Sphingosine-1-phosphate Receptor Research Articles

Fingolimod (FTY720), an FDA-approved sphingosine 1-phosphate (S1P) receptor agonist, restores endothelial hyperpermeability in cellular and animal models of dengue virus serotype 2 infection.

Extensive vascular leakage and shock is a major cause of dengue-associated mortality. At present, there are no specific treatments available. Sphingolipid pathway is a key player in the endothelial barrier integrity; and is mediated through the five sphingosine-1-phosphate receptors (S1PR1-S1PR5). Signaling through S1PR2 promotes barrier disruption; and in Dengue virus (DENV)-infection, there is overexpression of this receptor. Fingolimod (FTY720) is a specific agonist that targets the remaining barrier-protective S1P receptors, without targeting S1PR2. In the present study, we explored whether FTY720 treatment can alleviate DENV-induced endothelial hyperpermeability. In functional assays, in both in vitro systems and in AG129 animal models, FTY720 treatment was found effective. Upon treatment, there was complete restoration of the monolayer integrity in DENV serotype 2-infected human microvascular endothelial cells (HMEC-1). At the molecular level, the treatment reversed activation of the S1P pathway. It significantly reduced the phosphorylation of the key molecules such as PTEN, RhoA, and VE-Cadherin; and also, the expression levels of S1PR2. In DENV2-infected AG129 mice treated with FTY720, there was significant improvement in weight gain, in overall clinical symptoms, and in survival. Whereas 100% of the DENV2-infected, untreated animals died by day-10 post-infection, 70% of the FTY720-treated animals were alive; and at the end of the 15-day post-infection observation period, 30% of them were still surviving. There was a significant reduction in the Evan's-blue dye permeability in the organs of FTY720-treated, DENV-2 infected animals; and also improvement in the hemogram, with complete restoration of thrombocytopenia and hepatic function. Our results show that the FDA-approved molecule Fingolimod (FTY720) is a promising therapeutic intervention in severe dengue.

Bioavailable central nervous system disease-modifying therapies for multiple sclerosis.

Disease-modifying therapies for relapsing multiple sclerosis reduce relapse rates by suppressing peripheral immune cells but have limited efficacy in progressive forms of the disease where cells in the central nervous system play a critical role. To our knowledge, alemtuzumab, fumarates (dimethyl, diroximel, and monomethyl), glatiramer acetates, interferons, mitoxantrone, natalizumab, ocrelizumab, ofatumumab, and teriflunomide are either limited to the periphery or insufficiently studied to confirm direct central nervous system effects in participants with multiple sclerosis. In contrast, cladribine and sphingosine 1-phosphate receptor modulators (fingolimod, ozanimod, ponesimod, and siponimod) are central nervous system-penetrant and could have beneficial direct central nervous system properties.

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.

Bile acids regulate SF-1 to alter cholesterol balance in adrenocortical cells via S1PR2

Glucocorticoid synthesis typically occurs in adrenal cortex and is influenced by cholesterol balance, since cholesterol is the sole precursor of steroids. Bile acids as the signaling molecules, have been shown to promote steroidogenesis in steroidogenic cells. However, whether bile acids directly regulate cholesterol balance remains elusive. In this study, we prepared cholestatic mouse models and cultured human adrenocortical cells (H295R) treated with taurochenodeoxycholic acid (TCDCA) to determine transcription levels of cholesterol metabolism associated genes and cholesterol concentrations in adrenocortical cells. Results showed that common bile duct ligation (CBDL) and chenodeoxycholic acid (CDCA) feeding elevated the mRNA levels of Abca1, Cyp51, Hmgcr, Srb1, and Mc2r in adrenals of mice. Meanwhile, the concentrations of total cholesterol and cholesteryl ester in adrenals of CBDL and CDCA-fed mice were dramatically lowered. The total and phosphorylation levels of HSL in adrenal glands of CBDL mice were also enhanced. Similarly, TCDCA treatment in H295R cells decreased intracellular concentrations of total cholesterol and cholesteryl ester and increased transcription levels of SRB1, MC2R, and HSL as well. Inhibition of bile acids’ receptor sphingosine 1‐phosphate receptor 2 (S1PR2), extracellular signal‐regulated kinase (ERK) phosphorylation, and steroidogenic factor 1 (SF-1) respectively successfully abolished effect of TCDCA on H295R cells. SF-1s was found to be phosphorylated at Thr75 in TCDCA-treated H295R cells. While a mild increase of intracellular cAMP concentration was detected upon TCDCA treatment, inhibition of PKA activity with Rp-Isomer in H295R cells failed to decrease the expression of SF-1 and its target genes. Our findings suggest that conjugated bile acids affect cholesterol balance through regulation of SF-1 in adrenocortical cells so as to provide an adequate cholesterol supply for glucocorticoid synthesis, which improves and enriches our understanding of the mechanism whereby bile acids regulate cholesterol balance to affect adrenal function.

