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Cholesterol is highly abundant within all human body cells and modulates critical cellular functions related to cellular plasticity, metabolism, and survival. The cholesterol-binding toxin pneumolysin represents an essential virulence factor of Streptococcus pneumoniae in establishing pneumonia and other pneumococcal infections. Thus, cholesterol scavenging of pneumolysin is a promising strategy to reduce S.pneumoniae induced lung damage. There may also be a second cholesterol-dependent mechanism whereby pneumococcal infection and the presence of pneumolysin increase hepatic sterol biosynthesis. Here we investigated a library of polymer particles varying in size and composition that allow for the cellular delivery of cholesterol and their effects on cell survival mechanisms following pneumolysin exposure. Intracellular delivery of cholesterol by nanocarriers composed of Eudragit E100–PLGA rescued pneumolysin-induced alterations of lipid homeostasis and enhanced cell survival irrespective of neutralization of pneumolysin.

Cholesterol is highly abundant within all human body cells and modulates critical cellular functions related to cellular plasticity, metabolism, and survival. The cholesterol-binding toxin pneumolysin represents an essential virulence factor of Streptococcus pneumoniae in establishing pneumonia and other pneumococcal infections. Thus, cholesterol scavenging of pneumolysin is a promising strategy to reduce S. pneumoniae induced lung damage. There may also be a second cholesterol-dependent mechanism whereby pneumococcal infection and the presence of pneumolysin increase hepatic sterol biosynthesis. Here we investigated a library of polymer particles varying in size and composition that allow for the cellular delivery of cholesterol and their effects on cell survival mechanisms following pneumolysin exposure. Intracellular delivery of cholesterol by nanocarriers composed of Eudragit E100–PLGA rescued pneumolysin-induced alterations of lipid homeostasis and enhanced cell survival irrespective of neutralization of pneumolysin.

Background Sphingosine 1-phosphate (S1P) is a signaling lipid essential in regulating processes involved in sepsis pathophysiology, including endothelial permeability and vascular tone. Serum S1P is progressively reduced in sepsis patients with increasing severity. S1P function depends on binding to its carriers: serum albumin (SA) and high-density lipoproteins (HDL). The aim of this single-center prospective observational study was to determine the contribution of SA- and HDL-associated S1P (SA-S1P and HDL-S1P) to sepsis-induced S1P depletion in plasma with regard to identify future strategies to supplement vasoprotective S1P. Methods Sequential precipitation of lipoproteins was performed with plasma samples obtained from 100 ICU patients: surgical trauma (n = 20), sepsis (n = 63), and septic shock (n = 17) together with healthy controls (n = 7). Resultant fractions with HDL and SA were analyzed by liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-MS/MS) for their S1P content. Results Plasma S1P levels significantly decreased with sepsis severity and showed a strong negative correlation with increased organ failure, quantified by the Sequential Organ Failure Assessment (SOFA) score (rho − 0.59, P

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Sphingosine 1-phosphate (S1P) is a multifaceted lipid signaling molecule that activates five specific G protein-coupled S1P receptors. Despite the fact that S1P is known as one of the strongest barrier-enhancing molecules for two decades, no medical application is available yet. The reason for this lack of translation into clinical practice may be the complex regulatory network of S1P signaling, metabolism and transportation. In this review, we will provide an overview about the physiology and the network of S1P signaling with the focus on endothelial barrier maintenance in inflammation. We briefly describe the physiological role of S1P and the underlying S1P signaling in barrier maintenance, outline differences of S1P signaling and metabolism in inflammatory diseases, discuss potential targets and compounds for medical intervention, and summarize our current knowledge regarding the role of S1P in the maintenance of specialized barriers like the blood-brain barrier and the placenta.

Niemann–Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism. Fil: Newton, Jason. Virginia Commonwealth University School of Medicine; Estados Unidos Fil: Palladino, Elisa N.D.. Virginia Commonwealth University School of Medicine; Estados Unidos Fil: Weigel, Cynthia. Virginia Commonwealth University School of Medicine; Estados Unidos Fil: Maceyka, Michael. Virginia Commonwealth University School of Medicine; Estados Unidos Fil: Gräler, Markus H.. Universitätsklinikum Jena; Alemania Fil: Senkal, Can E.. Virginia Commonwealth University School of Medicine; Estados Unidos Fil: Enriz, Ricardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina Fil: Marvanova, Pavlina. Veterinární univerzita Brno; República Checa Fil: Jampilek, Josef. Univerzita Komenského v Bratislave; Eslovaquia Fil: Lima, Santiago. Virginia Commonwealth University; Estados Unidos Fil: Milstien, Sheldon. Virginia Commonwealth University School of Medicine; Estados Unidos Fil: Spiegel, Sarah. Virginia Commonwealth University School of Medicine; Estados Unidos

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid and ligand for five G protein-coupled cell-surface receptors designated S1PR1-5. The determination of low levels of S1P remains a challenge and usually requires sophisticated analytical instrumentation and methodology. This report describes a technique using the linear ion trap mode of a basic QTrap triple-quadrupole mass spectrometer. S1P was extracted from acidified biological samples using a modified Folch extraction procedure. After the addition of C17-sphingosine as an internal standard, a step gradient LC method was used to separate the analytes on a reversed-phase C18 MultoHigh analytical column. After the internal standard C17-sphingosine was detected by multiple reaction monitoring (MRM), the detection mode was switched to enhanced product ion (EPI) mode for the detection of S1P. The mode was switched back to MRM again for the detection of other analytes. Using this QTrap method, we reached a limit of detection of 1 nM and a limit of quantification of 3 nM for S1P, which was up to 30 times more sensitive than the MRM mode with the same instrument. Intra-day precision ranged between -3.8 and 6.3%, and inter-day precision was between -13.8 and 3.3%, depending on the spiked S1P concentration.

