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1.  The Sphingosine-1-Phosphate Analogue FTY720 Impairs Mucosal Immunity and Clearance of the Enteric Pathogen Citrobacter rodentium 
Infection and Immunity  2012;80(8):2712-2723.
The sphingosine-1-phosphate (S1P) analogue FTY720 is therapeutically efficacious in multiple sclerosis and in the prevention of transplant rejection. It prevents the migration of lymphocytes to sites of pathology by trapping them within the peripheral lymph nodes, mesenteric lymph nodes (MLNs), and Peyer's patches. However, evidence suggests that its clinical use may increase the risk of mucosal infections. We investigated the impact of FTY720 treatment on susceptibility to gastrointestinal infection with the mouse enteric pathogen Citrobacter rodentium. This attaching and effacing bacterium induces a transient bacterial colitis in immunocompetent mice that resembles human infection with pathogenic Escherichia coli. FTY720 treatment induced peripheral blood lymphopenia, trapped lymphocytes in the MLNs, and prevented the clearance of bacteria when mice were infected with luciferase-tagged C. rodentium. FTY720-treated C. rodentium-infected mice had enhanced colonic inflammation, with significantly higher colon mass, colon histopathology, and neutrophil infiltration than vehicle-infected animals. In addition, FTY720-treated infected mice had significantly lower numbers of colonic dendritic cells, macrophages, and T cells. Gene expression analysis demonstrated that FTY720-treated infected mice had an impaired innate immune response and a blunted mucosal adaptive immune response, including Th1 cytokines. The data demonstrate that the S1P analogue FTY720 adversely affects the immune response to and clearance of C. rodentium.
PMCID: PMC3434556  PMID: 22615252
2.  Treatment with the immunomodulator FTY720 does not promote spontaneous bacterial infections after experimental stroke in mice 
FTY720, an immunomodulator derived from a fungal metabolite which reduces circulating lymphocyte counts by increasing the homing of lymphocytes to the lymph nodes has recently gained interest in stroke research. The aim of this study was to evaluate the protective efficacy of FTY720 in cerebral ischemia in two different application paradigms and to gather first data on the effect of FTY720 on the rate of spontaneous bacterial infections in experimental stroke.
Middle cerebral artery occlusion (MCAO) in C57BL/6 mice (strain J, groups of 10 animals) was performed with two different durations of ischemia (90 min and 3 h) and FTY720 was applied 2 h after vessel occlusion to study the impact of reperfusion on the protective potency of FTY720. Lesion size was determined by TTC staining. Mice treated with FTY720 or vehicle were sacrificed 48 h after 90 min MCAO to determine the bacterial burden in lung and blood.
FTY720 1 mg/kg significantly reduced ischemic lesion size when administered 2 h after the onset of MCAO for 3 h (45.4 ± 22.7 mm3 vs. 84.7 ± 23.6 mm3 in control mice, p = 0.001) and also when administered after reperfusion, 2 h after the onset of MCAO for 90 min (31.1 ± 28.49 mm3 vs. 69.6 ± 27.2 mm3 in control mice, p = 0.013). Bacterial burden of lung homogenates 48 h after stroke did not increase in the group treated with the immunomodulator FTY720 while there was no spontaneous bacteremia 48 h after MCAO in treated and untreated animals.
Our results corroborate the experimental evidence of the protective effect of FTY720 seen in different rodent stroke models. Interestingly, we found no increase in bacterial lung infections even though FTY720 strongly reduces the number of circulating leukocytes.
PMCID: PMC3068098  PMID: 21388542
3.  Prevention, but not cure, of autoimmune diabetes in a NOD.scid transfer model by FTY720 despite effective modulation of blood T cells 
Autoimmunity  2010;44(2):115-128.
FTY720 modulates lymphocyte trafficking through blood (PBL) and peripheral lymph nodes (PLN). Treatment with FTY720 causes retention of most blood lymphocytes in PLN. Long-term treatment can slow and/or prevent type 1 diabetes in the non-obese diabetic (NOD) mouse model. B and T cells are both affected by FTY720 binding to S1P1 (sphingosine-1-phosphate receptor 1). However, little has been done to elucidate which T cell subsets are differentially affected by FTY720 under healthy conditions, and how this affects disease pathogenesis in Type 1 Diabetes (T1D). In healthy C57BL/6J (B6) mice, total CD4+ and CD8+ T cell subsets were diminished by FTY720, but recently activated and memory subsets were spared and a constituted significantly higher percentage of remaining T cells in blood. FTY720 also lowered PBL counts in NOD mice, but less severely than in B6 mice. This is consistent with a different ratio of naïve, activated, and memory cells in NOD mice compared to B6 mice, as well as alterations in S1P1 and S1P (sphingosine-1-phosphate) levels in PBLs and blood of NOD mice, respectively. To address the functional consequences of PBL T cell depletion, we studied the effects of FTY720 on disease progression in a timed adoptive transfer model of T1D. Continuous treatment with FTY720 eliminated T1D if treatment was started before splenocyte transfer. FTY20 treatment started after disease onset slowed disease progression. The inability to fully suppress memory and effector T cell circulation may explain why FTY720 is only partially effective in the NOD adoptive transfer model of T1D.
