Sphingosine-1-phosphate Levels Research Articles

BXQ-350: Modulating ceramide and sphingosine-1-phosphate for antitumor activity in patients with advanced CRC.

154 Background: Sphingolipids are a class of bioactive signaling molecules implicated in multiple cellular processes and molecular pathways. Many publications have demonstrated that ceramides are proapoptotic, synergize with cancer treatments, and mitigate chemoresistance. Findings also demonstrated that sphingosine-1-phosphate (S1P) is a key sphingolipid that promotes cancer cell proliferation, activates multiple oncogenic pathways, and stimulates immuno-suppressor cell populations promoting a pro-tumoral microenvironment. Several studies of colorectal cancer patients have shown high levels of ceramides being associated with improved survival, while high S1P levels are associated with a poor prognosis. Hence, modulation of sphingolipid metabolism continues to be a promising treatment approach. Methods: BXQ-350 is a nanovesicle of Saposin C, an allosteric activator of sphingolipid metabolism, that lowers systemic S1P and increases C18 ceramide. BXQ-350 was investigated in a Phase 1 dose-escalation safety study in an all-comer cancer patients with advanced solid malignancies ( NCT02859857 ) to determine its safety profile and potential clinical activity as monotherapy. Samples were collected to explore potential biomarkers. Results: 13 patients (~17.8% of evaluable patients) had a clinical benefit up to cycle 6 (PR, SD), with the majority experiencing a decrease in systemic S1P levels and an increase in C18 levels. 8 patients (~11% of evaluable patients) had PFS > 6 months, with 2 patients still on study six years after enrollment. Analysis of plasma samples also revealed an increase in anti-tumoral cytokines (IFNg, TNFa, IL-2) and a decrease in pro-tumoral ones (IL-6, 8, 10). Among patients with PFS > 6 months, there were 4 recurrent CRC patients (1PR, 3SD): 1 patient had a PFS of ~12 months, 2 of ~18 months, and 1 is still on study after 6 years. Conclusions: Results of this Phase 1 study in heavily pretreated patients show BXQ-350 was well tolerated and seem to generate a clinical benefit in CRC patients via modulation of S1P and ceramides. A phase 2 trial of BXQ-350 in combination with FOLFOX/Bevacizumab in newly diagnosed mCRC is on-going with plans to further investigate this novel mechanism of action. Clinical trial information: 02859857 .

Associations between multiple heavy metals exposure and neural damage biomarkers in welders: A cross-sectional study

BackgroundBoth occupational and environmental exposure to heavy metals are associated with various neurodegenerative diseases. However, limited evidence is available on the potential effects of exposure to metallic mixtures and neural damage. ObjectivesThis study aimed to evaluate the association between metal mixtures in urine and neural damage biomarkers in welders. MethodsIn this cross-sectional study, a total of 186 workers were recruited from steel mills. Twenty-three metals in urine were measured by inductively coupled plasma mass spectrometry. Serum neural damage biomarkers, including neurofilament light chain (NfL), sphingosine-1-phosphate (S1P), prolactin (PRL), and dopamine (DA) were detected using enzyme-linked immunosorbent assay kits. Multivariable linear regression, Bayesian kernel machine regression (BKMR), and Quantile g-computation (QG-C) were employed to estimate the association between metals exposure and neural damage biomarkers. ResultsInverted u-shaped associations of nickel with NfL, S1P, and DA were observed in the BKMR model. A non-linear relationship was also found between Fe and PRL. Urinary cobalt was positively associated with serum PRL and had the strongest positive weights in the QG-C model. Urinary lead was associated with higher serum S1P levels. We also found the interaction among nickel, zinc, arsenic, strontium, iron, and lead with the neural damage biomarkers. ConclusionThis study provides new evidence of a direct association between metal mixture exposure and the serum biomarkers of neural damage. Several metals Ni, Co, Pb, Sr, As and Fe, may have adverse effects on the nervous system, while Zn may have neuroprotective effects.