Dysfunctional HDL is associated with coronary artery disease (CAD), but its effect on inflammation in vascular smooth muscle cells (VSMCs) in atherosclerosis is unknown. We investigated the effect of healthy human HDL and CAD-HDL on TNF-α-driven inflammation in VSMCs and examined whether HDL-associated sphingosine-1-phosphate (HDL-S1P) could modulate inflammation with the aim of designing novel HDL-based anti-inflammatory strategies. Healthy human HDL, human CAD-HDL, and mouse HDL were isolated by ultracentrifugation, S1P was measured by liquid chromatography-tandem mass spectrometry, and TNF-α-induced inflammation was characterized by gene expression and analysis of NF-κB-dependent signaling. Mechanisms of S1P interference with TNF-α were assessed by S1P receptor antagonists, mouse knockouts, and short interfering RNA. We observed that healthy HDL potently inhibited the induction of TNF-α-stimulated inflammatory genes, such as iNOS (inducible NO synthase) and MMP9 (matrix metalloproteinase 9), a process that was entirely dependent on HDL-S1P, as evidenced by loss-of-function using S1P-less HDL and mimicked by genuine S1P. Inhibition was based on suppression of TNF-α-activated Akt signaling resulting in reduced IkBαSer32 and p65Ser534 NF-κB phosphorylation based on a persistent phosphatase and tensin homolog activation by S1P through the S1P receptor 2. Intriguingly, S1P suppressed inflammation even hours after initial TNF-α stimulation. The anti-inflammatory effect of healthy HDL correlated with HDL-S1P content and was superior to that of CAD-HDL featuring lower HDL-S1P. Nevertheless, therapeutic loading of HDL with S1P completely restored the anti-inflammatory capacity of CAD-HDL and greatly boosted that of both healthy and CAD-HDL. Suppression of inflammation by HDL-S1P defines a novel pathophysiologic characteristic that distinguishes functional from dysfunctional HDL. The anti-inflammatory HDL function can be boosted by S1P-loading and exploited by S1P receptor-targeting to prevent and even turn off ongoing inflammation.-Keul, P., Polzin, A., Kaiser, K., Gräler, M., Dannenberg, L., Daum, G., Heusch, G., Levkau, B. Potent anti-inflammatory properties of HDL in vascular smooth muscle cells mediated by HDL-S1P and their impairment in coronary artery disease due to lower HDL-S1P: a new aspect of HDL dysfunction and its therapy.

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38–GSK3β–β-catenin and WNT5A–LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases. Promoting more bone growth is of keen interest in the treatment of osteoporosis, and preventing the degradation of S1P offers a new therapeutic avenue for this approach.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator released from activated platelets by an adenosine triphosphate (ATP)-dependent export mechanism. A candidate transport protein is the multidrug resistance protein 4 (MRP4/ABCC4), an ATP-dependent transporter highly expressed in platelets. Furthermore, several statins are known to affect platelet functions and exhibit antithrombotic properties. This study determines the involvement of MRP4 in the transport of S1P and a possible interference by statins. Transport studies in membrane vesicles of Sf9 cells containing recombinant human MRP4 revealed that MRP4 mediates ATP-dependent transport of fluorescein- and tritium-labelled S1P. Also, ATP-dependent S1P transport in platelet membrane vesicles containing endogenous MRP4 was inhibited by the MRP inhibitor MK571 and the MRP4-selective compound Ceefourin-1. Confocal microscopy using fluorescein-labelled S1P as well as boron-dipyrromethene (BODIPY)-labelled sphingosine indicated association of S1P and MRP4 in human platelets. In MRP4-deficient mice, agonist-induced S1P secretion was reduced compared with matched wild-type C57Bl/6 mice and platelet S1P concentrations were lower. Fluvastatin and rosuvastatin interfered with MRP4 function inhibiting ATP-dependent cGMP (cyclic guanosine monophosphate) uptake into MRP4-containing vesicles, inhibited MRP4-mediated S1P transport in vitro and significantly attenuated endogenous S1P release from agonist-activated platelet ex vivo. These data suggest that release of S1P from platelets depends on MRP4 and statins can interfere with this transport process. Potentially, this may be relevant for the pleiotropic anti-inflammatory effects of statins and their effect on modulating atherothrombosis.