PMCID: PMC2994995  PMID: 20695767
Type 1 diabetes; T cells; immunomodulation; FTY720; adoptive transfer
4.  Evaluation of the Lymphocyte Trafficking Drug FTY720 in Vaginal Tissues 
Journal of medical primatology  2013;42(2):89-100.
FTY720 is an immunomodulatory agent that reduces lymphocytes in peripheral tissues and circulation. Such agents may be effective as vaginal microbicides for HIV prevention. Systemic or vaginal application of FTY720 may reduce lymphocyte concentrations in genital tissues, reducing HIV target cell numbers.
Five female pigtail macaques received topical vaginal gel FTY720 (n=2), intravenous (IV) FTY720 (n=2), or placebo gel (n=1) in this pilot study. Circulating and mucosal lymphocytes and genital mucosa, cytokines, and tissue histology were analyzed to document topical and IV FTY720 effects.
Topical and IV FTY720 appeared to decrease levels of cervicovaginal IL-8, IL-1ra, and genital inflammatory cells. Small sample size precluded statistical analysis. Topical administration had no overt adverse effects.
This study introduces FTY720 as an immunomodulatory agent for the vaginal mucosa, compares topical effects to those of IV administration, and provides the basis for future studies involving FTY720 for HIV prevention.
PMCID: PMC3594332  PMID: 23311598
Genital; nonhuman primates; topical gel; HIV
5.  Anticancer activity of FTY720: phosphorylated FTY720 inhibits autotaxin, a metastasis-enhancing and angiogenic lysophospholipase D 
Cancer letters  2008;266(2):203-208.
FTY720 is an immunomodulator that is phosphorylated in vivo and inhibits lymphocyte mobilization by targeting sphingosine 1-phospate receptors. At doses higher than required for immunomodulation, FTY720 inhibits tumor progression through an unknown mechanism. Here we show that FTY720-phosphate is a competitive inhibitor (Ki ~0.2 µM) of autotaxin (ATX or NPP2), a nucleotide phosphodiesterase/pyrophosphatase (NPP) that enhances metastasis and angiogenesis and acts as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA). FTY720-phosphate did no affect the activity of NPP1, the closest relative of ATX. After oral administration in mice, FTY720 (3 mg/kg) significantly reduced plasma LPA levels. These results suggest that FTY720 may exert its anticancer effects, at least in part, by targeting the ATX-LPA axis.
PMCID: PMC2943588  PMID: 18378389
FTY720; autotaxin; lysophosphatidic acid; tumor progression
6.  Treatment with a sphingosine analog does not alter the outcome of a persistent virus infection 
Virology  2009;397(2):260.
There is no known antiviral drug treatment that routinely terminates persistent virus infections. A recent provocative report indicated that low dosage of the sphingosine analog FTY720 caused lymphopenia in mice persistently infected with lymphocytic choriomeningitis virus (LCMV) -clone 13 (−Cl 13) and induced viral clearance within 30 days post-treatment (Premenko-Lanier et al., 2008). However, we find that low dosage of FTY720 fails to purge LCMV-Cl 13 infection and does not induce lymphopenia in LCMV-Cl 13-infected mice. In fact, infection with non-persistent LCMV-Arm53b or with persistent LCMV-Cl 13 induces an equivalent lymphopenia, demonstrating that the quantity of circulating cells has little bearing on viral persistence. In addition, treatment with FTY720 or the sphingosine-1-phosphate receptor 1 (S1P1) -specific agonist, AUY954, does not alleviate T cell exhaustion and exacerbates disruption of the CD8+ T cells response following LCMV-Cl 13 infection. Therefore, treatment with a sphingosine analog does not ameliorate persistent LCMV-Cl 13 infection.
PMCID: PMC2821988  PMID: 19962171
viral persistence; FTY720; lymphopenia; lymphocytic choriomeningitis virus; sphingosine; influenza; sphingosine-1-phosphate receptor 1; AUY954
7.  The Immunosuppressant FTY720 (Fingolimod) enhances Glycosaminoglycan depletion in articular cartilage 
FTY720 (Fingolimod) is a novel immunosuppressive drug investigated in clinical trials for organ transplantation and multiple sclerosis. It acts as a functional sphingosine-1-phosphate (S1P) receptor antagonist, thereby inhibiting the egress of lymphocytes from secondary lymphoid organs. As S1P is able to prevent IL-1beta induced cartilage degradation, we examined the direct impact of FTY720 on cytokine induced cartilage destruction.
