Activation Of Sphingosine-1-phosphate Receptors Research Articles

Role of the S1P Signaling Pathway in the Pathogenesis of Angioimmunoblastic T-Cell Lymphoma

Peripheral T cell lymphomas (PTCLs) are a group of aggressive lymphoid malignancies originating from mature T cells. Angioimmunoblastic T-cell lymphomas (AITL) represent 20% to 30% of all PTCL diagnoses and are associated with autoimmune features, poor response to chemotherapy, and dismal prognosis. Molecular profiling of AITL has identified T-follicular helper (TFH) cells as the cell of origin of AITL, while mutational analysis has identified frequent alterations in epigenetic regulators ( TET2, DNMT3A, and IDH2) and elements of the TCR pathway as drivers of AITL transformation. Our group identified the highly recurrent RHOA G17V mutation as a defining hallmark of AITL and other T-cell lymphomas of TFH-cell origin (Palomero et al., 2014). Using a novel conditional knockin mouse model, we demonstrated that expression of Rhoa G17V in CD4+ T-cells induces TFH cell specification and promotes AITL lymphomagenesis in the context of loss of Tet2 (Cortes et al., 2018). Recently, we have identified that Rhoa G17V regulates the expression of the sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) one of the five members of the receptor family of S1P, which plays an essential role in immune response and lymphocyte trafficking. During thymic T-cell development, expression of Rhoa G17V in CD4+ cells inhibited S1PR1 downregulation after the double positive stage leading to altered positive selection, reduced CD4+ single positive cell numbers and premature thymic egress of autorreactive T-cells which results in the development of systemic inflammation and autoimmunity. In our Tet2 −/− RHOA G17V AITL tumor model, constitutive activation of S1P receptors supported activation of the STAT3 and NFkB pathway and enhanced migration of malignant cells to peripheral organs. Mechanistically, expression of Rhoa G17V in mature CD4+ T-cells impaired re-phosphorylation of the Ezrin-Radoxin-Moesin (ERM) complex independently of ROCK signaling and led to the activation and increased migration of CD4+ T-cells. Importantly, blockade of S1PR1 signaling by fingolimod induced anti-tumor activity in Tet2 −/− RHOA G17V lymphoma-bearing mice in vivo with significant reductions in tumor burden associated with increased apoptosis and decreased STAT3 phosphorylation. Furthermore, analysis of a panel of primary tumors from AITL patients using a multiplexed staining panel and PhenoImager platform revealed significantly higher S1PR1 expression in AITL tumor cells than normal TFH cells from benign reactive lymph nodes. Our findings highlight the role of S1P signaling pathway in the pathogenesis of RHOA G17V-driven AITLs and the potential therapeutic benefit of targeting this pathway.

Abstract 11818: The Beneficial Effects of Ceramide on Human Microvascular Endothelial Function is Dependent on S1P/S1PR1 Signaling Axis

Despite the known damaging effects of elevated intracellular ceramides on human microvascular function, our recent work showed that loss of ceramide-formation through neutral sphingomyelinase (NSmase) promotes endothelial dysfunction in arterioles from healthy adults. This presents as a switch in the endothelial-derived mediator responsible for eliciting dilation to flow (flow-induced dilation; FID), from the vasoprotective nitric oxide (NO) to pro-inflammatory hydrogen peroxide (H 2 O 2 ). The mechanism through which ceramide exerts beneficial effects on the vasculature remains poorly understood. One of ceramide’s metabolites, sphingosine-1-phosphate (S1P), has been shown to stimulate NO production through the activation of S1P receptor 1 (S1PR1). Thus, we hypothesize that ceramide promotes NO-mediated FID through its metabolism to S1P and activation of S1PR1. Arterioles (100-250μm) from healthy adults were dissected from discarded surgical adipose tissue. Vessels were cannulated in organ chambers, pre-constricted with endothelin-1, and changes in internal diameter in response to increases in flow or C2-ceramide was measured via videomicroscopy. Difference between mean % dilation ±SE with two-way ANOVA is reported. Agonism of S1PR1 (CYM5442, 1μM, 30min) during NSmase inhibition (GW4869, 4μM, 30min) restored dependency on NO for FID, as L-NAME (100μM; NO synthase inhibitor) diminished FID (GW+CYM, n=4 vs GW+CYM+L-NAME, n=4: -70.3±20.0, p=0.01). A similar switch to NO-mediated FID was seen with C2-ceramide (10μM)+GW4869 treatment (GW+C2, n=5 vs GW+C2+L-NAME, n=4: -52.5±5.9, p<0.001). Inhibition of S1PR1 (W146, 10μM, 30min) induced a switch from NO to H 2 O 2 mediated FID, as catalase (500U/ml; W146, n=5 vs W146+Catalase, n=4: -34.0±7.9; p=0.01) but not L-NAME (W146, n=5 vs W146+L-NAME, n=4; -1.4±9.9, p=0.89) impaired FID. Acute ceramide-induced dilation (1min) was diminished in the presence of L-NAME (C2, n=5 vs C2+L-NAME, n=4: -33.40±10.06, p=0.02) and W146 (C2, n=5 vs C2+W146, n=4; -39.7±5.9, p=0.01). Together, these data suggest that ceramide induces NO-signaling through the activation of S1PR1 and provide mechanistic insight into how ceramide may exert beneficial effects within the human microvascular endothelium.

