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

1-磷酸鞘氨醇 (S1P) 及其受体在 Notch1 介导的胸腺 T 细胞发育中的作用：TEC 依赖性和非依赖性信号通路

• 批准号: 396772280
• 负责人: Professor Dr. Bodo Levkau
• 金额: --
• 依托单位: Institut für Molekulare Medizin III
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/396772280?language=en
• 关键词: role; sphingosine; phosphate; S1P; its

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.

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。这是批准功能性S1PR1拮抗剂FTY720来治疗多发性硬化症的基础。急性S1PR1拮抗作用抑制单阳性（SP）胸腺细胞的出口，而慢性FTY720给药可导致所有淋巴细胞含量降低约20％。此外，S1P降解酶S1P裂解酶的缺失会导致胸腺萎缩，输出量大量降低。细胞适应性免疫系统的这种急剧减少的原因，机制和后果在很大程度上尚不清楚。胸腺对于功能性T细胞库是必不可少的，其年龄依赖性的涉及治疗性免疫耗竭后的免疫系统的恢复。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后果。我们的初步数据表明，S1P裂解酶的药理抑制和遗传缺失都导致胸腺萎缩，这是由于迄今未知的未知发育T细胞缺陷引起的主要影响双阳性胸腺细胞（DPS）。谱系追踪实验确定了双阴性DN2/DN3胸腺细胞以及SPS与DPS的分化期间的缺陷。通过观察到：a）在S1P裂解酶抑制下，脑膜上皮细胞（TECS）的表型明显改变的DNS少得多的DNS少得多的DNS表达NOTCH1的DNS少得多。在该提案中，我们将解决基本机制，并表征S1P和S1PR在TEC依赖性t-cell发育阶段的作用。我们将首先研究哪种S1PR介导S1P对Notch1表达的抑制作用以及如何介导。我们将在与DLL4-OP9基质细胞的共同培养系统中解决它，其中S1P激动剂，拮抗剂，S1P裂解酶抑制剂以及S1PR和S1P裂解酶KOS的DNS将被使用。然后，我们将研究全球和血管性S1PR KOS以及具有S1P裂解酶抑制的骨髓嵌合体中的胸腺发育。此外，我们将把TEC表型变化作为胸腺细胞发育缺陷的第二个，非冗余的原因。为此，我们将分别通过S1P裂解酶抑制，缺失和S1PR KOS分析Notch1配体DLL4和关键的细胞因子SCF，CXCL12，CCL25和IL-7以及FOXN1调节的转录。我们还将生成TEC特异性KO，以确定S1PR在TEC中的作用，以分化，成熟和功能。最后，我们将使用3D细胞培养模型研究S1P对TECS的直接胸腺交叉谈话无关的作用。结果将阐明S1P和S1PR在早期胸腺稳态中的作用，并引起可能的治疗意义。