Bovine chondrocytes were treated with the bioactive phosphorylated form of FTY720 (FTY720-P) in combination with IL-1beta or TNF-alpha. Expression of MMP-1,-3.-13, iNOS and ADAMTS-4,-5 and COX-2 was evaluated using quantitative real-time PCR and western blot. Glycosaminoglycan depletion from cartilage explants was determined using a 1,9-dimethylene blue assay and safranin O staining.
FTY720-P significantly reduced IL-1beta and TNF-alpha induced expression of iNOS. In contrast FTY720-P increased MMP-3 and ADAMTS-5 mRNA expression. Furthermore depletion of glycosaminoglycan from cartilage explants by IL-1beta and TNF-alpha was significantly enhanced by FTY720-P in an MMP-3 dependent manner.
Our results suggest that FTY720 may enhance cartilage degradation in pro-inflammatory environment.
PMCID: PMC3258222  PMID: 22151889
chondrocyte; fingolimod; FTY720; interleukin-1β; tumor necrosis factor-α; inducible nitric oxide synthase; glycosaminoglycan
8.  FTY720 stimulates multidrug transporter– and cysteinyl leukotriene–dependent T cell chemotaxis to lymph nodes 
Journal of Clinical Investigation  2003;111(5):627-637.
FTY720 is a sphingosine-derived immunosuppressant. Phosphorylated FTY720 promotes T cell homing from spleen and peripheral blood to LNs by acting as an agonist for sphingosine-1-phosphate (S1P) receptors. Here we demonstrate that FTY720 enhances the activity of the sphingosine transporter Abcb1 (Mdr1) and the leukotriene C4 transporter Abcc1 (Mrp1). Both transporters must be active for FTY720-mediated T cell migration and LN homing. Migration and homing driven by FTY720, phosphorylated FTY720, or S1P also require 5-lipoxygenase–mediated synthesis of cysteinyl leukotrienes and their efflux from the cell. FTY720-mediated LN homing events further downstream are dependent on CCL19, CCL21, VLA-4α, and CD44. Use of T cells deficient in 5-lipoxygenase, Abcb1, and Abcc1, and comparison of the effects of FTY720 with those of S1P, suggest a model of sequential engagement of Abcb1, SP1 receptors, 5-lipoxygenase, and Abcc1 to enhance T cell migration and homing.
PMCID: PMC151892  PMID: 12618517
9.  Targeting Lymphangiogenesis After Islet Transplantation Prolongs Islet Allograft Survival 
Transplantation  2011;92(1):25-30.
Lymphatics are important for their conduit functions of transporting antigen, immune cells, and inflammatory mediators to draining lymph nodes and to the general circulation. Lymphangiogenesis is involved in many pathologic processes; however, the roles for lymphatic responses in transplantation have not been thoroughly investigated.
Mice were made diabetic by a single high dose of streptozotocin and then received islet allografts. Animals were treated with three different lymphatic inhibitors. FTY720, an analog of sphingosine 1-phosphate, inhibited lymphocyte migration into afferent and efferent lymphatics. Sunitinib, a kinase inhibitor, blocked several receptors, including vascular endothelial growth factor receptor 3 (VEGFR3), the major growth factor receptor for lymphatic endothelial cells. Anti-VEGFR3 monoclonal antibody specifically inhibited VEGFR3. Diabetes was determined by daily monitoring of blood glucose levels. Inflammation within islet grafts was assessed by immunohistochemistry for insulin, T cells (CD3), and lymphatics (LYVE-1).
After transplantation, lymphangiogenesis occurred in islet allografts and in draining lymph nodes. FTY720, sunitinib, and anti-VEGFR3 each inhibited lymphangiogenesis in the islets and significantly prolonged allograft survival. Immunofluorescent staining demonstrated that administration of each of the lymphatic inhibitors resulted in preservation of islets and β-cells along with a markedly reduced infiltration of T cells into the grafts.
Lymphangiogenesis occurs in islet allografts in response to inflammation and plays a key role in the islet inflammation in alloimmunity. Interfering with lymphatic function leads to inhibition of lymphangiogenesis and prolonged or indefinite allograft survival. These observations suggest new therapeutic targets for rejection and tolerance.
PMCID: PMC3703312  PMID: 21508896
Allograft; Lymphangiogenesis; Islet; Tolerance
10.  Fingolimod (FTY720) Stimulates Ca2+/Calcineurin Signaling in Fission Yeast 
PLoS ONE  2013;8(12):e81907.