Renal Medullary Overexpression of Sphingosine-1-Phosphate Receptor 1 Transgene Attenuates Deoxycorticosterone Acetate (DOCA)-Salt Hypertension.

Our previous studies showed that renal medullary sphingosine-1-phosphate receptor 1 (S1PR1) mediated sodium excretion, high salt intake increased S1PR1 level, deoxycorticosterone acetate (DOCA) blocked high salt-induced S1PR1 in the renal medulla, and that conditional knockout of S1PR1 in the collecting duct aggravated DOCA-salt hypertension. The present study tested the hypothesis that overexpression of S1PR1 transgene in the renal medulla attenuates the sodium retention and hypertension in DOCA-salt mouse model. Male C57BL/6J mice received renal medullary transfection of control or S1PR1-expressing plasmids and then DOCA-salt treatment. Renal sodium excretion and arterial pressure were compared between control and S1PR1-overexpressed mice in response to high salt loading or pressure natriuresis. S1PR1-transfected mice showed significantly enhanced urinary sodium excretion in response to acute sodium loading (0.93 ± 0.27 in control vs. 4.72 ± 1.12 µmol/min/gKW in S1PR1-overexpressed mice, P < 0.05) and the pressure natriuresis (3.58 ± 1.77 vs. 9.52 ± 1.38, P < 0.05), less positive sodium balance in response to chronic high-salt intake (3.05 ± 0.39 vs. 1.65 ± 0.39 mmol/72 hr, P < 0.05), and consequently, the attenuation of DOCA-salt hypertension (134.2 ± 6.79 vs. 109.8 ± 3.54 mm Hg, P < 0.05). The αENaC protein amount in the renal medulla was not changed, however, the βENaC was significantly decreased and the γENaC was significantly increased in S1PR1-overexpressed mice. The immunostaining showed apical membrane translocation of γENaC, while no change of αENaC and βENaC in control mice, and that the apical membrane translocation of γENaC was blocked in S1PR1-treasffected mice. These results suggested that activation of S1PR1 in the renal medulla attenuates DOCA-induced sodium retention and salt-sensitive hypertension associated with inhibition of ENaC.

Sphingosine 1-Phosphate Activates S1PR3 to Induce a Proinflammatory Phenotype in Human Myometrial Cells.

One of the common mechanisms responsible for obstetric complications, affecting millions of women every year, is abnormal uterine contractility. Despite the critical importance of this process for women's health, the mechanisms of uterine contraction regulation remain poorly understood. The initiation of uterine smooth muscle (myometrial) contraction is an inflammatory process, accompanied by upregulation of proinflammatory genes and cytokine release. In this study, we show that sphingolipid metabolism is activated during human labor and that sphingosine 1-phosphate (S1P), the main bioactive sphingolipid, may modify the myometrial proinflammatory phenotype. Our data in both primary and immortalized human myometrial cells show that exogenous S1P induces a proinflammatory gene signature and upregulates the expression of known inflammatory markers of parturition, such as IL8 and COX2. Using expression of IL8 as a readout for S1P activity in myometrial cells, we established that these S1P effects are mediated through the activation of S1P receptor 3 (S1PR3) and downstream activation of ERK1/2 pathways. Inhibition of S1PR3 in human myometrial cells attenuates upregulation of IL8, COX2, and JUNB both at the mRNA and protein levels. Furthermore, activation of S1PR3 with a receptor-specific agonist recapitulated the effects seen after treatment with exogenous S1P. Collectively, these results suggest a signaling pathway activated by S1P in human myometrium during parturition and propose new targets for development of novel therapeutics to alter uterine contractility during management of preterm labor or labor dystocia.