Receptor-dependent effects of sphingosine-1-phosphate (S1P) in COVID-19: the black side of the moon.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection leads to hyper-inflammation and amplified immune response in severe cases that may progress to cytokine storm and multi-organ injuries like acute respiratory distress syndrome and acute lung injury. In addition to pro-inflammatory cytokines, different mediators are involved in SARS-CoV-2 pathogenesis and infection, such as sphingosine-1-phosphate (S1P). S1P is a bioactive lipid found at a high level in plasma, and it is synthesized from sphingomyelin by the action of sphingosine kinase. It is involved in inflammation, immunity, angiogenesis, vascular permeability, and lymphocyte trafficking through G-protein coupled S1P receptors. Reduction of the circulating S1P level correlates with COVID-19 severity. S1P binding to sphingosine-1-phosphate receptor 1 (S1PR1) elicits endothelial protection and anti-inflammatory effects during SARS-CoV-2 infection, by limiting excessive INF-α response and hindering mitogen-activated protein kinase and nuclear factor kappa B action. However, binding to S1PR2 opposes the effect of S1PR1 with vascular inflammation, endothelial permeability, and dysfunction as the concomitant outcome. This binding also promotes nod-like receptor pyrin 3 (NLRP3) inflammasome activation, causing liver inflammation and fibrogenesis. Thus, higher expression of macrophage S1PR2 contributes to the activation of the NLRP3 inflammasome and the release of pro-inflammatory cytokines. In conclusion, S1PR1 agonists and S1PR2 antagonists might effectively manage COVID-19 and its severe effects. Further studies are recommended to elucidate the potential conflict in the effects of S1P in COVID-19.

Association of gut microbiota in the pathogenesis of acute respiratory distress syndrome and possible pharmacotherapy by sphingosine-1-phosphate

The Acute Respiratory Distress Syndrome (ARDS) is one of the major heterogeneous clinical manifestations of severe respiratory failure developing in response to pathogenetic mechanisms and several inflammatory insults. The translocation of gut microbiota has a crucial impact on the pathogenesis of ARDS. Hence, a deeper understanding of the interplay of the gut microbiota would allow shedding valuable insights into both the pathogenesis of ARDS and the development of effective therapeutic interventions. Moreover, the modulation of gut-lung axis could also play a pivotal role in minimizing the lung dysbiosis with gut microbiota. There is little question that greater study of the gut-lung axis in critically ill patients is required to establish causal links between the shifted microbiota, infections, inflammation, and acute lung damage. It is worth mentioning that the lack of effective preventative measures is one of the main reasons for the increased mortality rate of 30-40% in ARDS patients. Some antibiotics and nanosized drugs showed positive results in ARDS management to some extent in pre-clinical or even first stages of clinical trials but large-scale results had been controversial. However, Sphingosine-1-Phosphate (S1P) showed hopeful results in ARDS patients by facilitating both systemic and endothelial integrity. Numerous investigations have shown the immunological connection between the gastrointestinal tract and the respiratory system. As the SphKs /S1P /S1PL metabolic pathway is associated with a wide variety of human illnesses (including respiratory diseases), it should come as no surprise that influencing intracellular S1P levels would have therapeutic promise in reducing the severity of lung diseases like ARDS.

Safety and Clinical Effects of a Muse Cell-Based Product in Patients With Amyotrophic Lateral Sclerosis: Results of a Phase 2 Clinical Trial

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of motor neurons. Multilineage-differentiating stress-enduring (Muse) cells are unique endogenous stem cells that show therapeutic effects on motor function in ALS mouse models. We conducted a single-center open phase II clinical trial to evaluate the safety and clinical effects of repeated intravenous injections of an allogenic Muse cell-based product, CL2020, in patients with ALS. Five patients with ALS received CL2020 intravenously once a month for a total of six doses. The primary endpoints were safety and tolerability, and the secondary endpoint was the rate of change in the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score. In addition, serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), sphingosine-1-phosphate (S1P), cerebrospinal fluid chitotriosidase-1 (CHIT-1), and neurofilament light chain (NfL) levels were evaluated. The CL2020 treatment was highly tolerated without serious side effects. The ALSFRS-R score change trended upward at 12 months post-CL2020 treatment compared with that at 3 months pre-administration, but the difference was not statistically significant. Among five patients diagnosed with ALS, three exhibited a decrease in the rate of ALSFRS-R score change, one demonstrated an increase, and another showed no change. In addition, the patients’ serum IL-6 and TNF-α levels and cerebrospinal fluid CHIT-1 and NfL levels increased for up to 6 months post-treatment; however, their serum S1P levels continuously decreased over 12 months. These findings indicate a favorable safety profile of CL2020 therapy. In the near future, a double-blind study of a larger number of ALS patients should be conducted to confirm the efficacy of ALS treatment with CL2020.