Fingolimod hydrochloride (FTY720) is the first in class of sphingosine 1-phosphate (S1P) receptor modulator approved to treat multiple sclerosis via down-regulation of G protein-coupled S1P receptor 1 by its phosphorylated form (FTY720-P). Many studies have revealed that FTY720 exerts various biological effects, including antitumor activities, angiogenesis inhibition, Ca2+ mobilization and apoptosis, independently of S1P receptors. However, the exact mechanisms underlying their effects or signaling pathways mediated by FTY720 have not been completely established. To gain further insights into molecular mechanisms of FTY720 action, the effect of FTY720 on Ca2+ signaling in fission yeast was analyzed. The addition of Ca2+ enhanced the sensitivity induced by FTY720, and mutants lacking genes required for calcium homeostasis, including calcineurin and its downstream transcription factor, Ppb1-responsive zinc finger protein (Prz1), were hypersensitive to FTY720 and CaCl2. The effect of FTY720 on calcineurin signaling was monitored by utilizing a luciferase reporter construct fused to three tandem repeats of the calcineurin-dependent response element (CDRE), which gives an accurate measure of calcineurin activity. The addition of FTY720 increased calcineurin activity as well as Ca2+ influx in a concentration-dependent manner. Notably, the FTY720-mediated Ca2+ influx and calcineurin activation were reduced markedly by the deletion of yam8+ or cch1+ encoding putative subunits of a Ca2+ channel. Consistently, the deletion of Pmk1 mitogen-activated protein kinase (MAPK), which plays an important role in the activation of the Yam8/Cch1 channel, markedly decreased the intracellular Ca2+ levels upon FTY720 treatment. These results suggest that the FTY720-stimulated Ca2+/calcineurin signaling activation partly involves the Yam8/Cch1 channel in fission yeast.
PMCID: PMC3849299  PMID: 24312601
11.  Gilenya (FTY720) inhibits acid sphingomyelinase by a mechanism similar to tricyclic antidepressants 
The immunomodulator drug Gilenya (FTY720), marketed as the first oral sphingosine 1-phosphate receptor (S1P-R) modulator for treatment of Multiple Sclerosis (MS) also inhibits lysosomal acid sphingomyelinase (ASMase). Treatment of cultured cells for 24h with FTY720 (up to 10μM) inhibited ASMase by >80% and this could be reversed by pre-treatment with the cathepsin protease inhibitor leupeptin (5μM). In contrast, neutral sphingomyelinase activity was unaffected and sphingosine-1-phosphate treatment had no effect on ASMase. RT-PCR revealed no inhibition of ASMase mRNA and there was no direct (in vitro) inhibition of ASMase by either FTY720 or FTY720-phosphate. This suggests that its mechanism of inhibition is similar to that of tricyclic anti-depressants such as desipramine, which are also amphiphilic cationic drugs. Both Desipramine and FTY720 treatment reduced ASMase without significant inhibition of other lysosomal hydrolases but most hydrolases showed increased secretion (up to a 50% increase) providing more evidence of lysosomal disruption by these drugs.
PMCID: PMC3031124  PMID: 21130737
Galinya; myriocin analog; acid sphingomyelinase; lysosomal hydrolases; amphiphilic cation; tricyclic anti-depressant
12.  Sphingosine 1-Phosphate Receptor 1 as a Useful Target for Treatment of Multiple Sclerosis 
Pharmaceuticals  2012;5(5):514-528.
Sphingosine 1-phosphate (S1P), a lysophospholipid mediator, is generated from sphingosine by sphingosine kinases and binds five known cell surface receptors. S1P receptor 1 (S1P1) plays an essential role in lymphocyte egress from secondary lymphoid organs (SLO), as evinced by the inability of lymphocytes to exit from the SLO in mice lacking lymphocytic S1P1. Fingolimod hydrochloride (FTY720) is a first-in-class, orally active, S1P receptor modulator with a structure closely related to sphingosine. FTY720 was first synthesized by chemical modification of a natural product, myriocin. FTY720 is effectively converted to an active metabolite, FTY720 phosphate (FTY720-P) by sphingosine kinases. FTY720-P shows high affinity to 4 of the S1P receptors (S1P1, S1P3, S1P4, and S1P5). In particular, FTY720-P strongly induces internalization and degradation of S1P1, inhibits S1P responsiveness of lymphocytes in the SLO, and acts as a functional antagonist at lymphocytic S1P1. Consequently, FTY720 inhibits S1P1-dependent lymphocyte egress from the SLO to decrease circulation of lymphocytes including autoreactive Th17 cells and is highly effective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Because FTY720 shows a superior efficacy in relapsing remitting MS patients compared to intramuscular interferon-β-1a (Avonex®), S1P1 is presumed to be a useful target for the therapy of MS.