S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with unclear etiology and limited treatment options. The median survival time for IPF patients is approximately 2-3 years and there is no effective intervention to treat IPF other than lung transplantation. As important components of lung tissue, endothelial cells (ECs) are associated with pulmonary diseases. However, the role of endothelial dysfunction in pulmonary fibrosis (PF) is incompletely understood. Sphingosine-1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor highly expressed in lung ECs. Its expression is markedly reduced in patients with IPF. Herein, we generated an endothelial-conditional S1pr1 knockout mouse model which exhibited inflammation and fibrosis with or without bleomycin (BLM) challenge. Selective activation of S1PR1 with an S1PR1 agonist, IMMH002, exerted a potent therapeutic effect in mice with bleomycin-induced fibrosis by protecting the integrity of the endothelial barrier. These results suggest that S1PR1 might be a promising drug target for IPF therapy.

Selective depletion of CD11b-positive monocytes/macrophages potently suppresses bleomycin-induced pulmonary fibrosis

The understanding of pathogenesis underlying idiopathic pulmonary fibrosis (IPF) is still limited presently. Monocytes or macrophages are involved in progression of the pulmonary injury and repair. The aim of this study is to investigate the roles of CD11b+ monocytes/macrophages in the progression of pulmonary fibrosis. In this study, the expression levels of CD11B gene and inflammatory genes in the IPF patients are evaluated using the available datasets. CD11b cells are conditionally depleted in a CD11b-diptheria toxin receptor (CD11b-DTR) mouse by administration of diptheria toxin (DT). Pulmonary fibrosis in mice is induced using intranasalbleomycin. The mRNAs and proteins expression in lung tissues are determined by quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining and Western-blot assays. It shows that the expression of CD11B mRNA is up-regulated in fibrotic lungs and alveolar macrophages of IPF patients and bleomycin-treated rodents. Selective depletion of CD11b+ monocytes/macrophages in CD11b-DTR mice potently halts bleomycin-induced pulmonary fibrosis progression. CD11b depletion inhibits the polarization of macrophages in the fibrotic lungs. Mechanically, CD11b deficiency represses the activation of sphingosine 1-phosphate receptor 2 (S1PR2)/sphingosine kinase 2 (SphK2) signaling during pulmonary fibrosis. In conclusion, our data suggest that CD11b+ monocytes/macrophages contribute to pulmonary fibrosis and represent a potential therapeutic target for IPF.

Abstract 1312: Pro-survival lipid metabolism activates intracellular complement signaling to induce inflammasome-mediated tumor metastasis

Abstract Tumor metastasis remains one of the major causes of cancer-related deaths in patients with solid tumors. Mechanisms by which metabolic alterations regarding pro-survival lipid signaling activation in inducing cancer cell migration and metastasis are largely unknown. Here, we demonstrate that sphingosine 1-phosphate (S1P) metabolism activates endogenous complement signaling for inducing tumor metastasis via inducing inflammasome-mediated pro-inflammation. Our studies using molecular, pharmacologic, and genetic tools showed that activation of S1P and S1P receptor 1 (S1P/S1PR1) induces tumor metastasis via enhanced intracellular complement signaling by the regulation of the C3-PPIL1 complex and subsequent inflammasome activation in cancer cells and in vivo xenograft-derived tumors. Citation Format: Alhaji H. Janneh, Besim Ogretmen. Pro-survival lipid metabolism activates intracellular complement signaling to induce inflammasome-mediated tumor metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1312.

Pro‐survival lipid metabolism activates intracellular complement signaling to induce inflammasome‐mediated tumor metastasis

Tumor metastasis remains one of the major causes of cancer‐related deaths in patients with solid tumors. Mechanisms by which metabolic alterations regarding pro‐survival lipid signaling activation in inducing cancer cell migration and metastasis are largely unknown. Here, we demonstrate that sphingosine 1‐phosphate (S1P) metabolism activates endogenous complement signaling for inducing tumor metastasis via inducing inflammasome‐mediated pro‐inflammation. Our studies using molecular, pharmacologic, and genetic tools showed that activation of S1P and S1P receptor 1 (S1P/S1PR1) induces tumor metastasis via enhanced intracellular complement signaling by the regulation of the C3‐PPIL1 complex and subsequent inflammasome activation in cancer cells and in vivo xenograft‐derived tumors.

