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

描述（由申请人提供）：多发性硬化症（MS）是一种中枢神经系统（CNS）的炎症性攻击，导致神经元功能丧失并可能导致死亡。实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis， EAE）是多发性硬化症的小鼠模型，与多发性硬化症一样，是一种由促炎免疫细胞浸润中枢神经系统并对髓磷脂成分产生免疫反应介导的自身免疫性疾病，导致神经元损伤，可导致瘫痪甚至死亡。CD73 （Ecto-5'-核苷酸酶）是一种糖基磷脂酰肌醇（GPI）连接的膜蛋白，可催化AMP胞外去磷酸化为腺苷。在免疫应答过程中，受损细胞释放的ATP主要通过CD73的酶促作用转化为腺苷。这种细胞外腺苷对表达腺苷受体的淋巴细胞起到负反馈信号的作用，并从本质上关闭免疫反应，以防止过度的细胞损伤。初步证据表明，CD73生成的腺苷是淋巴细胞向中枢神经系统的有效迁移和EAE发展所必需的。本提案的目标是：1)确定腺苷信号在调节淋巴细胞向中枢神经系统迁移导致自身免疫性疾病中的作用；2)定义受腺苷信号调节的粘附分子和趋化因子，以影响脉络膜丛淋巴细胞的迁移。为了实现这些目标，我们将：检测野生型和CD73-/-小鼠EAE诱导前和诱导过程中的细胞外腺苷浓度；研究EAE发生前和发生过程中脑内腺苷受体的表达模式和水平；确定EAE期间腺苷受体阻断对淋巴细胞浸润和疾病发展的影响；确定腺苷受体信号缺失小鼠是否易受EAE诱导；确定腺苷受体信号传导对淋巴细胞进入中枢神经系统的影响，是否阻断CD73酶的能力可以阻止野生型小鼠EAE的发展或稳定已有EAE小鼠的疾病；并探讨用PEG-ADA增强腺苷降解是否可以预防野生型小鼠EAE的发展或稳定疾病。为了确定受腺苷信号调节的粘附分子和趋化因子影响脉络膜丛淋巴细胞迁移，我们将使用小鼠脉络膜丛细胞系来确定脉络膜丛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EAE过程中，在腺苷受体信号存在或不存在的情况下，量化脉络膜丛上粘附分子的表达；我们将量化在体内和体外存在或不存在腺苷受体信号的脉络丛和淋巴细胞上粘附分子的表达。最后，我们将研究在EAE过程中腺苷受体信号对脉络膜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中枢神经系统的慢性炎症性疾病。它是通过免疫细胞渗入中枢神经系统（CNS）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生成的腺苷调节EAE小鼠的淋巴细胞进入大脑。这一发现为开发旨在阻断免疫细胞入侵中枢神经系统的治疗方法提供了潜力，这些治疗方法可用于阻止MS等神经炎性疾病的进展，甚至可能逆转MS引起的损伤。

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.