The role of sphingosine-1-phosphate (S1P) and its receptors in Notch1-mediated T-cell development in the thymus: TEC-dependent and -independent signaling pathways

S1 P具有重要的免疫、炎症和心血管功能。在免疫系统中，S1 P通过S1 P受体（S1 PR）调节淋巴细胞的循环、浸润和局部定位。这是批准功能性S1 PR 1拮抗剂FTY 720用于治疗多发性硬化症的基础。急性S1 PR 1拮抗作用抑制单阳性（SP）胸腺细胞的退出，而慢性FTY 720给药导致所有淋巴细胞含量减少约20%。此外，S1 P降解酶S1 P裂解酶的缺失导致胸腺萎缩，产量大幅减少。这种细胞适应性免疫系统急剧减少的原因、机制和后果在很大程度上是未知的。胸腺对于功能性T细胞库是不可或缺的，并且其年龄依赖性退化损害了治疗性免疫耗竭后免疫系统的恢复。我们的重点在于S1 P在心血管和代谢疾病中的作用及其对免疫系统的影响。我们的初步数据表明，药理学抑制和基因缺失的S1 P裂解酶导致胸腺萎缩所造成的迄今未知的发育T细胞缺陷，主要影响双阳性胸腺细胞（DP）。谱系追踪实验确定了双阴性DN 2/DN 3胸腺细胞产生过程中以及SP与DP分化过程中的缺陷。关键的机制提示通过以下观察提供：a）表达Notch 1作为T细胞发育的关键受体的DN少得多，和B）在S1 P裂解酶抑制下胸腺上皮细胞（TEC）的表型明显改变。在这个提议中，我们将解决潜在的机制和特点的作用，S1 P和S1 PR在早期，TEC依赖性阶段的T细胞development.We将首先检查哪些S1 PR对DN介导的抑制作用S1 P对Notch 1的表达和如何。我们将在DN与Dll 4-OP 9基质细胞的共培养系统中解决它，其中将分别使用S1 P激动剂、拮抗剂、S1 P裂解酶抑制剂以及来自S1 PR和S1 P裂解酶科斯的DN。然后，我们将研究胸腺发育的全球和造血S1 PR科斯以及骨髓嵌合体与S1 P裂解酶抑制和不抑制。此外，我们还将TEC表型变化作为胸腺细胞发育缺陷的第二个非冗余原因。为此，我们将分别分析Notch 1配体Dll 4和关键细胞因子SCF、CXCL 12、CCL 25和IL-7以及FoxN 1调节的转录与S1 P裂解酶抑制、缺失和S1 PR科斯。我们还将产生TEC特异性科斯，以确定S1 PR对TEC分化，成熟和功能的作用。最后，我们将研究直接的，胸腺串扰独立的影响S1 P对TEC使用三维细胞培养模型。这些结果将阐明S1 P和S1 PR在早期胸腺稳态中的作用，并引出可能的治疗意义。

S1P has important immunological, inflammatory and cardiovascular functions. In the immune system, S1P regulates the circulation, infiltration and local positioning of lymphocytes through S1P receptors (S1PR). This has been the basis for the approval of the functional S1PR1 antagonist FTY720 for the treatment of multiple sclerosis. Acute S1PR1 antagonism inhibits the exit of single-positive (SP) thymocytes, whereas chronic FTY720 administration leads to a ~20% reduction of all lymphocyte content. Furthermore, deletion of the S1P degrading enzyme S1P lyase causes thymus atrophy with massively reduced output. The reasons, mechanisms and consequences of this dramatic reduction of the cellular adaptive immune system are largely unknown. The thymus is indispensable for the functional T-cell repertoire, and its age-dependent involution impairs the recovery of the immune system after therapeutic immune depletion. Our focus lies on the roles of S1P in cardiovascular and metabolic diseases and their consequences for the immune system. Our preliminary data show that both pharmacological inhibition and genetic deletion of the S1P lyase lead to thymus atrophy caused by a so far unknown developmental T-cell defect impacting mainly on double-positive thymocytes (DPs). Lineage tracing experiments identified defects during the generation of double-negative DN2/DN3 thymocytes as well as during the differentiation of SPs from DPs. The key mechanistic hints were delivered by observations of: a) much less DNs expressing Notch1 as the crucial receptor for T-cell development, and b) a clearly altered phenotype of thymic epithelial cells (TECs) under S1P lyase inhibition. In this proposal, we will address the underlying mechanisms and characterize the role of S1P and S1PR in the early, TEC-dependent phase of T-cell development.We will first examine which S1PR on DNs mediate the inhibitory effect of S1P on Notch1 expression and how. We will address it in a co-culture system of DNs with Dll4-OP9 stroma cells, where S1P agonists, antagonists, S1P lyase inhibitors as well as DNs from S1PR and S1P lyase kos, respectively, will be employed. We will then study thymus development in global and hematopoetic S1PR kos as well as bone marrow chimera with and without S1P lyase inhibition. Furthermore, we will address the TEC phenotypic changes as a second, non-redundant cause for the thymocyte development defects. For this, we will analyze the Notch1 ligand Dll4 and the key cytokines SCF, CXCL12, CCL25 and IL-7 as well as FoxN1-regulated transcription with S1P lyase inhibition, deletion and in S1PR kos, respectively. We will also generate TEC-specific kos to identify the role of S1PR on TECs for differentiation, maturation and function. Finally, we will investigate direct, thymus cross talk-independent effects of S1P on TECs using a 3D cell culture model. The results will elucidate the role of S1P and S1PR in early thymus homeostasis and elicit possible therapeutic implications.