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

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

Comprehensive overview of novel chemical drugs for ulcerative colitis: focusing on phase 3 and beyond.

INTRODUCTION: Despite the growing number of highly efficacious biologics and chemical drugs for ulcerative colitis (UC), steroid-free disease control is still difficult to achieve in subgroups of patients due to refractoriness, adverse events, primary or secondary failure. New treatments are therefore still required in order to optimize clinical management of patients with UC. AREAS COVERED: The efficacy and safety of both currently available and newly developed small molecules have been summarized. The PubMed database and clinicaltrials.gov were considered in order to search for phase 2b and 3 trials on new chemical drugs for UC. The study drugs reviewed included Janus kinases (JAK) and sphingosine-1-phosphate receptor (S1Pr) inhibitors, α4 integrin antagonist, and micro-RNA-124 upregulators. EXPERT OPINION: Rapidity of onset, low immunogenicity, and safety are the main characteristics of small molecules currently available or under evaluation for treatment patients with UC. Among the currently available chemical drugs, the selective JAK and the S1Pr inhibitors are characterized by a good safety profile combined with the ability to induce clinical remission in UC. A relatively low frequency of endoscopic improvement and healing currently appears associated with their use, being higher in UC patients treated with S1Pr inhibitor Etrasimod. Overall, additional new safe and effective drugs are still required in order to optimize disease control in a larger majority of UC patients.

S1PR3 agonism and S1P lyase inhibition rescue mice in the severe state of experimental sepsis.

Sepsis is characterized as life-threatening organ dysfunction caused by a dysregulated host response to an infection. Despite numerous clinical trials that addressed this syndrome, there is still no causative treatment available to dampen its severity. Curtailing the infection at an early stage with anti-infectives is the only effective treatment regime besides intensive care. In search for additional treatment options, we recently discovered the inhibition of the sphingosine 1-phosphate (S1P) lyase and subsequent activation of the S1P receptor type 3 (S1PR3) in pre-conditioning experiments as promising targets for sepsis prevention. Here, we demonstrate that treatment of septic mice with the direct S1P lyase inhibitor C31 or the S1PR3 agonist CYM5541 in the advanced phase of sepsis resulted in a significantly increased survival rate. A single dose of each compound led to a rapid decline of sepsis severity in treated mice and coincided with decreased cytokine release and increased lung barrier function with unaltered bacterial load. The survival benefit of both compounds was completely lost in S1PR3 deficient mice. Treatment of the murine macrophage cell line J774.1 with either C31 or CYM5541 resulted in decreased protein kinase B (Akt) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) phosphorylation without alteration of the mitogen-activated protein kinase (MAPK) p38 and p44/42 phosphorylation. Thus, activation of S1PR3 in the acute phase of sepsis by direct agonism or S1P lyase inhibition dampened Akt and JNK phosphorylation, resulting in decreased cytokine release, improved lung barrier stability, rapid decline of sepsis severity and better survival in mice.