Dependence of IL-7R-Mediated Signaling on Sphingosine Kinase Activity in Acute Lymphoblastic Leukemia, but Not Healthy Lymphoid Cells, Is an Exploitable Therapeutic Vulnerability

Acute lymphoblastic leukemia (ALL) is an aggressive cancer affecting B or T cell precursors. Interleukin 7 (IL-7) is essential for normal lymphoid development but shown also to contribute to ALL development and resistance to glucocorticoids. IL7R (the gene that encodes for IL-7 receptor α) is a bona fide lymphoid oncogene that is mutated in around 10% T-ALL and 3% B-ALL patients. Mutational activation of IL7R or high levels of wild type IL7R can drive ALL development in vivo. Sphingosine kinases, of which there are two isoforms (SK1 and SK2), generate sphingosine-1-phosphate (S1P) from sphingosine, thus promoting cell survival. We previously identified SKs as potential targets for therapeutic intervention in ALL with IL7R gain-of-function mutations. However, how IL-7R-mediated signaling crosstalks with SKs, and whether there is a therapeutic window for the use of SK inhibitors in ALL without provoking excessive side effects, remains to be determined. Our thorough immunophenotypic characterization of CD2Cre.SK1 fl/fl.SK2 -/- mice, which display SK complete ablation exclusively in the lymphoid compartment, revealed no major impact on lymphoid development. Since, on the contrary, IL7R-null mice display severe B- and T-cell deficiency, this indirectly indicates that SKs are not involved in IL-7R-mediated signaling during lymphoid development. Accordingly, we found that healthy thymocytes and B-cell precursors did not require SK activity for IL-7-mediated signaling, as measured by the levels of STAT5 phosphorylation. In sharp contrast, we found compelling evidence that SKs are involved in IL-7R-mediated signaling and functional impact in the context of leukemia. First, SK deletion in the lymphoid compartment significantlydelayed the development of B-ALL in mice with IL7R mutation in lymphoid precursors (CD2Cre.Il7r cpt/wt mice). Second, SK deletion impaired both basal and IL-7-mediated JAK/STAT5 pathway activation in leukemia cells from the same mice. In agreement, CRISPR-Cas9 SK1/SK2 gene editing in the human IL-7R-responsive HPB-ALL cell line prevented IL-7-induced JAK/STAT5 pathway activation and leukemia cell viability. Finally, pharmacological inhibition of SKs blocked IL-7-triggered viability of human patient-derived xenograft T-ALL cells, without significantly impacting healthy thymocytes. We next sought to understand the reasons underlying IL-7R-mediated signaling aberrant dependence on SK activity in leukemia cells. Besides direct intracellular roles, S1P is exported to the extracellular compartment and able to induce autocrine or paracrine signaling via S1P receptors expressed at the cell surface. We found that S1PR4 transcript levels were upregulated in diagnostic T-ALL patient samples. Inhibition of S1PR4 downregulated IL-7-mediated JAK/STAT5 pathway activation in leukemia cells but not in healthy thymocytes. In agreement, S1P stimulation partially mimicked the effects of IL-7 exclusively in T-ALL cells. In addition, our analyses demonstrated that SK1 and SK2 are overexpressed in B- and T-ALL diagnostic patient samples. Importantly, we further report that SK1 co-immunoprecipitated with IL-7Rα in ALL cells and that SK1 overexpression upregulates IL-7R-mediated signaling in HeLa cells transfected with the IL-7R signaling machinery. Our results indicate that overexpression of SKs and/or S1PR4 may justify the augmented reliance of IL-7R-mediated signaling on SK activity displayed by ALL cells. Finally, we demonstrated the potential therapeutic impact of our findings. The SK pharmacological inhibitor compound 49 reversed IL-7-dependent pro-survival effects in human B- and T-ALL samples in vitro. We then conducted a phase 2-like preclinical trial with 7 distinct IL-7-responsive T-ALL patient samples xenografted into immunodeficient mice, which showed that compound 49 significantly reduced disease burden (p=0.0156, Wilcoxon matched pairs signed rank test), with highest effects in the samples that displayed higher IL-7 signaling responsiveness. Overall, our studies demonstrate that SKs are key players in oncogenic, but not in physiological, IL-7/IL-7R-mediated signaling. We uncover the importance of the SK-IL7R signaling crosstalk in ALL and pinpoint the therapeutic potential of SK inhibitors for IL-7R-dependent ALL.

