Administrative Supplement for DePaul-Silva K22 award

摘要/项目摘要 颞叶癫痫(TLE)是获得性癫痫最常见的形式。30%的患者是 治疗难治，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，但其机制是 炎症导致癫痫发作的原因尚不清楚。利用第一个病毒诱导的小鼠TLE模型， 我们实验室发现，急性癫痫的诱发与外周血巨噬细胞有关。 渗透到大脑和分泌促炎细胞因子，主要是IL-6。 通过对从TMEV感染的脑内分离的浸润性巨噬细胞的RNA测序(RNAseq) 在小鼠癫痫发作活动的高峰期，我发现一群中枢神经系统浸润性巨噬细胞高表达 髓系细胞-1(TREM1)上表达的触发受体的水平。TREM1的激活导致 促炎症细胞因子的分泌。TREM1表达增加与炎症相关 条件和抑制TREM1功能可以改善几种动物的炎症和疾病严重程度 炎症性疾病模型。然而，TREM1参与了急性癫痫的发展。 未知。在目标1中，我将测定TREM1在脑浸润性巨噬细胞中表达的时间进程，以及 TREM1的遗传和药物抑制是否会减少急性癫痫的发生 在感染TMEV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感染中渗透巨噬细胞，在诱发癫痫中起着关键作用；然而，IL-6如何 这是未知的。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 生物素化分析，以及IL-6途径(经典或反式信号)在急性癫痫发作中的作用。 由于脑部炎性巨噬细胞与TMEV感染的急性癫痫的发生有关 小鼠，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 通过视频脑电波观察急性癫痫的发展。我还将在脑内渗透的巨噬细胞中鉴定microRNAs 在TMEV感染的小鼠的急性癫痫发作过程中，它们是推动炎症的因素。视频脑电波 和受体生物素化试验是我在指导阶段(Aim1/Aim2a)学习的关键技术， 将是在第二阶段(Aim2b/Aim3)进行实验所必需的。这些新技能，结合到我以前的 培训，将使我处于一个独特的位置来解决与神经炎症和癫痫相关的关键问题 在我自己的实验室里使用尖端方法，并将对我的研究和职业生涯产生重大影响。 这些目的将提高对炎症和癫痫之间关系的认识，并具有 揭示炎性巨噬细胞作为神经炎症治疗新靶点的巨大潜力 允许开发新的干预措施来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 impacting 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.