Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P₁ and S1P₃ receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1−/−, or Sphk2−/− mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2−/− renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2−/− cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P₁ and S1P₃ antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P₁ and S1P₃. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P₁₊₃ to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.     

Scintillation Proximity Assay to Detect the Changes in Cellular Dihydrosphingosine 1-Phosphate Levels

Compounds that modulate the activity of sphingosine 1‐phosphate (S1P)‐metabolizing enzymes are expected to be potential therapeutic agents for various diseases. Investigation of their potencies requires not only cell‐free but also cell‐based assays in which intracellular accumulation/depletion of S1P could be monitored. However, conventional methods have limitations to their simplicity, mainly due to the necessity of a separation process that separates S1P from its related substances. Here, we describe a method utilizing a scintillation proximity assay (SPA) for semi‐quantifying intracellular [³H]‐labeled dihydroS1P ([³H]dhS1P), which is also a substrate for S1P‐metabolizing enzymes. We found that uncoated yttrium silicate SPA beads could selectively bind to and detect [³H]dhS1P rather than [³H]dihydrosphingosine (the non‐phosphorylated form of [³H]dhS1P). Based on this, we developed a novel cell‐based assay system which does not require any organic solvent extraction or chromatographic separation, and confirmed its practicality by using siRNA targeting S1P lyase (S1PL) and known S1PL inhibitors as models. Our results demonstrated that this assay is useful for rapid and easy evaluation of S1PL inhibitors, and could be potentially applicable for all compounds that modulate the activity of S1P‐metabolizing enzymes.     

n-3 Polyunsaturated Fatty Acids Improve Inflammation via Inhibiting Sphingosine Kinase 1 in a Rat Model of Parenteral Nutrition and CLP-Induced Sepsis

The sphingosine kinase 1 (SphK1)/sphingosine‐1‐phosphate (S1P) pathway plays a key role in inflammation. Parenteral nutrition containing n‐3 polyunsaturated fatty acids (n‐3 PUFA) may regulate inflammatory reactions. The aim of this study is to determine whether n‐3 PUFA may improve inflammatory responses by neutralizing SphK1 signaling. Rat models of parenteral nutrition, cecal ligation and puncture (CLP)‐induced sepsis were generated. Male Sprague–Dawley rats were operated for CLP on day 2 after venous catheterization. The rats were randomized to receive normal saline (NS; n = 20), parenteral nutrition (PN; n = 20), or PN + fish oil (FO; n = 20) for 5 days. The daily intake of fish oil (1.25–2.82 g EPA and 1.44–3.09 g DHA per 100 ml) in the FO group was approximately 1.8 g/kg body weight/day. Rats in the control group (n = 10) were subjected to sham operation and received a chow diet. Spleen tissues were collected for SphK1 and S1P receptor expression analysis. Our data showed that n‐3 PUFA ameliorated the survival rate. SphK1 expression and its enzymatic activity were significantly upregulated in sepsis rats. Furthermore, mRNA and protein levels of S1PR3, but not S1PR1, were also facilitated after CLP. However, PN + FO dramatically decreased SphK1 mRNA level and its enzymatic activity. S1PR3 expression was also attenuated by FO addition. In conclusion, the anti‐inflammatory effect of n‐3 PUFA may be linked to the inhibition of the SphK1/S1P pathway in a rat model of parenteral nutrition and CLP‐induced sepsis.     

Exogenous Sphingosine 1-Phosphate Protects Murine Splenocytes Against Hypoxia-Induced Injury