Etrasimod (Velsipity) for ulcerative colitis.

The oral sphingosine 1-phosphate (S1P) receptor modulator etrasimod (Velsipity – Pfizer) has been approved by the FDA for treatment of moderately to severely active ulcerative colitis in adults. It is the second oral S1P receptor modulator to be approved in the US for this indication; ozanimod (Zeposia) was the first.

S1PR2 inhibition mitigates cognitive deficit in diabetic mice by modulating microglial activation via Akt-p53-TIGAR pathway

Cognitive deficit is one of the challenging complications of type 2 diabetes. Sphingosine 1- phosphate receptors (S1PRs) have been implicated in various neurodegenerative and metabolic disorders. The association of S1PRs and cognition in type 2 diabetes remains elusive. Microglia-mediated neuronal damage could be the thread propagating cognitive deficit. The effects of S1PR2 inhibition on cognition in high-fat diet and streptozotocin-induced diabetic mice were examined in this work. We further assessed microglial activation and putative microglial polarisation routes. Cognitive function loss was observed after four months of diabetes induction in Type 2 diabetes animal model. JTE013, an S1PR2 inhibitor, was used to assess neuroprotection against cognitive decline and neuroinflammation in vitro and in vivo diabetes model. JTE013 (10 mg/kg) improved synaptic plasticity by upregulating psd95 and synaptophysin while reducing cognitive decline and neuroinflammation. It further enhanced anti-inflammatory microglia in the hippocampus and prefrontal cortex (PFC), as evidenced by increased Arg-1, CD206, and YM-1 levels and decreased iNOS, CD16, and MHCII levels. TIGAR, TP53-induced glycolysis and apoptosis regulator, might facilitate the anti-inflammatory microglial phenotype by promoting oxidative phosphorylation and decreasing apoptosis. However, since p53 is a TIGAR suppressor, inhibiting p53 could be beneficial. S1PR2 inhibition increased p-Akt and TIGAR levels and reduced the levels of p53 in the PFC and hippocampus of type 2 diabetic mice, thereby decreasing apoptosis. In vitro, palmitate was used to imitate sphingolipid dysregulation in BV2 cells, followed by conditioned media exposure to Neuro2A cells. JTE013 rescued the palmitate-induced neuronal apoptosis by promoting the anti-inflammatory microglia. In the present study, we demonstrate that the inhibition of S1PR2 improves cognitive function and skews microglia toward anti-inflammatory phenotype in type 2 diabetic mice, thereby promising to be a potential therapy for neuroinflammation.

