Die Bedeutung der Hämoxygenase 1 und Kohlenmonoxid für die Erythrophagozytose
Klinische Neurologie; Neurochirurgie und Neuroradiologie
Zusammenfassung der Projektergebnisse
In this project, we defined two novel roles for microglial HO-1 and local CO-production in hemorrhagic brain injury. Our study in primary microglia, mice and human CSF samples shows that HO-1 expression in microglia regulates the neuroinflammatory response in response to hemorrhage and catalyzes the phagocytosis of erythrocytes via a pathway involving ROS, AMPK and CD36. We demonstrated in in vitro studies that the load of heme-containing blood components determines neuronal cell death and that microglia regulate neuroinflammation in response to hemorrhage via heme oxygenase-1 (HO-1, Hmox-1) and endogenous CO-release. In murine hippocampal HT22 cells, damage depended on the amount and duration of exposure to heme-containing blood components. HO-1 was induced in primary microglia (PMG) following blood exposure. Neuronal cells were protected from blood-induced cell death by microglia cell medium conditioned with blood. This was associated with a HO-1-dependent change in the cytokines released by wild-type compared to tissue-specific HO-1-deficient PMG (LyzM-Cre- Hmox1fl/fl). These in vitro findings correlated with results from a clinical study involving patients with SAH (subarachnoid hemorrhage): high HO-1 expression in relation to the actual hematoma size was associated with improved functional outcome at hospital discharge. Regarding the mechanisms microglial erythrophagocytosis, a burst in ROS-production was seen after CO-exposure, which led to an increased AMPK-phosphorylation with subsequently enhanced CD36-expression. Naïve PMG from Hmox1-deficient mice (LyzM-Cre-Hmox1fl/fl) showed reduced ROS-production and CD36-expression and failed to respond to CO with increased CD36-expression. From a functional standpoint, lack of HO-1 and CD36 resulted in markedly reduced erythrophagocytosis that could not be rescued with CO. Erythrophagocytosis was enhanced in BV-2 cells in the presence of exogenous CO, which was abolished in cells treated with siRNA against AMPK. CD36-/- mice subjected to SAH showed enhanced neuronal injury compared to wildtype, which resulted in impaired spatial memory function. Collectively, we demonstrate that neuronal cell death after hemorrhage is attributable to the deposition of heme-containing blood components. We further show that microglial HO-1 induction with subsequent endogenous CO production functions as a crucial signaling pathway in response to blood-induced inflammation, determining the local inflammatory response and the extent of neuronal cell death. We further demonstrate that the phagocytic function of microglia partly depends on a pathway involving ROS, AMPK and CD36. CD36 was identified as a crucial component in erythrophagocytosis and blood clearance after hemorrhage that ultimately determines neuronal outcome. These results give further insight into the pathophysiology of neuronal damage after SAH and how HO-1 in humans is associated with improved functional outcome. They demand further clinical investigations studying the potential neuroprotective properties of CO.
