Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1.

Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.

Gradients of the signaling lipid S1P in lymph nodes position natural killer cells and regulate their interferon-γ response.

The lymph node periphery is an important site for many immunological functions, from pathogen containment to the differentiation of helper T cells, yet the cues that position cells in this region are largely undefined. Here, through the use of a reporter for the signaling lipid S1P (sphingosine 1-phosphate), we found that cells sensed higher concentrations of S1P in the medullary cords than in the T cell zone and that the S1P transporter SPNS2 on lymphatic endothelial cells generated this gradient. Natural killer (NK) cells are located at the periphery of the lymph node, predominantly in the medulla, and we found that expression of SPNS2, expression of the S1P receptor S1PR5 on NK cells, and expression of the chemokine receptor CXCR4 were all required for NK cell localization during homeostasis and rapid production of interferon-γ by NK cells after challenge. Our findings elucidate the spatial cues for NK cell organization and reveal a previously unknown role for S1P in positioning cells within the medulla.

CD8αα intraepithelial lymphocytes arise from two main thymic precursors.

TCRαß+CD4-CD8α+CD8ß- intestinal intraepithelial lymphocytes (CD8αα IELs) are an abundant population of thymus-derived T cells that protect the gut barrier surface. We sought to better define the thymic IEL precursor (IELp) through analysis of its maturation, localization and emigration. We defined two precursor populations among TCRß+CD4-CD8- thymocytes by dependence on the kinase TAK1 and rigorous lineage-exclusion criteria. Those IELp populations included a nascent PD-1+ population and a T-bet+ population that accumulated with age. Both gave rise to intestinal CD8αα IELs after adoptive transfer. The PD-1+ IELp population included more strongly self-reactive clones and was largely restricted by classical major histocompatibility complex (MHC) molecules. Those cells localized to the cortex and efficiently emigrated in a manner dependent on the receptor S1PR1. The T-bet+ IELp population localized to the medulla, included cells restricted by non-classical MHC molecules and expressed the receptor NK1.1, the integrin CD103 and the chemokine receptor CXCR3. The two IELp populations further differed in their use of the T cell antigen receptor (TCR) α-chain variable region (Vα) and ß-chain variable region (Vß). These data provide a foundation for understanding the biology of CD8αα IELs.

Sphingosine-1-phosphate and its receptors: structure, signaling, and influence.

The sphingosine-1-phosphate (S1P) receptor signaling system has biological and medical importance and is the first lipid G protein-coupled receptor (GPCR) structure to be solved to 2.8-Å resolution. S1P binds to five high-affinity GPCRs generating multiple downstream signals that play essential roles in vascular development and endothelial integrity, control of cardiac rhythm, and routine oral treatment of multiple sclerosis. Genetics, chemistry, and now structural biology have advanced this integrated biochemical system. The S1P receptors have a novel N-terminal fold that occludes access to the binding pocket from the extracellular environment as well as orthosteric and bitopic ligands with very different physicochemical properties. S1P receptors and metabolizing enzymes have been deleted, inducibly deleted, and knocked in as tagged or altered receptors in mice. An array of genetic models allows analysis of integrated receptor function in vivo. We can now directly understand causal relationships among protein expression, signal, and control points in physiology and pathology.

A map of the distribution of sphingosine 1-phosphate in the spleen.

Despite the importance of signaling lipids, many questions remain about their function because few tools are available for charting lipid gradients in vivo. Here we generated a sphingosine 1-phosphate (S1P) reporter mouse and used this mouse to define the distribution of S1P in the spleen. Unexpectedly, the presence of blood did not serve as a predictor of the concentration of signaling-available S1P. Large areas of the red pulp had low concentrations of S1P, while S1P was sensed by cells inside the white pulp near the marginal sinus. The lipid phosphate phosphatase LPP3 maintained low S1P concentrations in the spleen and enabled efficient shuttling of marginal zone B cells. The exquisitely tight regulation of S1P availability might explain how a single lipid can simultaneously orchestrate the movements of many cells of the immune system.

Where Have All the T Cells Gone?

Glioblastoma are highly immunosuppressive brain tumors that are known for their T cell paucity. In a recent issue of Nature Medicine, Chongsathidkiet et al. (2018) discovered a brain-specific mechanism of tumors to escape immunosurveillance by trapping T cells in the bone marrow through the loss of sphingosine-1-phosphate (S1P) receptor on the T cell surface.

Fine-tuning activation specificity of G-protein-coupled receptors via automated path searching.

