Sphingosine-1-phosphate Receptor Research Articles

Targeting SphK1/S1PR3 axis ameliorates sepsis-induced multiple organ injury via orchestration of macrophage polarization and glycolysis.

Sepsis is a heterogeneous and imprecise disorder characterized by aberrant response to infection which has been accredited for detrimental impact on immune homeostasis. Recently, macrophage metabolism has been recognized as attractive targets to develop novel immunomodulatory therapy for sepsis research. However, the fine-tuning regulators dictating macrophage functions and the specific mechanisms underlying macrophage metabolic reprogramming remain largely obscure. Sphingosine-1-phosphate (S1P), a metabolic mediator of sphingolipid catabolism, predominantly formed through sphingosine kinase 1 (SphK1) catalyzing, mediates inflammation in sepsis by binding to S1P receptor 3 (S1PR3) expressed in macrophages. Here we demonstrate that SphK1/S1PR3 axis was upregulated in lipopolysaccharide (LPS)-induced macrophages and septic mice lungs, cascading the activation of proglycolytic signaling such as HIF-1α, HK2 and PFKFB3. Targeted inhibition of Sphk1 by PF-543 effectively abrogated upregulated SphK1/S1PR3 axis in vitro and in vivo. In addition, PF-543 significantly suppressed sepsis-related inflammation and multi-organ injury in vivo. Furthermore, PF-543 not only blunted key glycolytic enzymes HIF-1α, HK2, and PFKFB3 in LPS-treated macrophages but also inhibited HK2 and PFKFB3 in septic mice. Silencing or inhibiting SphK1 tempered pro-inflammatory M1 macrophages while boosted anti-inflammatory M2 macrophages. Intriguingly, S1PR3 knockdown proficiently dampened glycolysis-associated markers, retrieved LPS-modulated M1/M2 polarization and attenuated NF-κB p65 activation. In conclusion, our study provides the first evidence that PF-543 orchestrates proportional imbalance of macrophage polarization and the Warburg effect in a SphK1/S1PR3 dependent manner during sepsis, mitigating both hyperinflammation and multi-organ failure, adding a novel puzzle piece to pharmacologically exploitable therapy for sepsis.

Abstract 4120623: Sphingosine Kinase 1 Is Integral For Elastin Deficiency-induced Arterial Hypermuscularization

Introduction: Defective elastic lamellae and smooth muscle cell (SMC) accumulation are characteristics of diverse obstructive arterial diseases (e.g., atherosclerosis, pulmonary hypertension, and supravalvular aortic stenosis [SVAS]) as well as physiological closure of the ductus arteriosus (DA). Mechanistic links between defective elastin and SMC proliferation are not well elucidated. Methods: Immunostaining for proliferation marker Ki67 was performed on wild-type (WT) and Eln (-/-) mouse aortas at embryonic day (E) 13.5 and E15.5 because elastin (ELN) is expressed in the mouse aorta from E14. Bulk RNA-seq was conducted on mouse aortic SMCs isolated from WT or Eln (-/-) embryos at E15.5. As sphingosine kinase 1 (SPHK1) was found as a highly promising candidate, its expression was evaluated in human SVAS patient aortas and in mouse Eln (-/-) aortas and WT DA. S1P receptor 1 (S1PR1) activity was assessed in aortic SMCs from S1pr1 (knock-in/knock-in) , H2B-GFP mice. Genetic deletion of Sphk1 in SMCs was performed on the elastin mutant background. Pharmacological SPHK1 inhibition was evaluated on both elastin mutants and WT embryos. Regulatory mechanisms of SPHK1 were assessed by mRNA stability assay and TRANSFAC database analysis. Results: SMC hyperproliferation was first observed in Eln (-/-) aorta at E15.5, prior to morphological differences. Bulk RNA-seq revealed that Sphk1 is the most upregulated transcript in Eln (-/-) aortic SMCs at E15.5. Reduced ELN increases SPHK1 levels in SMCs of human patient aortas and mouse aorta and DA. S1PR1 expression and activity are increased by elastin insufficiency. SMC Sphk1 deletion attenuates SMC proliferation and muscularization in the elastin-defective aorta, leading to extended viability of Eln (-/-) mice. Similarly, pharmacological SPHK1 inhibition ameliorates elastin aortopathy but leads to patent DA in WT mice. mRNA stability assay indicated Sphk1 is upregulated by enhanced transcription. TRANSFAC and bulk RNA-seq data suggested that transcription factor early growth response 1 (Egr1) induces Sphk1 transcription. Indeed, EGR1 was upregulated in elastin mutant aortas and DA. Conclusions: Elastin deficiency upregulates SPHK1 transcription, leading to SMC proliferation and hypermuscularization in elastin aortopathy as well as during physiological DA closure. Inhibiting SPHK1 is a promising therapeutic strategy for elastin aortopathy and for select congenital heart diseases in which patent DA maintains circulation.

Sphingosine-1-phosphate (S1P) receptor type 1 signaling in macrophages reduces atherosclerosis in LDL receptor-deficient mice.