S1P lyase inhibition prevents lung injury following high pressure-controlled mechanical ventilation in aging mice

Ventilator-induced Lung Injury (VILI) is characterized by hypoxia, inflammatory cytokine influx, loss of alveolar barrier integrity, and decreased lung compliance. Aging influences lung structure and function and is a predictive factor in the severity of VILI; however, the mechanisms of aging that influence the progression or increased susceptibility remain unknown. Aging impacts immune system function and may increase inflammation in healthy individuals. Recent studies suggest that the bioactive sphingolipid mediator sphingosine-1-phosphate (S1P) and the enzyme that degrades it S1P lyase (SPL) may be involved in lung pathologies including acute lung injury. It is unknown whether aging influences S1P and SPL expression that have been implicated in lung inflammation, injury, and cell apoptosis. We hypothesized that aging and injurious mechanical ventilation synergistically impair S1P levels and enhance S1P lyase (SPL) expression that amplifies alveolar barrier damage and diminishes pulmonary function. Young (2–3 mo) and old (20–25 mo) C57BL/6 mice were mechanically ventilated for 2 h using pressure-controlled mechanical ventilation (PCMV) at 45 cmH2O and 35 cmH2O, respectively. We assessed the impact of aging and PCMV on several indications of acute lung injury, immune cell recruitment, S1P levels and SPL activity. Furthermore, we evaluated the protective effects of inhibiting SPL by tetrahydroxybutylimidazol (THI) administration on the negative outcomes associated with aging and mechanical injury. PCMV exacerbated lung injury in old mice and increased neutrophil influx that was further exacerbated due to aging. SPL expression increased in the young and old ventilated mice and the old nonventilated group. THI treatment reduced several of the indicators of lung injury and resulted in elevated S1P levels in lung tissue and plasma from mice that were injured from mechanical ventilation. CD80 and CD206 activation markers of alveolar and interstitial macrophages were also influenced by THI. SPL inhibition may be a viable therapeutic approach for patients requiring mechanical ventilation by preventing or regulating the exaggerated inflammatory response and reducing lung injury.

Apolipoprotein D modulates lipid mediators and osteopontin in an anti-inflammatory direction.

HDL has been proposed to possess anti-inflammatory properties; however, the detail mechanisms have not been fully elucidated. We investigated the roles of Apolipoprotein D (ApoD) in the pathogenesis of inflammation in the mouse model of diet-induced obesity and that of lipopolysaccharide-induced sepsis and the in vitro experiments. Furthermore, we analyzed serum ApoD levels in human subjects. The overexpression of human ApoD decreased the plasma IL-6 and TNF-a levels in both mice models. Lipidomics analyses demonstrated association of ApoD with increase of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, as well as of their metabolites, and of the anti-inflammatory molecule sphingosine 1-phosphate, and decrease of proinflammatory lysophosphatidic acids and lysophosphatidylinositol. ApoD-containing lipoproteins might directly bind eicosapentaenoic acid and docosahexaenoic acid. The modulations of the lysophosphatidic acid and sphingosine 1-phosphate levels resulted from the suppression of autotaxin expression and elevation of apolipoprotein M (ApoM), respectively. Moreover, ApoD negatively regulated osteopontin, a proinflammatory adipokine. The activation of PPARg by ApoD might suppress autotaxin and osteopontin. Serum ApoD levels were negatively correlated with the serum osteopontin and autotaxin levels and, positively with serum ApoM levels. ApoD is an anti-inflammatory apolipoprotein, which modulates lipid mediators and osteopontin in an anti-inflammatory direction.

Plasma sphingolipid abnormalities in neurodegenerative diseases.