Physics-based simulation methods can grant atomistic insights into the molecular origin of the function of biomolecules. However, the potential of such approaches has been hindered by their low efficiency, including in the design of selective agonists where simulations of myriad protein-ligand combinations are necessary. Here, we describe an automated input-free path searching protocol that offers (within 14 d using Graphics Processing Unit servers) a minimum free energy path (MFEP) defined in high-dimension configurational space for activating sphingosine-1-phosphate receptors (S1PRs) by arbitrary ligands. The free energy distributions along the MFEP for four distinct ligands and three S1PRs reached a remarkable agreement with Bioluminescence Resonance Energy Transfer (BRET) measurements of G-protein dissociation. In particular, the revealed transition state structures pointed out toward two S1PR3 residues F263/I284, that dictate the preference of existing agonists CBP307 and BAF312 on S1PR1/5. Swapping these residues between S1PR1 and S1PR3 reversed their response to the two agonists in BRET assays. These results inspired us to design improved agonists with both strong polar head and bulky hydrophobic tail for higher selectivity on S1PR1. Through merely three in silico iterations, our tool predicted a unique compound scaffold. BRET assays confirmed that both chiral forms activate S1PR1 at nanomolar concentration, 1 to 2 orders of magnitude less than those for S1PR3/5. Collectively, these results signify the promise of our approach in fine agonist design for G-protein-coupled receptors.

Get me out of here: Sphingosine 1-phosphate signaling and T cell exit from tissues during an immune response.

During an immune response, the duration of T cell residence in lymphoid and non-lymphoid tissues likely affects T cell activation, differentiation, and memory development. The factors that govern T cell transit through inflamed tissues remain incompletely understood, but one important determinant of T cell exit from tissues is sphingosine 1-phosphate (S1P) signaling. In homeostasis, S1P levels are high in blood and lymph compared to lymphoid organs, and lymphocytes follow S1P gradients out of tissues into circulation using varying combinations of five G-protein coupled S1P receptors. During an immune response, both the shape of S1P gradients and the expression of S1P receptors are dynamically regulated. Here we review what is known, and key questions that remain unanswered, about how S1P signaling is regulated in inflammation and in turn how S1P shapes immune responses.

Lymphatic Endothelial Cells Control Initiation of Lymph Node Organogenesis.

Lymph nodes (LNs) are strategically situated throughout the body at junctures of the blood vascular and lymphatic systems to direct immune responses against antigens draining from peripheral tissues. The current paradigm describes LN development as a programmed process that is governed through the interaction between mesenchymal lymphoid tissue organizer (LTo) cells and hematopoietic lymphoid tissue inducer (LTi) cells. Using cell-type-specific ablation of key molecules involved in lymphoid organogenesis, we found that initiation of LN development is dependent on LTi-cell-mediated activation of lymphatic endothelial cells (LECs) and that engagement of mesenchymal stromal cells is a succeeding event. LEC activation was mediated mainly by signaling through receptor activator of NF-κB (RANK) and the non-canonical NF-κB pathway and was steered by sphingosine-1-phosphate-receptor-dependent retention of LTi cells in the LN anlage. Finally, the finding that pharmacologically enforced interaction between LTi cells and LECs promotes ectopic LN formation underscores the central LTo function of LECs.

Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection.

Cytokine storm during viral infection is a prospective predictor of morbidity and mortality, yet the cellular sources remain undefined. Here, using genetic and chemical tools to probe functions of the S1P(1) receptor, we elucidate cellular and signaling mechanisms that are important in initiating cytokine storm. Whereas S1P(1) receptor is expressed on endothelial cells and lymphocytes within lung tissue, S1P(1) agonism suppresses cytokines and innate immune cell recruitment in wild-type and lymphocyte-deficient mice, identifying endothelial cells as central regulators of cytokine storm. Furthermore, our data reveal immune cell infiltration and cytokine production as distinct events that are both orchestrated by endothelial cells. Moreover, we demonstrate that suppression of early innate immune responses through S1P(1) signaling results in reduced mortality during infection with a human pathogenic strain of influenza virus. Modulation of endothelium with a specific agonist suggests that diseases in which amplification of cytokine storm is a significant pathological component could be chemically tractable.

Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells.

Invasion of brain endothelial cells (BECs) is central to the pathogenicity of Neisseria meningitidis infection. Here, we established a key role for the bioactive sphingolipid sphingosine-1-phosphate (S1P) and S1P receptor (S1PR) 2 in the uptake process. Quantitative sphingolipidome analyses of BECs infected with N. meningitidis revealed elevated S1P levels, which could be attributed to enhanced expression of the enzyme sphingosine kinase 1 and its activity. Increased activity was dependent on the interaction of meningococcal type IV pilus with the endothelial receptor CD147. Concurrently, infection led to increased expression of the S1PR2. Blocking S1PR2 signaling impaired epidermal growth factor receptor (EGFR) phosphorylation, which has been shown to be involved in cytoskeletal remodeling and bacterial endocytosis. Strikingly, targeting S1PR1 or S1PR3 also interfered with bacterial uptake. Collectively, our data support a critical role of the SphK/S1P/S1PR axis in the invasion of N. meningitidis into BECs, defining a potential target for adjuvant therapy.

