Potential sphingosine-1-phosphate-related therapeutic targets in the treatment of cerebral ischemia reperfusion injury

Life Sciences 249 (2020) 117542

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Potential sphingosine-1-phosphate-related therapeutic targets in the treatment of cerebral ischemia reperfusion injury

T

Mengtao Hana, Tao Suna, Haijun Chena, Mingzhi Hanb, Donghai Wanga,

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a

Department of Neurosurgery, Qilu Hospital of Shandong University and Brain Science Research Institute, Shandong University, Key Laboratory of Brain Functional Remodeling, Shandong, 107# Wenhua Xi Road, Jinan 250012, China b K.G. Jebsen Brain Tumor Research Center, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway

ARTICLE INFO

ABSTRACT

Keywords: Sphingosine-1-phosphate (S1P) Cerebral ischemia reperfusion (IR) injury Ischemia FTY720 Inflammation

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that regulates lymphocyte trafficking, glial cell activation, vasoconstriction, endothelial barrier function, and neuronal death pathways in the brain. Research has increasingly implicated S1P in the pathology of cerebral ischemia reperfusion (IR) injury. As a high-affinity agonist of S1P receptor, fingolimod exhibits excellent neuroprotective effects against ischemic challenge both in vivo and in vitro. By summarizing recent progress on how S1P participates in the development of brain IR injury, this review identifies potential therapeutic targets for the treatment of brain IR injury.

1. Introduction Acute ischemic stroke is the leading cause of mortality and morbidity in both developed and developing countries. The most important factor in mitigating the impact of stroke and reducing the risk of mortality is to achieve reperfusion (removal of the clot and the restoration of blood flow) as soon as possible after the stroke event. To improve reperfusion, intravenous injection of recombinant tissue plasminogen activator (rtPA) and catheter-based intraarterial approaches have been used [1]. However, these advances do not facilitate the reconstruction of neuronal functionality immediately after blood flow is reestablished. Indeed, these relatively modern therapies are frequently associated with severe brain damage, including cerebral ischemia reperfusion (IR) injury [2]. Cerebral IR injury is a pathological condition characterized by two stages: (1) initial ischemia and the consequent severe shortages of oxygen and nutrient supply activate cell death programs, and (2) cell debris and microglial activation trigger an inflammatory cascade that damages vessels and the parenchyma within minutes to hours of the stroke event [2]. Subsequent reperfusion is concomitant with an exacerbation of autoimmune responses. The upregulation of cerebral proinflammatory cytokines, activation of systemic lymphocytes, and invasion of leukocytes account for this effect [3]. Immune interventions that restrict brain inflammation, vascular permeability, and tissue edema must be performed rapidly to reduce acute immune-mediated destruction and avoid immunosuppression [4,5]. Therefore,

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interventions that target the immune system to restrict inflammation are being explored as therapies for neural IR injury. Similar to cranial IR injury, multiple sclerosis (MS) is an inflammatory and autoimmune disorder of the CNS [6]. Some drugs used to treat MS, such as enlimomab, minocycline, and fingolimod, have been proven to attenuate inflammation and improve clinical outcomes for patients with acute stroke [4]. Among the aforementioned drugs, fingolimod – also called FTY720 – demonstrates neuroprotective effects against ischemic challenge [7–9]. A small-scale clinical trial that included 22 patients with acute and anterior cerebral circulation showed that the oral administration of fingolimod within 72 h of stroke onset limited secondary tissue injury between baseline and 7 days following the stroke, decreased microvascular permeability, attenuated neurological deficits, and promoted recovery [8]. Such findings have engendered multiple ongoing clinical trials of FTY720 alone or in combination with a thrombolytic agent to treat ischemia. Fingolimod is a high-affinity agonist of sphingosine-1-phosphate (S1P) receptor [10]. Found at its highest concentration in the blood, S1P is a type of bioactive sphingolipid derived from the cell membrane that plays a critical role in lymphocyte trafficking, glial cell activation, vasoconstriction, endothelial barrier function, and neuronal death pathways [11]. The current paradigm for the mechanism underlying the participation of S1P in these functions is generally referred as “inside-out signaling”: intracellularly produced S1P must be exported out of cells, where it can in an autocrine or paracrine manner by binding to its receptors present on endothelial and inflammatory cells [12].

Corresponding author at: 107# Wenhua Xi Road, Jinan 250012, Shandong, China. E-mail address: [email protected] (D. Wang).

https://doi.org/10.1016/j.lfs.2020.117542 Received 17 December 2019; Received in revised form 29 February 2020; Accepted 9 March 2020 Available online 10 March 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.

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Fig. 1. S1P metabolism and distribution. A simplified metabolic process of sphingolipids in endothelial cells is shown, along with the key metabolic enzymes involved in their formation and degradation. S1P primarily binds with ApoM on HDL in circulation, where its concentration is highest and is a strong attractant of mature lymphocytes. S1P (sphingosine-1-phosphate); S1PR (S1P receptor); Spn2 (spinster homolog 2); CDase (ceramidase); SphK (sphingosine kinase); SPL (S1P lyase); CerS (ceramide synthase); SPPase (S1P phosphohydrolases); NK cell (natural killer cell); ApoM (apolipoprotein M); HDL (high-density lipoprotein).