Sphingosine 1-phosphate (S1P) is a lysosphingolipid with anti-atherogenic properties, but mechanisms underlying its effects remain unclear. We here investigated atherosclerosis development in cholesterol-rich diet-fed LDL receptor-deficient mice with high or low overexpression levels of S1P receptor type 1 (S1P1) in macrophages. S1P1-overexpressing macrophages showed increased activity of transcription factors PU.1, IRF8, and LXR and were skewed towards a M2-distinct phenotype characterized by enhanced production of IL-10, IL-1RA, and IL-5, increased ATP-binding cassette transporter A1- and G1-dependent cholesterol efflux, increased expression of MerTK and efferocytosis, and reduced apoptosis due to elevated Bcl6 and MafB. A similar macrophage phenotype was observed in mice administered S1P1-selective agonist KRP203. Mechanistically, the enhanced PU.1, IRF8, and LXR activity in S1P1-overexpressing macrophages led to down-regulation of the cAMP-dependent protein kinase A and activation of the signaling cascade encompassing protein kinases Akt and mTOR complex 1 (mTORC1) as well as the late endosomal/lysosomal adaptor MAPK and mTOR activator 1 (Lamtor-1). Atherosclerotic lesions in aortic roots and brachiocephalic arteries were profoundly or moderately reduced in mice with high and low S1P1 overexpression in macrophages, respectively. We conclude that S1P1 signaling polarizes macrophages towards an anti-atherogenic functional phenotype and countervails the development of atherosclerosis in mice.

TMIC-57. EFFECT OF HYPOXIA ON SPHINGOSINE-1-PHOSPHATE (S1P) METABOLIZING ENZYMES IN DIFFUSE INTRINSIC PONTINE GLIOMA CELL LINES

Abstract Diffuse Midline Glioma H3K27 altered (DMG) is a devastating and incurable paediatric brain tumour. Previously a bioinformatic approach was taken to evaluate genes involved in the sphingosine-1-phosphate (S1P) biosynthesis pathway in diffuse intrinsic pontine gliomas (DIPG). S1P is known to interact with epigenetic partners (HDAC1) as well as play a role in proliferation, immune cell trafficking, angiogenesis and apoptosis. As a part of a broad bioinformatic approach evaluating the expression of genes associated with the sphingomyelin (SM) biosynthesis pathway, S1P metabolism, S1P receptors and downstream targets, 14 of 25 genes were found to be dysregulated in DIPG relative to normal brain (p<0.0002). Herein we focus on the imbalance of the S1P metabolic enzymes sphingosine kinase 2 (SPHK2) and sphingosine-1-phosphate lyase (SGPL1). Bioinformatic data revealed higher levels of SGPL1 and lower levels of SPHK2 compared with normal brain. DIPG cell lines (SU-DIPG-IV, VUMC-DIPG-08, VUMC-DIPG-A) were used to validate gene and protein expression and to evaluate changes following exposure to hypoxic conditions. Confocal images reveal an increase in nuclear SPHK2 staining in cells cultured for 24- and 48-hours under hypoxic conditions. HDAC1 immunostaining was localised in the nucleus. SGPL1 immunostaining, while mostly cytoplasmic, nuclear staining was observed. qRT-PCR assays were used to evaluate changes in mRNA expression in SPHK2, SGPL1 and HDAC1 in DIPG cell lines incubated under hypoxic conditions. The Pfaffl method was used to analyse gene expression relative to the housekeeping gene TBP. Under hypoxic conditions, the levels of SPHK2 and HDAC1 were higher in DIPG cells cultured in hypoxic conditions for 48 hours compared to DIPG cells cultured in normoxic conditions, whereas SGPL1 expression was lower in hypoxic treated cells. Current experiments targeting the modulation of these S1P-metabolizing enzymes are underway to determine the role of the S1P signalling pathway and determine the potential advantage of targeting this pathway in DIPG.

Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

BackgroundAbnormal sensory perception, particularly pain insensitivity (PAI), is a typical symptom of autism spectrum disorder (ASD). Despite the role of myelin metabolism in the regulation of pain perception, the mechanisms underlying ASD-related PAI remain unclear.MethodsThe pain-associated gene sphingosine-1-phosphate receptor 1 (S1PR1) was identified in ASD samples through bioinformatics analysis. Its expression in the dorsal root ganglion (DRG) tissues of BTBR ASD model mice was validated using RNA-seq, western blot, RT-qPCR, and immunofluorescence. Pain thresholds were assessed using the von Frey and Hargreaves tests. Patch-clamp techniques measured KCNQ/M channel activity and neuronal action potentials. The expression of S1PR1, KCNQ/M, mitogen-activated protein kinase (MAPK), and cyclic AMP/protein kinase A (cAMP/PKA) signaling proteins was analyzed before and after inhibiting the S1P-S1PR1-KCNQ/M pathway via western blot and RT-qPCR.ResultsThrough integrated transcriptomic analysis of ASD samples, we identified the upregulated gene S1PR1, which is associated with sphingolipid metabolism and linked to pain perception, and confirmed its role in the BTBR mouse model of ASD. This mechanism involves the regulation of KCNQ/M channels in DRG neurons. The enhanced activity of KCNQ/M channels and the decreased action potentials in small and medium DRG neurons were correlated with PAI in a BTBR mouse model of ASD. Inhibition of the S1P/S1PR1 pathway rescued baseline insensitivity to pain by suppressing KCNQ/M channels in DRG neurons, mediated through the MAPK and cAMP/PKA pathways. Investigating the modulation and underlying mechanisms of the non-opioid pathway involving S1PR1 will provide new insights into clinical targeted interventions for PAI in ASD.ConclusionsS1PR1 may contribute to PAI in the PNS in ASD. The mechanism involves KCNQ/M channels and the MAPK and cAMP/PKA signaling pathways. Targeting S1PR1 in the PNS could offer novel therapeutic strategies for the intervention of pain dysesthesias in individuals with ASD.

