Sphingosine-1-phosphate Signaling Research Articles

Imaging and molecular features of adenomyosis after menopause

ObjectivesTo explore the imaging features and the molecular characterization of adenomyosis after menopause. Study designAn observational cross-sectional study was performed in a group of postmenopausal patients undergoing a transvaginal ultrasound (TVUS) (n = 468). Among those presenting the US criteria for adenomyosis, also confirmed by magnetic resonance imaging (MRI), previous menstrual symptoms, gynecological and obstetric history were reviewed. In a subgroup undergoing hysterectomy, uterine specimens were analyzed by histology and expression of genes implicated in the epithelial-mesenchymal transition, inflammation and fibrosis, including the sphingosine-1-phosphate (S1P) pathway, was evaluated and compared to matched non-menopausal adenomyosis specimens. Main outcome measuresDirect and indirect US features of adenomyosis according to Morphological Uterus Sonographic Assessment at TVUS. Molecular characterization of postmenopausal versus pre-menopausal adenomyosis samples. ResultsAccording to TVUS and MRI, adenomyosis was identified in 49 patients (10.4 %). On US, diffuse adenomyosis was the most common phenotype, whereas internal adenomyosis with diffuse pattern and asymmetric type was the most prevalent on MRI. Molecular analysis showed that adenomyosis lesions express markers of epithelial-mesenchymal transition, inflammation and fibrosis also in postmenopausal women. By comparing the results with those from pre-menopausal samples, the expression of α smooth muscle actin (αSMA), a marker of fibrosis, was significantly greater after menopause, and altered S1P catabolism and signaling were observed. ConclusionsAdenomyosis may be identified in postmenopausal women by imaging, either TVUS or MRI, and fibrosis is one of the key features on molecular analysis.

Abstract 2659: Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response

Abstract Introduction: Immunotherapy has revolutionized the landscape of cancer treatment; however, to date, only limited aspects of the tumor-immune interaction have been exploited successfully, e.g., relief of immune checkpoints, increase of tumor-associated antigen presentation. Although it is well-known that trafficking and entry of T cells from the circulation into tumors is often impaired, leading to an immune “cold”, checkpoint inhibitor-refractory state, there are no available therapies that address this important aspect of antitumor immunity. Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive lipid that has been shown to regulate lymphocyte egress from lymph nodes through its interaction with one of its several receptors, S1P1. Clinically, tumor expression levels of the sphingosine-1-phosphate-producing enzyme sphingosine kinase 1 (SPHK1) strongly and negatively correlates with survival of melanoma patients after immune checkpoint inhibitor (ICI) therapy, and pre-clinically correlates with reduced tumor infiltrating lymphocytes (TILs). We therefore hypothesize that aberrant S1P signaling in tumors and in their draining lymph nodes (TDLN) causes immune exclusion by impeding lymphocyte egress from the TDLN, ultimately restricting immune cell infiltration of tumors. Methods: We tested the therapeutic efficacy of an engineered S1P-degrading enzyme, S1P lyase (S1PL), on established (~100mm^3) syngeneic B16, CT26, and MC38 murine tumors. Using flow cytometry, we performed immune profiling of systemic, TDLN, and intratumoral T cell populations, assessing both activation status and expression of the S1P1 receptor. Results: Treatment of tumor-bearing animals with S1PL depleted systemic S1P, and dramatically increased both trafficking of lymphocytes out of TDLNs and T cell infiltration of tumors relative to control-treated animals. Mice treated with S1PL evinced a higher proportion of S1P1+ circulating and intratumoral T cells relative to control-treated mice, suggesting that the increase in liberation of T cells from the TDLN was effected through the S1P/S1P1 axis. This increased lymphocyte trafficking correlated with strong inhibition of tumor growth when S1PL was administered as a single agent. Conclusions: Therapeutic administration of S1PL may represent an attractive strategy for modulating lymphocyte trafficking to improve the efficacy of therapies that rely on the adaptive immune system to exert antitumor activity. This approach may be particularly potent in settings wherein tumor-mediated immune exclusion is a dominant method of immune escape. Citation Format: Alessandra Araujo, Max Rodnick-Smith, Ranya Al-Khaledy, George Georgiou, Mark Badeaux, Everett Stone. Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2659.

Targeting Sphingosine-1-Phosphate Signaling in Breast Cancer.