However, an increasing body of evidence indicates that S1P directly binds to transcription factors, such as histone deacetylases (HDAC)1 and 2, in the nucleus [13]. This review expounds how S1P-related potential therapeutic targets (mainly S1P receptors and synthetic enzymes) of the immune system as a whole participate in cerebral IR injury as well as the associated underlying mechanisms.

diverse cell types, while ceramide and sphingosine are generally associated with growth arrest and cell death (Fig. 1). Hence, the three metabolites form a ‘sphingolipid rheostat’ to regulate cell fate [15]. S1P concentration in plasma is about 0.9 μmol/L. The majority of S1P is bound to high-density lipoprotein (HDL) particles (~60%) and albumin (~35%), while the minority resides in other lipoproteins, primarily low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) [16]. With a half-life four-fold that of the albumin-associated S1P, HDL-associated S1P is more stable. Research has revealed that the latter form of S1P is bound to the center of the calyx-like ligand-binding pocket of the apolipoprotein M (apoM) of HDL [17]. Furthermore, S1P is absent in HDL obtained from apoM-null mice, while transgenic mice overexpressing human apoM showed a significant increase of S1P in HDL particles [18]. These observations indicate that apoM determines the plasma concentrations of S1P. Contrastingly, lymph S1P only accounts for an approximate 25% of plasma concentrations, and the interstitial fluid levels of S1P present in the low

2. Synthesis, distribution, and transportation of S1P Sphingosine, a precursor of S1P that is produced by the deacylation of ceramide and not de novo via biosynthesis, can be reversibly phosphorylated by sphingosine kinase (SphK) 1 and 2 to form S1P [14], which is then processed through one of two metabolic pathways: (1) it can be dephosphorylated back to sphingosine via S1P phosphatase or (2) irreversibly degraded into hexadecenal and phosphoethanolamine via S1P lyase. S1P, sphingosine, and ceramide are interconvertible sphingolipid metabolites; S1P enhances the growth and survival of 2

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Fig. 2. Signaling pathways transduced by S1PRs. S1PR1 couples with Gi, whereas S1PR2 and S1PR3 couple with Gi, Gq and G12/13; S1PR4 and S1PR5 couple through Gi and G12/13. Signaling through Gi has been associated with activation of Ras/ERK to promote proliferation through PI3K/Akt to increase survival; through PI3K/Rac to promote enhance endothelial barrier function and vasodilation; through PKC or PLC to increase intracellular Ca2+; and inhibition of AC cAMP. Signaling through Gq primarily activates PLC pathways; while signaling through G12/13 can promote activation of Rho/ROCK to inhibit migration, reduce endothelial barrier function and induce vasoconstriction. Dotted red line represents negative regulation. PI3K (phosphatidylinositol 3-kinase); AC (adenylyl cyclase); PKC (protein kinase C); PLC (phospholipase C); ERK (extracellular signal-regulated kinase); Akt (protein kinase B); cAMP (cyclic adenosine monophosphate); ROCK (Rho-associated kinase). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

nanomolar range. While this concentration is low, it generates a crucial biological gradient for lymphocyte egress [19]. High levels of S1P in circulation are maintained by erythrocytes and endothelial cells, which are metabolically geared toward S1P secretion [20] (Fig. 1). Erythrocytes, which contain about half of all blood S1P, are considered the main source of plasma S1P. Indeed, the adoptive transfer of wild-type erythrocytes to irradiated SphK2-deficient mice with the conditional deletion of SphK1 could restore normal plasma S1P levels [21]. Mature erythrocytes barely express ceramidase, SphK2, S1P lyase, and S1P phosphohydrolases but host high SphK1 activity and can intake sphingosine from the plasma. These properties enable them to efficiently incorporate, store, and release S1P [21,22]. While the release of S1P from RBC is constitutive and does not require any stimulus, the transport of S1P increases with the addition of ATP, dATP, and nonhydrolysable ATP-analogues. Furthermore, the ATP-binding cassette (ABC) transporter inhibitor vanadate prevents the uptake of S1P, suggesting that the release of S1P by RBC depends on ABC transporters [23]. A major facilitator superfamily transporter 2b (Mfsd2b) has been identified as essential for S1P export from red blood cells and platelets [24]. Since plasma S1P levels were not appreciably altered in thrombocytopenic, anemic, or leukopenic mice, endothelial cells show a robust expression of the enzymes involved in the synthesis and degradation of S1P, indicating a high S1P metabolic rate in these cells,

were confirmed to be another contributor of plasma S1P [25], and they actually need spinster homolog 2 (Spns2) for normal S1P release [26]. Besides, although platelets contain high level of S1P due to lack of efficiently degradation, they do not significantly contribute to plasma S1P in steady state [27]. Of note, SphK2 is the principal S1P synthetic enzyme in platelets [28], and ABC transporters mediate the release of S1P in platelets [29]. 2.1. Signals transduced by S1PRs There are five specific G-protein-coupled receptors (GPCR) with high affinities for S1P: S1PR1-S1PR5. Although they overlap to a certain extent, each of these GPCRs prompts different signaling cascades indispensable to normal development. Among these receptors, S1PR1–3 is expressed ubiquitously [30]; S1PR4, in the hematopoietic system [31]; and S1PR5, in the white matter of the brain as well as natural killer (NK) cells [32,33]. By describing the current scope of elucidated functions of S1PRs in the CNS and immune system, this review aims to identify new investigative directions that might yield novel, effective treatments for cerebral IR injury. In the CNS, neurons primarily express S1PR1 and S1PR3, while astrocytes express a wide range of lipid-activated receptors that play important roles in their proliferation and gliosis [34]; apart from 3