Sphingosine-1-phosphate (S1P), a biologically active pleiotropic lipid, is involved in several physiological processes especially in the area of vascular biology and immunology encompassing cell survival, angiogenesis, vascular tone, immune response etc. by interacting with specific cell surface receptors. Hypoxia, a condition common to innumerable pathologies, is known to lethally affect cell survival by throwing off balance global gene expression, redox homeostasis, bioenergetics etc. Several molecular events of cellular adaptations to hypoxia have been closely linked to stabilization of hypoxia inducible factor-1α (HIF-1α). Signalling functions of S1P in physiological events central to hypoxia-induced pathologies led us to investigate efficacy of exogenous S1P in preconditioning murine splenocytes to sustain during cellular stress associated with sub-optimal oxygen. The present study recapitulated the pro-survival benefits of exogenous S1P under normobaric hypoxia. Results indicate a direct effect of S1P supplementation on boosting cellular adaptive responses via HIF-1α stabilization and, activation of pro-survival mediators ERK and Akt. Overwhelming anti-oxidative and anti-inflammatory benefits of S1P preconditioning could also be captured in the present study, as indicated by improved redox homeostasis, reduced oxidative damage, balanced anti/pro-inflammatory cytokine profiles and temporal regulation of nitric oxide secretion and intra-cellular calcium release. Hypoxia induced cell death and the associated stress in cellular milieu in terms of oxidative damage and inflammation could be alleviated with exogenous S1P preconditioning.     

Silencing of Sphingosine kinase 1 Affects Maturation Pathways in Mouse Neonatal Cardiomyocytes

Sphingosine kinase-1 (Sphk1) and its product, sphingosine-1-phosphate (S1P) are important regulators of cardiac growth and function. Numerous studies have reported that Sphk1/S1P signaling is essential for embryonic cardiac development and promotes pathological cardiac hypertrophy in adulthood. However, no studies have addressed the role of Sphk1 in postnatal cardiomyocyte (CM) development so far. The present study aimed to assess the molecular mechanism(s) by which Sphk1 silencing might influence CMs development and hypertrophy in vitro. Neonatal mouse CMs were transfected with siRNA against Sphk1 or negative control, and subsequently treated with 1 µM angiotensin II (AngII) or a control buffer for 24 h. The results of RNASeq analysis revealed that diminished expression of Sphk1 significantly accelerated neonatal CM maturation by inhibiting cell proliferation and inducing developmental pathways in the stress (AngII-induced) conditions. Importantly, similar effects were observed in the control conditions. Enhanced maturation of Sphk1-lacking CMs was further confirmed by the upregulation of the physiological hypertrophy-related signaling pathway involving Akt and downstream glycogen synthase kinase 3 beta (Gsk3β) downregulation. In summary, we demonstrated that the Sphk1 silencing in neonatal mouse CMs facilitated their postnatal maturation in both physiological and stress conditions.     

Sphingolipid metabolism in colorectal adenomas varies depending on histological architecture of polyps and grade of nuclear dysplasia

Incidence of colorectal cancer (CRC) is growing worldwide. Pathogenetic mechanisms responsible for its onset and progression need further clarification. Colorectal adenomatous polyps are precancerous lesions with malignant potential dependent on histological architecture and grade of nuclear dysplasia. One of the factors conditioning CRC development are abnormalities in sphingolipid metabolism. The aim of this study was to assess the levels of sphingolipids in human colorectal adenomas. The control group (C, n = 12) consisted of patients with no colonic polyps. The examined group consisted of patients with prior diagnosed colonic polyps, qualified to endoscopic polypectomy. This group was further divided due to histological architecture into tubular adenomas group (TA, n = 10), tubulovillous adenomas with low‐grade dysplasia (LGD‐TVA, n = 10), and tubulovillous adenomas group with high‐grade dysplasia (HGD‐TVA, n = 11). In tissue samples, sphingolipd metabolite contents were measured using high performance liquid chromatography (HPLC). In cases of polypoid lesions with low malignancy potential (tubular adenomas), concentration of ceramide, which is characterized by proapoptotic and anti‐proliferative properties, increases compared with control group (p < 0.05), whereas content of sphingosine‐1‐phosphate with anti‐apoptotic and stimulating cellular proliferation properties is reduced in comparison with control group (p < 0.05). On the contrary, in cases of more advanced form of adenomatous polyps (tubulovillous adenomas with high‐grade dysplasia), the ceramide level decreases compared with control group (p < 0.05) while sphingosine‐1‐phosphate concentration is elevated (p < 0.05). We found that concentrations of pro‐apoptotic ceramide are decreased and pro‐proliferative S1P levels are increased in polypoid lesions with high malignancy potential, and it was the opposite in those with low malignancy potential.     