Defective sphingosine 1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation.

Sphingosine 1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The sphingosine phosphate receptor 1 (S1P1) agonist FTY-720 (Gilenya) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring an S1pr1 gene encoding phosphorylation-deficient receptors (S1P1(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) in the peripheral immune and nervous system. S1P1 directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells.

Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (T(RM) cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of T(RM) cells is unknown. We found that CD8(+) T(RM) cells lacked expression of the transcription factor KLF2 and its target gene S1pr1 (which encodes S1P1, a receptor for sphingosine 1-phosphate). Forced expression of S1P1 prevented the establishment of T(RM) cells. Cytokines that induced a T(RM) cell phenotype (including transforming growth factor-ß (TGF-ß), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S1P1 provides a switch that dictates whether CD8(+) T cells commit to recirculating or tissue-resident memory populations.

Autoimmune Renal Disease Is Exacerbated by S1P-Receptor-1-Dependent Intestinal Th17 Cell Migration to the Kidney.

Th17 cells are most abundant in the gut, where their presence depends on the intestinal microbiota. Here, we examined whether intestinal Th17 cells contribute to extra-intestinal Th17 responses in autoimmune kidney disease. We found high frequencies of Th17 cells in the kidneys of patients with antineutrophil cytoplasmatic antibody (ANCA)-associated glomerulonephritis. We utilized photoconversion of intestinal cells in Kaede mice to track intestinal T cell mobilization upon glomerulonephritis induction, and we found that Th17 cells egress from the gut in a S1P-receptor-1-dependent fashion and subsequently migrate to the kidney via the CCL20/CCR6 axis. Depletion of intestinal Th17 cells in germ-free and antibiotic-treated mice ameliorated renal disease, whereas expansion of these cells upon Citrobacter rodentium infection exacerbated pathology. Thus, in some autoimmune settings, intestinal Th17 cells migrate into target organs, where they contribute to pathology. Targeting the intestinal Th17 cell "reservoir" may present a therapeutic strategy for these autoimmune disorders.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

BACKGROUND: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. METHODS: Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. RESULTS: In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. CONCLUSIONS: Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Sphingosine 1-phosphate receptor signaling.

The sphingosine 1-phosphate (S1P) receptor signaling system is a productive model system. A hydrophobic zwitterionic lysophospholipid ligand with difficult physical properties interacts with five high-affinity G protein-coupled receptors to generate multiple downstream signals. These signals modulate homeostasis and pathology on a steep agonist concentration-response curve. Ligand presence is essential for vascular development and endothelial integrity, while acute increases in ligand concentrations result in cardiac death. Understanding this integrated biochemical system has exemplified the impact of both genetics and chemistry. Developing specific tools with defined biochemical properties for the reversible modulation of signals in real time has been essential to complement insights gained from genetic approaches that may be irreversible and compensated. Despite its knife-edge between life and death, this system, based in part on receptor subtype-selectivity and in part on differential attenuation of deleterious signals, now appears to be on the cusp of meaningful therapy for multiple sclerosis.

Sphingosine-1-phosphate and its receptors in vascular endothelial and lymphatic barrier function.

The vascular and lymphatic systems both comprise a series of structurally distinct vessels lined with an inner layer of endothelial cells that function to provide a semipermeable barrier to blood and lymph. Regulation of the endothelial barrier is critical for maintaining vascular and lymphatic barrier homeostasis. One of the regulators of endothelial barrier function and integrity is sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite secreted into the blood by erythrocytes, platelets, and endothelial cells and into the lymph by lymph endothelial cells. Binding of S1P to its G protein-coupled receptors, known as S1PR1-5, regulates its pleiotropic functions. This review outlines the structural and functional differences between vascular and lymphatic endothelium and describes current understanding of the importance of S1P/S1PR signaling in regulation of barrier functions. Most studies thus far have been primarily focused on the role of the S1P/S1PR1 axis in vasculature and have been summarized in several excellent reviews, and thus, we will only discuss new perspectives on the molecular mechanisms of action of S1P and its receptors. Much less is known about the responses of the lymphatic endothelium to S1P and the functions of S1PRs in lymph endothelial cells, and this is the major focus of this review. We also discuss current knowledge related to signaling pathways and factors regulated by the S1P/S1PR axis that control lymphatic endothelial cell junctional integrity. Gaps and limitations in current knowledge are highlighted together with the need to further understand the role of S1P receptors in the lymphatic system.