Elevated levels of free fatty acids (FFAs) have been related to pancreatic beta-cell failure in type 2 diabetes (T2DM), though the underlying mechanisms are not yet fully understood. FFAs have been shown to dysregulate formation of bioactive sphingolipids, such as ceramides and sphingosine-1 phosphate (S1P) in beta-cells. The aim of this study was to analyze the role of sphingosine-1 phosphate lyase (SPL), a key enzyme of the sphingolipid pathway that catalyzes an irreversible degradation of S1P, in the sensitivity of beta-cells to lipotoxicity. To validate the role of SPL in lipotoxicity, we modulated SPL expression in rat INS1E cells and in human EndoC-βH1 beta-cells. SPL overexpression in INS1E cells (INS1E-SPL), which are characterized by a moderate basal expression level of SPL, resulted in an acceleration of palmitate-mediated cell viability loss, proliferation inhibition and induction of oxidative stress. SPL overexpression affected the mRNA expression of ER stress markers and mitochondrial chaperones. In contrast to control cells, in INS1E-SPL cells no protective effect of oleate was detected. Moreover, Plin2 expression and lipid droplet formation were strongly reduced in OA-treated INS1E-SPL cells. Silencing of SPL in human EndoC-βH1 beta-cells, which are characterized by a significantly higher SPL expression as compared to rodent beta-cells, resulted in prevention of FFA-mediated caspase-3/7 activation. Our findings indicate that an adequate control of S1P degradation by SPL might be crucially involved in the susceptibility of pancreatic beta-cells to lipotoxicity.

Introduction To correctly interpret plasma- or serum-sphingosine-1-phosphate (S1P) concentrations measured in clinical studies it is critical to understand all major determinants in healthy controls. Methods Serum- and plasma-S1P from 174 healthy blood donors was measured by liquid chromatography-tandem mass spectrometry and correlated to clinical laboratory data. Selected plasma samples, 10 with high and 10 with low S1P concentrations, were fractionated into very low-density lipoprotein (VLDL)-, low density lipoprotein (LDL)-, high density lipoprotein (HDL)-, and lipoprotein-free fractions. S1P was then measured in each fraction to determine its distribution. Results The mean S1P concentration in serum (1.04 ± 0.24 nmol/mL) was found 39% higher compared with plasma (0.75 ± 0.16 nmol/mL) and overall was not different between men and women. Only when stratified for age and gender, older women were found to exhibit higher circulatory S1P levels than men. In plasma, S1P levels correlate to red blood cell (RBC) counts but not to platelet counts. Conversely, serum-S1P correlates to platelet counts but not to RBC counts. In addition, eosinophil counts are strongly associated with serum-S1P concentrations. Both serum- and plasma-S1P correlate to total cholesterol but not to HDL-C. The distribution of S1P between VLDL-, LDL-, HDL-, and lipoprotein-free fractions is independent of total plasma-S1P concentrations. S1P concentrations in HDL but not in LDL are highly variable. Conclusion These data indicate S1P concentrations in plasma and serum to be differentially associated with cell counts and S1P carrier proteins. Besides platelets, eosinophil counts are identified as a novel determinant for serum-S1P concentrations further suggesting a role for S1P in eosinophil pathologies.

SUMMARYLipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid raft microdomains are essential components of the phagolysosomal membrane of macrophages. Genetic deletion of the lipidraft chaperons flotillin-1 and flotillin-2 demonstrate that the assembly of both major defense complexes vATPase and NADPH oxidase on the phagolysosomal membrane requires lipid rafts. Furthermore, we discovered a new virulence mechanism leading to the dysregulation of lipid-raft formation by melanized wild-type conidia of the important human-pathogenic fungusAspergillus fumigatus. This results in reduced phagolysosomal acidification. Phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ions as compared to phagolysosomes with melanin-free conidia. In agreement with a role of Ca2+for generation of functional lipid rafts, we show that Ca2+-dependent calmodulin activity is required for lipid-raft formation on the phagolysosome. We identified a single nucleotide polymorphism in the humanFLOT1gene that results in heightened susceptibility for invasive aspergillosis in hematopoietic stem-cell transplant recipients. Collectively, flotillin-dependent lipid rafts on the phagolysosomal membrane play an essential role in protective antifungal immunity in humans.

Sphingosine 1-phosphate (S1P) is an important immune modulator responsible for physiological cellular responses like lymphocyte development and function, positioning and emigration of T and B cells and cytokine secretion. Recent reports indicate that S1P does not only regulate immunity, but can also protect the function of organs by inducing disease tolerance. S1P also influences the replication of certain pathogens, and sphingolipids are also involved in pathogen recognition and killing. Certain carrier molecules for S1P like serum albumin and high density lipoproteins contribute to the regulation of S1P effects. They are able to associate with S1P and modulate its signaling properties. Similar to S1P, both carrier molecules are also decreased in sepsis patients and likely contribute to sepsis pathology and severity. In this review, we will introduce the concept of disease tolerance and the involvement of S1P. We will also discuss the contribution of S1P and its precursor sphingosine to host defense mechanisms against pathogens. Finally, we will summarize current data demonstrating the influence of carrier molecules for differential S1P signaling. The presented data may lead to new strategies for the prevention and containment of sepsis.

Background: Sphingosine-1-phosphate (S1P) is a constituent of high-density lipoproteins (HDL) that contributes to their beneficial effects. We have shown decreased HDL-S1P in coronary arter...