Mocravimod, a Selective Sphingosine-1-Phosphate Receptor Modulator, in Allogeneic Hematopoietic Stem Cell Transplantation for Malignancy.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the sole curative option for patients with acute myelogenous leukemia. Outcomes are limited by leukemia relapse, graft-versus-host disease (GVHD), and abnormal immune reconstitution. Mocravimod (KRP203) is an oral sphingosine-1-phosphate receptor (S1PR) modulator that blocks the signal required by T cells to egress from lymph nodes and other lymphoid organs. Mocravimod retains T cell effector function, a main differentiator to immunosuppressants. In preclinical models, mocravimod improves survival by maintaining graft-versus-leukemia (GVL) activity while reducing GVHD. In patients undergoing allo-HSCT for hematological malignancies, mocravimod is postulated to prevent GVHD by redistributing allogeneic donor T cells to lymphoid tissues while allowing a sufficient GVL effect in the lymphoid, where malignant cells usually reside. The primary objective of this study was to assess the safety and tolerability of mocravimod in patients undergoing allo-HSCT for hematologic malignancies. Secondary objectives were to determine the pharmacokinetic profiles of mocravimod and its active metabolite mocravimod-phosphate in this patient group, as well as to assess GVHD-free, relapse free survival at 6 months after the last treatment. In this 2-part, single- and 2-arm randomized, open-label trial, we evaluated the safety, tolerability, and pharmacokinetics of mocravimod in allo-HSCT recipients (ClinicalTrials.gov identifier NCT01830010). Patients received either 1 mg or 3 mg mocravimod per day on top of standard of care GVHD prophylaxis with either cyclosporine A/methotrexate or tacrolimus/methotrexate. We found that mocravimod can be safely added to standard treatment regimens in patients with hematologic malignancies requiring allo-HSCT. Mocravimod resulted in a significant reduction of circulating lymphocyte numbers and had no negative impact on engraftment and transplantation outcomes. Our results indicate that mocravimod is safe and support a larger study to investigate its efficacy in a homogeneous acute myelogenous leukemia patient population undergoing allo-HSCT.

In Vitro Characterization of Sphingosine 1-Phosphate Receptor 1 (S1P1) Expression and Mediated Migration of Primary Human T and B Cells in the Context of Cenerimod, a Novel, Selective S1P1 Receptor Modulator.

Cenerimod is a potent, selective sphingosine 1-phosphate receptor 1 (S1P1) modulator currently investigated in a Phase IIb study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including T and B lymphocytes) in the bloodstream and inflamed tissues, making them an effective therapeutic concept for autoimmune disorders. Although the effect of S1P receptor modulators in reducing circulating lymphocytes is well documented, the precise molecular role of the S1P1 receptor on these cell types is not fully understood. In this study, the mode of action of cenerimod on human primary lymphocytes in different activation states was investigated focusing on their chemotactic behavior towards S1P in real-time, concomitant to S1P1 receptor expression and internalization dynamics. Here, we show that cenerimod effectively prevents T and B cell migration in a concentration-dependent manner. Interestingly, while T cell activation led to strong S1P1 re-expression and enhanced migration; in B cells, an enhanced migration capacity and S1P1 receptor surface expression was observed in an unstimulated state. Importantly, concomitant treatment with glucocorticoids (GCs), a frequently used treatment for autoimmune disorders, had no impact on the inhibitory activity of cenerimod on lymphocytes.

The sphingosine 1-phosphate receptor 2/4 antagonist JTE-013 elicits off-target effects on sphingolipid metabolism.

Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors. Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P2 and S1P4) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P2/4, inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P2/4.

Discovery of Novel Sphingosine-1-Phosphate-1 Receptor Agonists for the Treatment of Multiple Sclerosis.

The sphingosine-1-phosphate-1 (S1P1) receptor agonists have great potential for the treatment of multiple sclerosis (MS) because they can inhibit lymphocyte egress through receptor internalization. We designed and synthesized triazole and isoxazoline derivatives to discover a novel S1P1 agonist for MS treatment. Of the two scaffolds, the isoxazoline derivative was determined to have excellent in vitro efficacy and drug-like properties. Among them, compound 21l was found to have superior drug-like properties as well as excellent in vitro efficacies (EC50 = 7.03 nM in ß-arrestin recruitment and EC50 = 11.8 nM in internalization). We also confirmed that 21l effectively inhibited lymphocyte egress in the peripheral lymphocyte count test and significantly improved the clinical score in the experimental autoimmune encephalitis MS mouse model.

Novel 5-fluorouracil sensitizers for colorectal cancer therapy: Design and synthesis of S1P receptor 2 (S1PR2) antagonists.