The Role of Sphingosine-1-Phosphate Receptor 2 in Mouse Retina Light Responses.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) acts as a ligand for a family of G protein-coupled S1P receptors (S1PR1-5) to participate in a variety of signaling pathways. However, their specific roles in the neural retina remain unclear. We previously showed that S1P receptor subtype 2 (S1PR2) is expressed in murine retinas, primarily in photoreceptors and bipolar cells, and its expression is altered by retinal stress. This study aims to elucidate the role of S1PR2 in the mouse retina. We examined light responses by electroretinography (ERG), structural differences by optical coherence tomography (OCT), and protein levels by immunohistochemistry (IHC) in wild-type (WT) and S1PR2 knockout (KO) mice at various ages between 3 and 6 months. We found that a- and b-wave responses significantly increased at flash intensities between 400~2000 and 4~2000 cd.s/m2, respectively, in S1PR2 KO mice relative to those of WT controls at baseline. S1PR2 KO mice also exhibited significantly increased retinal nerve fiber layer (RNFL) and outer plexiform layer (OPL) thickness by OCT relative to the WT. Finally, in S1PR2 KO mice, we observed differential labeling of synaptic markers by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). These results suggest a specific involvement of S1PR2 in the structure and synaptic organization of the retina and a potential role in light-mediated functioning of the retina.

Treatment of Antibiotic Refractory Chronic Pouchitis With JAK Inhibitors and S1P Receptor Modulators: An ECCO CONFER Multicentre Case Series.

Data regarding the effectiveness and safety of Janus kinase [JAK] inhibitors and sphingosine-1-phosphate [S1P] receptor modulators in antibiotic refractory chronic pouchitis [CARP] are lacking. This ECCO-CONFER project retrospectively collected data for JAK inhibitor or S1P receptor modulator treatments for CARP with at least 3 months of follow-up. The outcomes included corticosteroid- and antibiotic-free clinical response and remission at 3 and 12 months, and trends in modified pouchitis disease activity index [mPDAI], endoscopic PDAI, C-reactive protein, and calprotectin. Seventeen treatments in 15 patients were evaluated. Previous pouchitis treatments included infliximab [5/15], adalimumab [4/15], vedolizumab [9/15], and ustekinumab [5/15]. Pooling data on JAK inhibitors [eight tofacitinib, one filgotinib, and six upadacitinib] after 3 months [T3], steroid- and antibiotic-free clinical response was achieved in 53.3% [8/15], and steroid- and antibiotic-free clinical remission was achieved in 40% [6/15]. Of the patients with at least 12 months of follow-up, steroid- and antibiotic-free clinical response was achieved in 50% [3/6] and remission in one patient [16.7%], endoscopic response in 50% [3/6], and endoscopic remission in 50% [3/6]. Of the two ozanimod treatments at T3, steroid- and antibiotic-free clinical response was achieved in one patient, without remission; both discontinued ozanimod before T12. No side effects were reported. Small molecules may represent a suitable option for CARP refractory to multiple biologics, deserving further investigation.

TMIC-21. SPHINGOSINE-1-PHOSPHATE RECEPTOR 3 (S1PR3) IN THE GLIOBLASTOMA TUMOR MICROENVIRONMENT MAY PLAY A ROLE IN IMMUNOSUPPRESSION

Abstract Glioblastoma remains an intractable clinical problem with few efficacious treatments and poor patient outcomes because of the challenge in treating the tumor cells that have invaded into the normal brain. These invasive tumor cells typically remain after standard-of-care surgical resection and likely persist to generate recurrent tumors. In our previous studies of the glioma tumor microenvironment (TME), we identified sphingosine-1-phosphate receptor 3 (S1PR3) as upregulated in glioma TME regions with increased fluid flow. In order to efficiently study S1PR3 in the normal brain/ glioblastoma tumor interface, we engaged the Wake Forest University Brain Tumor Center of Excellence to construct a tissue microarray (TMA) including glioblastoma tissue cores specifically of the normal brain/ tumor interface as determined by pathologist review. The TMA included FFPE tissue cores from 42 GBM patients (average age 67.7 ± 10.9 years, 25 male, 17 female). Immunohistochemistry to detect S1PR3 was performed using an antibody that targets the amino-terminal domain followed by spatial profiling using neuroscience, neuroinflammation, and immune antibody panels (Akoya Biosciences). Preliminary analyses of the spatial profiling revealed 34 evaluable tissue cores, with no difference in the percentage of neurons, glial cells, vascular cells, or T cells between the S1PR3 positive and negative cores, however a significant difference in the percentage of M1 macrophages/ antigen presenting cells was found between the two groups (S1PR3-positive 0.646 ± 0.569%, n=13; S1PR3-negative 2.95 ± 3.28%, n=21, p=0.0178 two-tailed t-test. These findings suggest that the presence of S1PR3 may be associated with reduced M1 macrophages/ antigen presenting cells and therefore S1PR3 may contribute to glioblastoma tumor associated immunosuppression.

