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

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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.