PMCID: PMC3763654  PMID: 24281561
sphingosine 1-phasphate (S1P); S1P receptor 1 (S1P1); fingolimod (FTY720); lymphocyte egress; immunomodulator; experimental autoimmune encephalomyelitis (EAE); multiple sclerosis (MS); therapy
13.  B Lymphocytes Exit Lymph Nodes through Cortical Lymphatic Sinusoids Near to Lymph Node Follicles by a Mechanism Independent of S1P-Mediated Chemotaxis 
Immunity  2009;30(3):434-446.
Sphingosine-1 Phosphate (S1P) helps mediate lymphocyte egress from lymph nodes, yet significant mechanistic questions remain. Here we show that B lymphocyte egress sites exist close to lymph node follicles. Recent B cell emigrants localize towards follicle centers, while longer-term residents tend towards cortical sinusoids. Exiting B lymphocytes squeeze through apparent portals in the lymphatic endothelium. Treatment with the S1P receptor agonist FTY720 empties the cortical sinusoids of lymphocytes, blocks lymphatic endothelial penetration, and displaces B lymphocytes into the T cell zone. S1P3−/− B cells, which lack chemoattractant responses to S1P, transit lymph nodes normally, while Gnai2−/− B cells, which have impaired responses to chemokines and S1P, transit more rapidly than do wild type cells. This study identifies a major site of B lymphocyte lymph node egress, shows that FTY720 treatment blocks passage through the cortical lymphatic endothelium, and argues against a functional role for S1P chemotaxis in B lymphocyte egress.
PMCID: PMC2744325  PMID: 19230723
14.  Protective Effect of FTY720 on Several Markers of Liver Injury Induced by Concanavalin A in Mice 
2-Amino-2-[2-(4-octylphenyl)ethyl] propane-1,3-diol hydrochloride (FTY720) is a novel agent with protective effect on several markers of liver injury. It is a chemical substance derived by modifying myriocin from the ascomycete Isaria sinclairii. It has been reported that FTY720 is able to treat autoimmune encephalomyelitis, renal cancer, asthma, and multiple sclerosis. More potent clinical applications of FTY720 need to be investigated.
The aim of this study was to evaluate the protective effect of FTY720 on several markers of experimental liver injury and to investigate the possible mechanism of action.
Concanavalin A (Con A) at a dose of 15 mg/kg was intravenously. injected in mice, and 10 days before the Con A challenge, 1 mg/kg, 3 mg/kg, and 6 mg/kg of FTY720 were administered to mice. The liver injury was monitored biochemically by measuring serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and tumor necrosis factor-α (TNF-α) levels. TNF-α and nuclear factor-κB (NF-κB) in liver tissue were detected by Western blot analysis.
FTY720, when administered intragastrically for 10 days in mice with Con A–induced liver injury, dose-dependently reduced serum ALT and AST and TNF-α levels. The differences were statistically significant (P ≤ 0.05). It was also found that FTY720 decreases TNF-α and NF-κB protein expression in liver tissue.
FTY720 is able to improve several markers of Con A–induced liver injury in mice, including serum ALT, serum AST, TNF-α, and NF-κB, which might be at least in part related to its ability to reduce TNF-α/NF-κB cascade activity.
PMCID: PMC3954012  PMID: 24653516
concanavalin A; FTY720; liver injury; nuclear factor-κB; tumor necrosis factor-α
15.  FTY720 blocks egress of T cells in part by abrogation of their adhesion on the lymph node sinus 
Egress of lymphocytes from lymphoid tissues is a complex process in which Gαi-mediated signals play a decisive role. We show here that although FTY720, an agonist of the sphingosine 1-phosphate 1 receptor (S1P1R), induces S1P1R internalization sufficiently in the presence or absence of Gαi2 or Gαi3, the drug blocks egress of wild type (WT) and Gαi3-deficent T cells, but not Gαi2-deficient T cells in both WT and Gαi2-deficient hosts. Intravital imaging of lymph nodes revealed that all three groups of T cells approached and engaged cortical sinusoids similarly in the presence or absence of FTY720. The cells also entered and departed the sinus at an almost identical frequency in the absence of the drug. However, after engagement of the sinus, most WT and Gαi3-deficient T cells retracted and migrated back into the parenchyma in FTY720-treated animals, due to a failure of the cells to establish adhesion on the sinus, whereas Gαi2-deficient T cells adhered firmly on the sinus that prevented their retraction, facilitating their transmigration of the lymphatic endothelial barrier. These data confirm egress of Gαi2−/− T cells independent of S1P-mediated chemotaxis and failure of FTY720 to close lymphatic stromal channels, and argue for the first time that FTY720 induces lymphopenia in part by impairing T cell adhesion to the sinus in a manner dependent on Gαi2.