Sphingosine 1-phosphate (S1P) is a lipid metabolite and a ligand of five G protein-coupled cell surface receptors S1PR1 to S1PR5. These receptors are expressed on various cells and cell types of the immune, cardiovascular, respiratory, hepatic, reproductive, and neurologic systems, and S1P has an impact on many different pathophysiological conditions including autoimmune, cardiovascular, and neurodegenerative diseases, cancer, deafness, osteogenesis, and reproduction. While these diverse signalling properties of S1P have been extensively reviewed, the particular role of S1P in blood is still a matter of debate. Blood contains the highest S1P concentration of all body compartments, and several questions are still not sufficiently answered: Where does it come from and how is it metabolized? Why is the concentration of S1P in blood so high? Are minor changes of the high blood S1P concentrations physiologically relevant? Do blood cells and vascular endothelial cells that are constantly exposed to high blood S1P levels still respond to S1P via S1P receptors? Recent data reveal new insights into the functional role and the metabolic fate of blood-borne S1P. This review aims to summarize our current knowledge regarding the source, secretion, transportation, function, metabolism, and fate of S1P in blood.

Sphingosine-1-Phosphate (S1P) is a bioactive sphingolipid with important functions in immunity, inflammation and cardiovascular biology. S1P is associated with prevalence and severity of coronary artery disease and myocardial infarction. However, its relevance in ischemic cardiomyopathy is unknown. We aimed to investigate associations of plasma S1P and other sphingolipids with the extent of heart failure in patients with ischemic heart disease.74 patients with ischemic heart disease were investigated in this observational study. Plasma concentrations of S1P, C16 ceramide and sphingomyelin (SM) were measured using liquid chromatography/tandem mass-spectrometry and associated with objective (echocardiography) and subjective (dyspnea) signs of heart failure. Plasma S1P and SM but not C16 ceramide concentrations were negatively associated with left ventricular ejection fraction (LVEF) and dyspnea (ranked by New York Heart Association; LVEF: S1P standardized coefficient beta: -0.25; 95%CI: -273 to -13nM, p=0.03; SM beta: -0.24; 95%CI: -16,310 to -413nM, p=0.04; NYHA: S1P beta: -0.3; 95%CI: -174 to -26nM, p=0.009; SM beta: -0.46; 95%CI: -13,462 to -5013nM, p0.001). ROC analysis revealed that S1P and SM predicted impaired LVEF with optimal cut-off levels below 843nM and 77μM, respectively.S1P is associated with the impairment of LVEF and dyspnea. Considering the major effects of S1P on cardiac and vascular functions in experimental models, we put forward the hypothesis that S1P is causally involved in the pathophysiology of heart failure. Interfering pharmacologically with S1P receptors may have an impact on ischemic cardiomyopathy.

Sepsis is an acute life-threatening multiple organ failure caused by a dysregulated host response to infection. Endothelial dysfunction, particularly barrier disruption leading to increased vascular permeability, edema, and insufficient tissue oxygenation, is critical to sepsis pathogenesis. Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates important pathophysiological processes including vascular endothelial cell permeability, inflammation, and coagulation. It is present at high concentrations in blood and lymph and at very low concentrations in tissues due to the activity of the S1P-degrading enzyme S1P-lyase in tissue cells. Recently, four preclinical observational studies determined S1P levels in serum or plasma of sepsis patients, and all found reduced S1P levels associated with the disease. Based on these findings, this review summarizes S1P-regulated processes pertaining to endothelial functions, discusses the possible use of S1P as a marker and possibilities how to manipulate S1P levels and S1P receptor activation to restore endothelial integrity, dampens the inflammatory host response, and improves organ function in sepsis.

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.

Well-defined gradients of the lipid mediator sphingosine-1-phosphate (S1P) direct chemotactic egress of mature thymocytes from the thymus into the circulation. Although it is known that these gradients result from low S1P levels in the thymic parenchyma and high S1P concentrations at the exit sites and in the plasma, the biochemical mechanisms that regulate these differential S1P levels remain unclear. Several studies demonstrated that ceramide synthase 2 (Cers2) regulates the levels of the S1P precursor sphingosine. We, therefore, investigated whether Cers2 is involved in the regulation of S1P gradients and S1P-dependent egress into the circulation. By analyzing Cers2-deficient mice, we demonstrate that Cers2 limits the levels of S1P in thymus and blood to maintain functional S1P gradients that mediate thymocyte emigration into the circulation. This function is specific for Cers2, as we also show that Cers4 is not involved in the regulation of thymic egress. Our study identified Cers2 as an important regulator of S1P-dependent thymic egress, and thus contributes to the understanding of how S1P gradients are maintained in vivo.