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S1PR1, 3, 4, and 5, these include protease-activated receptors (PAR) 1–4 and lysophosphatidic acid receptors (LPA) 1–3. In the white matter and myelin, which are rich in lipids and sphingomyelin, S1PR5 is expressed throughout the lives of oligodendrocytes and directs their maturation and retraction [32]. As the resident immune cells in the brain, microglia predominantly express S1PR1 [35]. Individual S1P receptor subtypes are bound to the different subunits of the heterotrimeric G proteins and transduce different signaling pathways. S1PR1 normally couples with Gi; S1PR2 and S1PR3, with Gi, Gq, and G12/13; and S1PR4 and S1PR5, with Gi and G12/13. Signaling through Gi has been associated with 1) the promotion of proliferation via the activation of the small guanosine triphosphatase (GTPase) Ras and the extracellular signal-regulated kinase (ERK); 2) the enhancement of cellular survival through the activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt); 3) the promotion of cytoskeletal rearrangement, enhancement of endothelial barrier function, and induction of vasodilation via the activation of PI3K and Rac; 4) the increase of intracellular Ca2+ required for many cellular responses through the activation of protein kinase C (PKC) and phospholipase C (PLC); and 5) the reduction of cyclic adenosine monophosphate (cAMP) via the inhibition of adenylyl cyclase (AC) activity. Signaling through Gq primarily activates PLC pathways, while signaling through G12/13 can inhibit migration, reduce endothelial barrier function, and induce vasoconstriction by promoting the activation of the small GTPase Rho and the Rho-associated kinase (ROCK) [30] (Fig. 2). FTY720 is an immunosuppressant that reversibly sequesters circulating lymphocytes away from inflamed lesions by inhibiting their exit from secondary lymphoid organs, such as lymph nodes, thereby inducing peripheral lymphopenia [36]. FTY720 is a prodrug that requires the phosphorylation of SphKs – mainly SphK2 – to become active. Activated p-FTY720 mimics S1P to function as an agonist to S1P receptors, excluding S1PR2 [10]. However, the binding of p-FTY720 to S1PR1 could induce receptor internalization, ubiquitination, and subsequent degradation, thereby resulting in the sustained desensitization of the S1PR1-mediated signaling pathway [36,37]. The down-regulation of S1PR1 on T cells renders them unable to respond to the chemotactic cue of S1P, which may account for most of the immunosuppressive effect of fingolimod. While S1P also causes receptor internalization, the receptors are recycled back to the plasma membrane and resensitized thereafter [38]. S1PR3, 4, and 5 are also internalized upon binding with p-FTY720; however, akin to the S1P-induced S1PR1 internalization, these receptors are redistributed back to the cell surface. Hence, FTY720 still functions as an agonist for these receptors [10]. The function of FTY720, SEW2871, and other S1P analogues remains controversial in the literature: some researchers consider them S1PR1 agonists, while others view them as functional antagonists. While these drugs could bind to S1PR1 to mediate transient agonist signaling, they also induce receptor internalization to desensitize S1PR1. Hence, whether S1P analogues function agonistically or antagonistically largely depends on the cell to which they bind. For example, FTY720 functions as an antagonist when it binds to S1PR1 on T cells, but its effects vary when it binds to receptors on endothelial cells. Mullershausen et al. found that – even after being internalized in vesicular structures by p-FTY720 – S1PR1 is not degraded by S1P lyases and thus remains bioactive. This latent bioactivity allows for persistent intrinsic signal delivery in human umbilical vein endothelial cells (HUVECs) [38]: the Gi-coupled inhibition of AC and the consequent reduction of cAMP production and increase in ERK phosphorylation were shown to have been persistently induced by p-FTY720-bound internalized S1PR1, whereas calcium responses were abrogated as a result of receptor internalization. Similarly, Healy et al. also observed continued cAMP signaling and a transient increase of Ca2+, but also the subsequent inhibition of Ca2+ signaling, as a result of FTY720 administration to human and rat astrocytes [39]. Moreover, the sustained treatment of neurons with FTY720 does not influence the expression level of S1PR1 [40]. These results suggest that p-FTY720 might function

as both an agonist and antagonist in different S1PR1-mediated signaling pathways and cells. Recent literature thus tends to refer to them as “modulators” rather than “agonists” or “antagonists.” 2.2. Multiple roles of S1PR1 in different cell types S1PR1 is important for vascular maturation, lymphocyte migration, and endothelial barrier function. The expression of S1PR1 is reportedly the highest in the brain, followed by the lungs, spleen, heart or vasculature, and kidney [41]. During embryonic development, intense signals indicating S1PR1 are detected in the forebrain and heart, and mice with the deletion of genes encoding S1PR1 die between E12.5 and E14.5 due to the absence of supporting vascular smooth muscle cells and consequent severe bleeding [42]. S1P signaling reportedly modulates the trafficking of T and B lymphocytes, macrophages, osteoclasts, and hematopoietic progenitor cells. This activity is primarily dependent on S1PR1 [43] and clinically observed effects of FTY720 are largely due to lymphocyte sequestration. T cells are considered a detrimental post-ischemic conductor, as they upregulate the expression of adhesion receptors, such as lymphocyte function antigen-1 (LFA-1), macrophage antigen-1 (Mac-1), and very late antigen-4 (VLA-4), in the brain endothelium. This causes disruption of the primary endothelial barrier, impaired capillary reperfusion, and subsequent microvascular obstruction, further inducing the “no-reflow phenomenon” [44]. Mice deficient of recombination activating gene 1 (Rag1−/−), which induces the lack of functional lymphocytes, displayed lower brain infarct volume, neurological deficits, as well as leukocyte and platelet adhesion after the induction of transient middle cerebral artery occlusion (tMCAO) with the stringent preservation of microvascular integrity [45]. Thus, lymphocytopenia could help ameliorate brain damnification caused by IR injury. FTY720 could not reduce infarct size or improve neurological outcome in Rag1−/− mice, suggesting that its post-stroke neuroprotective function primarily depends on T-cell quantity. In vitro experiments have also demonstrated that FTY720 administration did not directly protect neuronal cells from death under hypoxic conditions, even when the specimens were treated with high FTY720 concentrations [46]. Notably, T helper 17 (TH17) might be critical in both the onset and progression of brain autoimmunity because increased numbers of IL-17expressing cells can be found in the peripheral blood of patients with ischemic stroke relative to healthy individuals. Furthermore, the characterization of CNS-infiltrating immune cells in mouse models with experimental autoimmune encephalomyelitis (EAE) featured significantly higher numbers of CD4+ T cells, many of which expressed IL17. Further providing support for the T cell-dependent efficacy of FTY720, Rag1−/− mice transferred with IL-17-deficient T lymphocytes after tMCAO induction exhibited significantly less brain damage relative to those transferred with wide-type T lymphocytes [47]. IL-17 participates in TH17 polarization and is naturally secreted by TH17. Garris et al. found that S1PR1 signaling in T cells directly modulates IL17 expression and contributes to TH17 differentiation, a process likely dependent on the IL-6-JAK-STAT3 pathway [48]. Choi et al. also demonstrated that the genetic and pharmacological loss of CNS S1PR1 signaling could help reduce IL-17 production and attenuate inflammation [49]. Microglia are another important source of IL-17. They typically differentiate into two distinct populations after cytokine stimulation: M1 and M2 phenotypes. The “classically activated” M1 microglia release destructive proinflammatory mediators, while the “alternatively activated” M2 phenotype exerts neuroprotective properties [50]. During brain ischemia, inflammatory cytokines, including IFN-γ and TNF-α, could induce M1 polarization. Subsequently, damage-associated molecular patterns originating from cellular debris and destructed extracellular matrix components activate Toll-like receptor2 (TLR2) on M1 microglia. These cells then upregulate IL-23 expression, which further induces IL-17 production. The microglia-associated TLR2/IL4

