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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Trends Pharmacol Sci. Author manuscript; available in PMC 2012 January 1.
Published in final edited form as:
PMCID: PMC3017656
NIHMSID: NIHMS253392

Sphingosine 1-phosphate and immune regulation: trafficking and beyond

Abstract

Sphingosine 1-phosphate (S1P) is a bioactive lipid with important functions in the immune system. S1P levels are regulated by the balance between its synthesis through sphingosine kinases and its degradation by the S1P lyase. S1P signals through plasma membrane G protein-coupled receptors (S1P1 – S1P5) or directly acts on intracellular targets. Although the S1P–S1P1 axis has long been known to mediate T cell egress from lymphoid organs, recent studies have revealed intrinsic functions of S1P and its receptors in both innate and adaptive immune systems that are independent of immune cell trafficking. Here I summarize recent advances in understanding of the roles of S1P and S1P receptors in inflammatory and allergic responses and lymphocyte differentiation, which directly contribute to the regulation of inflammatory and autoimmune diseases. I also describe strategies to target S1P and S1P receptors for immune-mediated diseases, particularly the immunosuppressant FTY720 (fingolimod), which has recently become the first oral therapy for relapsing multiple sclerosis.

Keywords: sphingosine 1-phosphate, inflammation, T cell differentiation, FTY720, multiple sclerosis

Introduction

Sphingosine 1-phosphate (S1P) is a natural bioactive lysophospholipid generated from intracellular sphingosine, a product of the cell membrane component sphingomyelin (Figure 1). S1P is synthesized in most cells by the actions of sphingosine kinases (SphKs), SphK1 and SphK2, which exhibit both overlapping and distinct physiological functions 1. S1P regulates diverse physiological and immunological processes by activating G protein-coupled receptors (GPCRs), S1P1-S1P5, to engage downstream pathways including Ras-Erk, PI3K-Akt, and small G proteins Rac and Rho. S1P also functions as a second messenger to act upon intracellular targets. S1P is irreversibly degraded by S1P lyase or reversibly dephosphorylated by S1P phosphatases 1. The constitutive activity of S1P lyase results in low concentrations of S1P in most tissues, including lymphoid organs. Notable exceptions are found in circulation – the plasma and lymph – in which S1P levels are in the sub-to-low micromolar range 2. Plasma S1P is mainly produced by erythrocytes 3, with additional contributions from non-hematopoietic sources such as vascular endothelium 4, 5, whereas lymph S1P is mainly non-hematopoietic in origin 3, 6. The marked difference in S1P concentrations between the circulation and tissues constitutes the “S1P gradient” that drives the trafficking of various immune cells 7. As the roles of S1P in immune cell trafficking have been covered in depth by recent excellent reviews 1, 2, 8, 9, I will briefly discuss this topic below. I will then provide more thorough discussions on trafficking-independent roles of S1P and S1P receptors (S1PRs) in immune system function, with a particular focus on recent genetic studies. Finally, I will describe therapeutic targeting of S1P and S1PRs in inflammation and other immune-mediated diseases.

Figure 1
S1P synthesis and degradation

Immune cell trafficking

In 2002, Mandala et al. and Brinkmann et al. reported that FTY720, a new immunosuppressive drug for transplant rejection, caused lymphopenia and sequestrated T cells in lymphoid organs by acting on four of the five S1PRs (excluding S1P2) 10, 11. Following this seminal discovery, genetic approaches to alter the function of S1P1 have established that S1P1 is the main S1P receptor that regulates T cell trafficking: T cells from S1P1-deficient mice failed to egress from the thymus and peripheral lymphoid organs 12, 13, whereas S1P1-transgenic T cells preferentially distributed to the blood rather than lymphoid organs 14, 15. S1P1 facilitates T cell trafficking at multiple stages of T cell development and responses, including thymocyte egress into periphery, egress of mature T cells out of lymph nodes during systemic trafficking as well as after immune activation, and retention of T cells in non-lymphoid tissues 12, 13, 16.