Although predominantly expressed on lymphocytic and hematopoietic cells, the role of sphingosine-1-phospate receptor 4 (S1P(4)) in immune homeostasis is still poorly understood. In this report, we used a S1P(4)-deficient murine model to characterize the biological role of S1P(4)-mediated S1P signaling in the immune system. S1p(4)(-/-) animals showed normal peripheral lymphocyte numbers and a regular architecture of secondary lymphoid organs. Interestingly, S1P(4) only marginally affects T-cell function in vivo. In contrast, dendritic cell (DC) migration and cytokine secretion are profoundly affected by S1P(4) deficiency. Lack of S1P(4) expression on DCs significantly reduces T(H)17 differentiation of T(H) cells. Furthermore, in various in vivo models of T(H)1- or T(H)2-dominated immune reactions, S1P(4) deficiency consistently increased the amplitude of T(H)2-dominated immune responses, while those depending on T(H)1-dominated mechanisms were diminished. Finally, S1p(4)(-/-) mice showed decreased pathology in a model of dextran sulfate sodium-induced colitis. In summary, for the first time, we show that S1P(4) signaling is involved in the regulation of DC function and T(H)17 T-cell differentiation. S1P(4)-mediated S1P signaling also modifies the course of various immune diseases in a murine model. We propose that S1P(4) may constitute an interesting target to influence the course of various autoimmune pathologies.

Sphingosine 1-phosphate (S1P) is an important regulator of many different immune functions including lymphocyte circulation, antigen presentation, and T cell development. It stimulates five G protein-coupled receptors designated S1P(1-5), which are also expressed by immune cells. S1P receptors couple to different heterotrimeric G proteins including G alpha i, q, and 12/13, and elicit cellular signalling events by activating the small GTPases Rac and Rho and protein kinases Akt, ERK, and JNK, and by inducing cellular calcium flux and inhibiting cAMP accumulation, amongst others. S1P is the exit signal for lymphocytes leaving lymphoid organs and present in blood and lymph at high nanomolar concentrations due to the S1P-producing activity of sphingosine kinases (SK). The S1P-degrading enzyme S1P-lyase maintains low amounts of S1P in lymphoid organs. Disrupting this concentration difference by S1P receptor agonists and antagonists like FTY720, SEW2871, and VPC23019, by an anti-S1P antibody, or by inhibiting the S1P-lyase has therapeutic potential for autoimmune diseases like multiple sclerosis (MS) and rheumatoid arthritis and for many other disorders like cancer, fibrosis, inflammation, macular degeneration, diabetic retinopathy, and glaucoma. This report aims to provide a brief overview of concepts, approaches, pharmaceutical compounds, and targets that are currently used to modulate S1P-driven immune functions.

Sphingolipids are known to function as structural components in cell membranes, but they also have signalling properties. Sphingosine 1-phosphate (S1P), in particular, is an extracellular messenger that activates five G protein-coupled cell surface receptors, and regulates important physiological functions in the cardiovascular and immune system, in hearing, and fertility. Tight regulation of receptor-available S1P, establishment of concentration gradients and the close interplay between S1P and its receptors are necessary requirements in a multitude of processes. Pharmacological and genetic studies in animals have demonstrated that altered S1P concentrations in blood and organs, as well as mutated expression of S1P receptors in certain cell systems can be causative, but may also be efficacious medication in selected diseases. Sphingolipids and their receptors are, therefore, current targets for medical research.

Sphingosine 1-phosphate (S1P) is an extra- and intracellular messenger that specifically activates five G-protein-coupled cell surface receptors designated S1P(1-5). The S1P(1) receptor is particularly important for the maintenance of immune surveillance by regulating egress of lymphocytes from thymus and secondary lymphoid organs. S1P is generated through phosphorylation of sphingosine which is catalyzed by sphingosine kinase types 1 and 2. The immunosuppressant and sphingosine analog Fingolimod (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol, FTY720) can also be phosphorylated and induces lymphopenia by downregulating cell surface expression of the S1P(1) receptor on lymphocytes. To analyze the role of S1P in lymphocyte circulation and distribution we established a high-performance-liquid-chromatography-based method for parallel detection and quantification of Fingolimod, sphingosine, and dihydrosphingosine together with their phosphorylated derivatives Fingolimod-phosphate, S1P, and dihydrosphingosine 1-phosphate. Phosphorylated and nonphosphorylated lipids were efficiently isolated from biological samples such as cells, tissues, serum, plasma, and media by simple chloroform extraction. Fluorescence labeling with 9-fluorenylmethyl chloroformiate ensured high selectivity and enhanced sensitivity for sphingolipid detection. The described method provides an accurate approach to investigate phosphorylation, dephosphorylation, hydrolyzation, and dehydrolyzation of sphingolipids and analogs. In addition it works independently from enzymatic conversions, measuring actual concentrations rather than enzymatic activities.

Sphingosine 1-phosphate (S1P) type 1G protein-coupled receptors (S1P1 GPCRs) are specific high-affinity transducers for this lipid growth factor and cellular mediator. S1P1 GPCRs are widely-expressed and physiologically critical in the cardiovascular and immune systems. Functional rat monoclonal antibodies (MoAbs) have been generated against human S1P1 GPCRs expressed in rat null-cell transductants to provide bioavailable agents capable of stimulating or suppressing the S1P-S1P1 GPCR axis. The rat IgM-kappa anti-S1P1 GPCR MoAb designated 4B5.2 binds specifically to native human or mouse S1P1 GPCRs in cell membranes, but not to solubilized and denatured S1P1 GPCRs. Specific binding of 32P-S1P to cellular S1P1 GPCRs is not blocked by 4B5.2. T cell chemotactic responses to S1P and S1P suppression of T cell chemotaxis to chemokines both are inhibited selectively by 4B5.2. In contrast, generation of gamma-interferon by stimulated T cells is diminished by 4B5.2 as by S1P. T cell S1P1 GPCR-selective antagonist and agonist effects of 4B5.2 in vivo may alter immune responses as distinctively as the available poly-S1P GPCR-directed pharmacological agents, without the undesirable side-effects attributable to actions of these agents on other S1P GPCRs.