In recent years, there has been increasing evidence that several lipid metabolism abnormalities play an important role in the pathogenesis of neurodegenerative diseases. However, it is still unclear which lipid metabolism abnormalities play the most important role in neurodegenerative diseases. Plasma lipid metabolomics (lipidomics) has been shown to be an unbiased method that can be used to explore lipid metabolism abnormalities in neurodegenerative diseases. Plasma lipidomics in neurodegenerative diseases has been performed only in idiopathic Parkinson's disease (IPD) and Alzheimer's disease (AD), and comprehensive studies are needed to clarify the pathogenesis. In this study, we investigated plasma lipids using lipidomics in individuals with neurodegenerative diseases and healthy controls (CNs). Plasma lipidomics was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in those with IPD, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), AD, and progressive supranuclear palsy (PSP) and CNs. The results showed that (1) plasma sphingosine-1-phosphate (S1P) was significantly lower in all neurodegenerative disease groups (IPD, DLB, MSA, AD, and PSP) than in the CN group. (2) Plasma monohexylceramide (MonCer) and lactosylceramide (LacCer) were significantly higher in all neurodegenerative disease groups (IPD, DLB, MSA, AD, and PSP) than in the CN group. (3) Plasma MonCer levels were significantly positively correlated with plasma LacCer levels in all enrolled groups. S1P, Glucosylceramide (GlcCer), the main component of MonCer, and LacCer are sphingolipids that are biosynthesized from ceramide. Recent studies have suggested that elevated GlcCer and decreased S1P levels in neurons are related to neuronal cell death and that elevated LacCer levels induce neurodegeneration by neuroinflammation. In the present study, we found decreased plasma S1P levels and elevated plasma MonCer and LacCer levels in those with neurodegenerative diseases, which is a new finding indicating the importance of abnormal sphingolipid metabolism in neurodegeneration.

Erythrocyte Sphingosine 1 Phosphate Is Essential to Combat HIF1α-Dependent Creatine Shuttle, Macrophage Polarization and Fibrosis

Erythrocytes have long been considered as the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), which is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and polarization of tissue macrophages. Currently, emerging evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O2) delivery to counteract tissue hypoxia. However, whether O2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization, inflammation and fibrosis remains unrecognized. Here we report that genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte eSphK1-/-) resulted in less O2 delivery from erythrocytes, thus leading to severe hypoxic tissue microenvironment, kidney damage and fibrosis in a well-accepted pathological hypoxic model infused with angiotensin II (a potent vasoconstrictor known to be elevated in hypertensive patients). Surprisingly, although plasma S1P levels were reduced to 50% in Ang II-infused eSphK1-/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules (one of the most hypoxic sites with Ang II infusion). Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. Further studies demonstrated that no significant differences of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1-/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization in Ang II-infused eSphK1-/- mice. Instead, we revealed that reduced O2 offload from erythrocytes was previously unrecognized but essential to promote tissue M2 polarization and fibrosis by upregulating creatine shuttle to meet rapid intracellular energy needs when FAs, AAs and O2 were insufficient due to hypoxia in Ang II-infused eSphK1-/- mice. At cellular and molecular level, we demonstrated that hypoxia signaling via HIF-1a directly induced M2 polarization as well as expression of Slc6a8 (a gene encoding creatine transport), M2 specific marker gene and fibrotic marker gene in cultured murine BMDMs. Overall, our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, tissue macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for macrophage polarization and tissue fibrosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Clinical and Translational Exploration of the Role of S1P/SPHK1 Pathway in Non-Hodgkin Lymphoma with Obesity