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23/IL-17 axis has thus been implicated in neuronal damage following cerebral ischemia reperfusion [51]. Mice treated with FTY720 were found to exhibit a significantly reduced number of activated microglia and neutrophils in the brain infarct area relative to wild-type mice [9]. Moreover, after receiving the S1PR1-selective modulator AUY954, the significant attenuation of NF-κB activation and M1 polarization markers, including CD11b, CD16, CD32, and CD86, was observed after tMCAO induction in mouse microglia; contrastingly, M2 polarization markers, such as CD206, CCL22, Arg1, IL-10, TGF-β1, and Ym1, were upregulated in the same post-ischemic specimens, suggesting the importance of S1PR1 in promoting M1 functions and deterring M2 polarization [35]. It can thus be postulated that interrupting S1PR1 signaling in microglia might address effects of brain IR injury. Indeed, both the phenotypical shift of activated microglia from M1 to M2 and reduced secretion of inflammatory cytokines have become new therapeutic targets in research on brain IR injury. In recent years, the interaction between S1PR1 and STAT3 has been the focus in various oncological studies [52–54]. S1PR1 has been identified as a key element involved in the persistent activation of STAT3 [55]. Separately, STAT3 has been shown to induce S1PR1 expression [56]. In fact, hyperactivation of STAT3 is not only tumorpromoted, but also associated with increased predisposition to autoimmune diseases such as psoriasis and multiple sclerosis [57]. Individuals carrying STAT3 hyperactive mutations show reduced regulatory T lymphocytes (Tregs), consistent with roles for STAT3 in restraining FoxP3 expression and Treg development [55]. Thus, blocking S1PR1 to help boost Treg energy to mitigate inflammation may be a potential therapeutic strategy for cerebral IR injury. Besides, STAT3-stimulatory cytokines such as IL-6 [58], IL-10 [59], G-CSF or VEGF [54] are important mediators participating cerebral IR injury. Therefore, whether the therapeutic effect of S1PR1 blockade by FTY720 is partly mediated by STAT3 needs to be further determined. Although S1PR1 activation and the recruitment of immune cells to the site of an inflamed lesion seem to be detrimental during ischemia, the S1PR1 on endothelial cells play divergent roles that have been implicated in the formation and maintenance of the adherens junctions and the concomitant protection of vascular integrity. Numerous studies have observed that exogenous S1P or the S1PR1-selective agonist attenuates vascular leakage [60–62]; by contrast, the antagonist of S1PR1 abolishes the protective function of SEW2871 in the mouse lung and skin [63], suggesting that S1PR1 signaling plays a pivotal role in the stabilization of blood vessels. Subsequent immunofluorescent analysis and/or transmonolayer endothelial resistance (TER) also demonstrated that S1P exerts rapid and dose-dependent barrier-protective effects via S1PR1 activation [64–66]. While the mechanisms underlying the endothelial protective functions of S1PR1 remain incompletely understood, some studies have confirmed that S1PR1 signaling coupled to Gi promotes the rearrangement of cortical actin by activating the PI3K/ Rac pathway. This stabilizes the cell shape and stimulates cell adhesion molecules, such as vascular endothelial (VE)-cadherin and β-catenin, to translocate to the EC periphery and assemble into cell-cell junctions [63,64,67]. In addition, S1PR1-mediated activation of Akt and ERK, which constitutes part of the antiapoptotic pathway, could promote cell viability [68,69]. These effects are significant for stroke since they contribute to the maintenance of blood-brain barrier (BBB) integrity which could help attenuate hemorrhagic transformation and relieve tissue edema. It is reasonable to speculate that part of the FTY720 effect on cerebral IR injury might be due to S1PR1 activation on endothelial cells which prevents the degradation of cell adhesion molecules and help to preserve the intact BBB; however, further research found that it actually did not reduce IR-induced BBB leakage [70], which is consistent with the findings of another independent study [46]. Hence, it remains unclear whether pure S1PR1 activation on endothelial cells (without receptor endocytosis) possesses an endothelial-protective property. To directly explore the effect of S1P on vascular permeability, Camerer et al. used genetically modified mice with depleted circulating