Progressive accumulation of the amyloid β protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid β is generated during sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca(2+) from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.

Liquid chromatography coupled to tandem mass spectrometry has evolved as the method of choice for the detection of sphingolipid metabolites due to its high sensitivity and superior specificity compared to other methodological approaches. Here, we describe a simple and robust method for the detection and quantification of sphingosine-1-phosphate (S1P) and related sphingolipids in biological samples. Tissue homogenates, cells, supernatant, plasma, and whole blood are spiked with an internal standard to account for loss of material during sample handling. After chloroform extraction of lipids under acidified conditions, the solvent is evaporated, and the remaining lipid extracts are dissolved in 20% CHCl(3) and 80% methanol. Following reversed-phase high-performance liquid chromatography step-gradient separation of sphingolipids and positive electrospray ionization, detection is carried out with the AB Sciex QTrap triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Characteristic fragment ions of S1P and related sphingolipids are monitored and subsequently analyzed relative to known standard concentrations of the pure compounds. Known problems of S1P quantification, such as carryover and insufficient HPLC separation, are discussed.

It is well established that the lysophospholipid and signalling molecule sphingosine 1-phosphate (S1P) has many important functions in immune surveillance. S1P is produced from sphingosine by two distinct sphingosine kinases, SphK1 and SphK2, and acts as an intracellular messenger and as an extracellular ligand of five G protein-coupled cell surface receptors designated S1P(1)-S1P(5). S1P not only regulates peripheral lymphocyte circulation, but also influences their differentiation, activation, infiltration, and local positioning. The therapeutic value of modulating S1P metabolism and S1P receptor function is currently tested in clinical trials and holds great promise for treatment of different autoimmune diseases. Despite its obvious contribution to immune regulation, the analysis of S1P is still challenging. A major obstacle is the difficulty to analyze S1P locally in tissues and within cells due to its high metabolic turnover and the limited resolution of current analytical techniques like liquid chromatography and mass spectrometry. This review focuses on recent advancements to our understanding how different sources of S1P contribute to immune function, and how changes in production, secretion, and degradation of S1P can influence immune responses.

Rationale: The role of sphingosine-1-phosphate (S1P) and its receptors in the pathogenesis of atherosclerosis has not been investigated. Objective: We hypothesized that the S1P receptor 3 (S1P 3 ) plays a causal role in the pathogenesis of atherosclerosis. Methods and Results: We examined atherosclerotic lesion development in mice deficient for S1P 3 and apolipoprotein (Apo)E. Although S1P 3 deficiency did not affect lesion size after 25 or 45 weeks of normal chow diet, it resulted in a dramatic reduction of the monocyte/macrophage content in lesions of S1P 3 −/− /ApoE −/− double knockout mice. To search for putative defects in monocyte/macrophage recruitment, we examined macrophage-driven inflammation during thioglycollate-induced peritonitis. Elicited peritoneal macrophages were reduced in S1P 3 -deficient mice and expressed lower levels of tumor necrosis factor-α and monocyte chemoattractant protein-1. Bone marrow–derived S1P 3 -deficient macrophages produced less MCP-1 in response to lipopolysaccharide stimulation. In vitro, S1P was chemotactic for wild-type but not S1P 3 -deficient peritoneal macrophages. In vivo, S1P concentration increased rapidly in the peritoneal cavity after initiation of peritonitis. Treatment with the S1P analog FTY720 attenuated macrophage recruitment to the peritoneum. Studies in bone marrow chimeras showed that S1P 3 in both hematopoietic and nonhematopoietic cells contributed to monocyte/macrophage accumulation in atherosclerotic lesions. Finally, S1P 3 deficiency increased the smooth muscle cell content of atherosclerotic lesions and enhanced neointima formation after carotid ligation arguing for an antiproliferative/antimigratory role of S1P 3 in the arterial injury response. Conclusions: Our data suggest that S1P 3 mediates the chemotactic effect of S1P in macrophages in vitro and in vivo and plays a causal role in atherosclerosis by promoting inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior.