Sphingosine-1-phosphate receptor 2 (S1PR2) has been identified as a brand-new GPCR target for designing antagonists to reverse 5-FU resistance. We herein report the structural optimization and structure-activity relationship of JTE-013 derivatives as S1PR2 antagonists. Compound 9d was the most potent S1PR2 antagonist (KD = 34.8 nM) among developed compounds. Here, compound 9d could significantly inhibit the expression of dihydropyrimidine dehydrogenase (DPD) to reverse 5-FU-resistance in HCT116DPD and SW620/5-FU cells. Further mechanism studies demonstrated that compound 9d not only inhibited S1PR2 but also affected the transcription of S1PR2. In addition, compound 9d also showed acceptable selectivity to normal cells (NCM460). Importantly, compound 9d with suitable pharmacokinetic properties could significantly reverse 5-FU-resistance in the HCT116DPD and SW620/5-FU xenograft models without obvious toxicity, in which the inhibition rates of 5-FU were increased from 23.97% to 65.29% and 27.23% to 60.81%, respectively. Further immunohistochemistry and western blotting analysis also demonstrated that compound 9d significantly decreases the expression of DPD in tumor and liver tissues. These results indicated that compound 9d is a promising lead compound to reverse 5-FU-resistance for colorectal cancer therapy.

Arachnoid membrane as a source of sphingosine-1-phosphate that regulates mouse middle cerebral artery tone.

Growing evidence indicates that perivascular tissue is critical to modulate vessel function. We hypothesized that the arachnoid membrane surrounding middle cerebral artery (MCA) regulates its function via sphingosine-1-phosphate (S1P)-induced vasoconstriction. The MCA from 3- to 9-month-old male and female wild-type (Oncine France 1 and C57BL/6) mice and sphingosine kinase 2 knockout (SphK2-/-) mice in the C57BL/6 background was mounted in pressure myographs with and without arachnoid membrane. Raman microspectroscopy and imaging were used for in situ detection of S1P. The presence of arachnoid tissue was associated with reduced external and lumen MCA diameters, and with an increase in basal tone regardless of sex and strain background. Strong S1P-positive signals were detected in the arachnoid surrounding the MCA wall in both mice models, as well as in a human post-mortem specimen. Selective S1P receptor 3 antagonist TY 52156 markedly reduced both MCA vasoconstriction induced by exogenous S1P and arachnoid-dependent basal tone increase. Compared to 3-month-old mice, the arachnoid-mediated contractile influence persisted in 9-month-old mice despite a decline in arachnoid S1P deposits. Genetic deletion of SphK2 decreased arachnoid S1P content and vasoconstriction. This is the first experimental evidence that arachnoid membrane regulates the MCA tone mediated by S1P.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Promoted Osteogenesis by Increasing Vesicle Trafficking, Wnt/Ca2+, and BMP/Smad Signaling.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 µM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 µM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.

Discovery and Optimization of Orally Bioavailable Phthalazone and Cinnolone Carboxylic Acid Derivatives as S1P2 Antagonists against Fibrotic Diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease. Current treatments only slow down disease progression, making new therapeutic strategies compelling. Increasing evidence suggests that S1P2 antagonists could be effective agents against fibrotic diseases. Our compound collection was mined for molecules possessing substructure features associated with S1P2 activity. The weakly potent indole hit 6 evolved into a potent phthalazone series, bearing a carboxylic acid, with the aid of a homology model. Suboptimal pharmacokinetics of a benzimidazole subseries were improved by modifications targeting potential interactions with transporters, based on concepts deriving from the extended clearance classification system (ECCS). Scaffold hopping, as a part of a chemical enablement strategy, permitted the rapid exploration of the position adjacent to the carboxylic acid. Compound 38, with good pharmacokinetics and in vitro potency, was efficacious at 10 mg/kg BID in three different in vivo mouse models of fibrotic diseases in a therapeutic setting.

P-Rex1 Controls Sphingosine 1-Phosphate Receptor Signalling, Morphology, and Cell-Cycle Progression in Neuronal Cells.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation.

Design, synthesis and biological evaluation of sphingosine-1-phosphate receptor 2 antagonists as potent 5-FU-resistance reversal agents for the treatment of colorectal cancer.