PMCID: PMC3167479  PMID: 21788441
16.  Prevention of Diabetes by FTY720-Mediated Stabilization of Peri-Islet Tertiary Lymphoid Organs 
Diabetes  2010;59(6):1461-1468.
The nonobese diabetic (NOD) mouse is a well-established mouse model of spontaneous type 1 diabetes, which is characterized by an autoimmune destruction of the insulin-secreting pancreatic β-cells. In this study, we address the role of tertiary lymphoid organs (TLOs) that form in the pancreas of NOD mice during disease progression.
We developed a model designed to “lock” lymphocytes in the pancreatic lymph node (PLN) and pancreas by the use of FTY720, which blocks the exit of lymphocytes from lymph nodes. A combination of flow cytometry, immunofluorescence, and analysis of clinical scores was used to study the effects of long-term FTY720 treatment on TLO development and development of diabetes.
Continuous treatment of NOD mice with FTY720 prevented diabetes development even at a time of significant insulitis. Treatment withdrawal led to accelerated disease independent of the PLN. Interestingly, naive T-cells trafficked to and proliferated in the TLOs. In addition, morphological changes were observed that occurred during the development of the disease. Remarkably, although the infiltrates are not organized into T/B-cell compartments in 8-week-old mice, by 20 weeks of age, and in age-matched mice undergoing FTY720 treatment, the infiltrates showed a high degree of organization. However, in naturally and FTY720-induced diabetic mice, T/B-cell compartmentalization was lost.
Our data show that TLOs are established during diabetes development and suggest that islet destruction is due to a loss of TLO integrity, which may be prevented by FTY720 treatment.
PMCID: PMC2874707  PMID: 20299465
17.  FTY720 postconditions isolated perfused heart by a mechanism independent of sphingosine kinase 2 and different from S1P or ischemic postconditioning 
We investigated the hypothesis that postconditioning by FTY720 (FTY) in isolated perfused mouse hearts is independent of the sphingosine 1-phosphate (S1P) pathway.
Ex vivo hearts were exposed to postconditioning (POST) by either ischemia or FTY720. Protection against ischemia/reperfusion (IR) injury was measured by recovery of left ventricular developed pressure (LVDP) and infarct size.
FTY effectively postconditioned (POST) ex vivo hearts against ischemia/reperfusion (IR) injury as measured by recovery of LVDP and a low infarct size. FTY protection, unlike S1P but like sphingosine (Sph), was insensitive to inhibition of S1P G-Protein Coupled Receptors (GPCRs) or inhibition of PI3 kinase. Protection by FTY and Sph was however blocked by inhibitors of PKA and PKG. Thus, FTY follows the same cardioprotective pathway as Sph. This was further supported by studies of FTY POST in knockout (KO) mice lacking the SphK2 form of Sph kinase that is needed for phosphorylation of FTY to an S1P analog. In the absence of SphK2, FTY (and Sph) POST was still cardioprotective. This differed from the effect of SphK2 KO on protection by ischemic POST (IPOST). IPOST was not effective in KO hearts. To see if the GPCR signaling pathway to protection is normal in KO hearts, we looked at POST by GPCR agonists S1P and adenosine. Both provided effective protection even in KO hearts suggesting that the problem with IPOST in KO hearts is a low level of S1P available for release during IPOST. Thus, pharmacologic POST with FTY or Sph, like adenosine and S1P, is unaffected in the KO.
FTY720 administered in vivo might behave in a dual manner showing both S1P-like effects and sphingosine-like effects. It appears that the latter may have been overlooked and may be the more important in aging hearts.
PMCID: PMC3659128  PMID: 23567658
cardioprotection; FTY720; ischemia/reperfusion injury; postconditioning; sphingosine; sphingosine kinase 2; sphingosine 1-phosphate
Cellular signalling  2011;23(10):1590-1595.
Sphingosine kinase 2 (SK2) catalyzes the conversion of sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). We report here, the stereospecific synthesis of an analogue of FTY720 called (R)-FTY720-OMe, which we show is a competitive inhibitor of SK2. (R)-FTY720-OMe failed to inhibit sphingosine kinase 1 activity, thereby demonstrating specificity for SK2. Prolonged treatment of HEK 293 cells with (R)-FTY720-OMe also induced a reduction in SK2 expression. In addition, (R)-FTY720-OMe inhibited DNA synthesis and prevented S1P-stimulated rearrangement of actin in MCF-7 breast cancer cells. These findings demonstrate that SK2 functions as a pro-survival protein and is involved in promoting actin rearrangement into membrane ruffles/lamellipodia in response to S1P in MCF-7 breast cancer cells.
