Meningeal Mast Cells: Key effectors of stroke pathology

描述(由申请人提供)：中风是美国长期严重残疾的主要原因，需要减少损害和促进康复的治疗方法。卒中后炎症是损害和恢复的关键决定因素，因此是一个有希望的治疗靶点。肥大细胞(Mast cell，MC)在其他病理的炎症过程中起着关键作用，最近被认为在卒中后脑部炎症和损伤的加重中发挥了作用。这表明MCs可能是中风引起炎症的关键决定因素，因此是一个有吸引力的治疗靶点。这项建议的主要焦点是阐明肥大细胞在加重中风病理过程中的作用机制，长期目标是确定中风的新治疗策略。我们的总体假设是，驻留在脑膜中的肥大细胞是卒中病理的关键效应细胞。这在目标1中使用两种不同的方法进行了测试。功能获得法(Aim 1a)使用了一种“肥大细胞敲入小鼠”模型，在该模型中，通过植入体外培养的肥大细胞，选择性地修复了MC基因缺陷小鼠的MC缺陷。将MC直接植入这些MC缺陷小鼠的脑膜，将确定位于脑膜的MC参与中风的病理过程。这与功能丧失方法(Aim1b)相辅相成，在该方法中，脑膜MCs通过脑膜注射白喉毒素(DT)到一种新的小鼠模型中来耗尽MCs，该模型选择性地在MCs上表达DT受体。综上所述，这些小鼠模型的创新使用使我们能够确定脑膜MC对于中风后MC的有害影响是否必要和充分。目的2利用肥大细胞敲入小鼠模型研究肥大细胞作用的分子机制。通过植入不同受体敲除小鼠的MC，我们将研究脑膜MC激活的机制(Aim 2a)，并通过移植来自细胞因子敲除小鼠的MC，我们将识别肥大细胞分泌的因素，这些因素对MCs在卒中病理中的下游作用(Aim 2b)非常重要。明确脑膜MC在卒中后的关键作用将突出脑膜在调节脑病理中的重要性。由于脑膜相对容易进入(例如，通过鞘内注射)，这一概念可能为中风治疗提供一种新的策略，可能克服靶向药物靶向受损大脑的障碍，并减少全身免疫调节的不良副作用。此外，通过建立MC的作用机制，我们开始描绘参与调节对中风的反应的分子通路，这是寻找新的治疗靶点的关键步骤。总之，我们建议的研究解决了对卒中后炎症事件的理解方面的重大差距，这些事件有助于卒中病理，并可能确定卒中治疗的新策略。

DESCRIPTION (provided by applicant): Stroke, a leading cause of serious long-term disability in the US, is in need of therapeutics that reduce damage and promote recovery. Post-stroke inflammation is a critical determinant of damage and recovery and is thus a promising therapeutic target. Mast cells (MCs), which play critical roles in the development of inflammatory processes in other pathologies, were recently ascribed a role in the exacerbation of post-stroke brain inflammation and damage. This suggests that MCs could be key determinants of stroke-induced inflammation and hence are an attractive therapeutic target. The primary focus of this proposal is to elucidate the mechanism of action of mast cells in exacerbating stroke pathology, with the long-term goal of identifying novel therapeutic strategies for stroke. Our overall hypothesis is that mast cells residing in the meninges are key effectors of stroke pathology. This is tested in Aim 1 using two different approaches. The gain-of-function approach (Aim 1a) uses a "mast cell knock-in mouse" model in which the MC-deficiency of genetically MC- deficient mice is selectively repaired by engraftment of in vitro grown mast cells. Direct engraftment of MCs into the meninges of these MC-deficient mice will determine the involvement of meningeal-located MCs in stroke pathology. This is complemented by the loss-of-function approach (Aim1b) where meningeal MCs are depleted by meningeal injection of diphtheria toxin (DT) into a novel mouse model that selectively expresses the DT receptor on MCs. Together, innovative use of these mouse models enables us to determine if meningeal MCs are necessary and sufficient for the detrimental effects of MCs after stroke. Aim 2 addresses the molecular mechanism of action of MCs using the mast cell knock-in mouse model. Through engraftment of MCs from various receptor knock-out mice we will investigate the mechanism of meningeal MC activation (Aim 2a), and by engraftment of MCs from cytokine knock-out mice we will identify mast cell-secreted factors important for the downstream effects of MCs on stroke pathology (Aim 2b). Identifying a crucial role for meningeal MCs after stroke will highlight the importance o the meninges in modulating brain pathology. As the meninges are relatively accessible (e.g., by intrathecal injection) this concept could potentially present a new strategy for stroke therapeutic that may overcome the hurdle of targeting drugs to the injured brain and reduce unwanted side effects of systemic immunomodulation. Furthermore, by establishing the mechanism of action of MCs we begin to delineate the molecular pathways involved in modulating the response to stroke, an essential step to finding novel therapeutic targets. Together, our proposed studies address significant gaps in the understanding of post-stroke inflammatory events that contribute to stroke pathology and may identify new strategies for stroke therapeutics.