S1P receptor-1 signaling reduces arterial thrombosis by upregulation of endothelial thrombomodulin expression

Abstract Background Sphingosine-1-phosphate (S1P) is a bioactive lipid known for its cardioprotective effects. The extent to which S1P also has effects on arterial thrombosis has not yet been fully elucidated. Another important factor in arterial thrombosis is thrombomodulin (TM), an important endothelial antithrombotic factor. To what extent S1P, TM, and, in addition, platelet adhesion as an initial part influences arterial thrombosis is unclear and the aim of this work. Methods The influence of S1P on human umbilical cord venous endothelial cells (HUVECs) was investigated in cell culture. To investigate effects of S1P and TM on platelet adhesion, flow chamber experiments were performed. In addition, murine arterial thrombus formation was analyzed by intravital microscopy. Results S1P increased TM expression on HUVECs. This effect was abolished by S1P receptor1 (S1PR1) inhibition with W146 (S1P: 1.081 ± 0.091 vs. S1P + W146: 0.951 ± 0.073, normalized to control, p<0.0001). Sphingosine kinase-2 deficient mice (SphK1-/-) lack the kinase primarily producing sphingosine. Accordingly, these animals have less S1P. Here, TM expression was significantly lower in the aorta compared to wild-type mice (WT: 207.2 ± 45.8 pg/g aorta vs. SphK1-/- 124.5 ± 40.2 pg/g aorta, p<0.0001). S1P treatment decreased platelet adhesion from whole blood to endothelial cells in the flow chamber (Con: 100.00 ± 28.09% vs. S1P: 80.91 ± 22.85%, p<0.0001). W146 reversed this effect by S1PR1 inhibition (S1P: 80.91 ± 22.85%, vs. S1P + W146: 118.60 ± 33.65%, p=0.0067). Platelet adhesion to endothelial cells was assessed in the presence of a TM-neutralizing antibody. The TM antibody abolished the anti-adhesive effects of S1P (S1P: 54.79 ± 29.59% vs. S1P+AB: 105.54 ± 35.43%, p=0.0174). TM inhibited intravital murine arterial thrombus formation (time to first occlusion: con: 9.38 ± 1.02 min vs. TM: 60 ± 0 min, p=0.0286). Furthermore, the duration of the first occlusion was significantly shorter and the embolic rate as a marker for thrombus stability was higher. Conclusion We demonstrated that S1P inhibits arterial thrombosis via regulation of endothelial TM. In particular, platelet adhesion to endothelial cells is reduced. This effect is mediated via S1PR1. The extent to which S1PR1 represents a potential antithrombotic target must be clarified in future studies.

Analogs of FTY720 inhibit TRPM7 but not S1PRs and exert multimodal anti-inflammatory effects.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo.

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.

Sphingosine-1-Phosphate Receptor 3 Induces Endothelial Barrier Loss via ADAM10-Mediated Vascular Endothelial-Cadherin Cleavage.

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

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

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

MicroRNA-10a enrichment in factor VIIa–released endothelial extracellular vesicles: potential mechanisms

BackgroundFactor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa–released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. ObjectivesTo investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. MethodsActivation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. ResultsFVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa–driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa–EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM−/− mice against lipopolysaccharide-induced inflammation and vascular leakage. ConclusionOur data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.