PMCID: PMC3148273  PMID: 21620961
19.  FTY720 stimulates 27-hydroxycholesterol production and confers atheroprotective effects in human primary macrophages 
Circulation research  2010;106(4):720-729.
The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known.
We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages.
Methods and Results
FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann-Pick C1 (NPC1) and ATP-binding cassette transporter A1 (ABCA1) proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor (LXR), leading to LXR–induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not.
We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.
PMCID: PMC2871287  PMID: 20056921
sphingolipid; atherogenesis; foam cell; cholesterol transport
20.  Antitumor Effects of OSU-2S, a Non-immunosuppressive Analogue of FTY720, in Hepatocellular Carcinoma 
Hepatology (Baltimore, Md.)  2011;53(6):1943-1958.
Accumulating evidence suggests the therapeutic potential of the immunosuppressive agent FTY720 (fingolimod) in hepatocellular carcinoma (HCC). Based on our previous finding that FTY720 mediates apoptosis in HCC cells by activating reactive oxygen species (ROS)-protein kinase (PK)Cδ signaling independent of effects on sphingosine-1-phosphate (S1P) receptors, we embarked on the pharmacological exploitation of FTY720 to develop a non-immunosuppressive analogue with antitumor activity. This effort led to the development of OSU-2S, which exhibits higher potency than FTY720 in suppressing HCC cell growth through PKCδ activation. In contrast to FTY720, OSU-2S was not phosphorylated by sphingosine kinase (SphK)2 in vitro, and did not cause S1P1 receptor internalization in HCC cells or T lymphocyte homing in immunocompetent mice. Though devoid of S1P1 receptor activity, OSU-2S exhibited higher in vitro antiproliferative efficacy relative to FTY720 against HCC cells without cytotoxicity in normal hepatocytes. Several lines of pharmacological and molecular genetic evidence indicate that ROS-PKCδ-caspase-3 signaling underlies OSU-2S-mediated antitumor effects, and that differences in the antitumor activity between FTY720 and OSU-2S were attributable to SphK2-mediated phosphorylation of FTY720, which represents a metabolic inactivation of its antitumor activity. Finally, OSU-2S exhibited high in vivo potency in suppressing xenograft tumor growth in both ectopic and orthotopic models without overt toxicity. Conclusion: Using the molecular platform of FTY720, we developed OSU-2S, a novel PKCδ-targeted antitumor agent, which is devoid of S1P1 receptor activity and is highly effective in suppressing HCC tumor growth in vivo. These findings suggest that OSU-2S has clinical value in therapeutic strategies for HCC and warrants continued investigation in this regard.
PMCID: PMC3103615  PMID: 21391227
hepatocellular carcinoma; FTY720; OSU-2S; sphingosine 1-phosphate receptor; protein kinase Cδ
21.  Local delivery of FTY720 accelerates cranial allograft incorporation and bone formation 
Cell and tissue research  2011;347(3):553-566.
Endogenous stem cell recruitment to the site of skeletal injury is key to enhanced osseous remodeling and neovascularization. To this end, this study utilized a novel bone allograft coating of poly(lactic-co-glycolic acid) (PLAGA) to sustain the release of FTY720, a selective agonist for sphingosine 1-phosphate (S1P) receptors, from calvarial allografts. Uncoated allografts, vehicle-coated, low dose FTY720 in PLAGA (1:200 w:w) and high dose FTY720 in PLAGA (1:40) were implanted into critical size calvarial bone defects. The ability of local FTY720 delivery to promote angiogenesis, maximize osteoinductivity and improve allograft incorporation by recruitment of bone progenitor cells from surrounding soft tissues and microcirculation was evaluated. FTY720 bioactivity after encapsulation and release was confirmed with sphingosine kinase 2 assays. HPLC-MS quantified about 50% loaded FTY720 release of the total encapsulated drug (4.5 µg) after 5 days. Following 2 weeks of defect healing, FTY720 delivery led to statistically significant increases in bone volumes compared to controls, with total bone volume increases for uncoated, coated, low FTY720 and high FTY720 of 5.98, 3.38, 7.2 and 8.9 mm3, respectively. The rate and extent of enhanced bone growth persisted through week 4 but, by week 8, increases in bone formation in FTY720 groups were no longer statistically significant. However, micro-computed tomography (microCT) of contrast enhanced vascular ingrowth (MICROFIL®) and histological analysis showed enhanced integration as well as directed bone growth in both high and low dose FTY720 groups compared to controls.
PMCID: PMC3464023  PMID: 21863314
Bone tissue engineering; Drug delivery; Angiogenesis; Osseointegration; Massive Allograft
22.  FTY720 Reduces Post-Ischemic Brain Lymphocyte Influx but Does Not Improve Outcome in Permanent Murine Cerebral Ischemia 
PLoS ONE  2011;6(6):e21312.