levels of S1P. The study found that the pS1Pless (plasma S1Pless) mouse exhibited a severe basal lung vascular leak and elevated local response to platelet-activating factor (PAF) and histamine. While the administration of AUY954 reversed the increased sensitivity of pS1Pless mice to PAF [71], it failed to protect the wild-type mice – in which S1PR1 might be fully saturated by plasma S1P – against the leakage-inducer. These results prompted the reevaluation of whether activated S1PR1 signaling exerts an endothelial-protective function under normal conditions, as the normal concentrations of S1P in the plasma are high enough to fully activate S1PR1. Additionally, after brain ischemia, S1P augments locally and thereby draws immune cells, which might exacerbate local inflammation rather than play a barrier-protective role [72]. Finding that the inhibition of S1PR1 through the intracerebroventricular microinjection of shRNA lentivirus reduces tMCAO-induced brain damage [73], Gaire et al. have provided evidence indicating that S1PR1 may indeed be a pathogenic factor in cerebral ischemia. In general, however, much remains unknown concerning the roles of S1PR1 in cerebral IR injury, and more evidence – especially from studies using genetically modified animals – is required. 2.3. S1PR2 might function as a detrimental factor for brain IR injury In contrast to the protective effect of S1PR1 on the endothelium, S1PR2 is recognized as a detrimental factor because it activates RhoROCK-PTEN signaling, which disrupts the adherens junctions that increase paracellular permeability [74]. Moreover, as Rho, ROCK, and PTEN are all negative regulators of small GTPase Rac [75], their activation by S1PR2 opposes the barrier-protective function of S1PR1. However, S1PR2 remains necessary: S1pr2−/− mice were shown to be deaf on account of an early defective regulation of vascular tone in the stria vascularis, the compartment that harbors the main vasculature of the inner ear that is critical for hearing [76]. Many studies have noted that the mRNA and protein levels of S1PR2 significantly increase during IR injury, indicating the enhanced activation of S1PR2 signaling pathway. Contributing to cerebral edema and leading to the subsequent disruption of BBB and hemorrhagic transformation, the activation of S1PR2 could be pernicious at the early stage of ischemic stroke [1,77]. Having subjected mice with the genetic deletion of the genes encoding S1PR2 to tMCAO for 90 min and reperfusion 24 h later, an in vivo study found that the mice exhibited significantly improved neurological scores as well as dramatically decreased infarct sizes (~70%) and total cerebral edema (~60%) relative to their S1pr2+/+ littermates. Similar results were obtained in the group of mice to which the S1PR2 antagonist JTE013 was administered 10 min before reperfusion [78,79]. In an in vitro experiment, mouse brain microvascular endothelial cell lines bEnd3 and hBMVEC were exposed to oxygen and glucose deprivation (OGD) to simulate the metabolic and inflammatory conditions of IR injury. This intervention upregulated S1PR2 and matrix metalloproteinases-9 (MMP-9). MMP-9 is a kinase that plays a vital role in the degradation of extracellular matrix components [80] and thus contributes to the leakage of cerebral microvessels after IR injury. These activities of MM-9 can be markedly decreased by blockading S1PR2 signaling via JTE013 administration [78,79]. However, the role of S1PR2 in the regulation of responses to IR injury in mouse cortical primary neurons and mixed glial cells is not as clear as its effect in cerebral endothelial cells, in which JTE013 neither inhibits neuronal cell death induced by OGD nor downregulates MMP-9 activity in glial cells by TNF-α stimulation [79]. Research also indicates that S1PR2 inhibits myeloid macrophage motility via the cAMP/protein kinase A pathway and assists in the production of inflammatory cytokines IL-1β and IL-18 [81]. Karunakaran et al. have found that S1PR2 is the main receptor that mediates IL-6 secretion in microglia exposed to exogenous S1P and that S1RP2 may account for autophagy defects that could result in the accumulation of aggregate-prone proteins as well as the disruption of homeostasis [72]. However, the relationship between autophagy and inflammation through the S1P-S1PR2 axis remains 5

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clear; specifically, what is directly modulated by S1PR2 requires further study. The review above underscores the need to expand our understanding of the association between S1PR2 and microglia, as well as the role it plays during ischemic stroke, before the S1PR2 signaling pathway can be targeted as a potent treatment modality for cerebral IR injury. Indeed, this approach may offer an attenuated risk of infection relative to the administration of fingolimod, which attenuates peripheral lymphocytes.

in HUVECs via the mobilization of intracellular Ca2+ and Akt-mediated eNOS phosphorylation, which consequently induce vasodilation [92,93]. Although these findings conflict, they affirm that S1PR3 plays a pivotal role in the regulation of vascular tone; depending on the signaling pathway, this may involve contraction or dilation. Hence, interrupting S1PR3 signaling might help to mitigate vascular spasm and reduce inflammation following brain ischemia.

