediate brain Iron accumulation during LPS-induced brain Inflammation.

Introduction: Brain inflammation leads to an increase in the amount of iron in brain tissue; however, studies do not address the source of the iron that could lead to the accumulation. Most of the brain iron uptake is mediated through the blood-brain barrier (BBB), but studies have not examined whether inflammation increases or decreases iron flux across the BBB. Our recent in vitro study discovered a novel alternate mechanism that iron transport across the BBB is mediated via the extracellular vesicles (EVs). Herein, we investigated the impact of brain inflammation on iron release and iron transport via EVs from the brain microvasculature (BMV). Methods: For this study, we developed an in vivo brain inflammation model. We induced brain inflammation in three-month-old C57BL/6 by intracerebroventricular injection of lipopolysaccharide (LPS,12μg/mice). For in vitro, we used human blood-brain barrier endothelial cells derived from human-induced pluripotent stem cells (hiPSCs). We separated the BMV from brain parenchyma by using density gradient centrifugation. To inhibit the EVs synthesis, we injected intraperitoneally for 21 days GW4869 (60μg/mice), an inhibitor of neutral sphingomyelinase 2, a key regulatory enzyme necessary for EV formation. The brain EVs were isolated by ultracentrifugation. We measured the BMV and parenchyma iron concentration by Inductively coupled plasma mass spectrometry (ICP-MS). Furthermore, we performed immunoblotting to measure the protein expression in BMV and EVs. Results: The LPS injection activated microglia and astrocytes as well as increased the brain proinflammatory cytokines compared to the control mice. Furthermore, brain inflammation increased the iron levels in the brain parenchyma but decreased the iron levels in BMV. Brain inflammation was associated with the degradation of ferroportin (FPN1), an iron exporter, in the BMV. CD63, an EVs membrane protein, was increased in the BMV and associated with increased FTH1-iron release via EVs from BMVs to the brain. Moreover, brain inflammation induced iron deficiency in BMV as evidenced by an increase in the transferrin receptor and decreased FTH1, suggestive of increased iron uptake. Pharmacological reduction of EVs by GW4869 reduced iron accumulation in the inflamed brain parenchyma compared to control mice. Conclusion: This is the first study to demonstrate that EV inhibition decreases iron in the brain. Degradation of FPN1 in the BMV during inflammation did not limit iron accumulation but there was an increase in FTH1-iron-enriched EVs indicating these are responsible for brain iron accumulation during inflammation. Thus, in summary, we have discovered a novel mechanism that involves BMV-released EVs enriched with iron that is the mechanism for brain iron accumulation during inflammation.