Is myeloid-derived growth factor a ligand of the sphingosine-1-phosphate receptor 2?

Secretory myeloid-derived growth factor (MYDGF) exerts beneficial effects on organ repair, probably via a plasma membrane receptor; however, the identity of the expected receptor has remained elusive. In a recent study, MYDGF was reported as an agonist of the sphingosine-1-phosphate receptor 2 (S1PR2), an A-class G protein-coupled receptor that mediates the functions of the signaling lipid, sphingosine-1-phosphate (S1P). In the present study, we conducted living cell-based functional assays to test whether S1PR2 is a receptor for MYDGF. In the NanoLuc Binary Technology (NanoBiT)-based ß-arrestin recruitment assay and the cAMP-response element (CRE)-controlled NanoLuc reporter assay, S1P could efficiently activate human S1PR2 overexpressed in human embryonic kidney (HEK) 293T cells; however, recombinant human MYDGF, overexpressed either from Escherichia coli or HEK293 cells, had no detectable effect. Thus, the results demonstrated that human MYDGF is not a ligand of human S1PR2. Considering the high conservation of MYDGF and S1PR2 in evolution, MYDGF is also probably not a ligand of S1PR2 in other vertebrates.

Celastrol inhibits angiogenesis and the biological processes of MDA-MB-231 cells via the DEGS1/S1P signaling pathway.

Celastrol (Cel) shows potent antitumor activity in various experimental models. This study examined the relationship between Cel's antivascular and antitumor effects and sphingolipids. CCK-8 assay, transwell assay, Matrigel, PCR-array/RT-PCR/western blotting/immunohistochemistry assay, ELISA and HE staining were used to detect cell proliferation, migration and invasion, adhesion and angiogenesis, mRNA and protein expression, S1P production and tumor morphology. The results showed that Cel could inhibit proliferation, migration or invasion, adhesion and angiogenesis of human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 cells by downregulating the expression of degenerative spermatocyte homolog 1 (DEGS1). Transfection experiments showed that downregulation of DEGS1 inhibited the above processes and sphingosine-1-phosphate (S1P) production of HUVECs and MDA-MB-231 cells, while upregulation of DEGS1 had the opposite effects. Coculture experiments showed that HUVECs could promote proliferation, migration and invasion of MDA-MB-231 cells through S1P/sphingosine-1-phosphate receptor (S1PR) signaling pathway, while Cel inhibited these processes in MDA-MB-231 cells induced by HUVECs. Animal experiments showed that Cel could inhibit tumor growth in nude mice. Western blotting, immunohistochemistry and ELISA assay showed that Cel downregulated the expression of DEGS1, CD146, S1PR1-3 and S1P production. These data confirm that DEGS1/S1P signaling pathway may be related to the antivascular and antitumor effects of cel.

S1PR3 inhibition protects against LPS-induced ARDS by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation.

BACKGROUND: Inflammation and endothelial barrier dysfunction are the major pathophysiological changes in acute respiratory distress syndrome (ARDS). Sphingosine-1-phosphate receptor 3 (S1PR3), a G protein-coupled receptor, has been found to mediate inflammation and endothelial cell (EC) integrity. However, the function of S1PR3 in ARDS has not been fully elucidated. METHODS: We used a murine lipopolysaccharide (LPS)-induced ARDS model and an LPS- stimulated ECs model to investigate the role of S1PR3 in anti-inflammatory effects and endothelial barrier protection during ARDS. RESULTS: We found that S1PR3 expression was increased in the lung tissues of mice with LPS-induced ARDS. TY-52156, a selective S1PR3 inhibitor, effectively attenuated LPS-induced inflammation by suppressing the expression of proinflammatory cytokines and restored the endothelial barrier by repairing adherens junctions and reducing vascular leakage. S1PR3 inhibition was achieved by an adeno-associated virus in vivo and a small interfering RNA in vitro. Both the in vivo and in vitro studies demonstrated that pharmacological or genetic inhibition of S1PR3 protected against ARDS by inhibiting the NF-κB pathway and improving mitochondrial oxidative phosphorylation. CONCLUSIONS: S1PR3 inhibition protects against LPS-induced ARDS via suppression of pulmonary inflammation and promotion of the endothelial barrier by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation, indicating that S1PR3 is a potential therapeutic target for ARDS.