The role of Infiltrating Macrophages in Seizure Generation Following CNS Infection

摘要/项目摘要 颞叶癫痫（TLE）是获得性癫痫最常见的形式。30%的患者 治疗难治性，迫切需要新的治疗方法。中枢神经系统（CNS）病毒感染 是TLE的一个重要原因，虽然脑炎是一个重要的贡献者，但如何 炎症导致癫痫发作尚不清楚。使用第一个病毒诱导的TLE小鼠模型， 本实验室发现，急性癫痫的诱发与外周血巨噬细胞 浸润到脑中并分泌促炎细胞因子，主要是IL-6。 通过从TMEV感染的脑中分离的浸润性巨噬细胞的RNA测序（RNAseq）， 在癫痫发作活动高峰期的小鼠中，我发现CNS浸润巨噬细胞的群体表达高水平的 髓样细胞上表达的触发受体-1（TREM 1）的水平。TREM 1的激活导致 促炎细胞因子的分泌。TREM 1表达增加与炎症相关 在一些动物中，TREM 1功能的抑制改善了炎症和疾病严重程度 炎症性疾病的模型。然而，TREM 1参与急性癫痫发作的发展是不可能的。 未知在目的1中，我将确定脑浸润巨噬细胞中TREM 1表达的时间过程， 解决TREM 1的遗传和药理学抑制是否会减少急性癫痫发作的发生 在TMEV感染的小鼠中，通过视频脑电图。促炎细胞因子IL-6主要由大脑产生， 在TMEV感染期间浸润巨噬细胞，在诱导癫痫发作中起关键作用;然而，IL-6如何 它是未知的。在目标2中，我将确定IL-6是否通过以下途径诱导神经元GABA受体下调： 生物素化测定，并解决哪种IL-6途径（经典或反式信号传导）参与急性癫痫发作。 由于脑中的炎性巨噬细胞与TMEV感染的小鼠中急性癫痫发作的产生相关， 在目标3中，我将确定巨噬细胞向抗炎表型倾斜是否会影响 通过视频脑电图来观察急性癫痫的发展我也会在脑浸润性巨噬细胞中识别微小RNA 在TMEV感染的小鼠急性癫痫发作过程中驱动炎症。视频脑电图 和受体生物素化测定是我在指导阶段（Aim 1/Aim 2a）学习的关键技术， 将是必不可少的进行第二阶段（目标2b/目标3）的实验。这些新技能，结合我以前的 培训，将使我在一个独特的位置，以解决有关神经炎症和癫痫的关键问题 在我自己的实验室使用尖端的方法，并将大大影响我的研究和职业生涯。 这些目标将提高炎症和癫痫发作之间关系的知识， 揭示炎性巨噬细胞作为神经炎症的新治疗靶点的巨大潜力， 允许开发新的干预以预防和治疗获得性癫痫的发展。

Abstract/Project Summary Temporal lobe epilepsy (TLE) is the most prevalent form of acquired epilepsy. 30% of the patients are refractory to therapy and new treatments are urgently needed. Viral infection of the central nervous system (CNS) is a significant cause of TLE, and, while encephalitis is an important contributor, the mechanisms of how inflammation leads to seizures is unclear. Using the first mouse model of viral-induced TLE, which was developed by our laboratory, we found that induction of acute seizures is associated with blood peripheral macrophage infiltration into the brain and secretion of pro-inflammatory cytokines, primarily IL-6. Through RNA sequencing (RNAseq) of infiltrating macrophages isolated from the brains of TMEV-infected mice at the peak of seizure activity, I found that a population of CNS-infiltrating macrophages expresses high levels of the triggering receptor expressed on myeloid cells-1 (TREM1). Activation of TREM1 leads to the secretion of pro-inflammatory cytokines. Increased TREM1 expression is associated with inflammatory conditions, and inhibition of TREM1 function ameliorates inflammation and disease severity in several animal models of inflammatory diseases. However, the involvement of TREM1 in the development of acute seizures is unknown. In Aim 1 I will determine the time course of TREM1 expression in brain-infiltrating macrophages, and address whether genetic and pharmacological inhibition of TREM1 decreases the development of acute seizures in TMEV-infected mice, via videoEEG. The pro-inflammatory cytokine IL-6, which is mainly produced by brain infiltrating macrophages during TMEV infection, plays a pivotal role in inducing seizures; however how IL-6 does it is unknown. In Aim 2, I will determine whether IL-6 induces downregulation of neuronal GABA receptors, via biotinylation assay, and address which IL-6 pathway (classical or trans-signaling) is involved in acute seizures. Since inflammatory macrophages in the brain are associated with generation of acute seizures in TMEV-infected mice, in Aim 3 I will determine whether skewing macrophages towards an anti-inflammatory phenotype will affect the development of acute seizures, via videoEEG. I will also identify microRNAs in brain-infiltrating macrophages that are driving inflammation during the course of acute seizure generation in TMEV-infected mice. VideoEEG and receptor biotinylation assay are critical techniques I will learn during my mentored phase (Aim1/Aim2a), that will be essential to carry out experiments in Phase II (Aim2b/Aim3). These new skills, combined to my previous training, will put me in a unique position to address crucial questions related to neuroinflammation and epilepsy in my own laboratory using cutting edge approaches, and will substantially impact my research and career. These aims will improve the knowledge of the relationship between inflammation and seizures, and has the great potential of revealing inflammatory macrophages as a novel therapeutic target of neuroinflammation, allowing for the development of novel intervention to prevent and treat the development of acquired epilepsy.