Non-Hodgkin lymphoma (NHL) is among the top 10 malignant tumors in incidence and mortality. Accumulating evidence indicates that obesity may influence the prognosis of several types of lymphoma, but the underlying mechanism is largely unknown for contributing to the poor prognosis or the aggressiveness of NHL. Obesity can result in abnormal lipid metabolism, especially abnormal sphingolipid metabolism. Existing data indicate the carcinogenetic role of Sphingosine-1-phosphate (S1P)/ sphingosine kinases (SPHK) signaling, we hypothesized that SIP/SPHK signaling played an important role in lymphomagenesis. To test the hypothesis that obesity deteriorate the prognosis of NHL, we retrospectively investigated a cohort of 2094 patients who were diagnosed with lymphoma in the First Hospital of Jilin University from Jun 1st, 2011 to Jun 1st 2021. These patients were categorized into high Body Mass Index (BMI, defined as BMI≥25) group and low BMI (defined as BMI<25) group. The demographic information and imaging evidence of disease progression were extracted and analyzed. Survival data were follow-up till Jun 1st 2021. Progression-free survival (PFS) and overall survival (OS) of three main subtypes of lymphomas (namely Diffuse large B-cell lymphoma [DLBCL], follicular lymphoma [FL], peripheral T-cell lymphoma-not otherwise specified [PTCL-NOS]) in this cohort were compared with the time-to-event analysis. High BMI group had a negative impact on PFS and OS in all three groups (DLBCL [P < 0.05], FL [P < 0.05], and PTCL-NOS [P < 0.05]).（A） To investigate S1P signaling, we performed an IHC study to determine the levels of S1P in the tissue array-samples from various human lymphomas. By computer-imaging analysis, significantly increased S1P levels were detected in the tissues from DLBCL, follicular lymphoma (FL), and peripheral T-cell lymphoma (PTCL). Using a web-based database, Gene Expression Omnibus (GEO), the high throughput gene expression was profiled for SPHK1 and SPHK2 in the lymphomas (DLBCL, FL, and PTCL) and the respective normal controls. Significantly up-regulated expression of SPHK1 was found in DLBCL (n = 55) versus centroblast B-cells (n = 7), in FL (n = 14) versus normal B-cells (n = 5), and in PTCL (n = 80) versus normal T cells (n =10). Significantly up-regulated expression of SPHK2 were also detected in DLBCL but not in FL and PTCL in comparison with their respective normal controls.（B-C） To further investigate the potential mechanism of S1P-S1P receptors mediated lymphogenisis, we employed two human lymphoma cell lines (HH for T cell lymphoma and SU-DHL-4 for B cell lymphoma) for verification. The S1P mediated down-regulation of YAP phosphorylation was found in both HH cells and SU-DHL-4 cells. WB showed up-regulated S1PR1 and S1PR3, and down-regulated S1PR2, consistent with the qPCR results. Three S1PR inhibitors (W146 for S1PR1, CAY10444 for S1PR3, and FTY720 for S1PR1-5) were selected to confirm the hypothesis that S1P signaling could mediate the HIPPO pathway in lymphoma cells. XTT cell viability assay and a trans-well assay as well as WB were further performed to investigate cell proliferation, migration, and EMT. Consistently, blockage of S1PR1 and/or S1PR3 significantly inhibited the S1P induced cell proliferation and migration, down regulated the Cyclin D1 expression, and attenuated the alterations of E-cadherin/Vimentin in both HH cells and SU-DHL-4 cells. These results indicated that blockage of S1PR1 and/or S1PR3 significantly attenuated the S1P-induced decreases of YAP phosphorylation in both HH cells and SU-DHL-4 cells.（D） In conclusion, the NHL patients with obesity have a poor PFS and OS, which is associated with the elevated S1P-SPHK1 signaling. S1P-S1PR1/S1PR3-YAP signaling mediates lymphomagenesis contributing to tumor aggressive growth. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Crosstalk between FcεRI and Sphingosine Signaling in Allergic Inflammation.

Sphingolipid molecules have recently attracted attention as signaling molecules in allergic inflammation diseases. Sphingosine-1-phosphate (S1P) is synthesized by two isoforms of sphingosine kinases (SPHK 1 and SPHK2) and is known to be involved in various cellular processes. S1P levels reportedly increase in allergic inflammatory diseases, such as asthma and anaphylaxis. FcεRI signaling is necessary for allergic inflammation as it can activate the SPHKs and increase the S1P level; once S1P is secreted, it can bind to the S1P receptors (S1PRs). The role of S1P signaling in various allergic diseases is discussed. Increased levels of S1P are positively associated with asthma and anaphylaxis. S1P can either induce or suppress allergic skin diseases in a context-dependent manner. The crosstalk between FcεRI and S1P/SPHK/S1PRs is discussed. The roles of the microRNAs that regulate the expression of the components of S1P signaling in allergic inflammatory diseases are also discussed. Various reports suggest the role of S1P in FcεRI-mediated mast cell (MC) activation. Thus, S1P/SPHK/S1PRs signaling can be the target for developing anti-allergy drugs.