Nox2 Upregulation and p38α MAPK Activation in Right Ventricular Hypertrophy of Rats Exposed to Long-Term Chronic Intermittent Hypobaric Hypoxia

One of the consequences of high altitude (hypobaric hypoxia) exposure is the development of right ventricular hypertrophy (RVH). One particular type of exposure is long-term chronic intermittent hypobaric hypoxia (CIH); the molecular alterations in RVH in this particular condition are less known. Studies show an important role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex-induced oxidative stress and protein kinase activation in different models of cardiac hypertrophy. The aim was to determine the oxidative level, NADPH oxidase expression and MAPK activation in rats with RVH induced by CIH. Male Wistar rats were randomly subjected to CIH (2 days hypoxia/2 days normoxia; n = 10) and normoxia (NX; n = 10) for 30 days. Hypoxia was simulated with a hypobaric chamber. Measurements in the RV included the following: hypertrophy, Nox2, Nox4, p22phox, LOX-1 and HIF-1α expression, lipid peroxidation and H₂O₂ concentration, and p38α and Akt activation. All CIH rats developed RVH and showed an upregulation of LOX-1, Nox2 and p22phox and an increase in lipid peroxidation, HIF-1α stabilization and p38α activation. Rats with long-term CIH-induced RVH clearly showed Nox2, p22phox and LOX-1 upregulation and increased lipid peroxidation, HIF-1α stabilization and p38α activation. Therefore, these molecules may be considered new targets in CIH-induced RVH.     

Phospholipid Transfer Protein Deficiency Decreases the Content of S1P in HDL via the Loss of its Transfer Capability

Sphingosine‐1‐phosphate (S1P) is an amphiphilic signaling molecule, which is enriched in functional high density lipoprotein (HDL) and shows arterial protection. The distribution of S1P is changed with increased plasma phospholipid transfer protein (PLTP) activity and impaired HDL function in patients with coronary heart diseases. Therefore, we hypothesized that PLTP might transfer S1P among cells or lipoproteins. We found that plasma S1P contents were decreased by 60.1 % in PLTP knockout mice (PLTP?/?, N = 5) compared with their wild type littermates (WT, N = 5) (151.70 ± 38.59 vs. 379.32 ± 59.90 nmol/l, P<0.01). S1P content in HDL fraction (HDL‐S1P) from PLTP?/? was decreased by 64.7 % compared with WT (49.36 ± 1.49 vs. 139.76 ± 2.94 nmol/l, P<0.01). The results of the S1P transfer assay indicated that PLTP could facilitate S1P transport from erythrocytes to HDL at 37 °C in D‐Hanks buffer. Plasma content of apolipoprotein M, a specific adaptor of S1P, was not changed in PLTP?/? compared with WT. Therefore, we concluded that PLTP was a key factor to maintain plasma HDL‐S1P, and PLTP deficiency could decrease the S1P content in plasma lipoproteins, which involves its capability of transferring S1P from erythrocyte to HDL.     

Aberrant Sphingolipid Metabolism in the Human Fallopian Tube with Ectopic Pregnancy

Sphingosine 1-phosphate (S1P), a product of sphingomyelin metabolism, is generated via phosphorylation of sphingosine by sphingosine kinases (SphK). It acts via a family of G protein-coupled receptors or as an intracellular second messenger for agonists acting through the S1P receptors (S1P1–5). In our study, the expression of SphK1 and S1P1 was identified by immunohistochemistry and immunoblot. The concentration of S1P was measured using ELISA. The spontaneous contraction of isolated fallopian tube strips was determined by tension recording. Our results showed that SphK1 and S1P1 were localized in the fallopian tube epithelial cells. In addition, smooth muscle cells also contained S1P1. Compared with the intrauterine pregnancy group, SPHK1 and S1P1 were overexpressed in ectopic pregnancy. However, the S1P concentration within the human oviduct from ectopic pregnancy subjects was largely reduced than that from normal pregnancy subject. The results from tension recording indicated that exogenous and intracellularly generated S1P can regulate the spontaneous contraction of oviduct isolated from rats and human. In conclusion, the sphingolipid metabolism signal pathway functionally existed in the human fallopian tube. Aberrant sphingolipid metabolism in the human fallopian tube may be involved in ectopic pregnancy.     