In recent years, newly emerging therapies, such as immune checkpoint inhibitors and antibody-drug conjugates, have further improved outcomes for breast cancer patients. However, recurrent and metastatic breast cancer often eventually develops resistance to these drugs, and cure is still rare. As such, the development of new therapies for refractory breast cancer that differ from conventional mechanisms of action is necessary. Sphingosine-1-phosphate (S1P) is a key molecule with a variety of bioactive activities, including involvement in cancer cell proliferation, invasion, and metastasis. S1P also contributes to the formation of the cancer microenvironment by inducing surrounding vascular- and lymph-angiogenesis and regulating the immune system. In this article, we outline the basic mechanism of action of S1P, summarize previous findings on the function of S1P in cancer cells and the cancer microenvironment, and discuss the clinical significance of S1P in breast cancer and the therapeutic potential of targeting S1P signaling.

The β-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis.

Canonical Wnt and sphingosine-1-phosphate (S1P) signaling pathways are highly conserved systems that contribute to normal vertebrate development, with key consequences for immune, nervous, and cardiovascular system function; despite these functional overlaps, little is known about Wnt/β-catenin-S1P cross-talk. In the vascular system, both Wnt/β-catenin and S1P signals affect vessel maturation, stability, and barrier function, but information regarding their potential coordination is scant. We report an instance of functional interaction between the two pathways, including evidence that S1P receptor 1 (S1PR1) is a transcriptional target of β-catenin. By studying vascular smooth muscle cells and arterial injury response, we find a specific requirement for the β-catenin carboxyl terminus, which acts to induce S1PR1, and show that this interaction is essential for vascular remodeling. We also report that pharmacological inhibition of the β-catenin carboxyl terminus reduces S1PR1 expression, neointima formation, and atherosclerosis. These findings provide mechanistic understanding of how Wnt/β-catenin and S1P systems collaborate during vascular remodeling and inform strategies for therapeutic manipulation.

Rewiring Endothelial Sphingolipid Metabolism to Favor S1P Over Ceramide Protects From Coronary Atherosclerosis.

Growing evidence correlated changes in bioactive sphingolipids, particularly S1P (sphingosine-1-phosphate) and ceramides, with coronary artery diseases. Furthermore, specific plasma ceramide species can predict major cardiovascular events. Dysfunction of the endothelium lining lesion-prone areas plays a pivotal role in atherosclerosis. Yet, how sphingolipid metabolism and signaling change and contribute to endothelial dysfunction and atherosclerosis remain poorly understood. We used an established model of coronary atherosclerosis in mice, combined with sphingolipidomics, RNA-sequencing, flow cytometry, and immunostaining to investigate the contribution of sphingolipid metabolism and signaling to endothelial cell (EC) activation and dysfunction. We demonstrated that hemodynamic stress induced an early metabolic rewiring towards endothelial sphingolipid de novo biosynthesis, favoring S1P signaling over ceramides as a protective response. This finding is a paradigm shift from the current belief that ceramide accrual contributes to endothelial dysfunction. The enzyme SPT (serine palmitoyltransferase) commences de novo biosynthesis of sphingolipids and is inhibited by NOGO-B, an ER membrane protein. Here, we showed that NOGO-B is upregulated by hemodynamic stress in myocardial EC of ApoE-/- mice and is expressed in the endothelium lining coronary lesions in mice and humans. We demonstrated that mice lacking NOGO-B specifically in EC (Nogo-A/BECKOApoE-/-) were resistant to coronary atherosclerosis development and progression, and mortality. Fibrous cap thickness was significantly increased in Nogo-A/BECKOApoE-/- mice and correlated with reduced necrotic core and macrophage infiltration. Mechanistically, the deletion of NOGO-B in EC sustained the rewiring of sphingolipid metabolism towards S1P, imparting an atheroprotective endothelial transcriptional signature. These data demonstrated that hemodynamic stress induced a protective rewiring of sphingolipid metabolism, favoring S1P over ceramide. NOGO-B deletion sustained the rewiring of sphingolipid metabolism toward S1P protecting EC from activation under hemodynamic stress and refraining coronary atherosclerosis. These findings also set forth the foundation for sphingolipid-based therapeutics to limit atheroprogression.

A Crosstalk between the Receptor Tyrosine Kinase-Like Orphan Receptors ROR1/2 and S1P Signaling Pathways in Lung Cancer.