High-density lipoproteins (HDL) are the major plasma carriers for sphingosine 1-phosphate (S1P) in healthy individuals, but their S1P content is unknown for patients with coronary artery disease (CAD). The aim of the study was to determine whether the S1P levels in plasma and HDL are altered in coronary artery disease. S1P was determined in plasma and HDL isolated by ultracentrifugation from patients with myocardial infarction (MI, n = 83), stable CAD (sCAD, n = 95), and controls (n = 85). In our study, total plasma S1P levels were lower in sCAD than in controls (305 vs. 350 pmol/mL). However, normalization to HDL-cholesterol (a known determinant of plasma S1P) revealed higher normalized plasma S1P levels in sCAD than in controls (725 vs. 542 pmol/mg) and even higher ones in MI (902 pmol/mg). The S1P amount contained in isolated HDL from these individuals was lower in sCAD than in controls (S1P per protein in HDL: 132 vs. 153 pmol/mg). The amount of total plasma S1P bound to HDL was lower in sCAD and MI than in controls (sCAD: 204, MI: 222, controls: 335 pmol/mL), while the non-HDL-bound S1P was, accordingly, higher (sCAD: 84, MI: 81, controls: 10 pmol/mL). HDL-bound plasma S1P was dependent on the plasma HDL-C in all groups, but normalization to HDL-C still yielded lower HDL-bound plasma S1P in patients with sCAD than in controls (465 vs. 523 pmol/mg). The ratio of non-HDL-bound plasma S1P to HDL-C-normalized HDL-bound S1P was also higher in both sCAD (0.18 mg/mL) and MI (0.15 mg/mL) than in controls (0.02 mg/mL). Remarkably, levels of non-HDL-bound plasma S1P correlated with the severity of CAD symptoms as graded by Canadian Cardiovascular Score, and discriminated patients with MI and sCAD from controls. Furthermore, a negative association was present between non-HDL-bound plasma S1P and the S1P content of isolated HDL in controls, but was absent in sCAD and MI. Finally, MI patients with symptom duration of less than 12 h had the highest levels of total and normalized plasma S1P, as well as the highest levels of S1P in isolated HDL. The HDL-C-normalized plasma level of S1P is increased in sCAD and even further in MI. This may be caused by an uptake defect of HDL for plasma S1P in CAD, and may represent a novel marker of HDL dysfunction.

Sphingosine kinases (SKs) 1 and 2 produce high concentrations of sphingosine 1-phosphate (S1P) in blood and lymph. In contrast, S1P concentrations in lymphoid tissues are kept low by the S1P-degrading activity of the S1P-lyase. These differences in S1P concentrations drive lymphocyte circulation. Inhibition of the S1P-lyase prevents lymphocyte egress and causes lymphopenia because of increased S1P levels in lymphoid tissues. In this study, we investigated the source of this accumulating S1P in lymphoid tissues by using SK2-deficient (SK2−/−) mice. In contrast to wild-type mice, SK2−/− mice exhibited attenuated lymphopenia after S1P-lyase inhibition by 4-deoxypyridoxine (DOP). Consistently, S1P concentrations were only modestly increased in lymphoid tissues of SK2−/− mice compared with a significantly higher increase in wild-type mice after DOP treatment. Low S1P concentrations in lymphoid tissues of DOP-treated SK2−/− mice were accompanied by higher S1P concentrations in blood, suggesting that SK2−/− mice display defective S1P transport from blood into lymphoid tissues. To investigate this potential new role of SK2, RBCs loaded with traceable C17-S1P were transfused into wild-type and SK2−/− mice, resulting in much higher C17-S1P concentrations in blood of SK2−/− mice compared with wild-type mice 2 h after transfusion. Moreover, cocultures of RBCs with mouse splenocytes and endothelial cells demonstrated that SK2 regulated cellular uptake of S1P from RBCs. Collectively, our data suggest that S1P in lymphoid tissues derives from blood and point to an essential role of SK2 in S1P transport.

Sphingosine 1-phosphate (S1P) is the natural ligand for a specific family of G protein-coupled receptors (-Rs). The type 1 S1P-R (S1P1) is important for lymphocyte egress, and blood-borne S1P as the natural ligand for S1P1 is involved in the maintenance of lymphocyte circulation. This report reveals that extracellular S1P was cleared by all tested primary cells and cell lines with exponential progression. Clearance of S1P, but not sphingosine (Sph) was inhibited with the protein phosphatase inhibitor sodium orthovanadate. Fluorescence microscopy and flow cytometry using fluorescently labeled S1P and Sph showed a major cellular uptake of Sph, but not S1P. HPLC-analyses with C17-Sph demonstrated that cellular Sph accumulation was transient in tested cell lines, but enduring in mouse splenocytes. Sub cellular fractionation resulted in dephosphorylation of S1P to Sph by nuclear, membrane, and cytosolic fractions. Degradation of Sph however only occurred in combined membrane and cytosolic fractions. Inhibitors for Sph kinases 1/2, ceramide synthase, and S1P-lyase, as well as S1P-lyase deficiency did not block clearance of extracellular S1P. In vivo experiments revealed a transient increase in plasma S1P levels after single intravenous injection into C57BL/6 mice. This exogenously added S1P was cleared within 15-30 min in contrast to ex vivo incubation of whole blood which required more than 8 h for comparable clearance from plasma. Our data thus show that extracellular S1P is dephosphorylated and subsequently converted by cells, which appears to be important for clearance of the signaling molecule S1P in the local tissue environment after infections or injuries. J. Cell. Biochem. 104: 756-772, 2008. 2008 Wiley-Liss, Inc.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are distinctive mediators of immune cellular proliferation, survival and functions, which are present at highly active concentrations in blood, lymph, and tissue corridors of lymphocyte traffic [Moolenaar et al., 1997; Spiegel et al., 1998]. In the immune system, dendritic cells, macrophages, mast cells, and platelets are the principal sources of LPA and S1P. Each type of immune cell expresses a characteristic profile of one or more of the nine Edg-family G protein-coupled receptors (GPCRs) selective for either S1P or LPA, which is controlled developmentally and by diverse stimuli (Table I) [Goetzl et al., 2000; Chun et al., 2002]. S1P and, often less effectively, LPA are chemotactic for many types of immune cells, including T-cells, B-cells, dendritic cells, and NK cells at their normal ambient tissue levels of 3–300 nM [Zheng et al., 2000, 2001; Graeler et al., 2002; Idzko et al., 2002; Kveberg et al., 2002]. At blood and lymph concentrations of over 0.3–3 μM, however, S1P but not LPA nearly completely inhibits lymphocyte migration to chemokines and other stimuli, and suppresses less profoundly lymphocyte proliferation and secretion of some cytokines [Graeler et al., 2002; Dorsam et al., 2003]. That expression of LPA and S1P GPCRs in thymus is restricted to the last phases of development indicates possible roles in terminal differentiation and emigration of CD4 and CD8 T-cells, which remain to be elucidated definitively. As there are numerous and often redundant stimuli of T-cell migration and distinct immunological functions, but fewer specific suppressive influences, the immunoregulatory activities of S1P and its GPCRs are the principal focus of current interest. The potential importance of these immunoregulatory effects is emphasized by their magnitude and involvement in development, tissue distribution, and activities of lymphocytes. Some of the recent excitement in this field is also attributable to the strong possibility of discovering unique classes of immunoactive drugs that target S1P GPCRs. Newly established mouse genetic models of altered expression of T-cell S1P GPCRs and novel functional anti-S1P R monoclonal antibodies now have provided systems and reagents for analyzing further the separate immune contributions of each of the predominant T-cell S1P GPCRs. The results of such studies also may permit accurate predictions of the immune effects of individual S1P GPCR-selective agonists and antagonists. TABLE I Immune Cell Expression of mRNAs Encoding LPA and S1P GPCRs*