5-Fluorouracil (5-FU) and its prodrugs are the essential clinical drugs for colorectal cancer (CRC) treatment. However, the drug resistance of 5-FU has caused high mortality of CRC patients. Thus, it is urgent to develop reversal agents of 5-FU resistance. Sphingosine-1-phosphate receptor 2 (S1PR2) was proved to be a potential target for reversing 5-FU resistance, but the activity of known S1PR2 antagonists JTE-013 were weak in 5-FU-resistant cell lines. To develop more potent S1PR2 antagonists to treat 5-FU-resistant cancer, a series of JTE-013 derivatives were designed and synthesized. The most promising compound 40 could markedly reverse the resistance in 5-FU-resistant HCT116 cells and 5-FU-resistant SW620 cells via inhibiting the expression of dihydropyrimidine dehydrogenase (DPD). The key was that compound 40 with improved pharmacokinetic properties significantly increased the inhibitory rate of 5-FU in the SW620/5-FU cells xenograft model with no observable toxicity by inhibiting the expression of DPD in tumor and liver tissues. Altogether, these results suggest that compound 40 may be a promising drug candidate to reverse 5-FU resistance in the treatment of CRC.

Antagonizing S1P3 Receptor with Cell-Penetrating Pepducins in Skeletal Muscle Fibrosis.

S1P is the final product of sphingolipid metabolism, which interacts with five widely expressed GPCRs (S1P1-5). Increasing numbers of studies have indicated the importance of S1P3 in various pathophysiological processes. Recently, we have identified a pepducin (compound KRX-725-II) acting as an S1P3 receptor antagonist. Here, aiming to optimize the activity and selectivity profile of the described compound, we have synthesized a series of derivatives in which Tyr, in position 4, has been substituted with several natural aromatic and unnatural aromatic and non-aromatic amino acids. All the compounds were evaluated for their ability to inhibit vascular relaxation induced by KRX-725 (as S1P3 selective pepducin agonist) and KRX-722 (an S1P1-selective pepducin agonist). Those selective towards S1P3 (compounds V and VII) were also evaluated for their ability to inhibit skeletal muscle fibrosis. Finally, molecular dynamics simulations were performed to derive information on the preferred conformations of selective and unselective antagonists.

Ponesimod protects against neuronal death by suppressing the activation of A1 astrocytes in early brain injury after experimental subarachnoid hemorrhage.

As an important initiator and responder of brain inflammation in the central nervous system (CNS), astrocytes transform into two new reactive phenotypes with changed morphology, altered gene expression and secretion profiles, termed detrimental A1 and beneficial A2. Inflammatory events have been shown to occur during the phase of early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the phenotype transformation of astrocytes as well as its potential contribution to inflammatory status in the EBI of SAH has yet to be determined. In the present study, both in vivo and in vitro models of SAH were established, and the polarization of astrocytes after SAH was analyzed by RNA-seq, western blotting, and immunofluorescence staining. The effect of astrocytic phenotype transformation on neuroinflammation was examined by real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that astrocytes were transformed into A1 astrocytes and caused neuronal death through the release of pro-inflammatory factors in EBI after SAH. Importantly, Ponesimod, an S1PR1 specific modulator, exerted neuroprotective effects through the prevention of astrocytic polarization to the A1 phenotype as proved by immunofluorescence, neurological tests, and TUNEL study. We also revealed the role of Ponesimod in modulating astrocytic response was mediated by the signal transducer and activator of transcription 3 (STAT3) signaling. Our study suggested that Ponesimod may be a promising therapeutic target for the treatment of brain injury following SAH.

JTE-013 Alleviates Inflammatory Injury and Endothelial Dysfunction Induced by Sepsis In Vivo and In Vitro.