Availability of multimodal treatment strategies, including targeted therapies and immunotherapies, have improved the survival of non-small cell lung carcinoma (NSCLC). However, some patients still progress or respond poorly due to inherent resistance, acquired resistance, or lack of druggable driver mutations. Sphingosine-1-phosphate (S1P) and receptor tyrosine kinase-like orphan receptor (ROR1/2) signaling pathways are activated during lung carcinogenesis. In this study, we have evaluated the crosstalk of S1P and ROR1/2 signaling pathways in lung cancer cells. S1P treatment of lung cancer cells decreases ROR1 and ROR2 transcript levels. While treatment with PF-543, a pharmacological SphK1 inhibitor or genetic knockdown of SPHK1 by shRNA, raises ROR1 and ROR2. Furthermore, simultaneous inhibition of SphK1 along with ROR1 reduced the migration of lung cancer cells. These findings demonstrate the reciprocal regulation of both pathways, suggesting that both pathways have an inverse relation i.e, in the absence of one pathway, another pathway may take charge of the other pathway. Therefore, simultaneously targeting both pathways could serve as a potential therapeutic target for lung cancer treatment.

How do sphingosine-1-phosphate affect immune cells to resolve inflammation?

Inflammation is an important immune response of the body. It is a physiological process of self-repair and defense against pathogens taken up by biological tissues when stimulated by damage factors such as trauma and infection. Inflammation is the main cause of high morbidity and mortality in most diseases and is the physiological basis of the disease. Targeted therapeutic strategies can achieve efficient toxicity clearance at the inflammatory site, reduce complications, and reduce mortality. Sphingosine-1-phosphate (S1P), a lipid signaling molecule, is involved in immune cell transport by binding to S1P receptors (S1PRs). It plays a key role in innate and adaptive immune responses and is closely related to inflammation. In homeostasis, lymphocytes follow an S1P concentration gradient from the tissues into circulation. One widely accepted mechanism is that during the inflammatory immune response, the S1P gradient is altered, and lymphocytes are blocked from entering the circulation and are, therefore, unable to reach the inflammatory site. However, the full mechanism of its involvement in inflammation is not fully understood. This review focuses on bacterial and viral infections, autoimmune diseases, and immunological aspects of the Sphks/S1P/S1PRs signaling pathway, highlighting their role in promoting intradial-adaptive immune interactions. How S1P signaling is regulated in inflammation and how S1P shapes immune responses through immune cells are explained in detail. We teased apart the immune cell composition of S1P signaling and the critical role of S1P pathway modulators in the host inflammatory immune system. By understanding the role of S1P in the pathogenesis of inflammatory diseases, we linked the genomic studies of S1P-targeted drugs in inflammatory diseases to provide a basis for targeted drug development.

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

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. 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. 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. 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.

The Oncogenic Lipid Sphingosine-1-Phosphate Impedes the Phagocytosis of Tumor Cells by M1 Macrophages in Diffuse Large B Cell Lymphoma.

A total of 30-40% of diffuse large B cell lymphoma (DLBCL) patients will either not respond to the standard therapy or their disease will recur. The first-line treatment for DLBCL is rituximab and combination chemotherapy. This treatment involves the chemotherapy-induced recruitment of tumor-associated macrophages that recognize and kill rituximab-opsonized DLBCL cells. However, we lack insights into the factors responsible for the recruitment and functionality of macrophages in DLBCL tumors. We have studied the effects of the immunomodulatory lipid sphingosine-1-phosphate (S1P) on macrophage activity in DLBCL, both in vitro and in animal models. We show that tumor-derived S1P mediates the chemoattraction of both monocytes and macrophages in vitro and in animal models, an effect that is dependent upon the S1P receptor S1PR1. However, S1P inhibited M1 macrophage-mediated phagocytosis of DLBCL tumor cells opsonized with the CD20 monoclonal antibodies rituximab and ofatumumab, an effect that could be reversed by an S1PR1 inhibitor. Our data show that S1P signaling can modulate macrophage recruitment and tumor cell killing by anti-CD20 monoclonal antibodies in DLBCL. The administration of S1PR1 inhibitors could enhance the phagocytosis of tumor cells and improve outcomes for patients.

Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1 , S1P3 and S1P5 , three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3 , given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

P172 Spatial Transcriptomics Characterization of S1P Pathway Genes in Ulcerative Colitis and Crohn’s Disease Colon Tissues

Abstract Background Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory diseases of the gastrointestinal tract, related to the confluence of environmental and immunological factors in genetically predisposed individuals. Activated T and B cells migrate from the lymphoid tissue into the circulation through the interaction of Sphingosine-1-phosphate (S1P) and the S1P1 receptor (S1P1), which can perpetuate inflammation and lead to tissue damage. Targeting of cellular migratory pathways has emerged as a novel therapeutic paradigm in IBD. The aim of this study is to characterize the S1P pathway-related genes and signaling pathways driving the pathogenesis of UC and CD. Methods Spatial transcriptomics analysis was performed in health volunteer (HV, n=2), UC (n=4) and CD (n=2) FFPE colon tissues using the GeoMX platform and NanoString whole transcriptome atlas (WTA) assay. The WTA panel was run on 8 samples stained with antibodies to S1P1, pan Cytokeratin, and CD45. Six regions of interests (ROIs) were selected in each sample based on the staining and each ROI were split into two areas (CD45+ or CD45-). Differential gene expression and enriched pathways analysis including S1P pathway genes were performed in CD45+ and CD45- segments in HV, UC and CD colon tissues. Results A total of 6085 and 6054 differentially expressed genes were identified from the CD45+ segments in UC and CD respectively, compared to HV colon (P=0.05), including the genes that are involved in T cell activation (e.g., HLA-DPA1, CCDC13), immune cell migration (e.g., ADAM15, CCL5) and tissue damage (e.g., FABP6, PDK4). Interestingly, significant differences were identified in UC and CD in S1P pathway gene expression. Specifically, SMPD1, a gene involved in generating ceramide and regulating inflammatory responses, was the most significantly up-regulated gene from both CD45+ and CD45- compartments in UC compared to HV colon. While CERS2 and CERS6, genes related to S1P metabolism, were significantly down-regulated genes in CD45+ and CD45- compartment in UC respectively, as compared to HV colon. Additionally, RTN4 (participates in the intestinal epithelial barrier function) and PLPP3 (dephosphorylates extracellular S1P to sphingosine) were most significantly down-regulated genes from CD45+ segments in both UC and CD compared to HV colon. Conclusion In summary, our study successfully validated the GeoMx transcriptome analysis approach in FFPE colon tissue and assessed the S1P pathway gene signature difference in HV, UC and CD colon tissues supporting the pathway’s role in IBD. Results could also facilitate the identification of potential S1P pathway UC and CD disease biomarkers.

Sphingosine-1-phosphate promotes osteogenesis by stimulating osteoblast growth and neovascularization in a vascular endothelial growth factor-dependent manner.

Sphingosine-1-phosphate (S1P) plays multiple roles in bone metabolism and regeneration. Here, we have identified a novel S1P-regulated osteoanabolic mechanism functionally connecting osteoblasts (OBs) to the highly specialized bone vasculature. We demonstrate that S1P/S1PR3 signaling in OBs stimulates vascular endothelial growth factor a (VEGFa) expression and secretion to promote bone growth in an autocrine and boost osteogenic H-type differentiation of bone marrow endothelial cells in a paracrine manner. VEGFa-neutralizing antibodies and VEGF receptor inhibition by axitinib abrogated OB growth in vitro and bone formation in male C57BL/6J in vivo following S1P stimulation and S1P lyase inhibition, respectively. Pharmacological S1PR3 inhibition and genetic S1PR3 deficiency suppressed VEGFa production, OB growth in vitro, and inhibited H-type angiogenesis and bone growth in male mice in vivo. Together with previous work on the osteoanabolic functions of S1PR2 and S1PR3, our data suggest that S1P-dependent bone regeneration employs several nonredundant positive feedback loops between OBs and the bone vasculature. The identification of this yet unappreciated aspect of osteoanabolic S1P signaling may have implications for regular bone homeostasis as well as diseases where the bone microvasculature is affected such as age-related osteopenia and posttraumatic bone regeneration.

Response to Fingolimod in Multiple Sclerosis Patients Is Associated with a Differential Transcriptomic Regulation.