Association of apolipoprotein M and sphingosine-1-phosphate with brown adipose tissue after cold exposure in humans

The HDL-associated apolipoprotein M (apoM) and its ligand sphingosine-1-phosphate (S1P) may control energy metabolism. ApoM deficiency in mice is associated with increased vascular permeability, brown adipose tissue (BAT) mass and activity, and protection against obesity. In the current study, we explored the connection between plasma apoM/S1P levels and parameters of BAT as measured via 18F-FDG PET/CT after cold exposure in humans. Fixed (n = 15) vs personalized (n = 20) short-term cooling protocols decreased and increased apoM (− 8.4%, P = 0.032 vs 15.7%, P < 0.0005) and S1P (− 41.0%, P < 0.0005 vs 19.1%, P < 0.005) plasma levels, respectively. Long-term cooling (n = 44) did not affect plasma apoM or S1P levels. Plasma apoM and S1P did not correlate significantly to BAT volume and activity in the individual studies. However, short-term studies combined, showed that increased changes in plasma apoM correlated with BAT metabolic activity (β: 0.44, 95% CI [0.06–0.81], P = 0.024) after adjusting for study design but not BAT volume (β: 0.39, 95% CI [− 0.01–0.78], P = 0.054). In conclusion, plasma apoM and S1P levels are altered in response to cold exposure and may be linked to changes in BAT metabolic activity but not BAT volume in humans. This contrasts partly with observations in animals and highlights the need for further studies to understand the biological role of apoM/S1P complex in human adipose tissue and lipid metabolism.

The Impact of Sphingosine Kinases on Inflammation-Induced Cytokine Release and Vascular Endothelial Barrier Integrity.

Sphingosine kinases type 1 and 2 (SphK1/2) are required for the production of the immune modulator sphingosine 1-phosphate (S1P). SphK1 deficient mice (SphK1-/-) revealed 50% reduced S1P in plasma, while SphK2-/- mice demonstrated 2-3 times increased S1P levels in plasma. Since plasma S1P is a potent inducer of vascular endothelial cell (VEC) barrier stability, we hypothesized that higher and lower levels of S1P in SphK2-/- and SphK1-/- mice, respectively, compared to wild type (wt) mice should translate into decreased and increased severity of induced systemic inflammation due to improved or damaged VEC barrier maintenance. To our surprise, both SphK1-/- and SphK2-/- mice showed improved survival rate and earlier recovery from inflammation-induced weight loss compared to wt mice. While no difference was observed in VEC barrier stability by monitoring Evans blue leakage into peripheral tissues, SphK1-/- mice demonstrated a distinct delay and SphK2-/- mice an improved resolution of early pro-inflammatory cytokine release in plasma. Ex vivo cell culture experiments demonstrated that bone marrow-derived dendritic cells (BMDC) generated from SphK1-/- and SphK2-/- mice responded with decreased interferon-γ (IFN-γ) production upon stimulation with lipopolysaccharides (LPS) compared to wt BMDC, while activation-induced cytokine expression of lymphocytes and macrophages was not majorly altered. Ex vivo stimulation of macrophages with IFN-γ resulted in increased cytokine release. These results suggest that SphK1/2 are involved in production and secretion of IFN-γ by DC. DC-derived IFN-γ subsequently stimulates the production and secretion of a large panel of inflammatory cytokines by macrophages, which belong to the main cytokine-releasing cells of the early innate immune response. Inhibitors of SphK1/2 may therefore be attractive targets to dampen the early cytokine response of macrophages as part of the innate immune response.

New mechanism for mesenchymal stem cell microvesicle to restore lung permeability: intracellular S1P signaling pathway independent of S1P receptor-1