Modulating intestinal barrier function by sphingosine-1-phosphate receptor 1 specific agonist SEW2871 attenuated ANIT-induced cholestatic hepatitis via the gut-liver axis

Bile acid (BA) homeostasis throughout the enterohepatic circulation system is a guarantee of liver physiological functions. BA circulation disorders is one of the characteristic clinical manifestations of cholestasis, and have a closely relationship with intestinal barrier function, especially ileum. Here, our in vivo and in vitro studies showed that intestinal tight junctions (TJs) were disrupted by α-naphthylisothiocyanate (ANIT), which also down-regulated the protein expression of sphingosine-1-phosphate receptor 1 (S1PR1) in the top of villus of mice ileum. Activating S1PR1 by specific agonist SEW2871 could improve TJs via inhibiting ERK1/2/LKB1/AMPK signaling pathway in the ileum of ANIT-treated mice and ANIT-cultured Caco-2 cells. SEW2871 not only regained ileum TJs by activating S1PR1 in the epithelial cells of ileum mucosa, but also recovered ileum barrier function, which was further verified by the recovered BA homeostasis in mice ileum (content and tissue) by using of high-performance liquid chromatographytandem mass spectrometry (LC-MS/MS). Subsequently, the improved intestinal injury and inflammation further strengthened that SEW2871 modulated intestinal barrier function in ANIT-treated mice. Finally, our data revealed that along with the down-regulated levels of serum lipopolysaccharides (LPS), SEW2871 improved liver function and relieved hepatitis via blocking TLR4/MyD88/NF-kB signaling pathway in ANIT-treated mice. In conclusion, these results demonstrated that activating intestinal S1PR1 by SEW2871 could modulate intestinal barrier function, leading to the improvement of cholestatic hepatitis in ANIT-treated mice via the “gut-liver” axis.

CD8 T cell tolerance results from eviction of immature autoreactive cells from the thymus.

CD8 T cell tolerance is thought to result from clonal deletion of autoreactive thymocytes before they differentiate into mature CD8 T cells in the thymus. However, we report that, in mice, CD8 T cell tolerance instead results from premature thymic eviction of immature autoreactive CD8 thymocytes into the periphery, where they differentiate into self-tolerant mature CD8 T cells. Premature thymic eviction is triggered by T cell receptor (TCR)-driven down-regulation of the transcriptional repressor Gfi1, which induces expression of sphingosine-1-phosphate receptor-1 (S1P1) on negatively selected immature CD8 thymocytes. Thus, premature thymic eviction is the basis for CD8 T cell tolerance and is the mechanism responsible for the appearance in the periphery of mature CD8 T cells bearing autoreactive TCRs that are absent from the thymus.

Post-vaccination SARS-Cov-2 T-cell receptor repertoires in patients with multiple sclerosis and related disorders.