2.4. S1PR3 exerts proinflammatory activity and regulates vessel tone

Unlike S1PR1–3, which are expressed on various cell types, S1PR4 is preferentially expressed at low levels on hematopoietic organs and lymphocyte-containing tissues, such as bone marrow, the thymus, spleen, appendix, peripheral lymph nodes, and so forth. A recent study has reported that S1PR4 also plays a role in skeletal muscle cells. For example, S1PR4 expressed on satellite cells, the resident stem cells of skeletal muscle, appear to be involved in promoting their migration to damage tissues [94]. While homozygous S1pr4−/− mice seem to show normal phenotypes, they exhibit increases in morphologically aberrant megakaryocytes as well as reduced proplatelet formation in their blood, indicating that S1PR4 signaling is necessary for platelet maturation [95]. Although it is clear that S1PR4 deficiency decreases the number of circulating neutrophils, its effects on cell migration remain unclear. A recent meta-analysis identified the association between a rare missense variant of S1PR4 and neutrophil counts in human samples with an exome chip. Loss of S1pr4 function tends to lower the basal number and proportion of circulating neutrophils. Researchers have used S1pr4defecient mice to examine the neutrophil counts and percentage relative to wild type mice; both were found to be lower in S1pr4−/− mice (neutrophil count: 28.0% decrease, P = 0.11; percentage: 54.3% decrease, P = 0.03). Testing with the zebrafish variant yielded a similar result, confirming the role of S1PR4 in the regulation of neutrophil counts [96]. Similarly, Allende et al. found that, compared to S1P lyase deficiency alone, the combined deletion of S1P lyase and S1pr4 in mice could partially decrease neutrophilia and inflammation [97]. While it can be speculated that S1PR4 also participates in immune cell migration, its function may oppose that of S1PR1: specifically, it may promote the migration of neutrophils from tissues into the blood. The study then evaluated the expression of CD62L on the neutrophil cell surface, which shed neutrophils from extravasation into tissues; the level of expression was reduced by approximately by two-fold in S1pr4-null mice. However, tissue neutrophil numbers did not increase in S1pr4null mice relative to their wild-type counterparts [96]. This finding was unexpected in the context of a former study having not observed that S1PR4 deficiency resulted in the increased migration of CD4+ and CD8+T cells toward an S1P gradient, indicating that S1P signaling via S1PR4 might negatively regulate immune cells migration with S1PR1 [95]. Matsuyuki et al. also found evidence for the requirement of both S1PR1 and S1PR4 in the migration of mouse T Cells toward S1P, but their specific function remains to be identified [98]. In Jurkat T cells, the stimulation of S1PR4 enhances peripheral stress fiber formation and cell rounding, and the overexpression of S1PR4 induces pertussis toxinsensitive cell motility even in the absence of exogenously added S1P [99]. The most recent finding reaffirmed that S1PR1 and S1PR4 had different roles in regulating T cell motility and VCAM-1 binding [100]. Further in vivo and in vitro research, including the induction of the overexpression of S1PR4, might help to fully elucidate the underlying mechanism. The stimulation of S1PR4 leads to an attenuated inflammatory response in autoimmune pathologies, and the activation of S1PR4 results in the attenuated biosynthesis of leukotrienes (LTs) as well as decreases in the inflammatory response. The mechanism underlying these effects involves the Gi-coupled influx of Ca2+, which triggers 5-lipoxygenase (5-LO) perinuclear translocation and the subsequent suicide inactivation of 5-LO due to the lack of substrate arachidonic acid (AA) [101].

2.5. S1PR4 might have opposite functions to S1PR1

In the CNS, the level of S1PR3 expression is highest in astrocytes. S1PR3 reportedly exerts proinflammatory activity in response to cerebral IR injury and autoimmune diseases, such as MS and Sandhoff disease. Evidence indicative of this includes the considerable upregulation of S1PR3 within the lesions consequent of these conditions and the relief of the associated symptoms through S1PR3 and SphK deletion. Moreover, both thrombin and increased S1P reportedly stimulate S1PR3 to activate RhoA, which contributes to astrocyte proliferation, and mediate inflammation. S1P coupled to G12/13 activates RhoA to induce the production of IL-6, VEGFa, and cyclooxygenase-2 (COX-2) in mouse astrocytes [82]. Extracellular vesicles released by the apoptotic cells, termed “apoptotic exosome-like vesicles” (AEVs), are implicated in the pathogenesis of various inflammatory diseases through production of IL-1β. However, cotreatment with the S1PR3 antagonist TY52156 and AEVs almost completely blocked IL-1β induction in macrophages, implying that interference with S1PR3 signaling pathways is a promising strategy to alleviate inflammation [83].Furthermore, Nussbaum et al. found that S1PR3 activation induces a rapid Pselectin mobilization to the surface of endothelial cells, attracting leukocytes that express selectin ligands to bind with them, thereby leading to consecutive P-selectin-dependent leukocyte rolling [84]. Other mediators during inflammation, such as histamine, directly activating SphK1, and thrombin, stimulate AC to elevate intracellular cAMP, activate SphK1, produce abundant endogenous S1P, and employ subsequent S1PR3 signaling to further promote leukocyte rolling. The S1P axis is also a downstream component of protease-activated receptor 1 (PAR1) signaling, PAR1, SphK1 and S1P3 are coupled in an autocrine pathway and mediated dendritic cell (DC) migration. S1pr3−/− dendritic cells are sequestered in draining lymph nodes. The dissemination of inflammatory molecules such as IL-1β is thereby attenuated, largely decreasing sepsis lethality among experimental mice [85]. It can thus be assumed that the coupling of thrombin to PAR1 activates AC and elevates cAMP, further activating SphK1 and contributing to the endogenous production of S1P. S1P then activates S1PR3 in an autocrine manner and mediates immune cell trafficking. However, more study is needed to verify this postulation. Moreover, mice lacking S1PR3 on DCs are protected from kidney IR injury, as S1PR3 is necessary for DCs to release IL-12 and IL-23, which exacerbate kidney IR by activating NK cells and inducing the secretion of IFN-γ [86]. Recent study has also found that S1PR3 contributes to brain IR injury by promoting microglia M1 polarization through the activation of NF-κB signaling [87]. S1PR3 is also reported to mediate vessel tone, but its function in vasocontraction, vasodilation, or both is still controversial. Murakami et al. and Salomone et al. have both observed that the activation of S1PR3 by S1P induced vasoconstriction through the increase of Ca2+ and Rho activation [88,89]. Clot formation could activate platelets and induce them to release S1P. Elevated S1P has been shown to cause basilar artery spasm [90]; further, basilar arteries isolated from S1PR3null mice exhibit weak or no vasoconstriction in response to S1P [89]. In vivo, the intracarotid injection of S1P decreases cerebral blood flow [91]. It can thus be postulated that sustained vascular spasm following subarachnoid hemorrhage and cerebral infarction, along with thrombin formation, is due to aggravated local S1P. However, several studies have also demonstrated that activated S1PR3 induces the release of NO 6

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During systemic anaphylaxis, upregulated IL-33 and IgE could stimulate mast cell degranulation, thereby aggravating allergic reactions. Although S1PR4 is dispensable for mast cell degranulation, S1pr4-null mice exhibited exacerbated IgE-mediated systemic anaphylaxis, indicating its inhibitory role in allergic reactions [102] Additionally, S1PR4 signaling inhibits the generation and secretion of trophic cytokines of T cells, such as IL-4, IL-2 and IFN-γ, while enhancing the suppressive cytokine IL-10 to decrease T cell proliferation and immunosuppressive effects [103]. The deletion of S1pr4 in a murine model of allergic airway disease induces increased airway inflammation and hyperresponsiveness [104]. Thus, while increasing S1PR4 expression might become a promising means of treating autoimmune disease, its potential to address cerebral IR injury requires more support.

to cell proliferation [112]. Numerous studies have confirmed the positive correlation between SphK1 expression and tumor development and progression. It is therefore regarded as an oncogenic factor [113]. Relative to SphK1, SphK2 (molecular mass, 66 kDa [618AA]) seems to be more enigmatic. It is predominantly located in intracellular membranes, including those of the mitochondria, endoplasmic reticulum, and the nucleus, where it inhibits growth and enhances apoptosis [114]. Sequences in the N-terminal and middle regions of SphK2 diverge from those of SphK1; specifically, SphK2 features ~230 more amino acids. The sequences at the N-terminus typically include a nuclear localization signal (NLS) [115] and a 9-amino acid putative BH3 domain [116]. The former enables SphK2 to enter into nuclei and bind with HDAC1/2, while the latter facilitates the interaction between mitochondrial SphK2 and Bcl-2 proteins as well as SphK2-mediated apoptosis. In the nucleus, S1P produced by SphK2 binds to HDAC1/2 and inhibits their enzymatic activity, preventing the removal of acetyl groups from lysine residues within histone tails. SphK2 associates with HDAC1/2 in repressor complexes and is selectively enriched at promoters of the genes encoding the cyclin-dependent kinase inhibitor p21 or the transcriptional regulator c-fos; at these sites, it enhances the acetylation and transcription of local histone H3 and likely induces growth arrest [13]. In the mitochondria, SphK2 cooperates with the Bcl2 family through BH3-only proteins to regulate mitochondrial outermembrane permeabilization (MOMP). S1P and hex bind directly to Bak and Bax, respectively, promoting Bak/Bax function and creating proteolipid pores on mitochondrial outer membrane, which are responsible for cytochrome-c release and apoptosis induction [117]. SphK2 also contains a proline-rich region located in the center of the enzyme. The proline-rich region has a putative SH3-binding domain: a protein-protein interaction module required for regulation of cytoskeletal architecture, signal transduction cascades, and endocytosis [118]. The inter-relationship between SphK1 and SphK2 remains unclear. Studies have reported that they exert divergent roles in kidney IR injury, arthritis, and mast cell functions. Sphk1 null mice were viable, fertile, and lacked any obvious abnormalities; however, their serum S1P levels were markedly decreased (~65%) [119]. Mice with SphK2 knockout also had normal phenotypes. Interestingly, however, both blood and lymphatic S1P levels had increased. This observation may be ascribed to a compensatory increase in SphK1 expression [120]. In Sphk1−/− mice, FTY720 could be successfully phosphorylated and thereby be capable of inducing lymphopenia. By contrast, SphK2−/− mice could not, indicating that SphK2 is required for the functional activation of the sphingosine analog prodrug [10]. Interestingly, after kidney IR injury, SphK1 activity increases almost threefold without any significant change in SphK2 levels. Following kidney IR, SphK1−/− mice did not evidently differ from SphK1+/+ mice. Based on the sparse compensatory SphK mRNA expression, mice without SphK2 appeared to have had more severe kidney damage than did their SphK2+/tr counterparts [121]. In mouse models of collagen-induced arthritis, SphK1 siRNA administration downregulated serum levels of S1P, IL-6, TNF-α, IFN-γ, and IgG2a anti-collagen Ab, whereas mice that received SphK2 siRNA developed more aggressive diseases. These observations demonstrate that by regulating the release of proinflammatory cytokines, SphK1 and SphK2 exert distinct immunomodulatory roles [122]. In mast cells, SphK2 seems to fulfill an intrinsic role as a determinant of the modulation of calcium influx and downstream signaling, particularly PKCα and β, leading to the degranulation and production of eicosanoids and cytokines. As passive systemic anaphylaxis was reportedly impaired in SphK1−/− mice, but not in SphK2−/− mice, and plasma histamine levels were correlated with SphK1 expression and circulating levels of S1P [123,124], SphK1 seems to play an extrinsic role.