Abstract 470: Sphingosine-1-Phosphate Signaling In The Human Microvascular Endothelium During Health And Disease

The loss of nitric oxide (NO)-mediated flow-induced dilation (FID) in the microvasculature, or microvascular endothelial dysfunction, is strongly linked to future cardiovascular events. Our lab has previously shown that chronic inhibition of sphingosine-1-phosphate (S1P) formation induces the change in FID mediator from the vasoprotective NO to the pro-inflammatory, pro-atherosclerotic hydrogen peroxide. The same transition is observed in arterioles from patients with coronary artery disease (CAD). Since S1P promotes NO-mediated FID, we hypothesized that formation of S1P and activation of S1P receptor 1 (S1PR1) is critical for FID in microvessels from healthy adults as opposed to arterioles from patients with CAD. Videomicroscopy was used to perform vascular function studies on human resistance arterioles (100-250μm) dissected from discarded surgical adipose tissue. The vessels were pre-constricted with endothelin-1 and changes in internal diameter in response to flow was measured. In arterioles from healthy adults, treatment with a sphingosine kinase (SpK) inhibitor for 30 minutes abolished FID (7.7±8.0% of maximal dilator capacity, n=3; mean±SEM) compared to control (73.1±8.2% n=9, p&lt;0.05; *two-way ANOVA), whereas inhibition of S1PR1 appeared to delay the response to dilation (19.1±10.5% vs. 52.8±9.0% at 20mmHg pressure gradient, p&lt;0.05*). In microvessels from patients with disease, both inhibition of SpK (91.8±2.8%, n=5) and S1PR1 (72.5±6.4%, n=6) did not affect FID compared to control (90.9±2.4%, n=4). Interestingly, S1PR1 expression was similar in CAD and nonCAD arterioles. Together these data suggest that formation of S1P and potentially the activation of S1PR1 is critical for FID during health, however this pathway does not contribute to FID during disease. We conclude that while arterioles can compensate for loss of this pathway in disease to maintain tissue perfusion, formation of S1P and activation of its receptors may be necessary to promote NO formation during shear and may serve as potential targets to prevent future cardiovascular disease.

Functional and Molecular Studies of Renal Sphingosine 1 Phosphate Signaling Pathway in IUGR Benjamin Bhunu1 and Suttira Intapad1 Tulane University School of Medicine

Fetal Growth Restriction (FGR) is now regarded as a risk factor for adult renal diseases, however, the precise underlying mechanism that is responsible for this observation remains vague. Sphingosine 1 Phosphate (S1P) is a lysophospholipid with important functions in the regulation of renal physiology and the cardiovascular system. Previously, we demonstrated that in FGR but not control animals, acute activation of Sphingosine 1 Phosphate Receptor 1 (S1PR1) transiently reduces Blood Pressure (BP) in an endothelial nitric oxide synthase dependent manner. We have also shown that both male and female FGR mice have lower nephron number in the kidneys, however, unlike females, male FGR offspring have reduced renal function suggesting sex specific differences in fetal programing of kidney diseases. We have also shown that within 5 minutes of pharmacologically activating S1PR1 with SEW2871, GFR significantly reduces in male control but not IUGR animals. The objective of this study was to test the effect of longer stimulation of S1PR1 agonist on renal function and to determine the distribution of renal S1P signaling molecules. On their 13th day of gestation, pregnant C57BL mice went through a Reduced Uterine Pressure Perfusion (RUPP) or sham surgery to generate FGR and control offspring respectively. At 6 months, measurement of Glomerular Filtration Rate (GFR) and expression of proteins that are involved in S1P signaling was performed using plasma FITC‐Anulin decay, PCR, and immunohistochemistry. For immunohistochemistry, the Integrated Optical Densities (IDO) values from (Diaminobenzidine) DAB‐stained kidney sections were used to statistically quantify protein expression. GFR was lower in male FGR offspring than control (8.85 ±0.55 vs 13.86±1.13 µL/min/kg BW, P˂0.05, FGR vs control males) while in females there was no statistical difference (12.33±0.87 vs 15.92 ±1.94 L/min/kg BW, P˃ 0.05 control females vs FGR). Administration of SEW2871 and measurement of GFR after one hour confirmed that GFR decreases only in control males but not IUGR while in females both FGR and control groups had similar reduction response (6.00±0.46 vs 8.41±0.79 µL/min/kg BW, P˃ 0.05 control males vs FGR) (5. 97±0.48 vs 5.72 ±1.01 µL/min/kg BW, P˃ 0.05 control females vs FGR). Whole kidney transcriptomics showed no statistical differences in the expression profile of S1PR 1‐3 and Sphingosine 1 Phosphate Kinases 1 and 2 (SPHK1‐2) between FGR and control male and female animals. Immunohistochemistry analysis of the kidney medulla and cortex of male animals revealed no difference in the expression profile of S1PR1 in both FGR and control and increased expression of S1PR2 in both the medulla and cortex of FGR animals. S1PR3 was decreased in both the medulla and cortex of FGR mice. SPHK 1 and 2 were not differentially expressed in the medulla of control and FGR animal whereas in the cortex both the two receptors were downregulated in FGR. Our results indicate that acute activation of S1PR1 differentially regulates GFR between male and female FGR. Consequences of the upregulation of S1PR2 and the down regulation of S1PR 3 and SPHK 1‐2 proteins in the kidney of male FGR warrants further investigations.

