Novel Functions of S1P in Neuroinflammation

描述（由申请人提供）：白细胞介素1（IL-1）是一种促炎细胞因子，对人类多发性硬化（MS）和小鼠实验性自身免疫性脑脊髓炎（EAE）的发展和进展至关重要。尽管IL-1通过激活核因子κB诱导许多细胞因子的表达，但对MS和EAE重要的几种趋化因子（如RANTES和IP-10）的诱导需要激活额外的转录因子干扰素调节因子-1（IRF-1）。值得注意的是，IRF-1在MS患者的脑中被激活，IRF-1敲除小鼠受到保护免受EAE，并且EAE由中枢神经系统（CNS）中表达的IRF-1调节。我们已经表明，IL-1刺激和上调鞘氨醇激酶1（SphK 1），两种同工酶之一，产生鞘氨醇-1-磷酸（S1 P），脂质介质，调节不同的细胞过程中重要的炎症。MS患者CSF中S1 P增加并诱导星形胶质细胞增生。FTY 720是一种前药，其被SphK 2磷酸化为S1 P模拟物（FTY 720-P），分泌后靶向五种S1 P受体中的四种，除了S1 P2，目前正处于III期临床试验中，并有效降低MS的频率和严重程度。FTY 720减少MS症状，部分是通过下调淋巴细胞上的S1 P1，从而防止其S1 P依赖性从淋巴结排出。然而，FTY 720也在大脑中积累，在那里它减少了星形胶质细胞增生;然而，它在中枢神经系统中的作用还不完全清楚。我们做了两个有趣的观察：1）S1 P直接结合细胞内的细胞凋亡抑制因子2（cIAP 2），并刺激cIAP 2介导的K63调节IRF-1的泛素化及其活化。2)与细胞内S1 P相反，细胞外S1 P抑制IRF-1的核转位，并通过S1 P2在星形胶质细胞中诱导RANTES和IP-10表达。因此，我们假设“S1 P通过调节IRF-1功能的新型细胞内和细胞外机制调节脑炎症”。这一假说对于理解S1 P、FTY 720和IRFs在星形胶质细胞介导的神经炎症和MS中的作用机制具有重要意义。分析细胞内S1 P作为cIAP 2介导的IRF-1泛素化辅因子的作用。目标2.确定细胞外S1 P影响IRF-1激活的机制。目标3：定义IRF-1的S1 P调节在体内的作用。该提案将增强对星形胶质细胞中IL-1，S1 P及其受体之间复杂相互作用如何调节炎症的理解，并可能为设计更有效的MS疗法铺平道路。

DESCRIPTION (provided by applicant): Interleukin 1 (IL-1) is a proinflammatory cytokine that is critical for the development and progression of multiple sclerosis (MS) in humans and experimental autoimmune encephalomyelitis (EAE) in mice. Although IL-1 induces expression of many cytokines via the activation of nuclear factor κB, induction of several chemokines important for MS and EAE, such as RANTES and IP-10, requires the activation of an additional transcription factor, interferon regulatory factor-1 (IRF-1). Significantly, IRF-1 is activated in the brains of MS patients, IRF-1 knockout mice are protected from EAE, and EAE is regulated by IRF-1 expressed in the central nervous system (CNS). We have shown that IL-1 stimulates and upregulates sphingosine kinase 1 (SphK1), one of the two isoenzymes that generate sphingosine-1-phosphate (S1P), a lipid mediator that regulates diverse cellular processes important for inflammation. S1P is increased in the CSF of MS patients and induces astrogliosis. FTY720, a pro-drug that is phosphorylated by SphK2 to an S1P mimetic (FTY720-P) and after secretion targets four of the five S1P receptors, except S1P2, is currently in phase III clinical trials, and effectively reduces the frequency and severity of MS. FTY720 reduces MS symptoms, in part, by downregulating S1P1 on lymphocytes thus preventing their S1P-dependent egress from the lymph nodes. However, FTY720 also accumulates in the brain where it reduces astrogliosis; nevertheless, its effects in the CNS are incompletely understood. We made two intriguing observations: 1) S1P directly binds to cellular inhibitor of apoptosis 2 (cIAP2) inside cells and stimulates cIAP2-mediated K63 regulatory ubiquitination of IRF-1 and its activation. 2) In contrast to intracellular S1P, extracellular S1P suppresses nuclear translocation of IRF-1 and induction of RANTES and IP-10 expression via S1P2 in astrocytes. Thus, we hypothesize that "S1P modulates brain inflammation by novel intracellular and extracellular mechanisms that regulate IRF-1 functions". This hypothesis has significant implications for understanding mechanisms of S1P, FTY720, and IRFs actions in astrocyte-mediated neuroinflammation and MS. We propose the following specific aims: Aim 1. Analyze the role of intracellular S1P as a cofactor for cIAP2-mediated ubiquitination of IRF-1. Aim 2. Identify the mechanisms by which extracellular S1P affects IRF-1 activation. Aim 3. Define the roles of S1P regulation of IRF-1 in vivo. This proposal will enhance understanding of how the intricate interplay between IL-1, S1P and its receptors in astrocytes regulates inflammation and could pave the way for the design of even more effective MS therapies.