Fingolimod is an immunomodulatory sphingosine-1-phosphate (S1P) analogue approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). The identification of biomarkers of clinical responses to fingolimod is a major necessity in MS to identify optimal responders and avoid the risk of disease progression in non-responders. With this aim, we used RNA sequencing to study the transcriptomic changes induced by fingolimod in peripheral blood mononuclear cells of MS-treated patients and their association with clinical response. Samples were obtained from 10 RRMS patients (five responders and five non-responders) at baseline and at 12 months of fingolimod therapy. Fingolimod exerted a vast impact at the transcriptional level, identifying 7155 differentially expressed genes (DEGs) compared to baseline that affected the regulation of numerous signaling pathways. These DEGs were predominantly immune related, including genes associated with S1P metabolism, cytokines, lymphocyte trafficking, master transcription factors of lymphocyte functions and the NF-kB pathway. Responder and non-responder patients exhibited a differential transcriptomic regulation during treatment, with responders presenting a higher number of DEGs (6405) compared to non-responders (2653). The S1P, NF-kB and TCR signaling pathways were differentially modulated in responder and non-responder patients. These transcriptomic differences offer the potential of being exploited as biomarkers of a clinical response to fingolimod.

The role of sphingosine-1-phosphate in the development and progression of Parkinson's disease.

Parkinson's disease (PD) could be viewed as a proteinopathy caused by changes in lipids, whereby modifications in lipid metabolism may lead to protein alterations, such as the accumulation of alpha-synuclein (α-syn), ultimately resulting in neurodegeneration. Although the loss of dopaminergic neurons in the substantia nigra is the major clinical manifestation of PD, the etiology of it is largely unknown. Increasing evidence has highlighted the important role of lipids in the pathophysiology of PD. Sphingosine-1-phosphate (S1P), a signaling lipid, has been suggested to have a potential association with the advancement and worsening of PD. Therefore, better understanding the mechanisms and regulatory proteins is of high interest. Most interestingly, S1P appears to be an important target to offers a new strategy for the diagnosis and treatment of PD. In this review, we first introduce the basic situation of S1P structure, function and regulation, with a special focus on the several pathways. We then briefly describe the regulation of S1P signaling pathway on cells and make a special focused on the cell growth, proliferation and apoptosis, etc. Finally, we discuss the function of S1P as potential therapeutic target to improve the clinical symptoms of PD, and even prevent the progression of the PD. In the context of PD, the functions of S1P modulators have been extensively elucidated. In conclusion, S1P modulators represent a novel and promising therapeutic principle and therapeutic method for PD. However, more research is required before these drugs can be considered as a standard treatment option for PD.

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.

Bone marrow mesenchymal stem cells therapy regulates sphingolipid and glycerophospholipid metabolism to promote neurological recovery in stroke rats: A metabolomics analysis

Bone marrow mesenchymal stem cells (BMSCs) have therapeutic potential in the subacute/chronic phase of acute ischemic stroke (AIS), but the underlying mechanisms are not yet fully elucidated. There is a knowledge gap in understanding the metabolic mechanisms of BMSCs in stroke therapy. In this study, we administered BMSCs intravenously 24 h after reperfusion in rats with transient cerebral artery occlusion (MCAO). The treatment with BMSCs for 21 days significantly reduced the modified neurological severity score of MCAO rats (P < 0.01) and increased the number of surviving neurons in both the striatum and hippocampal dentate gyrus region (P < 0.01, respectively). Moreover, BMSCs treatment resulted in significant enhancements in various structural parameters of dendrites in layer V pyramidal neurons in the injured hemispheric motor cortex, including total length (P < 0.05), number of branches (P < 0.05), number of intersections (P < 0.01), and spine density (P < 0.05). Then, we performed plasma untargeted metabolomics analysis to study the metabolic changes of BMSCs on AIS. There were 65 differential metabolites identified in the BMSCs treatment group. Metabolic profiling analysis revealed that BMSCs modulate abnormal sphingolipid metabolism and glycerophospholipid metabolism, particularly affecting core members such as sphingomyelin (SM), ceramide (Cer) and sphingosine-1-phosphate (S1P). The metabolic network analysis and pathway-based compound-reaction-enzyme-gene network analysis showed that BMSCs inhibited the Cer-induced apoptotic pathway and promoted the S1P signaling pathway. These findings suggest that the enhanced effects of BMSCs on neuronal survival and synaptic plasticity after stroke may be mediated through these pathways. In conclusion, our study provides novel insight into the potential mechanisms of BMSCs treatment in stroke and sheds light on the possible clinical translation of BMSCs.