Structural insights into sphingosine-1-phosphate receptor activation

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step “shallow to deep” transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.

A G-protein-biased S1P1 agonist, SAR247799, improved LVH and diastolic function in a rat model of metabolic syndrome.

AimHeart failure with preserved ejection fraction (HFpEF) is a major cause of death worldwide with no approved treatment. Left ventricular hypertrophy (LVH) and diastolic dysfunction represent the structural and functional components of HFpEF, respectively. Endothelial dysfunction is prevalent in HFpEF and predicts cardiovascular events. We investigated if SAR247799, a G-protein-biased sphingosine-1-phosphate receptor 1 (S1P1) agonist with endothelial-protective properties, could improve cardiac and renal functions in a rat model of metabolic syndrome LVH and diastolic function.Methods31- and 65-week-old obese ZSF1 (Ob-ZSF1) rats, representing adult and aged animals with LVH and diastolic dysfunction, were randomized to a chow diet containing 0.025% (w/w) of SAR247799, or control (CTRL) chow for 4 weeks. Age-matched lean ZSF1 (Le-ZSF1) rats were fed control chow. Echocardiography, telemetry, biochemical and histological analysis were performed to evaluate the effect of SAR247799.ResultsEchocardiography revealed that Ob-ZSF1 rats, in contrast to Le-ZSF1 rats, developed progressive diastolic dysfunction and cardiac hypertrophy with age. SAR247799 blunted the progression of diastolic dysfunction in adult and aged animals: in adult animals E/e’ was evaluated at 21.8 ± 1.4 for Ob-ZSF1-CTRL, 19.5 ± 1.2 for Ob-ZSF1-SAR247799 p<0.01, and 19.5 ± 2.3 for Le-ZSF1-CTRL (median ± IQR). In aged animals E/e’ was evaluated at 23.15 ± 4.45 for Ob-ZSF1-CTRL, 19.5 ± 5 for Ob-ZSF1-SAR247799 p<0.01, and 16.69 ± 1.7 for Le-ZSF1-CTRL, p<0.01 (median ± IQR). In aged animals, SAR247799 reduced cardiac hypertrophy (g/mm mean ± SEM of heart weight/tibia length 0.053 ± 0.001 for Ob-ZSF1-CTRL vs 0.046 ± 0.002 for Ob-ZSF1-SAR247799 p<0.01, Le-ZSF1-CTRL 0.035 ± 0.001) and myocardial perivascular collagen content (p<0.001), independently of any changes in microvascular density. In adult animals, SAR247799 improved endothelial function as assessed by the very low frequency bands of systolic blood pressure variability (mean ± SEM 67.8 ± 3.41 for Ob-ZSF1-CTRL 55.8 ± 4.27 or Ob-ZSF1-SAR247799, p<0.05 and 57.3 ± 1.82 Le-ZSF1-CTRL), independently of any modification of arterial blood pressure. In aged animals, SAR247799 reduced urinary protein/creatinine ratio, an index of glomerular injury, (10.3 ± 0.621 vs 8.17 ± 0.231 for Ob-ZSF1-CTRL vs Ob-ZSF1-SAR247799, respectively, p<0.05 and 0.294 ± 0.029 for Le-ZSF1-CTRL, mean ± SEM) and the fractional excretion of electrolytes. Circulating lymphocytes were not decreased by SAR247799, confirming lack of S1P1 desensitization.ConclusionsThese experimental findings suggest that S1P1 activation with SAR247799 may be considered as a new therapeutic approach for LVH and diastolic dysfunction, major components of HFpEF.

