CD-73 adenosine signaling in the central nervous system in disease and health

描述(申请人提供)：多发性硬化症(MS)是一种对中枢神经系统(CNS)的炎症性攻击，导致神经功能丧失，可导致死亡。实验性自身免疫性脑脊髓炎(EAE)是MS的小鼠模型，与MS一样，是一种由促炎症免疫细胞介导的自身免疫性疾病，这些细胞渗透到中枢神经系统，启动针对髓鞘成分的免疫反应，导致神经元损伤，可导致瘫痪甚至死亡。CD73(胞外-5‘-核苷酸酶)是一种糖基磷脂酰肌醇(GPI)连接的膜蛋白，催化细胞外AMP脱磷为腺苷。在免疫反应中，受损细胞释放的ATP主要通过CD73的酶作用转化为腺苷。这种细胞外的腺苷对表达腺苷受体的淋巴细胞起到负反馈信号的作用，基本上关闭了免疫反应，以防止过度的细胞损伤。初步证据表明，CD73产生的腺苷是淋巴细胞高效迁移到中枢神经系统和发展为EAE所必需的。该方案的目的是1)确定腺苷信号在调节淋巴细胞迁移到中枢神经系统从而引起自身免疫性疾病中的作用；2)确定由腺苷信号调节的影响脉络丛淋巴细胞迁移的黏附分子和趋化因子。为了实现这些目标，我们将：检测野生型和CD73-/-小鼠在EAE诱导前和诱导过程中细胞外腺苷的浓度；研究腺苷受体在EAE发生前和期间脑内的表达模式和水平；确定在EAE期间阻断腺苷受体对淋巴细胞浸润和疾病发展的影响；确定腺苷受体信号转导缺陷的小鼠是否对EAE诱导敏感；确定腺苷受体信号对淋巴细胞进入中枢神经系统的影响；阻断CD73的酶能力是否可以防止野生型小鼠EAE的发展或稳定已有EAE的小鼠的疾病；并研究用PEG-ADA促进腺苷降解是否可以防止野生型小鼠的EAE发展或稳定疾病。为了确定受腺苷信号调节的黏附分子和趋化因子影响脉络丛淋巴细胞的迁移，我们将使用小鼠脉络丛细胞系来确定脉络丛上的腺苷信号是否可以在体外触发淋巴细胞迁移；我们将在体外和有或没有腺苷受体信号的情况下对脉络丛上黏附分子的表达进行定量；我们将在有或没有腺苷受体信号的情况下定量在体内和体外脉络丛和淋巴细胞上黏附分子的表达。最后，我们将研究腺苷受体信号对EAE期间脉络丛趋化因子分泌的影响。公共卫生相关性：多发性硬化症是一种慢性中枢神经系统炎症性疾病。它是通过免疫细胞渗入中枢神经系统(CNS)而介导的，引起炎症，导致神经元损伤，导致瘫痪甚至死亡。EAE是MS的动物模型。我们有证据表明，CD73产生的腺苷调节淋巴细胞进入EAE小鼠的大脑。这一发现为开发旨在阻止MS等神经炎性疾病的中枢神经系统免疫细胞入侵的疗法提供了潜力，此类疗法可用于阻止MS的进展，甚至可能逆转MS造成的损害。

DESCRIPTION (provided by applicant): Multiple Sclerosis (MS) is an inflammatory attack on the central nervous system (CNS) that results in loss of neuronal function and can lead to death. Experimental Autoimmune Encephalomyelitis (EAE) is a mouse model for MS, and like MS, is an autoimmune disease mediated by proinflammatory immune cells that infiltrate the CNS and mount immune responses against myelin components, resulting in neuronal damage that can cause paralysis and even death. CD73 (Ecto-5'-nucleotidase) is a glycosyl phosphatidylinositol (GPI)-linked membrane protein that catalyzes the extracellular dephosphorylation of AMP to adenosine. During an immune response, ATP released from damaged cells is converted to adenosine, mainly through the enzymatic action of CD73. This extracellular adenosine acts as a negative feedback signal to lymphocytes that express adenosine receptors and essentially turns off the immune response to prevent excessive cellular damage. Preliminary evidence shows CD73 generated adenosine is required for efficient lymphocyte migration into the CNS and for EAE development. The goals of this proposal are to 1) determine the role of adenosine signaling in regulating lymphocyte migration into the CNS to cause autoimmune disease; and 2) define the adhesion molecules and chemokines that are regulated by adenosine signaling to influence lymphocyte migration at the choroid plexus. To achieve these goals we will: test the concentration of extracellular adenosine before and during EAE induction in wild type and CD73-/- mice; investigate the expression pattern and level of adenosine receptors in the brain before and during EAE development; determine the consequences of adenosine receptor blockade during EAE on lymphocyte infiltration and disease development; determine whether mice deficient in adenosine receptor signaling are susceptible to EAE induction; determine the effect of adenosine receptor signaling on lymphocyte passage into the CNS, whether blockade of CD73 enzymatic ability can prevent development of EAE in wild type mice or stabilize disease in mice with preexisting EAE; and investigate whether enhancing adenosine degradation with PEG-ADA can prevent development of EAE in wild type mice or stabilize the disease. To define the adhesion molecules and chemokines that are regulated by adenosine signaling to influence lymphocyte migration at the choroid plexus, we will use a mouse choroid plexus cell line to determine whether adenosine signaling at the choroid plexus can trigger lymphocyte migration in vitro; we will quantify the expression of adhesion molecules on the choroid plexus in vitro and during EAE in the presence or absence of adenosine receptor signaling; we will quantify the expression of adhesion molecules on the choroid plexus and lymphocytes in vivo and in vitro in the presence or absence of adenosine receptor signaling. Finally, we will investigate the impact of adenosine receptor signaling on chemokine secretion at the choroid plexus during EAE. PUBLIC HEALTH RELEVANCE: Multiple sclerosis is a chronic inflammatory disease of the central nervous system. It is mediated by infiltration of immune cells into the central nervous system (CNS), causing inflammation which results in damage to neurons, leading to paralysis and even death. EAE is the animal model for MS. We have evidence suggesting that CD73-generated adenosine regulates the entry of lymphocytes into the brains of mice with EAE. This finding provides potential for development of therapies aimed at blocking immune cell invasion of the CNS in neuroinflammatory diseases such as MS. Such therapies could be used to stop the progress of, or even possibly reverse the damage caused by MS.