Computational Analysis of S1PR1 SNPs Reveals Drug Binding Modes Relevant to Multiple Sclerosis Treatment

Background/Objectives: Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS) characterized by myelin and axonal damage with a globally rising incidence. While there is no known cure for MS, various disease-modifying treatments (DMTs) exist, including those targeting Sphingosine-1-Phosphate Receptors (S1PRs), which play important roles in immune response, CNS function, and cardiovascular regulation. This study focuses on understanding how nonsynonymous single nucleotide polymorphisms (rs1299231517, rs1323297044, rs1223284736, rs1202284551, rs1209378712, rs201200746, and rs1461490142) in the S1PR1’s active site affect the binding of endogenous ligands, as well as different drugs used in MS management. Methods: Extensive molecular dynamics simulations and linear interaction energy (LIE) calculations were employed to predict binding affinities and potentially guide future personalized medicinal therapies. The empirical parameters of the LIE method were optimized using the binding free energies calculated from experimentally determined IC50 values. These optimized parameters were then applied to calculate the binding free energies of S1P to mutated S1PR1, which correlated well with experimental values, confirming their validity for assessing the impact of SNPs on S1PR1 binding affinities. Results: The binding free energies varied from the least favorable −8.2 kcal/mol for the wild type with ozanimod to the most favorable −16.7 kcal/mol for the combination of siponimod with the receptor carrying the F2055.42L mutation. Conclusions: We successfully demonstrated the differences in the binding modes, interactions, and affinities of investigated MS drugs in connection with SNPs in the S1PR1 binding site, resulting in several viable options for personalized therapies depending on the present mutations.

Sphingosine 1-phosphate receptor subtype 1 (S1P1) activity in the course of Alzheimer's disease

Some specific lipid molecules in the brain act as signaling molecules, neurotransmitters, or neuromodulators, by binding to specific G protein-coupled receptors (GPCR) for neurolipids. One such receptor, sphingosine 1-phosphate receptor subtype 1 (S1P1), is coupled to Gi/o proteins and is involved in cell proliferation, growth, and neuroprotection. S1P1 constitutes an interesting target for neurodegenerative diseases like multiple sclerosis and Alzheimer's disease (AD), in which changes in the sphingolipid metabolism have been observed. This study analyzes S1P1 receptor-mediated activity in healthy brains and during AD progression using postmortem samples from controls and patients at different Braak's stages. Additionally, the distribution of S1P1 receptor activity in human brains is compared to that in commonly used rodent models, rats and mice, through functional autoradiography, measuring [35S]GTPγS binding stimulated by the S1P1 receptor selective agonist CYM-5442 to obtain the distribution of functional activity of S1P1 receptors.S1P1 receptor-mediated activity, along with that of the cannabinoid CB1 receptor, is one of the highest recorded for any GPCR in many gray matter areas of the brain, reaching maximum values in the cerebellar cortex, specific areas of the hippocampus and the basal forebrain. S1P1 signaling is crucial in areas that regulate learning, memory, motor control, and nociception, such as the basal forebrain and basal ganglia. In AD, S1P1 receptor activity is increased in the inner layers of the frontal cortex and underlying cortical white matter at early stages, but decreases in the hippocampus in advanced stages, indicating ongoing brain impairment. Importantly, we identified significant correlations between S1P1 receptor activity and Braak stages, suggesting that S1P1 receptor dysfunction is associated to disease progression, particularly in memory-related regions. The S1P signaling via S1P1 receptor is a promising neurological target due to its role in key neurophysiological functions and its potential to modify the progression of neurodegenerative diseases. Finally, rats are suggested as a preferred experimental model for studying S1P1 receptor-mediated responses in the human brain.

Fingolimod, a sphingosine-1-phosphate receptor modulator, prevents neonatal bronchopulmonary dysplasia and subsequent airway remodeling in a murine model.