The role of vitamin D through SphK1/S1P in the regulation of MS progression

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-β, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-β, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

Revealing concealed cardioprotection by platelet Mfsd2b-released S1P in human and murine myocardial infarction

Abstract Background/Introduction Platelet activation on ruptured or eroded atherosclerotic lesions leads to thrombus formation and ischemic events. Hence, platelet inhibition is the cornerstone of primary and secondary prevention in patients with cardiovascular disease. Recently, non-canonical effects of platelets in multiple disease processes were revealed. However, others have demonstrated the opposite: a protective and regenerative role of platelets during myocardial healing. Sphingosine-1-phosphate (S1P) is an important bioactive lipid released from platelets upon activation and has potent cardioprotective properties in AMI when acutely administered or kept constitutively high due to pharmacological or genetic inhibition of its degradation. In humans, S1P levels in plasma are dynamically regulated during AMI. Purpose We hypothesized that platelets may be an important source of the S1P burst during AMI and that platelet-derived S1P is relevant for the extent of myocardial damage due to AMI. Methods We have addressed these questions in experimental AMI in mice lacking components of the S1P synthesis, release and signaling pathways. In additon, we conducted a hypothesis generating, prospective, monocentric, time-series, translational analysis in 127 patients with ST-elevation myocardial infarction (STEMI) to analyze the association between S1P plasma concentration and infarct size. Results Injection of cell-free supernatant of activated platelets (SNT+) prior AMI in mice led to decreased infarct size after 24 h of reperfusion as compared to supernatant of non-activated platelets (SNT-). This reduction of infarct size by SNT+ was associated with reduced neutrophil accumulation and reduced apoptosis after AMI. S1P-concentrations in SNT+ was 25% higher as compared to SNT-. S1P concentrations in SNT- of mice lacking Sphingosinkinase-1 (SphK1) were 70% lower and did not increase in SNT+. Accordingly, SNT+ of SphK1- deficient platelets failed to reduce infarct size. Deficiency of platelet S1P exporter Mfsd2b shows increased infarct size during in vivo AMI compared to littermate controls. SNT+ of WT mice led to improved outcome after ischemia in a model of Langendorff-perfused hearts. This was blunted by inhibition of S1P receptor 1 and genetic cardiomyocyte deficiency of S1PR1. Platelet S1P release during activation was reduced by P2Y12 inhibition but preserved during GPIIb/IIIa (tirofiban) inhibition. This translated to blunted cardioprotection by SNT+ of cangrelor treated platelets but sustained cardioprotection during tirofiban treatment. Plasma S1P concentrations was associated with cardiovascular death and infarct size in patients with ST-elevation myocardial infarction. Conclusion We have identified the mechanisms underlying platelet-associated cardioprotection and finally, revealed the importance of platelet-released S1P on admission in patients with ST-elevation myocardial infarction (STEMI) regarding to infarct size and long-term prognosis.

Farnesoid X receptor prevents neutrophil extracellular traps via reduced sphingosine-1-phosphate in chronic kidney disease.

Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity. Renal NETosis and sphingosine kinase 1 (Sphk1) expression are increased in diseased mice, and they are reduced by OCA in both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly, kidney S1P levels in Alport mice are two-fold higher than controls, and FXR agonism restores them back to baseline. Short-term inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (six male, nine female), and NETosis severity correlates with clinical markers of kidney disease. This suggests the potential clinical relevance of the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis.NEW & NOTEWORTHY Many preclinical studies have shown that the farnesoid X receptor (FXR) reduces renal inflammation, but the mechanism is poorly understood. This report identifies FXR as a novel regulator of neutrophilic inflammation and NETosis via the inhibition of sphingosine-1-phosphate signaling. Additionally, NETosis severity in human Alport kidney biopsies correlates with clinical markers of kidney disease. A better understanding of this signaling axis may lead to novel treatments that prevent renal inflammation and chronic kidney disease.

Density of Sphingosine-1-Phosphate Receptors Is Altered in Cortical Nerve-Terminals of Insulin-Resistant Goto-Kakizaki Rats and Diet-Induced Obese Mice

Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction.