Understanding and interfering with resident microglia activation following subarachnoid hemorrhage (SAH)

在我们之前的工作中，我们已经将蛛网膜下腔出血（SAH）后的中枢神经系统免疫反应描述为先天免疫系统的激活。蛛网膜下腔室的血液产生促炎环境，导致脑基底巨噬细胞的激活，并诱导血管周围空间的炎症反应。随后，这以一种由外而内的方式激活了常驻的中枢神经系统特化小胶质细胞（如嵌合体实验所示）。此外，我们已经证明SAH后继发性神经轴突损伤与小胶质细胞激活有关。肿瘤坏死因子-α (TNF-α)已被确定为介导这种炎症反应的主要候选因子。此外，我们的初步工作表明，SAH后，细胞外RNA （eRNA）和DNA在脑内和周围积聚，并可能介导小胶质细胞释放TNF-α。eRNA已被证明可以在不同病理条件下（在中枢神经系统外）从细胞中释放出来，并诱导促炎和血管通透性诱导过程。因此，eRNA是调节髓细胞效应功能的靶标。在本文提出的项目中，我们首先旨在更详细地了解SAH后活化小胶质细胞的表型和功能。因此，我们将表征它们的炎症表型，并进一步研究这些效应细胞对SAH病理标志的影响，即早期血脑屏障（BBB）功能丧失和延迟的神经轴突损伤。这将通过结合表达谱、体外细胞、电生理和组织分析实验来实现。其次，我们将评估SAH后eRNA在蛛网膜下腔和大脑中积累的动力学和机制。在这里，我们将对小鼠和人类尸检标本进行染色，以量化eRNA的含量，并将在一系列体内和体外实验中确定eRNA的来源。第三部分，我们将在体外研究小胶质细胞释放TNF-α的机制及其下游信号传导。我们假设，SAH后释放的eRNA能够刺激中枢神经系统巨噬细胞/小胶质细胞释放TNF-α，并发挥效应功能，如血脑屏障损伤和神经轴突细胞死亡。在第四部分，我们将在体内干扰eRNA的功能，并将评估RNase1和RNase Inhibitor作为血管稳态调节剂来控制SAH脑损伤后eRNA的功能。为了验证这一点，我们将使用外源性RNase1/RNase Inhibitor或转基因ec特异性条件敲除RNase1和RNase Inhibitor。最后，我们将评估新的治疗策略，以干扰先天免疫系统的激活和实验性SAH后继发性脑损伤。在这里，我们将把重点放在针对(i)小胶质细胞和（ii） TNF-α的药理学策略上，它们有可能迅速转化为临床环境。

In our previous work, we have characterized CNS immune response after subarachnoid hemorrhage (SAH) as an activation of the innate immune system. Blood in the subarachnoid compartment creates a pro-inflammatory environment, leads to an activation of macrophages at the brain base and induces an inflammatory response at the perivascular space. This subsequently activates exclusively the resident CNS-specialized microglia (as shown by chimera experiments) in an outside-in fashion. Furthermore, we have demonstrated that secondary neuro-axonal injury after SAH is linked to microglia activation. Tumor-necrosis factor- α (TNF-α) has been identified as a primary candidate for mediating this inflammatory response. Besides, our preliminary work indicates that extracellular RNA (eRNA) and DNA accumulate in and around the brain after SAH and may mediate TNF-α liberation from microglia. eRNA has been shown to become released from cells under diverse pathological conditions (outside the CNS) and induces pro-inflammatory and vessel permeability-inducing processes. Thus, eRNA is a target for modulating myeloid cell effector functions. In the herein presented project, we first aim at understanding the phenotype and function of activated microglia after SAH in more detail. Therefore, we will characterize their inflammatory phenotype and further study the impact of these effector cells on the pathological hallmarks of SAH, i.e. early loss of blood-brain barrier (BBB) function and delayed neuro-axonal injury. This will be achieved by a combination of expression profiling, in vitro cellular, electrophysiological and histoanalytical experiments. Secondly, we will assess the dynamics and mechanisms of eRNA accumulation in the subarachnoid space and brains after SAH. Here, we will stain mouse and human autopsy specimens for quantified content of eRNA and will identify the source of eRNA in a set of in vivo and in vitro experiments. In the third part, we will study study the mechanism of TNF-α liberation by microglia and its downstream signaling in vitro. We hypothesis that eRNA, released after SAH, is capable of stimulating the release of TNF-α from CNS macrophages/microglia and exerting effector functions, such as BBB damage and neuro-axonal cell death. In the fourth part, we will interfere with eRNA functions in vivo and will evaluate RNase1 and RNase Inhibitor as modulators of vascular homeostasis to control the function of eRNA after brain injury following SAH. To test this, we will use exogenous RNase1/RNase Inhibitor or transgenic EC-specific conditional knockouts of Rnase1 and RNase Inhibitor. Finally, we will evaluate novel therapeutic strategies to interfere with the activation of the innate immune system and the subsequent secondary brain injury following experimental SAH. Here, we will put our emphasis on pharmacological strategies that target (i) microglial cells and (ii) TNF-α, which have the potential to be rapidly translated into a clinical setting.