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

S1P具有重要的免疫、炎症和心血管功能。在免疫系统中，S1P通过S1P受体（S1PR）调节淋巴细胞的循环、浸润和局部定位。这是功能性S1PR1拮抗剂FTY720被批准用于治疗多发性硬化症的基础。急性S1PR1拮抗剂抑制单阳性胸腺细胞的退出，而慢性FTY720可导致所有淋巴细胞含量减少约20%。此外，S1P降解酶S1P裂解酶的缺失导致胸腺萎缩，产量大量减少。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，其年龄依赖性的退化会损害治疗性免疫耗竭后免疫系统的恢复。我们的重点在于S1P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，S1P裂解酶的药物抑制和基因缺失都会导致胸腺萎缩，这是一种迄今为止未知的发育性t细胞缺陷，主要影响双阳性胸腺细胞（DPs）。谱系追踪实验发现，在双阴性DN2/DN3胸腺细胞的产生过程中，以及在SPs从dp分化的过程中，存在缺陷。关键的机制提示是通过观察：a)表达Notch1作为t细胞发育的关键受体的DNs少得多，b)胸腺上皮细胞（TECs）在S1P裂解酶抑制下表型明显改变。在本提案中，我们将探讨潜在的机制，并描述S1P和S1PR在t细胞发育的早期tec依赖阶段的作用。我们将首先研究DNs上的哪个S1PR介导了S1P对Notch1表达的抑制作用以及如何介导。我们将在Dll4-OP9基质细胞的DNs共培养系统中解决这个问题，其中将分别使用S1P激动剂、拮抗剂、S1P裂解酶抑制剂以及来自S1PR和S1P裂解酶的DNs。然后，我们将研究全球和造血S1PR kos以及骨髓嵌合体在S1P裂解酶抑制和不抑制情况下的胸腺发育。此外，我们将讨论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Notch1配体Dll4和关键细胞因子SCF、CXCL12、CCL25和IL-7，以及foxn1在S1P裂解酶抑制、缺失和S1PR kos中调控的转录。我们还将生成tec特异性ko，以确定S1PR在tec分化、成熟和功能中的作用。最后，我们将使用3D细胞培养模型研究S1P对TECs的直接、胸腺串音无关的影响。这些结果将阐明S1P和S1PR在早期胸腺稳态中的作用，并引发可能的治疗意义。

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.