Signaling through the S1P-S1PR Axis in the Gut, the Immune and the Central Nervous System in Multiple Sclerosis: Implication for Pathogenesis and Treatment.

Sphingosine 1-phosphate (S1P) is a signaling molecule with complex biological functions that are exerted through the activation of sphingosine 1-phosphate receptors 1–5 (S1PR1–5). S1PR expression is necessary for cell proliferation, angiogenesis, neurogenesis and, importantly, for the egress of lymphocytes from secondary lymphoid organs. Since the inflammatory process is a key element of immune-mediated diseases, including multiple sclerosis (MS), S1PR modulators are currently used to ameliorate systemic immune responses. The ubiquitous expression of S1PRs by immune, intestinal and neural cells has significant implications for the regulation of the gut–brain axis. The dysfunction of this bidirectional communication system may be a significant factor contributing to MS pathogenesis, since an impaired intestinal barrier could lead to interaction between immune cells and microbiota with a potential to initiate abnormal local and systemic immune responses towards the central nervous system (CNS). It appears that the secondary mechanisms of S1PR modulators affecting the gut immune system, the intestinal barrier and directly the CNS, are coordinated to promote therapeutic effects. The scope of this review is to focus on S1P−S1PR functions in the cells of the CNS, the gut and the immune system with particular emphasis on the immunologic effects of S1PR modulation and its implication in MS.

Sphingosine-1-phosphate receptor 1 agonist SEW2871 alters membrane properties of late-firing somatostatin expressing neurons in the central lateral amygdala

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide spectrum of biological processes including apoptosis, immune response and inflammation. Here, we sought to understand how S1P signaling affects neuronal excitability in the central amygdala (CeA), which is a brain region associated with fear learning, aversive memory, and the affective dimension of pain. Because the G-protein coupled S1P receptor 1 (S1PR1) has been shown to be the primary mediator of S1P signaling, we utilized S1PR1 agonist SEW2871 and S1PR1 antagonist NIBR to determine a potential role of S1PR1 in altering the cellular physiology of neurons in the lateral division of the CeA (CeL) that share the neuronal lineage marker somatostatin (Sst). CeL-Sst neurons play a critical role in expression of conditioned fear and pain modulation. Here we used transgenic breeding strategies to identify fluorescently labeled CeL-Sst neurons for electrophysiological recordings. Using principal component analysis, we identified two primary subtypes of Sst neurons within the CeL in both male and female mice. We denoted the two types regular-firing (type A) and late-firing (type B) CeL-Sst neurons. In response to SEW2871 application, Type A neurons exhibited increased input resistance, while type B neurons displayed a depolarized resting membrane potential and voltage threshold, increased current threshold, and decreased voltage height. NIBR application had no effect on CeL Sst neurons, indicating the absence of tonic S1P-induced S1PR1. Our findings reveal subtypes of Sst neurons within the CeL that are uniquely affected by S1PR1 activation, which may have implications for how S1P alters supraspinal circuits.

Constitutive activation of S1P receptors at the trans-Golgi network is required for surface transport carrier formation

SummaryThe importance of the G-protein βγ subunits in the regulation of cargo transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is well accepted; however, the molecular mechanism underlying the G-protein activation at the TGN remains unclear. We show here that sphingosine 1-phosphate (S1P) receptors at the PM were trafficked to the TGN in response to a surface transport cargo, temperature-sensitive vesicular stomatitis virus glycoprotein tagged with green fluorescent protein accumulation in the Golgi. The receptor internalization occurred in an S1P-independent manner but required phosphorylation by G-protein receptor kinase 2 and β-arrestin association before internalization. Continuously activated S1P receptors in a manner dependent on S1P at the TGN kept transmitting G-protein signals including the βγ subunits supply necessary for transport carrier formation at the TGN destined for the PM.