Neonatal bronchopulmonary dysplasia (BPD) is associated with alveolar simplification and airway remodeling. Airway remodeling leads to deformation of airways characterized by peribronchial collagen deposition and hypertrophy of airway smooth muscle, which contribute to the narrowing of airways. Poorly developed lungs contribute to reduced lung function that deteriorates with the passage of time. We have earlier shown that sphingosine kinase 1 (SPHK 1)/sphingosine-1-phosphate (S1P)/S1P receptor1 (S1PR1) signaling plays a role in the pathogenesis of BPD. In this study, we investigated the role of fingolimod or FTY720, a known S1PR1 modulator approved for the treatment of multiple sclerosis in the treatment of BPD. Fingolimod promotes the degradation of S1PR1 by preventing its recycling, thus serving as the equivalent of an inhibitor. Exposure of neonatal mice to hyperoxia enhanced the expression of S1PR1 in both airways and alveoli as compared with normoxia. This increased expression of S1PR1 in the airways persisted into adulthood, accompanied by airway remodeling and airway hyperreactivity (AHR) after neonatal hyperoxia. Intranasal fingolimod at a much lower dose compared with the intraperitoneal route of administration during neonatal hyperoxia improved alveolarization in neonates and reduced airway remodeling and AHR in adult mice associated with improved lung function. The intranasal route was not associated with the lymphopenia seen with the intraperitoneal route of administration of the drug. An increase in S1PR1 expression in the airways was associated with an increase in the expression of enzyme lysyl oxidase (LOX) in the airways following hyperoxia, which was suppressed by fingolimod. This association warrants further investigation.NEW & NOTEWORTHY The role of the S1P receptor1 modulator, fingolimod, as an FDA-approved drug in preventing the recurrence of multiple sclerosis is established. Fingolimod prevented bronchopulmonary dysplasia (BPD) and its sequela of airway remodeling in a neonatal murine model. This protection was associated with the downregulation of lysyl oxidase signaling pathway. Fingolimod could be repurposed for the therapy of BPD.

The effect of immunosuppressive agent FTY-720 on coagulation, nitroxidergic lung activity and water balance in experimental antiphospholipid syndrome

Antiphospholipid syndrome (APS) is a systemic acquired autoimmune thrombophilia that leads to impaired non-respiratory lung functions due to the circulation of antiphospholipid antibodies, shifting the balance between the coagulation link of hemostasis and the anti-coagulation system towards hypercoagulation. The research examines the modulating effect of FTY-720 on the water balance, coagulation and NO-activity of the lungs under conditions of experimental APS. The functional activity of FTY-720 is largely due to its ability to bind to S1P1 receptors and block signaling pathways mediated by the interaction of the mediator sphingosine-1-phosphate with them. The main effect of FTY-720 is immunosuppression, which develops as a result of the drug’s receptor interaction predominantly with S1P1 receptors, resulting in the retention of autoreactive lymphocytes in lymph nodes, and prevents further spread of inflammation and damage. The experiments were conducted on 85 male rats, divided into groups as follows: The first group of animals (n = 30) was injected with cardiolipin antigen every other day for three weeks for APS modeling; the rats of the second group (n = 25), which served as a control group, were similarly injected with 0.9% NaCl; APS modeling and FTY-720 injection were combined on the remaining animals (n = 30). Three weeks later, arterial (art) and venous (ven) blood was taken, in which APTT and PT were determined using a hemocoagulometer CGL 2110 “Solar” and the corresponding coefficients of APPT of ven/art, PT of ven/ art were calculated. NO-activity was assessed by the content of plasma NO and NO bronchoalveolar lavage obtained by washing the extraction of the bronchopulmonary complex in anesthetized animals. To study the water balance of the lungs, the mass of moist and dried lungs was measured, were determined the total, extra- and intravascular fluid of the lungs. In APS, an increase in APTT and PT of arterial and venous blood was observed, an increase in the level of NO in bronchoalveolar lavages against the background of worsening nitroxidergic activity of the lungs – the NO index of ven/art decreased. There was a positive effect of FTY- 720 on the restoration of non-respiratory lung functions in APS.

Role of Sphingosine-1-Phosphate Signaling Pathway in Pancreatic Diseases.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolic product produced via the phosphorylation of sphingosine by sphingosine kinases (SPHKs), serving as a powerful modulator of various cellular processes through its interaction with S1P receptors (S1PRs). Currently, this incompletely understood mechanism in pancreatic diseases including pancreatitis and pancreatic cancer, largely limits therapeutic options for these disorders. Recent evidence indicates that S1P significantly contributes to pancreatic diseases by modulating inflammation, promoting pyroptosis in pancreatic acinar cells, regulating the activation of pancreatic stellate cells, and affecting organelle functions in pancreatic cancer cells. Nevertheless, no review has encapsulated these advancements. Thus, this review compiles information about the involvement of S1P signaling in exocrine pancreatic disorders, including acute pancreatitis, chronic pancreatitis, and pancreatic cancer, as well as prospective treatment strategies to target S1P signaling for these conditions. The insights presented here possess the potential to offer valuable guidance for the implementation of therapies targeting S1P signaling in various pancreatic diseases.