BACKGROUND: Nowadays, there is no approved targeted agent for lung injury induced by sepsis. S1PR2 is confirmed to be a promising diagnosis and treatment target. JTE-013 as S1PR2 antagonists may be an agent of great potential. In this research, we sought to determine the functional role of JTE-013 in lung injury induced by sepsis. MATERIALS AND METHODS: Seventy-two rats were assigned into normal group, sepsis model group and JTE-013 group. The animal model of lung injury induced by sepsis was constructed by cecal ligation and puncture. The human pulmonary microvascular endothelial cells (HPMECs) were divided into control, LPS and LPS + JTE-013 group. HPMECs induced by LPS served as the cell model of lung injury induced by sepsis. HE staining assay was performed for assessment of the pathological condition and Evans blue was applied for assessment of pulmonary tissue permeability. Wet/dry ratio was measured as indicators of pulmonary edema degree and neutrophil count was measured as indicators of infection status. The levels of inflammatory factors were detected by corresponding kits, cell survival by CCK-8 assay and protein expression level by western blot. RESULTS: S1PR2 was highly expressed in vivo model of lung injury induced by sepsis. It was observed that JTE-013 as antagonist of S1PR2 alleviated the lung tissue injury, endothelial dysfunction and pulmonary edema induced by sepsis. In addition, JTE-013 reduced neutrophil count and levels of inflammatory factors. Moreover, results confirmed that JTE-013 enhanced cell viability and mitigated inflammatory response in cell model of sepsis. CONCLUSIONS: Overall, JTE-013 as an antagonist of S1PR2 could relieve inflammatory injury and endothelial dysfunction induced by sepsis in vivo and vitro, resulting in attenuation of lung injury. These findings elucidated that JTE-013 may be a promising targeted agent for lung injury induced by sepsis.

Discovery of the S1P2 Antagonist GLPG2938 (1-[2-Ethoxy-6-(trifluoromethyl)-4-pyridyl]-3-[[5-methyl-6-[1-methyl-3-(trifluoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea), a Preclinical Candidate for the Treatment of Idiopathic Pulmonary Fibrosis.

Mounting evidence from the literature suggests that blocking S1P2 receptor (S1PR2) signaling could be effective for the treatment of idiopathic pulmonary fibrosis (IPF). However, only a few antagonists have been so far disclosed. A chemical enablement strategy led to the discovery of a pyridine series with good antagonist activity. A pyridazine series with improved lipophilic efficiency and with no CYP inhibition liability was identified by scaffold hopping. Further optimization led to the discovery of 40 (GLPG2938), a compound with exquisite potency on a phenotypic IL8 release assay, good pharmacokinetics, and good activity in a bleomycin-induced model of pulmonary fibrosis.

Targeting S1PRs as a Therapeutic Strategy for Inflammatory Bone Loss Diseases-Beyond Regulating S1P Signaling.

As G protein coupled receptors, sphingosine-1-phosphate receptors (S1PRs) have recently gained attention for their role in modulating inflammatory bone loss diseases. Notably, in murine studies inhibiting S1PR2 by its specific inhibitor, JTE013, alleviated osteoporosis induced by RANKL and attenuated periodontal alveolar bone loss induced by oral bacterial inflammation. Treatment with a multiple S1PRs modulator, FTY720, also suppressed ovariectomy-induced osteoporosis, collagen or adjuvant-induced arthritis, and apical periodontitis in mice. However, most previous studies and reviews have focused mainly on how S1PRs manipulate S1P signaling pathways, subsequently affecting various diseases. In this review, we summarize the underlying mechanisms associated with JTE013 and FTY720 in modulating inflammatory cytokine release, cell chemotaxis, and osteoclastogenesis, subsequently influencing inflammatory bone loss diseases. Studies from our group and from other labs indicate that S1PRs not only control S1P signaling, they also regulate signaling pathways induced by other stimuli, including bacteria, lipopolysaccharide (LPS), bile acid, receptor activator of nuclear factor κB ligand (RANKL), IL-6, and vitamin D. JTE013 and FTY720 alleviate inflammatory bone loss by decreasing the production of inflammatory cytokines and chemokines, reducing chemotaxis of inflammatory cells from blood circulation to bone and soft tissues, and suppressing RANKL-induced osteoclast formation.

