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

除了研究得非常充分的鞘氨醇（Sph）-1-磷酸（S1 P）受体介导的作用外，细胞内鞘脂代谢酶的重要作用变得越来越明显;特别是在细胞内和细胞外区室之间的定位和转运方面。因此，SphK 1和2，磷酸酶1和2以及S1 P裂解酶，它们都位于细胞内的不同位置，可能特异性地调节S1 P浓度区室。自己的数据指出，S1 P调节树突状细胞（DC）的基本功能，但也必须以某种方式参与酶效应（迁移，成熟，IL-12，IL-23，IL-10的细胞因子谱）。在定义的DC子集中，应进行这些酶的单一或组合调节。关于S1 P受体独立的，细胞内鞘脂酶的免疫调节作用，我们的目标集中在DC相关的功能和分化过程的调查。在这种方法中，值得注意的是我们能够准确地确定酶活性，并将其与用LC-MS/MS或色谱法在不同细胞隔室中测量的鞘氨醇/S1 P浓度相关联。SphK 1/2缺陷的转基因小鼠系统是可用的。对于剩余的SphL酶，我们将应用特异性慢病毒转导的shRNA与泰特/TRE诱导机制的组合来敲低酶。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样和浆细胞样DC中的免疫调节影响。

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.