Targeting Sphingosine-1-Phosphate Signaling to Prevent the Progression of Aortic Valve Disease.

Aortic valve disease (AVD) is associated with high mortality and morbidity. To date, there is no pharmacological therapy available to prevent AVD progression. Because valve calcification is the hallmark of AVD and S1P (sphingosine-1-phosphate) plays an important role in osteogenic signaling, we examined the role of S1P signaling in aortic stenosis disease. AVD progression and its consequences for cardiac function were examined in a murine wire injury-induced AVD model with and without pharmacological and genetic modulation of S1P production, degradation, and receptor signaling. S1P was measured by LC-MS. Calcification of valvular interstitial cells and their response to biomechanical stress were analyzed in the context of S1P signaling. Human explanted aortic valves from patients undergoing aortic valve replacement and cardiovascular magnetic resonance imaging were analyzed for S1P by LC-MS. Raising S1P concentrations in mice with injury-induced AVD by pharmacological inhibition of its sole degrading enzyme S1P lyase vastly enhanced AVD progression and impaired cardiac function resembling human disease. In contrast, low S1P levels caused by SphK1 (sphingosine kinase 1) deficiency potently attenuated AVD progression. We found S1P/S1PR2 (S1P receptor 2) signaling to be responsible for the adverse S1P effect because S1PR2-deficient mice were protected against AVD progression and its deterioration by high S1P. It is important to note that pharmacological S1PR2 inhibition administered after wire injury successfully prevented AVD development. Mechanistically, biomechanical stretch stimulated S1P production by SphK1 in human valvular interstitial cells as measured by C17-S1P generation, whereas S1P/S1PR2 signaling induced their osteoblastic differentiation and calcification through osteogenic RUNX2/OPG signaling and the GSK3β-Wnt-β-catenin pathway. In patients with AVD, stenotic valves exposed to high wall shear stress had higher S1P content and increased SphK1 expression. Increased systemic or local S1P levels lead to increased valvular calcification. S1PR2 antagonists and SphK1 inhibitors may offer feasible pharmacological approaches to human AVD in prophylactic, disease-modifying or relapse-preventing manners.

Adipose tissue responds to stress-induced immunosuppression affecting immune response partially by miR-145-5p/S1PR1 pathway

Stress-induced immunosuppression (SIIS) is one of the most common problems in intensive poultry production, which can cause immunized chickens to still develop diseases and bring huge losses to production. Recently, adipose tissue, as an immunomodulatory organ, has become a hot topic of attention. However, the function and mechanism of adipose tissue involved in SIIS and its influence on the immune response are still unclear. In this study, we dynamically analyzed the correlations between the T cells migration and change of sphingosine-1-phosphate receptor 1(S1PR1) gene in adipose tissue using chicken models with different immune states, and further explored the regulatory mechanisms and application. The results showed that SIIS could significantly change the expressions of lymphocytes migration related S1PR1 gene, and SIIS could inhibit the Newcastle disease virus (NDV) immune response partially by affecting the migration and proliferation of TCRα+ T cells in adipose tissue. Moreover, the miR-145-5p/S1PR1 pathway was a potential key mechanism to regulate T cells migration in adipose tissue, and circulating miR-145-5p had potential value as a molecular marker. This research can provide innovative reference for in-depth studying the immunoregulatory function and mechanism of adipose tissue.

Ozanimod: A Review in Relapsing Forms of Multiple Sclerosis.

Ozanimod (Zeposia®), an orally administered sphingosine 1-phosphate (S1P) receptor modulator (S1PRM) that is selective for the S1P1 and S1P5 receptor subtypes, is approved in the USA for relapsing forms of multiple sclerosis (RMS). In pivotal phase III clinical trials in patients with RMS, ozanimod significantly reduced annualised relapse rates and the number of new or enlarging T2 lesions and gadolinium-enhancing lesions, and was associated with reduced brain volume loss, compared with interferon (IFN)-β1a. However, there were no significant differences in 3- and 6-month disability progression between the groups. Ozanimod was generally well tolerated, with the most common adverse reactions including upper respiratory tract infection and hepatic transaminase elevation. Efficacy and tolerability were sustained over more than 6 years with continued treatment. S1PRM-related adverse events seen with ozanimod are generally manageable with screening and/or monitoring. Notably, ozanimod does not require first-dose cardiac monitoring in the USA. In conclusion, ozanimod is a valuable once-daily oral disease-modifying therapy that extends the available treatment options for patients with RMS.

Explore the Role of the Sphingosine-1-Phosphate Signalling as a Novel Promising Therapeutic Target for the Management of Parkinson's Disease.