Attenuation in post-vaccination SARS-CoV-2 humoral responses has been demonstrated in people treated with either anti-CD20 therapies or sphingosine-1-phosphate (S1P) receptor modulators. In the setting of disease modifying therapy (DMT) use, humoral response may not correlate with effective immunity, and analysis of vaccine-mediated SARS-CoV-2-specific memory T-cell responses is crucial. While vaccination in patients treated with anti-CD20 agents leads to deficient antibody production, emerging data from live cell assays suggests intact T-cell responses to vaccination. We evaluated post-vaccination SARS-CoV-2 T-cell receptor (TCR) repertoires in DMT-treated patients using the ImmunoSeqR assay, an assay that does not require live cells. Adults 18-80 years old without prior COVID-19, with neuroimmune conditions, who had been vaccinated with two doses of Pfizer-BioNTech or Moderna mRNA vaccines at least 3 weeks and up to 6 months prior, were recruited. Whole blood was obtained for immunosequencing, and matched serum was obtained for humoral analysis. Immunosequencing of the CDR3 regions of human TCRβ chains was completed using the immunoSEQR Assay (Adaptive Biotechnologies). TCR sequences were mapped across a set of TCR sequences reactive to SARS-CoV-2. Clonal diversity (breadth) and frequency (depth) of TCRs specific to SARS-CoV-2 spike protein were calculated and relationships with clinical variables were assessed. Forty patients were recruited into the study, aged 25-77, and 27 female. 37 had MS, 2 had neuromyelitis optica spectrum disorder (NMOSD), and 1 had hypophysitis. Subjects treated with anti-CD20 agents and S1P receptor modulators had severely attenuated humoral responses, but SARS-CoV-2-spike-specific TCR clonal depth and breadth were robust across all treatment classes except S1P modulators. No spike-specific or non-spike-specific SARS-CoV-2-associated TCRs were found in those treated with S1P modulators (p=0.002 for both breadth and depth). Subjects treated with fumarates exhibited somewhat lower spike TCR breadth than subjects treated with other or no DMTs (median 2.27×10^-5 for fumarates and 4.96×10^-5 for all others, p=0.008), but no statistically significant difference was demonstrated with spike TCR depth. No other significant associations with DMT type were found. We found no significant correlations between depth or breadth and age, duration of treatment, type of vaccination, or time interval since vaccination. This is the first study to characterize post-vaccination SARS-CoV-2 TCR repertoires in DMT-treated individuals. We demonstrated a dichotomous response to SARS-CoV-2 vaccination in anti-CD20-treated patients, with severely attenuated humoral response but intact TCR depth and breadth. It is unclear to what degree each arm of the adaptive immune system impacts post-vaccine immunity, both from the standpoint of incidence of post-vaccine infections and that of infection severity, and further clinical studies are necessary. S1P modulator-treated subjects exhibited both severely attenuated humoral responses and absent spike-specific TCR depth and breadth, information which is crucial for counseling of patients on these agents. Our methodology can be used in larger studies to determine the benefit of repeated vaccination doses, including those that are modified to better target modern or seasonal variants, without the use of live cell assays.

Tertiary lymphoid structures as local perpetuators of organ-specific immune injury: implication for lupus nephritis.

In response to inflammatory stimuli in conditions such as autoimmune disorders, infections and cancers, immune cells organize in nonlymphoid tissues, which resemble secondary lymphoid organs. Such immune cell clusters are called tertiary lymphoid structures (TLS). Here, we describe the potential role of TLS in the pathogenesis of autoimmune disease, focusing on lupus nephritis, a condition that incurs major morbidity and mortality. In the kidneys of patients and animals with lupus nephritis, the presence of immune cell aggregates with similar cell composition, structure, and gene signature as lymph nodes and of lymphoid tissue-inducer and -organizer cells, along with evidence of communication between stromal and immune cells are indicative of the formation of TLS. TLS formation in kidneys affected by lupus may be instigated by local increases in lymphorganogenic chemokines such as CXCL13, and in molecules associated with leukocyte migration and vascularization. Importantly, the presence of TLS in kidneys is associated with severe tubulointerstitial inflammation, higher disease activity and chronicity indices, and poor response to treatment in patients with lupus nephritis. TLS may contribute to the pathogenesis of lupus nephritis by increasing local IFN-I production, facilitating the recruitment and supporting survival of autoreactive B cells, maintaining local production of systemic autoantibodies such as anti-dsDNA and anti-Sm/RNP autoantibodies, and initiating epitope spreading to local autoantigens. Resolution of TLS, along with improvement in lupus, by treating animals with soluble BAFF receptor, docosahexaenoic acid, complement inhibitor C4BP(β-), S1P1 receptor modulator Cenerimod, dexamethasone, and anti-CXCL13 further emphasizes a role of TLS in the pathogenesis of lupus. However, the mechanisms underlying TLS formation and their roles in the pathogenesis of lupus nephritis are not fully comprehended. Furthermore, the lack of non-invasive methods to visualize/quantify TLS in kidneys is also a major hurdle; however, recent success in visualizing TLS in lupus-prone mice by photon emission computed tomography provides hope for early detection and manipulation of TLS.