Deoxycholic acid enhancement of lymphocyte migration through direct interaction with the intestinal vascular endothelium.

BACKGROUND AND AIM: The small intestine plays a central role in gut immunity, and enhanced lymphocyte migration is involved in the pathophysiology of various enteropathy. Bile acid (BA) is closely related to lipid metabolism and gut microbiota and essential for gut homeostasis. However, the effects of BA on gut immunity have not been studied in detail, especially on the small intestine and lymphocyte migration. Therefore, we aimed to investigate the effect of BA on small intestinal lymphocyte microcirculation. METHODS: The effect of deoxycholic acid (DCA), taurocholic acid (tCA), or cholic acid (CA) on the indomethacin (IND)-induced small intestinal enteropathy in mice was investigated. Lymphocyte movements were evaluated after exposure to BA using intravital microscopy. The effects of BA on surface expression of adhesion molecules on the vascular endothelium and lymphocytes through BA receptors were examined in vitro. RESULTS: IND-induced small intestinal enteropathy was histologically aggravated by DCA treatment alone. The expression of adhesion molecules ICAM-1 and VCAM-1 was significantly enhanced by DCA. Exposure to DCA increased lymphocyte adhesion in the microvessels of the ileum, which was partially blocked by anti-α4ß1 integrin antibody in vivo. The expression of ICAM-1 and VCAM-1 was significantly enhanced by DCA in vitro, which was partially suppressed by the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist. The S1PR2 antagonist significantly ameliorated IND-induced and DCA-exaggerated small intestinal injury. CONCLUSION: DCA exacerbated IND-induced small intestinal enteropathy. DCA directly acts on the vascular endothelium and enhances the expression levels of adhesion molecules partially via S1PR2, leading to enhanced small intestinal lymphocyte migration.

Sphingosine-1-phosphate receptor 3 is implicated in BBB injury via the CCL2-CCR2 axis following acute intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a catastrophic cerebrovascular disease with high morbidity and mortality. Evidence demonstrated that sphingosine-1-phosphate receptor (S1PR) plays a vital role in inflammatory damage via the upregulation of CCL2 expression. However, whether S1PR3 is involved in blood-brain barrier (BBB) breakdown via CCL2 activation after ICH has not been described. METHODS: We investigated the expression profiles of all S1PRs using high-throughput RNA-seq analysis and RT-PCR. The potential role of S1PR3 and interaction between S1PR3 and CCL2 were evaluated via Western blotting, immunofluorescence, and flow cytometry. BBB disruption was examined via magnetic resonance imaging, transmission electron microscopy, and Evans blue extravasation. Microglial activation, proliferation, and polarization were assessed via histopathological analysis. The expression levels of CCL2, p-p38 MAPK, ICAM-1, and ZO-1 were examined in vitro and in vivo. RESULTS: The present results showed that the levels of S1PR3 and its ligand, sphingosine 1-phosphate (S1P), were dramatically increased following ICH, which regulated the expression of CCL2 and p38MAPK. Moreover, reductions in brain edema volume, amelioration of BBB integrity, and improvements in behavioral deficits were achieved after the administration of CAY10444, an S1PR3 antagonist, to rats. Remarkably increased CCL2, p-p38MAPK, and ICAM-1 expression and decreased ZO-1 expression were observed in cocultured human astrocytes (HAs) and hCMEC/D3 cells after S1P stimulation. However, the expression levels of CCL2, p-p38 MAPK, and ICAM-1 were decreased and ZO-1 expression was increased after S1PR3 inhibition. In addition, microglial proliferation and M1 polarization were attenuated after CAY10444 administration. CONCLUSION: To the best of our knowledge, this is the first demonstration of the neuroprotective role of S1PR3 modulation in maintaining BBB integrity by inhibiting the S1PR3-CCL2 axis after ICH, providing a novel treatment for ICH by targeting S1PR3.