The lysophospholipid (LPL) growth factors sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are generated by macrophages, dendritic cells, mast cells, and platelets, which leads to lymph and plasma concentrations of 0.1–1 μM. Distinctive profiles of G protein-coupled receptors (GPCRs) for S1P and LPA are expressed by each type of immune cell and are regulated by cellular activation. At 1–100 nM, S1P signals T cells through their principal S1P1 GPCRs with consequent protection from apoptosis, enhancement of chemotaxis, and facilitation of optimal regulatory activity of CD4+25+ T cells. At 0.3–3 μM, S1P inhibits T cell chemotaxis and to a lesser extent other functions. These S1P–S1P1 GPCR signals suppress homing of blood and spleen T cells to secondary lymphoid tissues. S1P1 GPCR antagonists evoke lymphopenia by permitting blood T cells to enter lymph nodes and blocking S1P1 GPCR-dependent T cell efflux from lymph nodes. Inversely, there is a decrease in lymphoid tissue traffic of T cells in transgenic mice, which overexpress lymphocyte S1P1 GPCRs. The immunotherapeutic activity of S1P1 GPCR antagonists, which limits T cell access to organ grafts and autoimmune antigens, does not reduce other functional capabilities of T cells. LPLs and their GPCRs thus constitute an immunoregulatory system of sufficient prominence for pharmacological targeting in transplantation, autoimmunity, and immunodeficiency.

FTY720 is an immunosuppressant that reduces circulating levels of naive lymphocytes by increasing their localization and sequestration in secondary lymphoid organs. It is considered to be an agonist for sphingosine 1-phosphate (S1P) G protein-coupled receptors (GPCRs) after phosphorylation at micromolar concentrations. We now describe its nonagonist and noncompetitive inhibitory activity at low nanomolar concentrations for types 1 and 5 S1P-GPCRs and of moderate potency for type 2 S1P-GPCRs. FTY720 blocks S1P signaling through S1P1,2,5 by inducing their internalization and intracellular partial degradation without affecting S1P3 or S1P4. S1P-R internalization is maximal several hours after only seconds of incubation with FTY720 at 37 degrees C and washing, and continues for days before recovery of surface expression and functions. The timing and extent of S1P-R internalization are highly dependent on FTY720 concentration. FTY720 is therefore an S1P-GPCR-selective and noncompetitive inhibitor with a unique mechanism of action.

Among the family of lipid-derived mediators, the group of lysophospholipids including lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have growth-related and -unrelated effects on diverse cell types including lymphocytes, macrophages, smooth muscle cells, endothelial cells, and neuronal cells. This review summarizes the known effects of lysophospholipids and their G protein-coupled receptors (GPCRs) in inflammation and immunity. Lysophospholipids have the capacity to evoke and modulate immune responses by attracting and activating T-cells, B-cells and macrophages directly and influencing their interactions with other cell types. Immune cells express multiple subsets of lysophospholipid receptors, which are critical for specific cellular responses such as proliferation and migration that are fundamental to immunity. Investigation of the expression pattern of EDG-receptors on human T-cells revealed a dynamic transcriptional regulation influenced by both developmental stages and activation states. Other lipid mediators like psychosine and other GPCRs for lipid mediators like G2A also may be involved in the development of normal immune and inflammatory reactions and diseases. These observations suggest that agonists and antagonists for lysophospholipid receptors may influence immune responses.