The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms.
Methodology/Principal Findings
FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls.
In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.
PMCID: PMC3119049  PMID: 21701599
23.  Evidence-based assessment of potential use of fingolimod in treatment of relapsing multiple sclerosis 
Core Evidence  2011;6:13-21.
Multiple sclerosis is an autoimmune inflammatory demyelinating disease of the central nervous system and represents one of the most common causes of chronic neurologic disability in young adults. All the current disease-modifying drugs are administered parenterally, and can be associated with varying degrees of injection site or infusion-related reactions. Together with other side effects, the parenteral route of administration is one of the key factors affecting adherence to therapy in multiple sclerosis. Fingolimod (FTY720) is an immunomodulator that acts on sphingosine 1-phosphate (S1P) receptors and is the first oral drug approved by the US Food and Drug Administration for the treatment of relapsing-remitting multiple sclerosis. Downmodulation of S1P receptor type 1 (S1P1) slows the egress of lymphocytes from lymph nodes and recirculation to the central nervous system, reduces astrogliosis, and inhibits angiogenesis during chronic neuroinflammation. Fingolimod also regulates the migration of B cells and dendritic cells, and enhances endothelial barrier function. Results from Phase II and III clinical trials provide robust evidence of the efficacy of fingolimod in relapsing-remitting multiple sclerosis. While some caution should be exercised in terms of safety issues, the introduction of fingolimod represents a great advance in the treatment of relapsing-remitting multiple sclerosis. The pharmacologic data on fingolimod and its efficacy and safety in multiple sclerosis are reviewed in this paper.
PMCID: PMC3065557  PMID: 21468239
FTY720; fingolimod; S1P receptors; oral treatment; multiple sclerosis
24.  S1P1 receptor signaling overrides retention mediated by Gαi-coupled receptors to promote T cell egress 
Immunity  2007;28(1):122-133.
The mechanism by which sphingosine-1-phosphate receptor-1 (S1P1) acts to promote lymphocyte egress from lymphoid organs is not defined. Here we showed that CCR7-deficient T cells left lymph nodes more rapidly than wild-type cells whereas CCR7 overexpressing cells were retained for longer. After treatment with FTY720, an agonist that causes down-modulation of lymphocyte S1P1, CCR7-deficient T cells were less effectively retained than wild-type T cells. Moreover, treatment with pertussis toxin to inactivate signaling via Gαi-protein coupled receptors restored egress competence to S1P1 deficient lymphocytes. We also found that T cell accumulation in lymph node cortical sinusoids required intrinsic S1P1 expression and was antagonized by CCR7. These findings suggest a model where S1P1 acts in the lymphocyte to promote lymph node egress by overcoming retention signals mediated by CCR7 and additional Gαi-coupled receptors. Furthermore, by simultaneously upregulating S1P1 and downregulating CCR7, T cells that have divided multiple times switch to a state favoring egress over retention.
PMCID: PMC2691390  PMID: 18164221
25.  Therapeutic dosing of FTY720 (Fingolimod) prevents cell infiltrate, rapidly suppresses ocular inflammation and maintains blood-ocular barrier 
The American journal of pathology  2011;180(2):672-681.
Fingolimod (FTY720) is a FDA-approved therapeutic, with efficacy demonstrated in experimental models of MS, and phase III human MS trials. FTY720 prevents T-cell migration to inflammatory sites, by down-regulating expression of the sphingosine-1 phosphate receptor normally required for egress from secondary lymphoid tissue. Experimental autoimmune uveoretinitis (EAU) serves as a preclinical model of human uveitis, permitting assessment of immunotherapeutic efficacy. Murine EAU is initiated by activation of retinal–antigen specific CD4+ T cells that infiltrate the eye, and previous studies demonstrate that high dose FTY720 treatment administered before disease onset reduces ocular infiltrate within hours of administration and suppresses clinico-pathological expression of EAU. The present study investigates the efficacy of FTY720 treatment for established disease. Single dose treatment is effective and its immunosuppressive ability is maintained through a dose range of FTY720, demonstrating significant and rapid reduction in the CD4+ cell infiltrate at clinically relevant therapeutic doses. Furthermore, a repeated treatment regimen using a comparable dose to current MS patient protocols significantly reduces infiltrate within 24 hours of administration, and importantly repeated doses do not compromise the vascular integrity of the blood-ocular barrier. Upon withdrawal of FTY720, drug induced remission is lost and recrudescence of clinical disease is observed. These results support the great therapeutic potential for FTY720 as an acute rescue therapy for the treatment of ocular immune-mediated inflammation.
PMCID: PMC3796282  PMID: 22119714
FTY720; Experimental autoimmune uveoretinitis; immuno-modulation; autoimmunity

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