Sphingosine-1-phosphate (S1P) is a cellular signalling molecule derived from sphingosine, which is a pro-apoptotic sphingolipid. Sphingolipids control various cellular actions like growth, homeostasis, and stress-related responses. The main sources of S1P in our body are erythrocytes. S1P controls both cellular mediators and other second messengers intracellularly. The S1P receptor also helps in inflammatory and neuroprotective effects (required to manage of Parkinson's). A large number of anti-Parkinson drugs are available, but still, there is a need for more effective and safer drugs. S1P and its receptors could be targeted as novel drugs due to their involvement in neuro-inflammation and Parkinson's. The present review effort to explore the biological role of S1P and related receptors, for their possible involvement in PD; furthermore. Overall, S1P and other related metabolizing enzymes have significant therapeutic opportunities for Parkinson's disease along with other neurological disorders.

Is FAM19A5 an adipokine? Peripheral FAM19A5 in wild-type, FAM19A5 knockout, and LacZ knockin mice

FAM19A5 is a novel secretory protein expressed primarily in the brain. However, a recent study reported that FAM19A5 is an adipocyte-derived adipokine that regulates vascular smooth muscle function through sphingosine-1-phosphate receptor 2 (S1PR2). In our study, we investigated FAM19A5 transcript and protein levels in peripheral tissues, including adipose tissues, from wild-type, FAM19A5 knockout, and FAM19A5 LacZ knockin mice. We found that the FAM19A5 transcript levels in the central nervous system were much greater than those in any of the peripheral tissues, including adipose tissues. Furthermore, the FAM19A5 protein levels in adipose and reproductive tissues were below detectable limits for Western blot analysis and ELISA. Additionally, we found that the FAM19A5 protein did not interact with S1PR2 in terms of G protein-mediated signal transduction, β-arrestin recruitment, or ligand-mediated internalization. Taken together, our findings revealed basal levels of FAM19A5 transcripts and proteins in peripheral tissues, confirming its primary expression in the central nervous system and lack of significant interaction with S1PR2.

Evaluation of proximod, a selective agonist of sphingosine-1-phosphate receptor-1, in healthy volunteers and patients with rheumatoid arthritis: a phase 1, double-blind, randomised, placebo-controlled, ascending dose trial

Proximod is a selective agonist of sphingosine-1-phosphate receptor-1 (S1PR1). It acts by redirecting lymphocytes from the circulation to secondary lymph nodes, and is under development as an immunomodulator for rheumatoid arthritis. We aimed to evaluate the safety, pharmacokinetics, and preliminary efficacy of proximod in healthy volunteers and patients with rheumatoid arthritis. We did a two part, phase 1, double-blind, randomised, placebo-controlled, ascending dose trial at a single centre in China. Eligible participants were adults aged 18-50 years with a BMI of 18-28 kg/m2 for healthy volunteers and aged 18-70 years with a BMI of 18-30 kg/m2 for patients with rheumatoid arthritis. In part 1, healthy volunteers were randomly assigned within ten cohorts to receive a single oral dose of proximod (0·125 mg, 0·25 mg, 0·5 mg, 1 mg, 1·5 mg, 2 mg, 3 mg, 5 mg, 10 mg, or 15 mg in cohorts 1-10) or placebo. In part 2, healthy volunteers were randomly assigned to receive once-daily doses of proximod 5 mg or placebo, and patients with rheumatoid arthritis were randomly assigned to receive once-daily doses of proximod 5 mg, proximod 10 mg, or placebo, for 28 days. Patients and investigators were masked to treatment assignment. The primary outcomes were safety, tolerability, and pharmacokinetic profile of proximod for 72 days in healthy volunteers and for 48 days in patients with rhematoid arthritis, assessed in all treated participants. This trial is registered with ClinicalTrials.gov (NCT06361199, NCT06361186), and is complete. Between Nov 1, 2017, and June 22, 2021, 124 healthy volunteers were randomly assigned in part 1 of the study and 124 were included in the analyses (mean age 34·3 years [SD 6·9], 62 [50%] of 124 participants were women and 62 [50%] were men, and 116 [94%] were Han Chinese ethnicity). Between Feb 16, 2022, and Oct 8, 2023, 113 participants were screened for inclusion in part 2 (80 healthy volunteers and 33 patients with rheumatoid arthritis). 79 participants were excluded and 34 were randomly assigned (10 healthy participants and 24 patients with rheumatoid arthritis), 34 of whom were included in the analyses. Ten (100%) of ten healthy participants were Han Chinese ethnicity, with a mean age of 39·9 years (SD 7·3). Five (50%) of ten healthy volunteers were women and five (50%) were men). 22 (92%) of 24 participants with rheumatoid arthritis were Han Chinese ethnicity, with a mean age of 52·7 years (SD 6·8). 22 (92%) of 24 patients with rheumatoid arthritis were women and two (8%) were men. In part 1, all doses of proximod were well tolerated, with no dose-related adverse reactions or serious adverse events observed. In part 2, 74 adverse reactions were reported in eight (80%) of ten healthy volunteers and 22 (92%) of 24 patients with rheumatoid arthritis. Adverse events associated with proximod were predominantly mild or moderate. In part 2, the concentration of proximod and its active metabolite, proximod-phosphate, gradually increased in all three groups receiving proximod and the EC50 of the S1PR1 agonist for proximod-phosphate (6·1 ng/mL) was reached on day 14 for both 5 mg groups, and on day 7 for the 10 mg group. The mean Ctrough values for proximod-phosphate on day 28 were 7·7 ng/mL and 10·2 ng/mL for 5 mg in healthy volunteers and patients with rheumatoid arthritis, respectively, and 15·3 ng/mL for 10 mg in patients with rheumatoid arthritis. In patients with rheumatoid arthritis, lymphocyte count decreased after treatment in all proximod groups reaching nadir at approximately day 28, with a corresponding percentage decline from baseline of 65·25% in the 5 mg group, 71·64% in the 10 mg group, and 20·57% in the placebo group. Proximod exhibited good tolerability over the 28-day treatment period, demonstrating its potential in reducing blood lymphocyte count. These results highlight the promise of the S1PR1 agonist proximod as a potential candidate for rheumatoid arthritis treatment, warranting further investigation in subsequent clinical studies. Beijing Union Pharmaceutical Factory and Jian Kuan (Suzhou) Biotechnology.