S1P–S1P1-dependent T cell egress is mainly regulated at three levels. The first is ligand availability. Because of the essential role of the S1P gradient, disruption of this gradient by eliminating S1P lyase activity causes altered distribution and aberrant development of T cells 7, 17, 18. Recent studies have revealed that lymphatic endothelial cells are an in vivo source of S1P that is required for lymphocyte egress from lymph nodes and Peyer's patches 6. In contrast, neural crest-derived perivascular cells, a specialized type of vessel-ensheathing cells, provide S1P to promote thymic egress 5. The second is the receptor surface expression. S1P1 can be internalized by its natural ligand S1P and upregulated in its absence, and this cyclical ligand-induced modulation of S1P1 on circulating lymphocytes has been proposed to contribute to establishing the lymphoid organ transit time 8. Consistently, when the endogenous S1P1 gene was replaced with a mutant form resistant to internalization, the T cells exhibited significantly delayed lymphopenia after S1P1 agonist administration or disruption of the S1P gradient, indicating that surface residency of S1P1 is a primary determinant of lymphocyte egress kinetics 19. Also, S1P1 can interact with the transmembrane C-type lectin CD69 in a mutually antagonizing manner 20. The third is transcriptional regulation of S1P1 expression, with the transcription factor KLF2 serving as the primary factor to drive S1P1 transcription in T cells 21.

Although the role of S1P1 in T cell trafficking was initially revealed by the use of FTY720 10, 11, it remains controversial whether FTY720, after being phosphorylated into FTY720-P in vivo, acts as an agonist or a functional antagonist or both to regulate lymphocyte trafficking. Also, whether the critical effect of the drug is on lymphocytes themselves or endothelial cells is under debate 12, 22. Moreover, despite rapid internalization, S1P1 receptors treated by FTY720 retain persistent signaling activity for hours 23. Finally, FTY720 possesses immunomodulatory activities independent of S1P receptors 24. Whereas S1P might regulate T cell trafficking by acting on both lymphocytes and endothelial cells, an intrinsic requirement for S1P1 in T cells is undisputable from the genetic evidence 12, 13. S1P1 also facilitates migration of B cells 25-27 and osteoclasts 28. By contrast, trafficking of natural killer cells and dendritic cells (DCs) requires S1P5 and S1P3 respectively 29-31. S1P2 negative regulates macrophage migration and recruitment to sites of inflammation 32. These studies illustrate that S1P has key roles in immune cell trafficking, a function that appears to be mediated exclusively by signaling through GPCRs.

Inflammatory and allergic responses mediated by the innate immune system

The innate and adaptive immune systems are two integral components of protective immunity. The innate immune system provides the first line of defense against invading pathogens. Innate immune responses are generally initiated by the engagement of pattern recognition receptors (PRRs) that bind highly conserved structures expressed by microorganisms, and can be further shaped by other receptor systems such as cytokine receptors. However, excessive or prolonged activation of the innate immune system can cause inflammation and severe immunopathology. Recent studies have identified essential functions of S1P and S1PRs in inflammation via modulating signaling of certain innate receptors (Figure 2).

Figure 2
Function of SphK1 and S1P in innate immune receptor signaling

Toll-like receptor (TLR) signaling and proinflammatory responses

Activation of TLRs, a prominent group of PRRs, results in a potent inflammatory response characterized by the release of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6. Whereas this response is important for clearance of infectious organisms, exuberant production of these cytokines can lead to sepsis and death from septic shock. SphK1 was upregulated in stimulated human phagocytes and in peritoneal phagocytes of patients with severe sepsis 33. Blockade of SphK1 inhibited TLR-induced NF-κB activation and production of proinflammatory cytokines. More importantly, mice treated with a specific SphK1 inhibitor were protected from sepsis. TLR-dependent nuclear factor (NF)-κB activation required protein kinase C (PKC)δ, whose activation was enhanced by S1P, thus placing sequential activation of SphK1 and PKCδ as important steps for NF-κB activation. Although the molecular detail of how SphK1 and S1P activate PKC remains to be established, these results have demonstrated a critical role for SphK1 in TLR signaling and established SphK1 as a new therapeutic target for sepsis 33.

TNF receptor (TNFR) dependent NF-κB activation

TNF-α is a pivotal pro-inflammatory cytokine implicated in a number of inflammatory and autoimmune diseases. Activation of the TNFR results in the assembly of a multi-component signaling complex, comprised of adaptors such as TNFR type 1-associated DEATH domain protein (TRADD), TNFR-associated factor 2 (TRAF2), and receptor-interacting protein 1 (RIP1), that is essential for transducing signals to trigger downstream NF-κB and MAP kinase pathways 34, 35. Activation of NF-κB by TNF-α requires the assembly of polyubiquitination chains linked by lysine-63 on the substrate RIP1 to recruit the TAK1 and IKK complexes, and by lysine-48 on the substrate IκB to mediate its proteasomal degradation 36. Although TRAF2 can function as a ubiquitin ligase, direct evidence that TRAF2 catalyzes RIP1 ubiquitination had been lacking. A recent study has shown that S1P produced by SphK1 is the missing cofactor for TRAF2 ubiquitin ligase activity 37. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its ligase activity to catalyze lysine-63-linked polyubiquitination of RIP1, which in turn activates downstream NF-κB signaling. These responses are mediated by intracellular S1P independently of its cell-surface GPCRs, indicating TRAF2 is a novel intracellular target of S1P. This elegant work establishes a new paradigm for the regulation of lysine-63-linked polyubiquitination, and highlights the key roles of SphK1 and its product S1P in the TNFR-dependent NF-κB pathway 37. Considering the roles of SphK1 in both TNF-α production and TNFR signaling 33, 37, inhibition of SphK1 is a promising strategy for sepsis and other inflammatory diseases involving TNF-α.

Protease-activated receptor 1 (PAR1) signaling in coagulation and inflammation

Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response in bacterial sepsis. The mechanism coupling coagulation and inflammation has been recently shown to involve PAR1; PAR1 deficiency attenuated coagulation and inflammation and protected mice from sepsis-induced lethality 38. DCs promote systemic coagulation and are the primary cells at which coagulation and inflammation intersect within the lymphatic compartment. In DCs, PAR1 signaling results in the activation of SphK1 and production of S1P, which signals through S1P3 in an autocrine/paracrine manner, a phenomenon known as “S1P inside-out signaling” 39. Loss of PAR1–S1P3 signaling sequesters DCs and inflammation into draining lymph nodes, and attenuates dissemination of IL-1β to the lungs. Thus, the SphK1–S1P–S1P3 axis mediates and amplifies PAR1 signaling in DCs to couple systemic inflammation and coagulation in innate immune responses 38.

FcεRI-dependent allergic responses in mast cells

Mast cells play pivotal roles in immediate-type and inflammatory allergic reactions and are the primary cell type for causing asthma 40. Mast cells are potent producers of intracellular and secreted S1P via SphK activation, following activation of the high-affinity Fc receptor for IgE (FcεRI). Although SphK activation and S1P production are clearly required for FcεRI-dependent allergic responses in mast cells, studies using mice deficient in SphK1 or SphK2 have yielded conflicting conclusions on the importance of the selective SphK isoenzyme 40, 41. Whereas one study has demonstrated that SphK2 is required for FcεRI-mediated responses 40, another study has found normal mast cell responses in either SphK1−/− or SphK2−/− mice 41. By contrast, results from siRNA-dependent silencing approaches have shown that SphK1 appears to have a dominant role in the generation of S1P in mast cells 41, 42. It is possible that both isoenzymes contribute to mast cell functions, and the overlap between them accounts for the differential phenotypes developed as a compensatory mechanism in genetically deficient mice 41.

Mast cells express S1P1 and S1P2 receptors, and export of S1P after FcεRI stimulation results in the rapid transactivation of S1P1 and S1P2, which is another example of “S1P inside-out signaling” 2, 39. Although S1P1 activation is important for cytoskeletal rearrangements and migration of mast cells, S1P1 is dispensable for FcεRI-triggered degranulation. Instead, S1P2, whose expression is upregulated by FcεRI cross-linking, is required for degranulation in vitro 2, 39, 43. In models of anaphylaxis (a severe allergic reaction mediated by mast cells), the S1P2 antagonist JTE-013 or S1P2 deficiency attenuated elevation of circulating histamine and the associated pulmonary edema in mice 43. However, in a separate study, S1P2-deficient mice showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis, which was ascribed to a mast cell-independent function for S1P244. Therefore, although SphKs and S1P2 are important regulators of mast cell responses, more defined systems such as tissue-specific or inducible knockout approaches are required to unambiguously determine their functions in vivo.

The vascular system and inflammation

S1P and S1PRs have potent effects on the vascular system that further impact inflammatory responses. Indeed, temporally and spatially regulated vascular leak is a central feature of inflammation. The importance of S1P in the vascular system has long been known 45, and mice deficient in S1P1 or in both SphK1 and SphK2 were embryonically lethal due to profound defects in vascular development 2. Nonetheless, the sources of S1P that regulates endothelial barrier function in vivo during inflammation were revealed only recently 46. Mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after inflammatory challenges. Such increased leak can be reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for S1P1. Therefore, S1P supplied to plasma from erythrocytes activates S1P1 signaling in endothelial cells to maintain vascular integrity and to prevent vascular leak during inflammatory responses 46.

In summary, S1P, either acting intracellularly or through surface GPCRs, generally elicits proinflammatory responses by modulating diverse receptor signaling. Animal models with diminished activities of SphKs are protected from or showed attenuated responses to various inflammatory insults such as bacterial sepsis 33, inflammatory arthritis 47, 48, colitis 49, allergic asthma 50, and anaphylaxis 43. Therefore, pharmacological blockade of the S1P–S1PR axis is a promising approach to treat these inflammatory and allergic diseases. However, it is important to note that S1P and S1PRs also mediate protective effects during inflammation such as maintaining vascular integrity and recovery from anaphylaxis 44, 46. Care should be taken in the design of therapeutic approaches to target S1P to control excessive inflammation.

Lymphocyte differentiation: the interface between immunity and tolerance

Compared with innate immune responses, adaptive immunity is slow to develop and mediates protection only several days or more post-infection. CD4 helper T cells are central regulators of adaptive immune responses and can either promote immune activation or induce tolerance or non-responsiveness, the balance of which is crucial to elicit immune defense, as well as to prevent autoimmune diseases such as type 1 diabetes and lupus 51. CD4 T cells orchestrate the decision between immunity and tolerance through their ability to differentiate into diverse effector and regulatory populations (Figure 3A). Some of these populations are determined when they emerge from the thymus, such as Foxp3+ natural regulatory T (nTreg) cells and natural killer T cells (NKT cells). The bulk of CD4 T cells develop from the thymus as conventional or naïve T cells, but can further differentiate into separate lineages in the peripheral lymphoid organs. In response to antigen stimulation, naïve CD4 T cells proliferate and differentiate into T helper type 1 (Th1) cells, Th2 cells, and Th17 cells to exert specific effector functions 51. Naïve precursors can also develop into antigen-specific Treg cells, known as induced Treg cells (iTreg), which act in synergy with nTreg cells to establish immune tolerance and counter-balance effector T cell functions. S1P1 has recently been implicated in the regulation of T cell differentiation and immune responses (Figure 3A).

Figure 3
S1P1 functions in lymphocyte differentiation

Differentiation and function of nTreg cells

Foxp3-expressing nTreg cells play a central role in the maintenance of immune tolerance and prevention of autoimmunity 52. However, the potent nTreg-mediated suppression could abrogate adaptive immune responses and render the host susceptible to infection and cancer. How are nTreg development and activity controlled to establish protective immunity toward pathogens and tumors without pathological anti-self reactivity? My colleagues and I have recently shown that S1P1 signaling delivers an intrinsic negative signal to restrain thymic generation, peripheral maintenance and suppressive activity of nTreg cells 53. S1P1 was expressed in both nTreg and naïve T cells, but upon activation, its expression was gradually reduced in nTreg cells as compared with a more pronounced downregulation in conventional T cells. Mice deficient in S1P1 contained an expanded nTreg cell population, whereas S1P1-transgenic mice showed diminished numbers of thymic nTreg cells. Moreover, S1P1 negatively affected the suppressive function of nTreg cells, and S1P1- transgenic Treg cells exhibited defective suppressive activity in vivo that contributed to the development of systemic autoimmunity. S1P1 impeded the differentiation and suppressive activity of nTreg cells by inducing the selective activation of the Akt-mTOR pathway. Notably, FTY720 has been shown to enhance the suppressive activity and numbers of nTreg cells 54, 55, similar to the effects observed in S1P1-deficient nTreg cells 53. Thus, FTY720 probably acts as a functional antagonist to down-modulate S1P1 in nTreg cells. Notably, the effects exerted by S1P1 are intrinsic to the development and function of nTreg cells, highlighting a key negative role of S1P1 signaling in this T cell subset 53.

Reciprocal differentiation of iTreg and Th1 cells

iTreg cells are a second subset of Treg cells that are preferentially generated in the mucosal environment in a process dependent upon transforming growth factor (TGF)-β stimulation 52. Also, iTreg cells generated in vitro represent a potent population for cellular immunotherapy of autoimmune diseases and transplant rejection. Consistent with a suppressive role for the development of nTreg cells, S1P1 also dampened the differentiation of iTreg cells 56. In addition, treatment of T cells with FTY720 upregulated iTreg cell generation in vitro and in vivo 56, 57. Moreover, S1P1 signaling drove Th1 cell generation in a reciprocal manner. Mechanistically, S1P1 signaled through mTOR (mammalian target of rapamycin) and antagonized TGF-β function by attenuating sustained Smad3 activity. Interestingly, TGF-β treatment upregulated the expression of SphKs in differentiating T cells, and experiments using SphK inhibitors indicated that S1P1 function is dependent upon endogenous sphingosine kinase activity, suggesting for the first time the involvement of S1P inside-out signaling in T cell differentiation. These studies establish an S1P1-mTOR axis that controls T cell lineage specification and immune homeostasis (Figure 3B) 56. Consistent with this observation, increased S1P1 expression is associated with autoimmune diseases 58, and S1P enhances IFN-γ production by human T cells from patients with the autoimmune disease primary Sjögren's syndrome 58.

Differentiation of other T cell lineages

S1P1 signaling has also been implicated in the regulation of Th2 and Th17 responses. T cells from transgenic mice expressing the human S1P1 cDNA showed higher levels of IL-4 and IL-17 59, 60. The reason for the distinct phenotypes observed in the transgenic mice expressing the mouse and human S1P1 genes is unclear 56, and whether this reflects species-specific functions of S1P1 or a different context of immune responses remains to be established. Also, unlike the role of S1P1 in Treg and Th1 responses, the role of S1P1 in Th2 and Th17 responses requires additional testing with the use of loss-of-function approaches. Interestingly, unlike S1P1, S1P4 mediates immunosuppressive effects of S1P by inhibiting proliferation and secretion of effector cytokines, while enhancing secretion of the suppressive cytokine IL-10 61.

Therapeutic targeting of S1P and S1PRs

FTY720

FTY720 (fingolimod), 2-amino-2-propane-1,3-diol hydrochloride, is the first-in-class S1PR modulator (Table 1). As a pro-drug, FTY720 is phosphorylated in vivo by SphK2 to form the active moiety FTY720-phosphate, a structural analog of S1P that binds to four of the five S1PR subtypes. FTY720 had initially been considered as a promising immunosuppressant for transplant rejection, but recent clinical trials failed to demonstrate a significant advantage of FTY720 over standard care 39. Serious adverse events included bradycardia that was likely caused by the activity of FTY720 on cardiac S1P3 receptors, because FTY720 failed to induce bradycardia in S1P3-deficient mice 62.

Table 1
Major compounds that target S1P and S1P receptors

Since 2002, a large number of studies have reported that FTY720 is effective in models of autoimmune diseases, particularly experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis (MS) 11, 63. In 2010, the results of two phase III clinical trials for MS (each enrolling more than 1000 patients with relapsing-remitting MS) were reported 64, 65. In one study (FREEDOMS – “FTY720 Research Evaluating Efects of Daily Oral Therapy in Multiple Sclerosis”), MS patients received oral FTY720 at a dose of 0.5 mg or 1.25 mg daily or placebo. As compared with placebo, both doses of oral FTY720 ameliorated the relapse rate, the risk of disability progression, and CNS lesions measured on magnetic resonance imaging (MRI) 64. In a second study (TRANSFORMS – “Trial Assessing Injectable Interferon versus FTY720 Oral in Relapsing–Remitting Multiple Sclerosis”), MS patients were randomized to receive either oral FTY720 or intramuscular interferon beta-1a, an established therapy for MS. This trial showed the superior efficacy of FTY720 with respect to relapse rates and MRI outcomes 65. Adverse events associated with FTY720 included bradycardia and atrioventricular block, certain infections, increased liver enzyme levels, hypertension and macular edema. Because of these encouraging clinical results, FTY720 was approved by FDA in September 2010 to become the first oral therapy for relapsing MS.

Mechanism of action for FTY720 include effects on lymphocyte trafficking, in which the initial activation and eventual downregulation and degradation of S1P1 prevent lymphocyte egress from lymphoid tissues, thereby reducing autoaggressive lymphocyte infiltration into cites of inflammation 63. Within this context, it is interesting to note that FTY720 differentially affects the recirculation of lymphocyte subsets; in MS patients, FTY720 primarily reduced the numbers of CCR7+CD45RA+ naive T cells and of CCR7+CD45RA central memory T cells in blood, whereas CCR7CD45RA and CCR7CD45RA+ effector memory T cell subsets remained largely unaffected 63. In addition, FTY720 exerts direct effects on T cell differentiation and function by enhancing the generation and function of Treg cells and inhibiting the differentiation of proinflammatory Th1 cells 54-57. Moreover, because of its lipophilic nature, FTY720 crosses the blood-brain barrier, and down-modulates S1P1 in neural cells/astrocytes to reduce astrogliosis, a phenomenon associated with neurodegeneration in MS. Additional effects might result from a modulation of S1P3 in astrocytes and of S1P1 and S1P5 in oligodendrocytes 63. Therefore, FTY720 has effects on lymphocyte trafficking, development and function of T cell subsets, and CNS cells, all of which may contribute to its immunosuppressive mechanisms.

S1P receptor selective modulators

Because FTY720-mediated immune regulation is mainly dependent upon its effects on S1P1, there is a distinct advantage to develop next-generation S1P receptor modulators by targeting S1P1 selectively. The S1P1-selective agonist SEW2871 is structurally unrelated to S1P but is capable of activating multiple signals that are triggered by S1P. Both SEW2871 and S1P activate Erk, Akt, and Rac signaling pathways and induced S1P1 internalization and recycling 66. SEW2871 stimulates lymphocyte trafficking in vitro and induces lymphopenia in mice via a S1P1-dependent mechanism 22, 62. In animal models, SEW2871 ameliorates renal ischemia/reperfusion injury by inhibiting lymphocyte egress and reducing pro-inflammatory molecules 9, 39.

Another S1P1 receptor-selective agonist, KRP-203, has structural similarity to FTY720. In animal models of organ transplantation, KRP-203 prolonged skin and heart allograft survival and attenuated chronic rejection 67. More recent preclinical studies using animal models for inflammatory and autoimmune diseases have revealed that KRP-203 treatment ameliorated the injury in concanavalin A-induced hepatitis, experimental autoimmune myocarditis, chronic colitis, and lupus pathogenesis 39, 68. KRP-203 sequestered circulating lymphocytes into lymphoid tissues and inhibited Th1 proinflammatory cytokine release. Notably, because KRP-203 selectively targets S1P1 (but not S1P3), the use of KRP-203 can potentially avoid the adverse effects associated with use of FTY720 39.

Several antagonists that interfere with S1P receptor activation by the physiological agonist S1P have recently been described, including 3-amino-4-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146), VPC23019, and JTE-013 69-71. Some of these compounds are useful for basic research but might not have potential use in humans (Table 1) 9, 39.

Sphingosine kinase inhibitors and silencing

Among the first compounds to be discovered as a competitive inhibitor of sphingosine kinase activity were DL-threo-dihydrosphingosine (DHS, Saphingol) and N,N-dimethylsphingosine (DMS) 39. Later, five non-lipid selective inhibitors that block the ATP binding site of sphingosine kinases were identified (compounds SKI I–V). Among them, SKI-II, 2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole, showed the highest selectivity against SphKs and promoted tumor cell apoptosis and in vivo antitumor activity 39, 72. One limitation for these early-stage SphK inhibitors is the lack of selectivity toward SphK1 and SphK2. Despite their sequence and functional similarity, these two isoenzymes can exhibit distinct immunomodulatory roles: for example, in vivo knockdown of SphK1 and SphK2 ameliorated and exacerbated inflammatory arthritis, respectively 73. As a result, more selective inhibitors are being developed. The first isoenzyme-specific inhibitor, (2R,3S,4E)-N-methyl-5-(4′-pentylphenyl)-2-aminopent-4-ene-1,3-diol, designated SK1-I (BML-258), acts on SphK1 but not SphK2 74. SK1-I induced apoptosis in tumor cells and inhibited growth of various xenograft tumors 74, 75. Another SphK1-selective compound, 5c, was efficacious in bacterial sepsis 33, 76. A SphK2-selective inhibitor, 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640), promoted tumor cell autophagy, leading to nonapoptotic cell death and delay of tumor growth in vivo 77, 78. Several natural products with inhibitory effects on SphK activity have also been described, although the specificity of these compounds remains unclear 72.