020 Sphingosine 1-phosphate receptor signalling promotes hair growth and inhibits perifollicular T-cell expansion and immune privilege collapse ex vivo

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that signals through five cell surface receptors (S1P1-5). Despite the role of S1P receptors in lymphocyte trafficking and epidermal keratinocyte proliferation and differentiation, their role in hair follicle (HF) physiology remains obscure. We hypothesized that S1P receptor activation may be involved in alopecia areata (AA), a T cell-mediated hair loss disorder characterized by immune privilege (IP) collapse in the hair bulb. RNAseq revelated S1P1,3,5 are expressed in human scalp HFs.

Abstract 29: Acute Systemic Treatment With Sphingosine -1- Phosphate Receptor 1 Agonist Diminishes Sex-difference In Renal Function Seen In Intrauterine Growth Restricted Mice.

IUGR is a risk factor for the early development of cardiorenal diseases in life. We previously showed that our IUGR mouse model exhibits sex differences in blood pressure (BP) and kidney functions as males have elevated BP and impaired kidney function while females are not. Sphingosine-1-Phosphate Receptor 1 (S1PR1) is reported to be involved in developing and progressing several cardiorenal diseases. We found that acute activation of S1PR1 transiently reduced BP in male IUGR; however, the effects of SIPR1 on renal function in IUGR are still unknown. We hypothesize that S1PR1 plays a role in the sex differences of impaired kidney function in IUGR. Here we investigated the acute effects of a specific S1PR1 agonist (SEW2178) on kidney function in IUGR offspring generated through placental insufficiency. Methods: C57BL IUGR or control offspring were obtained from the Reduced Uterine Perfusion Pressure (RUPP) or sham surgeries on the 13 th day of gestation. At 6 months, nephron number was measured by analyzing kidney histology while Glomerular Filtration Rate (GFR) was measured using FITC-Anulin decay after retroorbital injection. Mice were treated with 3mg/kg SEW2871 i.p before GFR measurement. Results: Both male and female IUGR had a decrease in nephron number compared to same-sex control (371.8± 28.8 vs 284.0± 11.3, P&lt;0.01, male control vs IUGR) and (333.6±13.3 vs 256± 13.0, P&lt;0.05, female control vs IUGR). Furthermore, IUGR impaired kidney function in male but not female mice as indicated by reduced GFR (8.9 ±0.6 vs 13.9±1.1 μL/min/kg BW, P&lt;0.05, IUGR vs control males) and (12.3±0.9 vs 15.9 ±1.9 L/min/kg BW, control females vs IUGR). Administration of SEW2871 diminished the difference of GFR between male control and IUGR (9.5±0.6 vs 9.7±1.1 μL/min/kg BW, P&gt; 0.05 control vs IUGR). While in females, both IUGR and control had a similar response to SEW2178 (7.4±1.0 vs 10.6 ±1.9 μL/min/kg BW, control vs. IUGR). Conclusion: IUGR impairs renal function in male offspring but not in females, indicating sex differences in fetal programming of kidney disease. Male and female IUGR offspring respond differently to acute systemic activation of S1PR1. Further studies are required to investigate the kidney-specific and cell-specific of S1PR1 activation/inhibition.

Activating Sphingosine-1-phospahte signaling in endothelial cells increases myosin light chain phosphorylation to decrease endothelial permeability thereby inhibiting cancer metastasis.

Targeting the metastatic process to prevent disease dissemination in cancer remains challenging. One step in the metastatic cascade involves cancer cells transiting through the vascular endothelium after inflammation has increased the permeability of this cellular layer. Reducing inflammation-mediated gaps in the vascular endothelium could potentially be used to retard metastasis. This study describes the development of a novel ASR396-containing nanoparticle designed to activate the Sphingosine-1-Phosphate Receptor 1 (S1PR1) in order to tighten the junctions between the endothelial cells lining the vascular endothelium thereby inhibiting metastasis. ASR396 was derived from the S1PR1 agonist SEW2871 through chemical modification enabling the new compound to be loaded into a nanoliposome. ASR396 retained S1PR1 binding activity and the nanoliposomal formulation (nanoASR396) made it systemically bioavailable upon intravenous injection. Studies conducted in microvessels demonstrated that nanoASR396 significantly attenuated inflammatory mediator-induced permeability increase through the S1PR1 activation. Similarly, nanoASR396 inhibited gap formation mediated by inflammatory agents on an endothelial cell monolayer by decreasing levels of phosphorylated myosin light chain protein thereby inhibiting cellular contractility. In animal models, nanoASR396 inhibited lung metastasis by up to 80%, indicating its potential for retarding melanoma metastasis. Thus, a novel bioavailable nanoparticle-based S1PR1 agonist has been developed to negate the effects of inflammatory mediators on the vascular endothelium in order to reduce the metastatic dissemination of cancer cells.

Novel compounds with dual S1P receptor agonist and histamine H3 receptor antagonist activities act protective in a mouse model of multiple sclerosis

The sphingosine 1-phosphate (S1P) receptor 1 (S1P1) has emerged as a therapeutic target for the treatment of multiple sclerosis (MS). Fingolimod (FTY720) is the first functional antagonist of S1P1 that has been approved for oral treatment of MS. Previously, we have developed novel butterfly derivatives of FTY720 that acted similar to FTY720 in reducing disease symptoms in a mouse model of experimental autoimmune encephalomyelitis (EAE).In this study, we have synthesized a piperidine derivative of the oxazolo-oxazole compounds, denoted ST-1505, and its ring-opened analogue ST-1478, and characterised their in-vitro and in-vivo functions. Notably, the 3-piperidinopropyloxy moiety resembles a structural motif of pitolisant, a drug with histamine H3R antagonistic/inverse agonist activity approved for the treatment of narcolepsy. Both novel compounds exerted H3R affinities, and in addition, ST-1505 was characterised as a dual S1P1+3 agonist, whereas ST-1478 was a dual S1P1+5 agonist. Both multitargeting compounds were also active in mice and reduced the lymphocyte numbers as well as diminished disease symptoms in the mouse model of MS. The effect of ST-1478 was dependent on SK-2 activity suggesting that it is a prodrug like FTY720, but with a more selective S1P receptor activation profile, whereas ST-1505 is a fully active drug even in the absence of SK-2.In summary, these data suggest that the well soluble piperidine derivatives ST-1505 and ST-1478 hold promise as novel drugs for the treatment of MS and other autoimmune or inflammatory diseases, and by their H3R antagonist potency, they might additionally improve cognitive impairment during disease.

Endothelial-protective effects of a G-protein-biased sphingosine-1 phosphate receptor-1 agonist, SAR247799, in type-2 diabetes rats and a randomized placebo-controlled patient trial.

AimsSAR247799 is a G‐protein‐biased sphingosine‐1 phosphate receptor‐1 (S1P1) agonist designed to activate endothelial S1P1 and provide endothelial‐protective properties, while limiting S1P1 desensitization and consequent lymphocyte‐count reduction associated with higher doses. The aim was to show whether S1P1 activation can promote endothelial effects in patients and, if so, select SAR247799 doses for further clinical investigation.MethodsType‐2 diabetes patients, enriched for endothelial dysfunction (flow‐mediated dilation, FMD <7%; n = 54), were randomized, in 2 sequential cohorts, to 28‐day once‐daily treatment with SAR247799 (1 or 5 mg in ascending cohorts), placebo or 50 mg sildenafil (positive control) in a 5:2:2 ratio per cohort. Endothelial function was assessed by brachial artery FMD. Renal function, biomarkers and lymphocytes were measured following 5‐week SAR247799 treatment (3 doses) to Zucker diabetic fatty rats and the data used to select the doses for human testing.ResultsThe maximum FMD change from baseline vs placebo for all treatments was reached on day 35; mean differences vs placebo were 0.60% (95% confidence interval [CI] −0.34 to 1.53%; P = .203) for 1 mg SAR247799, 1.07% (95% CI 0.13 to 2.01%; P = .026) for 5 mg SAR247799 and 0.88% (95% CI −0.15 to 1.91%; P = .093) for 50 mg sildenafil. Both doses of SAR247799 were well tolerated, did not affect blood pressure, and were associated with minimal‐to‐no lymphocyte reduction and small‐to‐moderate heart rate decrease.ConclusionThese data provide the first human evidence suggesting endothelial‐protective properties of S1P1 activation, with SAR247799 being as effective as the clinical benchmark, sildenafil. Further clinical testing of SAR247799, at sub‐lymphocyte‐reducing doses (≤5 mg), is warranted in vascular diseases associated with endothelial dysfunction.