Sphingolipidomics in Translational Sepsis Research-Biomedical Considerations and Perspectives.

Scientific Background: Sphingolipids are a highly diverse group of lipids with respect to physicochemical properties controlling either structure, distribution, or function, all of them regulating cellular response in health and disease. Mass spectrometry, on the other hand, is an analytical technique characterizing ionized molecules or fragments thereof by mass-to-charge ratios, which has been prosperingly developed for rapid and reliable qualitative and quantitative identification of lipid species. Parallel to best performance of in-depth chromatographical separation of lipid classes, preconditions of precise quantitation of unique molecular species by preprocessing of biological samples have to be fulfilled. As a consequence, "lipid profiles" across model systems and human individuals, esp. complex (clinical) samples, have become eminent over the last couple of years due to sensitivity, specificity, and discriminatory capability. Therefore, it is significance to consider the entire experimental strategy from sample collection and preparation, data acquisition, analysis, and interpretation. Areas Covered: In this review, we outline considerations with clinical (i.e., human) samples with special emphasis on sample handling, specific physicochemical properties, target measurements, and resulting profiling of sphingolipids in biomedicine and translational research to maximize sensitivity and specificity as well as to provide robust and reproducible results. A brief commentary is also provided regarding new insights of "clinical sphingolipidomics" in translational sepsis research. Expert Opinion: The role of mass spectrometry of sphingolipids and related species ("sphingolipidomics") to investigate cellular and compartment-specific response to stress, e.g., in generalized infection and sepsis, is on the rise and the ability to integrate multiple datasets from diverse classes of biomolecules by mass spectrometry measurements and metabolomics will be crucial to fostering our understanding of human health as well as response to disease and treatment.

Type 4 sphingosine 1-phosphate G protein-coupled receptor (S1P4) transduces S1P effects on T cell proliferation and cytokine secretion without signaling migration.

Sphingosine 1-phosphate (S1P) has diverse effects on T cells that are mediated by the predominant S1P1 and S1P4 G protein-coupled receptors (GPCRs). S1P4 is expressed principally by leukocytes, but little is known of its T cell effects in immunity. Two approaches were used to investigate S1P4 signals in T cells. First, S1P4 was introduced into D10G4.1 mouse Th2 cells and EL4.IL-2 mouse T cells lacking endogenous S1P GPCRs. Second, mouse splenic CD4 T cells were treated with FTY720 to suppress S1P1 and leave S1P4 GPCRs as the only functionally relevant S1P receptor. Unlike S1P1, S1P4 failed to transduce chemotactic responses of any of the S1P4-only T cells to S1P or the phyto-S1P ligand selective for S1P4, or to suppress their chemotactic responses to chemokines. The S1P-S1P4 axis significantly inhibited T cell proliferation in each of the S1P4-only T cells activated by anti-CD3 and anti-CD28 MoAbs. Secretion of IL-4 by S1P4-D10G4.1 cells, IL-2 by S1P4-EL4.IL-2, and IFN-gamma by FTY720-treated CD4 T cells were significantly inhibited by S1P. In contrast, S1P enhanced secretion of IL-10 by stimulated S1P4-D10G4.1 T cells. Thus, S1P4 mediates immunosuppressive effects of S1P by inhibiting proliferation and secretion of effector cytokines, while enhancing secretion of the suppressive cytokine IL-10.

Physiological sphingosine 1-phosphate requirement for optimal activity of mouse CD4+ regulatory T Cells.

Sphingosine 1-phosphate (S1P) evokes T cell chemotaxis at 1-100 nM and inhibits chemotaxis to chemokines at 300 nM-1 uM through their predominant S1P1 G protein-coupled receptor (R). Mouse CD4+25+ regulatory T cells now are shown to express the same pattern of S1P1 > S1P4 >>other S1P Rs as other CD4 T cells. CD4+25+ T cell suppression of 3H-thymidine uptake and IL-2 generation by CD4+25- T cells stimulated with anti-T cell receptor antibodies without S1P was enhanced significantly by S1P at normal blood and lymph concentrations. These levels of S1P also enhanced IL-10 generation by CD4+25+ T cells by up to threefold compared with that without S1P but decreased IL-10 from CD4+25- T cells. That IL-10 from CD4+25+ T cells incubated with S1P mediates suppressive activity was demonstrated by prevention with neutralizing anti-IL-10 or anti-IL-10 receptor antibodies. Extracellular fluid S1P thus is required for optimal activity of CD4+25+ T cells.

Transduction of multiple effects of sphingosine 1-phosphate (S1P) on T cell functions by the S1P1 G protein-coupled receptor.

Sphingosine 1-phosphate (S1P) in blood, lymph, and immune tissues stimulates and regulates T cell migration through their S1P(1) (endothelial differentiation gene encoded receptor-1) G protein-coupled receptors. We show now that S1P(1)Rs also mediate suppression of T cell proliferation and cytokine production. Uptake of [(3)H]thymidine by mouse CD4 T cells stimulated with anti-CD3 mAbs plus either anti-CD28 or IL-7 was inhibited up to 50% by 10(-9)-10(-6) M S1P. Suppression by S1P required Ca(2+) signaling and was reduced by intracellular cAMP. S1P decreased CD4 T cell generation of IFN-gamma and IL-4, without affecting IL-2. A Th1 line from D011.10 TCR transgenic mice without detectable S1P(1) was refractory to S1P until introduction of S1P(1) by retroviral transduction. S1P then evoked chemotaxis, inhibited chemotaxis to CCL-5 and CCL-21, and suppressed Ag-stimulated proliferation and IFN-gamma production. Thus, S1P(1) signals multiple immune functions of T cells as well as migration and tissue distribution.

Protein kinase C epsilon dependence of the recovery from down-regulation of S1P1 G protein-coupled receptors of T lymphocytes.

Sphingosine 1-phosphate (S1P) from mononuclear phagocytes and platelets signals T cells predominantly through S1P1 G protein-coupled receptors (Rs) to enhance survival, stimulate and suppress migration, and inhibit other immunologically relevant responses. Cellular S1P1 Rs and their signaling functions are rapidly down-regulated by S1P, through a protein kinase C (PKC)-independent mechanism, but characteristics of cell-surface re-expression of down-regulated S1P1 Rs have not been elucidated. T cell chemotactic responses (CT) to 10 and 100 nm S1P and inhibition of T cell chemotaxis to chemokines (CI) by 1 and 3 microm S1P were suppressed after 1 h of preincubation with 100 nm S1P, but recovered fully after 12-24 h of exposure to S1P. Late recovery of down-regulated CT and CI, but not early down-regulation, was suppressed by PKC and PKCepsilon-selective inhibitors and was absent in T cells from PKCepsilon-null mice. The same PKCepsilon inhibitors blocked S1P-evoked increases in T cell nuclear levels of c-Fos and phosphorylated c-Jun and JunD after 24 h, but not 1 h. A mixture of c-Fos plus c-Jun antisense oligonucleotides prevented late recovery of down-regulated CT and CI, without affecting S1P induction of down-regulation. Similarly, S1P-elicited threonine phosphorylation of S1P1 Rs was suppressed by a selective inhibitor of PKCepsilon after 24 h, but not 1 h. Biochemical requisites for recovery of down-regulated S1P1 Rs thus differ from those for S1P induction of down-regulation.