Anticancer Effects of BAF312 (Siponimod) in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is a lethal disease that is the fifth leading cause of cancer-related death in women. BAF312 (siponimod) is a potent and selective sphingosine-1-phosphate (S1P) receptor modulator that has been approved as a treatment for multiple sclerosis. In addition to its immunomodulatory action, BAF312 shows preclinical antitumor effects in several cancer types. This study sought to determine whether BAF312 had anticancer properties against EOC using in vitro and in vivo models. EOC cell lines A2780, SKOV3ip1, A2780-CP20, and SKOV3-TR were treated with BAF312 and tested for cell proliferation, apoptosis, and migration using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, fluorescence-activated cell sorting, and migration assays. We investigated the expression of sphingosine-1-phosphate receptor 1 (S1PR1) in most EOC cell lines through western blot analysis. To investigate potential mechanisms, western blot analysis was used to assess the expression of AKT serine/threonine kinase 1 (AKT) and extracellular-regulated kinase (ERK) after BAF312 treatment. We also created poly(D,L-lactide-co-glycolide) nanoparticles encapsulating BAF312 (PLGA-NP-BAF312) for in vivo therapy. The average size and zeta potential of PLGA-NP-BAF312 were determined using dynamic light scattering. The therapeutic efficacy of PLGA-NP-BAF312 was tested in an A2780 tumor-bearing orthotopic mouse model of EOC. S1PR1 was overexpressed in most EOC cell lines. BAF312 significantly reduced cell proliferation and migration while inducing significant apoptosis in all EOC cell lines. PLGA-NP-BAF312 treatment significantly reduced tumor weights in A2780 tumor-bearing mice. Furthermore, the anticancer effects of BAF312 were associated with reduced phosphorylation of ERK and AKT. Our findings show that BAF312 has significant anticancer effects in EOC cells by inhibiting the ERK and AKT pathways, and might potentially be used to treat EOCs.

Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE2 production

BackgroundMitochondria play a crucial role in shaping the macrophage inflammatory response during bacterial infections. Spinster homolog 2 (Spns2), responsible for sphingosine-1-phosphate (S1P) secretion, acts as a key regulator of mitochondrial dynamics in macrophages. However, the link between Spns2/S1P signaling and mitochondrial functions remains unclear.MethodsPeritoneal macrophages were isolated from both wild-type and Spns2 knockout rats, followed by non-targeted metabolomics and RNA sequencing analysis to identify the potential mediators through which Spns2/S1P signaling influences the mitochondrial functions in macrophages. Various agonists and antagonists were used to modulate the activation of Spns2/S1P signaling and its downstream pathways, with the underlying mechanisms elucidated through western blotting. Mitochondrial functions were assessed using flow cytometry and oxygen consumption assays, as well as morphological analysis. The impact on inflammatory response was validated through both in vitro and in vivo sepsis models, with the specific role of macrophage-expressed Spns2 in sepsis evaluated using Spns2flox/floxLyz2-Cre mice. Additionally, the regulation of mitochondrial functions by Spns2/S1P signaling was confirmed using THP-1 cells, a human monocyte-derived macrophage model.ResultsIn this study, we unveil prostaglandin E2 (PGE2) as a pivotal mediator involved in Spns2/S1P-mitochondrial communication. Spns2/S1P signaling suppresses PGE2 production to support malate-aspartate shuttle activity. Conversely, excessive PGE2 resulting from Spns2 deficiency impairs mitochondrial respiration, leading to intracellular lactate accumulation and increased reactive oxygen species (ROS) generation through E-type prostanoid receptor 4 activation. The overactive lactate-ROS axis contributes to the early-phase hyperinflammation during infections. Prolonged exposure to elevated PGE2 due to Spns2 deficiency culminates in subsequent immunosuppression, underscoring the dual roles of PGE2 in inflammation throughout infections. The regulation of PGE2 production by Spns2/S1P signaling appears to depend on the coordinated activation of multiple S1P receptors rather than any single one.ConclusionsThese findings emphasize PGE2 as a key effector of Spns2/S1P signaling on mitochondrial dynamics in macrophages, elucidating the mechanisms through which Spns2/S1P signaling balances both early hyperinflammation and subsequent immunosuppression during bacterial infections.

