Astrocytic control of lymphocyte migration and differentiation in CNS inflammatory lesions

多发性硬化（MS）的临床残疾是由免疫细胞浸润到CNS实质中驱动的。了解CNS屏障细胞调节免疫细胞激活和进入的机制，在这个浸润过程中，可能会确定翻译策略，对MS和其他自身免疫性CNS疾病。 在CNS炎症期间，免疫细胞从血液通过称为神经血管单位的双屏障结构运输。虽然许多人都专注于通过第一个屏障，一个专门的内皮细胞壁称为血脑屏障，但对免疫细胞如何与第二个屏障相互作用知之甚少，第二个屏障是一层星形胶质细胞末端足，称为胶质细胞界膜。在MS病变中，免疫细胞在血脑屏障和胶质细胞界膜之间的空间中循环，该空间称为血管周围空间。在这里，星形胶质细胞可以与浸润的免疫细胞相互作用以调节它们的功能。使用基因表达分析，我们确定了在MS病变中用白细胞介素-1 β（一种细胞因子）治疗时星形胶质细胞上调的免疫细胞受体。我们最强的候选者之一，连接粘附分子-A（JAM-A），是一种免疫细胞受体，调节其他组织中的促炎途径。我的试验数据显示，星形胶质细胞JAM-A结合T细胞，改变蛋白酶和细胞因子水平的模式，已知加剧MS的动物模型。进一步的试验数据表明，星形胶质细胞JAM-A缺失阻断免疫细胞渗透的胶质细胞界膜，并防止在该模型中的临床残疾。 我推测星形胶质细胞JAM-A驱动免疫细胞进入CNS，形成炎性病变和临床残疾。在目标1中，我将测试星形胶质细胞JAM-A信号传导到共培养的T细胞如何使用RNA测序改变两种细胞类型的基因表达网络，然后我将确定JAM-A和下游候选物如何调节与自身免疫攻击相关的效应子途径。在目标2中，使用转基因和药理学工具，我将建立星形胶质细胞JAM-A如何控制中枢神经系统炎症的两种动物模型中的病变形成。翻译策略阻断星形胶质细胞JAM-A和效应器，然后将测试对MS的动物模型中的临床残疾。在目的3中，我将测量T细胞如何与胶质界膜相互作用，使用活的双光子成像的炎症脊髓病变。然后，我将测试星形胶质细胞JAM-A和效应器如何调节这些相互作用。 这个K 08奖的目标是在Gareth John博士的指导下提高我在神经免疫学和胶质生物学方面的专业知识，Peter Calabresi，医学博士。和迪米特里奥斯·达瓦洛斯博士获得使用免疫和神经胶质细胞共培养和体内多光子成像的技术技能，并建立我自己的研究小组，专注于星形胶质细胞-免疫细胞信号传导在自身免疫攻击中的作用。该K 08计划研究了一种新型的接触介导的反应性星形胶质细胞和浸润性免疫细胞之间的相互作用，该相互作用在CNS的一个未充分研究的免疫调节区室中，并可能确定针对CNS自身免疫性疾病的新型治疗策略。

Clinical disability in multiple sclerosis (MS) is driven by immune cell infiltration into the CNS parenchyma. Understanding the mechanisms by which CNS barrier cells regulate immune cell activation and entry during this infiltrative process may identify translational strategies against MS and other autoimmune CNS diseases. During CNS inflammation, immune cells traffic from the blood through a two-barrier structure termed the neurovascular unit. While many have focused on entry through the first barrier, a specialized endothelial wall known as the blood-brain barrier, less is known about how immune cells interact with the second barrier, a layer of astrocyte end feet termed the glia limitans. In MS lesions, immune cells circulate in the space between the blood-brain barrier and the glia limitans, a compartment called the perivascular space. Here, astrocytes may interact with infiltrating immune cells to regulate their function. Using gene expression analysis, we identified immune cell receptors upregulated by astrocytes when treated with interleukin-1β, a cytokine in MS lesions. One of our strongest candidates, Junctional Adhesion Molecule-A (JAM-A), is an immune cell receptor that regulates pro-inflammatory pathways in other tissues. My pilot data reveals that astrocytic JAM-A Binds T cells and alters protease and cytokine levels in patterns known to exacerbate an animal model of MS. Further pilot data show that astrocytic JAM-A deletion blocks immune cell penetration of the glia limitans and protects against clinical disability in this model. I hypothesize that astrocytic JAM-A drives CNS entry of immune cells, inflammatory lesion formation and clinical disability. In Aim 1, I will test how astrocytic JAM-A signaling to T cells in co-culture modifies gene expression networks of both cell types using RNA sequencing, and then I will determine how JAM-A and downstream candidates regulate effector pathways relevant to autoimmune attack. In Aim 2, using transgenic and pharmacologic tools, I will establish how astrocytic JAM-A controls lesion formation in two animal models of CNS inflammation. Translational strategies blocking astrocytic JAM-A and effectors will then be tested against clinical disability in an animal model of MS. In Aim 3, I will measure how T cells interact with the glia limitans using live two-photon imaging of inflammatory spinal cord lesions. I will then test how astrocytic JAM-A and effectors regulate these interactions. Goals of this K08 award are to advance my expertise in neuroimmunology and glial biology under the mentorships of Gareth John, PhD., Peter Calabresi, MD. and Dimitrios Davalos, PhD., to acquire technical skills using immune and glial cell co-cultures and in vivo multiphoton imaging, and to establish my own research group focused on the role of astrocyte-immune cell signaling during autoimmune attack. This K08 plan investigates a novel contact-mediated interaction between reactive astrocytes and infiltrating immune cells within an understudied immunomodulatory compartment of the CNS and may identify novel therapeutic strategies against CNS autoimmune disease.