Sphingosine-1-Phosphate-Triggered Expression of Cathelicidin LL-37 Promotes the Growth of Human Bladder Cancer Cells

It has been proven that tumour growth and progression are regulated by a variety of mediators released during the inflammatory process preceding the tumour appearance, but the role of inflammation in the development of bladder cancer is ambiguous. This study was designed around the hypothesis that sphingosine-1-phosphate (S1P), as a regulator of several cellular processes important in both inflammation and cancer development, may exert some of the pro-tumorigenic effects indirectly due to its ability to regulate the expression of human cathelicidin (hCAP-18). LL-37 peptide released from hCAP-18 is involved in the development of various types of cancer in humans, especially those associated with infections. Using immunohistological staining, we showed high expression of hCAP-18/LL-37 and sphingosine kinase 1 (the enzyme that forms S1P from sphingosine) in human bladder cancer cells. In a cell culture model, S1P was able to stimulate the expression and release of hCAP-18/LL-37 from human bladder cells, and the addition of LL-37 peptide dose-dependently increased their proliferation. Additionally, the effect of S1P on LL-37 release was inhibited in the presence of FTY720P, a synthetic immunosuppressant that blocks S1P receptors. Together, this study presents the possibility of paracrine relation in which LL-37 production following cell stimulation by S1P promotes the development and growth of bladder cancer.     

To Be or Not to Be: The Divergent Action and Metabolism of Sphingosine-1 Phosphate in Pancreatic Beta-Cells in Response to Cytokines and Fatty Acids

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid with multiple functions conveyed by the activation of cell surface receptors and/or intracellular mediators. A growing body of evidence indicates its important role in pancreatic insulin-secreting beta-cells that are necessary for maintenance of glucose homeostasis. The dysfunction and/or death of beta-cells lead to diabetes development. Diabetes is a serious public health burden with incidence growing rapidly in recent decades. The two major types of diabetes are the autoimmune-mediated type 1 diabetes (T1DM) and the metabolic stress-related type 2 diabetes (T2DM). Despite many differences in the development, both types of diabetes are characterized by chronic hyperglycemia and inflammation. The inflammatory component of diabetes remains under-characterized. Recent years have brought new insights into the possible mechanism involved in the increased inflammatory response, suggesting that environmental factors such as a westernized diet may participate in this process. Dietary lipids, particularly palmitate, are substrates for the biosynthesis of bioactive sphingolipids. Disturbed serum sphingolipid profiles were observed in both T1DM and T2DM patients. Many polymorphisms were identified in genes encoding enzymes of the sphingolipid pathway, including sphingosine kinase 2 (SK2), the S1P generating enzyme which is highly expressed in beta-cells. Proinflammatory cytokines and free fatty acids have been shown to modulate the expression and activity of S1P-generating and S1P-catabolizing enzymes. In this review, the similarities and differences in the action of extracellular and intracellular S1P in beta-cells exposed to cytokines or free fatty acids will be identified and the outlook for future research will be discussed.     

A Rheostat of Ceramide and Sphingosine-1-Phosphate as a Determinant of Oxidative Stress-Mediated Kidney Injury

Reactive oxygen species (ROS) modulate sphingolipid metabolism, including enzymes that generate ceramide and sphingosine-1-phosphate (S1P), and a ROS-antioxidant rheostat determines the metabolism of ceramide-S1P. ROS induce ceramide production by activating ceramide-producing enzymes, leading to apoptosis, while they inhibit S1P production, which promotes survival by suppressing sphingosine kinases (SphKs). A ceramide-S1P rheostat regulates ROS-induced mitochondrial dysfunction, apoptotic/anti-apoptotic Bcl-2 family proteins and signaling pathways, leading to apoptosis, survival, cell proliferation, inflammation and fibrosis in the kidney. Ceramide inhibits the mitochondrial respiration chain and induces ceramide channel formation and the closure of voltage-dependent anion channels, leading to mitochondrial dysfunction, altered Bcl-2 family protein expression, ROS generation and disturbed calcium homeostasis. This activates ceramide-induced signaling pathways, leading to apoptosis. These events are mitigated by S1P/S1P receptors (S1PRs) that restore mitochondrial function and activate signaling pathways. SphK1 promotes survival and cell proliferation and inhibits inflammation, while SphK2 has the opposite effect. However, both SphK1 and SphK2 promote fibrosis. Thus, a ceramide-SphKs/S1P rheostat modulates oxidant-induced kidney injury by affecting mitochondrial function, ROS production, Bcl-2 family proteins, calcium homeostasis and their downstream signaling pathways. This review will summarize the current evidence for a role of interaction between ROS-antioxidants and ceramide-SphKs/S1P and of a ceramide-SphKs/S1P rheostat in the regulation of oxidative stress-mediated kidney diseases.     