Sphingosine-1-phosphate receptor type 4 is critically involved in the regulation of peritoneal B-1 cell trafficking and distribution in vivo.

B-1 cells are crucially involved in immune defense and regulation of inflammation and autoimmunity. B-1 cells are predominantly located in the peritoneal and pleural cavities, although body cavity B-1 cells recirculate systemically under steady-state conditions. The chemokines CXCL12 and CXCL13 have been identified as the main regulators of peritoneal B-cell trafficking. In mice deficient for sphingosine-1-phosphate receptor 4 (S1PR4), B-1a and B-1b cell numbers are reduced in the peritoneal cavity by an unknown mechanism. In this study, we show that S1PR4-mediated S1P signaling modifies the chemotactic response of peritoneal B cells to CXCL13 and CXCL12 in vitro. In vivo, S1PR4-mediated S1P signaling affects both immigration into and emigration from the peritoneal cavity. Long-term reconstitution experiments of scid mice with wt or s1pr4 -/- peritoneal B cells revealed a distinct distributional pattern in secondary lymphoid organs. As a functional consequence, both plasmatic and mucosal IgM levels, the main product of B-1a cells, are reduced in mice reconstituted with s1pr4 -/- peritoneal cells. In summary, our data identify S1PR4 as the second S1P receptor (besides S1PR1), which is critically involved in the regulation of peritoneal B-1 cell function.

Effectiveness of electro-acupuncture for cognitive improvement on Alzheimer's disease quantified via PET imaging of sphingosine-1-phosphate receptor 1.

Electro-acupuncture (EA) has demonstrated potential in improving mild-to-moderate dementiain clinics, but the underlying scientific target remains unclear. EA was administered to APP/PS1 Alzheimer's disease (AD) mice, with untreated AD, and wild type (WT) mice serving as controls. The efficacy of EA was assessed by the Morris water mazecognitive functional tests. Brain magnetic resonance imaging-positron emission tomography (PET) scans using [18F]TZ4877 targeting sphingosine-1-phosphate receptor 1 (S1PR1) and [18F]AV45 targeting amyloid beta fibrils were conducted. The correlation between regionalbrain PET quantificationsand cognitive functions was analyzed. EA significantlyimproved cognitive and memoryfunctions of AD (p = 0.04) and reduced the uptake of [18F]TZ4877 in the cortex (p = 0.02) and hippocampus (p = 0.03). Immunofluorescence confirmed colocalizations of S1PR1 with glial fibrillary acidic protein and ionized calcium-binding adaptor molecule-1. Furthermore, immunohistochemistry showed a significant reduction of interleukin 1β and tumor necrosis factor α after EA treatment. EA may reverse AD by suppressing neuroinflammation, and the PET imaging of S1PR1 seemed potent in evaluating the treatment for AD patients HIGHLIGHTS: Electro-acupuncture (EA) was administered to APP/PS1 Alzheimer's disease (AD) mice, with untreated AD, and wild type (WT) mice serving as controls. The efficacy of EA was assessed by the Morris water maze cognitive functional tests and positron emission tomography (PET) imaging quantifications. PET tracer [18F]AV45 was used to detect amyloid beta deposition. An increased uptake of [18F]AV45 was found in AD compared to WT mice, with significance observed only in the cortex and not in the hippocampus. EA treatment exhibited a trend toward reduced [18F]AV45 uptake in AD mouse brains post-treatment. However, statistical difference was not attained in most brain regions. EA "Baihui (DU20) and Sishencong (EX-HN1)" significantly improved cognitive and memory functions of AD (p = 0.04). Brain magnetic resonance imaging p(MRI)-positron emission tomography (PET) quantifications revealed that significantly reduced the uptake of [18F]TZ4877 in the cortex (p = 0.02) and hippocampus (p = 0.03) after EA treatment. The correlation between PET quantifications and cognitive functions was analyzed and the most notable correlations were found between escape latency (reaction cognitive and memory behavior) and volume distribution (VT) quantifications of [18F]TZ4877. VT quantifications of [18F]TZ4877 in key brain regions for cognitive and memory ability, such as the cortex and hippocampus, positively correlated with platform latency (cortex p < 0.01, r = 0.7102; hippocampus p < 0.01, r = 0.6891). Immunofluorescence confirmed colocalizations of S1PR1 with glial fibrillary acidic protein and ionized calcium-binding adaptor molecule-1 in the AD brain. And the EA treatment significantly reduced the signals in the cortex and hippocampus. Immunohistochemistry showed a significant reduction of interleukin 1β and tumor necrosis factor α after EA treatment. EA reversed AD by suppressing neuroinflammation in the cortex and hippocampus. The S1PR1 targeting PET tracer [18F]TZ4877 showed promise in evaluating the pathological progression of AD in clinical settings.