BackgroundMicrovesicles (MVs) derived from human bone marrow mesenchymal stem cell (MSC) were demonstrated to restore lung protein permeability and attenuate acute lung injury. In our previous study, we found that MSC MV increased sphingosine-1-phosphate (S1P) kinase1 mRNA levels in injured human lung microvascular endothelial cells (HLMVEC) significantly. However, the role of S1P signaling in MSC MV to restore lung protein permeability is unknown.MethodsIn this study, we hypothesized that MSC MV might restore lung permeability in part through increasing intracellular S1P signaling pathway in injured HLMVEC independent of S1P receptors. We used the transwell co-culture system to study the effect of MSC MV on protein permeability of Lipopolysaccharide (LPS) damaged HLMVEC.ResultsOur results showed that LPS significantly increased the permeability of HLMVEC to FITC-dextran (70 kDa) within 24 h. MSC MV restores this permeability and, to a large extent, prevents the cytoskeleton protein F-actin from recombining into “actin stress fibers,” and restores the positions of tight junctions and adhesion junctions in the damaged HLMVEC. This therapeutic effect of MSC MV was related to the increase in the S1P level in injured HLMVEC and was not eliminated when adding the antagonist of S1P receptor, suggesting that MSC MV to restore lung permeability was independent of S1P receptors on HLMVEC. Laser confocal further observed that Ca2+ mobilization and Rac1 activation in LPS injured HLMVEC were increased in parallel with the increase in intracellular S1P level after MSC MV treatment.ConclusionsIn short, MSC MV partially restored protein permeability across HLMVEC through the intracellular S1P signaling pathway independent of S1P receptor-1.

Postnatal deletion of Spns2 prevents neuroinflammation without compromising blood vascular functions.

Protein Spinster homolog 2 (Spns2) is a sphingosine-1-phosphate (S1P) transporter that releases S1P to regulate lymphocyte egress and trafficking. Global deletion of Spns2 (Spns2-/-) has been shown to reduce disease severity in several autoimmune disease models. To examine whether Spns2 could be exploited as a drug target, we generated and characterized the mice with postnatal knockout of Spns2 (Spns2-Mx1Cre). Our results showed that Spns2-Mx1Cre mice had significantly low number of lymphocytes in blood and lymphoid organs similar to Spns2-/- mice. Lymph but not plasma S1P levels were significantly reduced in both groups of knockout mice. Our lipidomic results also showed that Spns2 releases different S1P species into lymph. Interestingly, lymphatic vessels in the lymph nodes (LNs) of Spns2-/- and Spns2-Mx1Cre mice exhibited morphological defects. The structures of high endothelial venules (HEV) in the LNs of Spns2-Mx1Cre mice were disorganized. These results indicate that lack of Spns2 affects both S1P secretion and LN vasculatures. Nevertheless, blood vasculature of these Spns2 deficient mice was not different to controls under homeostasis and vascular insults. Importantly, Spns2-Mx1Cre mice were resistant to multiple sclerosis in experimental autoimmune encephalomyelitis (EAE) models with significant reduction of pathogenic Th17 cells in the central nervous system (CNS). This study suggests that pharmacological inhibition of Spns2 may be exploited for therapeutic applications in treatment of neuroinflammation.

Characterization of the Sphingolipidome of the Peri-Infarct Tissue during Hemorrhagic Transformation in a Mouse Model of Cerebral Ischemia.

Cardiovascular diseases like stroke cause changes to sphingolipid mediators like sphingosine 1-phosphate (S1P) or its ceramide analogs, which bear the potential to either alleviate or exacerbate the neurological damage. Therefore, the precise identification of alterations within the sphingolipidome during ischemic stroke (IS) and hemorrhagic transformation (HT) harbors a putative therapeutic potential to orchestrate local and systemic immunomodulatory processes. Due to the scarcity of research in this field, we aimed to characterize the sphingolipidome in IS and HT. C57BL/6 mice underwent middle cerebral artery occlusion (MCAO) and specimens of the peri-infarct tissue were taken for sphingolipid profiling. Ischemic stroke resulted in reduced S1P whilst ceramides were elevated six hours post ischemia onset. However, these differences were nearly revoked at 24 hours post ischemia onset. Moreover, the topmost S1P and ceramide levels were linked to the presence of HT after MCAO. In this study we show the characterization of the sphingolipidomic landscape of the peri-infarct tissue after ischemic stroke and HT. Especially, highest values of S1P, C 18 lactosylceramide, C 18 glucosylceramide, and C 24:1 ceramide were nearly entirely expressed by mice with HT. Our results warrant further investigations into the immunomodulatory consequences of altered sphingolipid species for the development of HT after IS.

Resveratrol Affects Sphingolipid Metabolism in A549 Lung Adenocarcinoma Cells.

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity—apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)—in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies.

ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response

The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated, and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1’s kinase substrate(s) and their functions are largely unknown. Here, we discovered that sphingosine 1-phosphate (S1P) lyase (SPL), the enzyme that degrades S1P, is a substrate for the mammalian IRE1 kinase. Our data show that IRE1-dependent SPL phosphorylation inhibits SPL’s enzymatic activity, resulting in increased intracellular S1P levels. S1P has previously been shown to induce the activation of mitochondrial UPR (UPRmt) in nematodes. We determined that IRE1 kinase-dependent S1P induction during ER stress potentiates UPRmt signaling in mammalian cells. Phosphorylation of eukaryotic translation initiation factor 2α (eif2α) is recognized as a critical molecular event for UPRmt activation in mammalian cells. Our data further demonstrate that inhibition of the IRE1-SPL axis abrogates the activation of two eif2α kinases, namely double-stranded RNA-activated protein kinase (PKR) and PKR–like ER kinase upon ER stress. These findings show that the IRE1-SPL axis plays a central role in coordinating the adaptive responses of ER and mitochondria to ER stress in mammalian cells.

P11.03.B BXQ-350: Modulating ceramide and Sphingosine-1-Phosphate for anti-tumor activity and potential mitigation of chemotherapy induced peripheral neuropathy

Abstract Background Sphingolipids are a class of bioactive signaling molecules implicated in multiple cellular processes and molecular pathways. Many publications have indicated that among these sphingolipids, sphingosine-1-phosphate (S1P) is a key sphingolipid that promotes cancer cell survival and proliferation, activates multiple oncogenic pathways, and stimulates immuno-suppressor cell populations promoting a pro-tumoral microenvironment. Many of these publications have also implicated S1P with chemotherapy induced peripheral neuropathy, a debilitating and serious side effect that may impact therapeutic treatments and patient’s quality of life. Material and Methods BXQ-350 is a nanovesicle of Saposin C, an allosteric activator of sphingolipid metabolism, that has broad anticancer activity, potentially in part by lowering systemic S1P levels. BXQ-350 was investigated in a Phase 1 dose-escalation safety study in an all-comer cancer patients with advanced solid malignancies, including CNS tumors (NCT02859857) to determine its safety profile and its potential clinical activity as a single agent in cancer patients. Samples were collected to determine pharmacokinetics and explore potential biomarkers. Results 11 patients (~15% of evaluable patients) with advanced disease had a clinical benefit and amongst these, 8 patients (~11% of evaluable patients) had PFS&amp;gt; 6 months with 2 patients still on study five years after enrollment. Analysis of patient’s samples revealed that BXQ-350 decreases systemic S1P levels in the majority of the patients experiencing a clinical benefit. In addition, several patients with established CIPN spontaneously reported improvements of their symptoms shortly after BXQ-350 administration. Analysis of these patients’ samples also revealed that S1P systemic levels had decreased, as well as pro-inflammatory cytokines associated with CIPN. Conclusion While these results are exploratory and preliminary in nature, they suggest that BXQ-350’s mechanism of action may represent a novel anti-cancer approach that may also mitigate CIPN.

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Aggravates Atherosclerosis and Induces Plaque Rupture in ApoE-/-Mice.

Altered plasma sphingosine-1-phosphate (S1P) concentrations are associated with clinical manifestations of atherosclerosis. However, whether long-term elevation of endogenous S1P is pro- or anti-atherogenic remains unclear. Here, we addressed the impact of permanently high S1P levels on atherosclerosis in cholesterol-fed apolipoprotein E-deficient (ApoE−/−) mice over 12 weeks. This was achieved by pharmacological inhibition of the S1P-degrading enzyme S1P lyase with 4-deoxypyridoxine (DOP). DOP treatment dramatically accelerated atherosclerosis development, propagated predominantly unstable plaque phenotypes, and resulted in frequent plaque rupture with atherothrombosis. Macrophages from S1P lyase-inhibited or genetically deficient mice had a defect in cholesterol efflux to apolipoprotein A-I that was accompanied by profoundly downregulated cholesterol transporters ATP-binding cassette transporters ABCA1 and ABCG1. This was dependent on S1P signaling through S1PR3 and resulted in dramatically enhanced atherosclerosis in ApoE−/−/S1PR3−/− mice, where DOP treatment had no additional effect. Thus, high endogenous S1P levels promote atherosclerosis, compromise cholesterol efflux, and cause genuine plaque rupture.