Pharmacophore‐Based Design of Novel Oxadiazoles as Selective Sphingosine‐1‐phosphate (S1P) Receptor Agonists with in vivo Efficacy

Sphingosine‐1‐phosphate (S1P) receptor agonists have shown promise as therapeutic agents for multiple sclerosis (MS) due to their regulatory roles within the immune, central nervous system, and cardiovascular system. Here, the design and optimization of novel [1,2,4]oxadiazole derivatives as selective S1P receptor agonists are described. The structure–activity relationship exploration was carried out on the three dominant segments of the series: modification of the polar head group (P), replacement of the oxadiazole linker (L) with different five‐membered heterocycles, and the use of diverse 2,2′‐disubstituted biphenyl moieties as the hydrophobic tail (H). All three segments have a significant impact on potency, S1P receptor subtype selectivity, physicochemical properties, and in vitro absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of the compounds. From these optimization studies, a selective S1P₁ agonist, N‐methyl‐N‐(4‐{5‐[2‐methyl‐2′‐(trifluoromethyl)biphenyl‐4‐yl]‐1,2,4‐oxadiazol‐3‐yl}benzyl)glycine ( 45 ), and a dual S1P_1,5 agonist, N‐methyl‐N‐(3‐{5‐[2′‐methyl‐2‐(trifluoromethyl)biphenyl‐4‐yl]‐1,2,4‐oxadiazol‐3‐yl}benzyl)glycine ( 49 ), emerged as frontrunners. These compounds distribute predominantly in lymph nodes and brain over plasma and induce long lasting decreases in lymphocyte count after oral administration. When evaluated head‐to‐head in an experimental autoimmune encephalomyelitis mouse model, together with the marketed drug fingolimod, a pan‐S1P receptor agonist, S1P_1,5 agonist 49 demonstrated comparable efficacy while S1P₁‐selective agonist 45 was less potent. Compound 49 is not a prodrug, and its improved property profile should translate into a safer treatment of relapsing forms of MS.     

Extracellular Sphingosine-1-Phosphate Downstream of EGFR Increases Human Glioblastoma Cell Survival

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ; moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.     

Recognition and Excision Properties of 8‐Halogenated‐7‐Deaza‐2′‐Deoxyguanosine as 8‐Oxo‐2′‐Deoxyguanosine Analogues and Fpg and hOGG1 Inhibitors

Cellular DNA continuously suffers various types of damage, and unrepaired damage increases disease progression risk. 8‐Oxo‐2′‐deoxyguanine (8‐oxo‐dG) is excised by repair enzymes, and their analogues are of interest as inhibitors and as bioprobes for study of these enzymes. We have developed 8‐halogenated‐7‐deaza‐2′‐deoxyguanosine derivatives that resemble 8‐oxo‐dG in that they adopt the syn conformation. In this study, we investigated their effects on Fpg (formamidopyrimidine DNA glycosylase) and hOGG1 (human 8‐oxoguanine DNA N‐glycosylase 1). Relative to 8‐oxo‐dG, Cl‐ and Br‐deaza‐dG were good substrates for Fpg, whereas they were less efficient substrates for hOGG1. Kinetics and binding experiments indicated that, although hOGG1 effectively binds Cl‐ and Br‐deaza‐dG analogues with low K_m values, their lower k_cat values result in low glycosylase activities. The benefits of the high binding affinities and low reactivities of 8‐oxo‐dG analogues with hOGG1 have been successfully applied to the competitive inhibition of the excision of 8‐oxoguanine from duplex DNA by hOGG1.     

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) ({"type":"clinical-trial","attrs":{"text":"NCT03742037","term_id":"NCT03742037"}}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.