Subcellular characterization and modulation of sphingolipid-metabolizing enzymes as key regulators of dendritic cell differentiation and their immune deviating function

鞘磷脂(Sph)-1-磷酸（S1P）受体介导的作用已得到很好的研究，鞘脂代谢酶在细胞内的重要作用也越来越明显；特别是在细胞内和细胞外的定位和运输的背景下。因此，SphK激酶1和2、磷酸酶1和磷酸酶2以及S1P裂解酶可能在细胞内的不同位置特异调控S1P浓度区。自身数据表明，S1P调节树突状细胞（DC）的基本功能，但一定也参与了酶的作用（迁移、成熟、细胞因子IL-12、IL-23、IL-10的谱）。在已定义的DC亚群中，应对这些酶进行单一或联合调节。关于细胞内鞘脂酶的S1P受体独立的免疫调节作用，我们的目的是研究dc相关的功能和分化过程。在这种方法中，值得注意的是，我们能够准确地确定酶活性，并将其与LC-MS/MS或色谱在不同细胞区室中测量的鞘氨醇/S1P浓度相关联。缺乏SphK1/2基因的转基因小鼠系统是可用的。对于剩余的SphL酶，我们将使用特定的慢病毒转导shRNA结合TET/TRE诱导机制来敲除这些酶。通过这种直接的策略和对细胞内靶点的关注，我们将研究内源性鞘脂酶及其产物在髓细胞和浆细胞样DC中的免疫调节作用。

Besides the very well investigated Sphingosine(Sph)-1-phosphate(S1P)-receptor-mediated effects the important roles of intracellular sphingolipid metabolising enzymes become more and more evident; especially in the context of localization and transport between intra- and extracellular compartments. Therefore Sph-kinase (SphK) 1 and 2, phosphatase 1 and 2 as well as S1P lyase, which are all differently located within the cell, probably regulate the S1P concentration compartment specifically. Own data point out that S1P modulates essential functions of dendritic cells (DC) but also enzyme effects must somehow be involved (migration, maturation, cytokine profile of IL-12, IL-23, IL-10). In defined DC subsets a single or combined modulation of those enzymes shall be done. With regard to the S1P receptor independent, immune modulating effects of intracellular sphingolipid enzymes, our aims focus on the investigation of DC-relevant functions and differentiation processes. Remarkably new in this approach is our capability to determine exactly the enzymatic activity and to correlate this with Sphingosine/S1P concentrations measured with LC-MS/MS or chromatographic in different cell compartments. Transgenic mice systems, deficient for SphK1/2, are available. For the remaining SphL enzymes we will apply specific lentivirally transduced shRNA in combination with a TET/TRE inducible mechanism to knockdown the enzymes. By means of this straight forward strategy and the focus on intracellular targets we will investigate the immune modulating influence of endogenous sphingolipid enzymes and their products in myeloid and plasmacytoid DC.