Inhibition of SphKs has been shown to affect immune cell functions in vitro and animal models of immune-mediated diseases. DMS modulated functions of murine DCs 39. Both DMS and SKI had the effects to promote iTreg generation and inhibit Th1 cell differentiation in vitro 56. More importantly, in vivo application of SphK inhibitors ameliorated murine disease models of allergic asthma, inflammatory arthritis and bacterial sepsis 33, 48, 50, 73. Some of these therapeutic effects were recapitulated by siRNA-mediated silencing of SphK1 in vivo, suggesting an important role for SphK1 to promote inflammation. Therefore, SphK1 inhibition represents a promising tool to treat inflammatory and immune-mediated diseases.

Other strategies to target S1P and S1PRs

Sabbadini and colleagues developed a specific monoclonal antibody to S1P, which reduced tumor progression and eliminated measurable tumors in xenograft and allograft models 79. The anti-S1P antibody also neutralized S1P-induced proliferation, the release of cytokines, and the ability of S1P to protect tumor cells from apoptosis in several tumor cell lines 79. A humanized form of the monoclonal S1P-specific antibody, LT1009, has entered Phase I clinical trials for cancer and age-related macular degeneration 80. The effects of anti-S1P antibody on inflammatory diseases have not been reported.

S1P lyase can be inhibited by treatment with the food colorant 2-acetyl-4-tetrahydroxybutylimidazole (THI), deoxypyridoxine, or the compound LX2931 of Lexicon Pharmaceuticals. This resulted in lymphopenia and ablation of the S1P gradient. The described effect of S1P lyase inhibition is similar to that observed after treatment with the FTY720, suggesting a shared mechanism at targeting the S1P–S1P1 axis 7, 81.

Given the specific signaling pathways activated by S1P receptors, targeting the downstream pathways can show therapeutic effects. Recently, my colleagues and I found that S1P1 signals through mTOR to control Th1 and iTreg cell differentiation 56. Treatment of S1P1-transgenic mice with the mTOR-selective inhibitor rapamycin restored the normal development of T cell subsets in these mice, similar as the treatment with FTY720. Notably, although both drugs are effective in a number of preclinical models of autoimmune diseases and graft rejection, FTY720 and rapamycin were thought to affect distinct molecular and cellular pathways in T cells, by acting on S1P1 to induce lymph node sequestration and on mTOR to block cell cycle entry, respectively. Therefore, our results suggest that these two immunosuppressants target the same S1P1–mTOR axis that likely contribute to their immunosuppressive functions in vivo.

Concluding remarks

The SphK–S1P–S1PR axis has been implicated to regulate immune responses by affecting lymphocyte trafficking, activating innate immune cells and inflammation, and directing T cell differentiation. S1P can be produced by the actions of SphKs to directly act on intracellular targets, independent of surface receptors; alternatively, S1P can be secreted out of the cells to activate surface receptors in an autocrine/paracrine manner, or activate upon other cells to regulate immune cell trafficking. Currently, drugs that target the ligand production and receptor function and signaling have been developed and are at various stages of preclinical and clinical development. The remarkable advancement of this area is facilitated by both genetic systems and pharmacological approaches, and further integration of these complementary tools will likely lead to even greater discoveries in this future.

Acknowledgments

I acknowledge the support from the US National Institutes of Health (K01 AR053573 and administrative supplement, R01 NS064599, and Cancer Center Support Grant CA021765), the Arthritis Foundation, the Lupus Research Institute, the Hartwell Foundation, and the American Lebanese Syrian Associated Charities. I apologize to those authors whose work I was unable to cite because of space limitations.

Footnotes

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