2.6. S1PR5 is indispensable for migration and maturation of NK cells While its expression is mainly restricted to oligodendrocytes and NK cells, S1PR5 is also reportedly expressed on brain ECs. Results obtained by examining samples from human brain tissues and immortalized human brain EC line hCMEC/D3 indicate that S1PR5 significantly contributes to BBB maintenance [105], which was consistent with the conclusion of Alba et al. [106]. However, a study that used both healthy and MS brain samples did not find any S1PR5 expression on endothelial cells [107], which is consistent with previous findings [32,108]. Contrastingly, research on mouse models and cultures of human oligodendroglia suggests that S1PR5 expression exhibits an oligodendrocyte restricted pattern. While the use of different cell lines may account for such discrepancies, additional studies are needed to resolve the controversy. Unlike T and B cells that sense S1P gradient through S1PR1, NK cells are primarily regulated by S1PR5. In S1pr5−/− mice, NK cells tend to be retained in the lymph nodes and bone marrow and are consequently depleted in the blood, spleen, and lungs. In vitro research has also found that NK cells isolated from S1PR5-deficient mice were unresponsive to S1P, indicating the pivotal role of S1PR5 in NK-cell distribution [33]. NK cells can be classified into three types according to the differential expression of CD11b and CD27, which also represent the different stages of NK-cell maturation: from CD11bdull NK cells, through CD27hiCD11bhi NK cells, to CD27dull NK cells. S1PR5 expression gradually increases across maturation. Altered NK cell distribution in S1P5-deficient mice is reportedly mainly, not exclusively, due to the altered distribution of CD27dull NK cells, suggesting that S1PR5 expression level profoundly influences NK-cell trafficking. Moreover, FTY720 treatment neither induces S1PR5 down-modulation nor removes NK cells from circulation. A further study revealed that CD69, a negative regulator of S1PR1 during lymphocyte maturation, does not influence S1PR5 expression; rather, the final maturation of NK cells requires the promotion of S1PR5 expression by T-bet, a T-box–containing transcription factor necessary for TH1 cell development [109] (Table 1). 3. SphKs and their characterization SphK1 and SphK2 are isoforms that bind to the primary hydroxyl group of sphingosine to catalyze its transformation into S1P in an ATPdependent manner. Containing five conserved domains, these isoforms are highly homologous; however, they are actually two distinct enzymes encoded by different genes and exhibit different subcellular localization as well as biochemical properties. The genes encoding SphK1 are situated on chromosome 17 (17q25.2), while those encoding SphK2 are located on chromosome 19 (19q13.2) [111]. Human SphK1 (molecular mass, 42 kDa [384AA]) mainly fulfills cytosolic and pro-survival functions. SphK1 overexpression promotes the cell-cycle transition from G1 to S, inhibits the apoptotic response to serum deprivation or ceramide treatment, and consequently contributes

3.1. The interplay between SphK1 and HIF SphK1 is upregulated after stroke [125–128]. This process is 7

8

Endothelial cell

S1PR5

S1PR4

Mast cell Endothelial cells NK cells

T cell

Neutrophil

Neuron Macrophage

Endothelial cell

Inhibits the generation and secretion of trophic cytokines of T cells, such as IL-4, IL-2 and IFN-γ, while enhances the suppressive cytokine IL-10 to decrease T cell proliferation Attenuates biosynthesis of LTs through suicide inactivation of 5-LO Might contribute to BBB maintenance Mediates migration of NK cells from lymphoid tissues to peripheral organ Final maturation of NK cells requires the expression of S1PR5

Mediates vessel tone, but its function in vasocontraction (through the increase of Ca2+ and Rho activation), vasodilation (increase NO release via Ca2+ and Akt-mediated eNOS phosphorylation), or both remains controversial Regulates neutrophil counts (S1pr4-defecient mouse has ~28% lower neutrophil counts than wild type) Might negatively regulate immune cell migration with S1PR1

S1PR3 is necessary for DCs to release IL-12 and IL-23, which could activate NK cells and induce the secretion of IFN-γ Induces a rapid P-selectin mobilization to the surface

Astrocyte/ macroglia Dendritic cell (DC)

S1PR3

S1PR2

Mediates phenotypical shift from anti-inflammatory M2 type to proinflammatory M1 type through TLR2/IL-23/IL-17 axis S1PR1-induced STAT3 hyperactivation could hamper Treg development through restraining of FoxP3 expression Forms and maintains the adherens junctions and protects vascular integrity by activating the PI3K/Rac pathway Promotes cell viability through S1PR1-mediated activation of Akt and ERK, which constitutes part of the antiapoptotic pathway Disrupts the adherens junctions that increase paracellular permeability by activation of RhoROCK-PTEN signaling Unclear Inhibits myeloid macrophage motility via the cAMP/protein kinase A pathway and assists in the production of inflammatory cytokines IL-1β and IL-18 Mediates IL-6 secretion and account for autophagy defects that could result in the accumulation of aggregate-prone proteins as well as the disruption of homeostasis Induces the production of IL-6, VEGFa, and COX-2 by G12/13-mediated activation of RhoA Promotes microglia M1 polarization through the activation of NF-κB signaling Mediates DCs migration from draining lymph nodes to inflammatory sites

Microglia

Regulatory T cell (Treg) Endothelial cell

Modulates the trafficking of T lymphocytes from peripheral lymph node to circulation and inflammatory site Increases the expression of IL-17 and induces TH17 polarization

T cell

S1PR1

Receptor function and related mechanism

Cell type

Receptor

Table 1 Summarizing table of potential targets of five S1PRs in different cell types related to cerebral IR injury.

Up-regulation/stimulation impedes immune cell migration from circulation to inflammatory sites Up-regulation/stimulation inhibits pro-inflammatory cytokine generation such as IL-4, IL-2 and IFN- γ; enhancing the level of anti-inflammatory cytokine IL-10 Up-regulation/stimulation has inhibitory role in allergic reactions Uncertain Lack of related research in the field of brain IR injury

–

Down-regulation/inactivation could reduce P-selectin expression on endothelium and lessen leukocyte attraction Uncertain (might help to mitigate vascular spasm and reduce tissue damage following brain ischemia)

[101] [105,107] [33] [109]

[103,104]

[95,100]

[96]

[88,89,92,93]

[84]

[86]

[82] [87] [85]

[72]

Down-regulation/inactivation could decrease IL-6 secretion Down-regulation/inactivation could decrease the production of IL-6, VEGFa, and COX-2 Down-regulation/inactivation could prevent microglia from M1 polarization Down-regulation/inactivation could sequester DCs in draining lymph nodes to attenuate dissemination of inflammatory molecules such as IL-1β Down-regulation/inactivation could attenuate the release of IL-12 and IL-23

[79] [81]

[78,79]

[68,69]

[63,64,67]

[55,58,59]

[35,51]

[48,49]

[43,110]

Reference

– Down-regulation/inactivation could decrease IL-1β and IL-18 production

Down-regulation/inactivation could reduce leakage of cerebral micro-vessels after IR injury

Down-regulation/inactivation could boost Treg energy which could mitigate inflammation through secretion of anti-inflammatory cytokines and restraint of function of effector T cells It remains unclear whether activated S1PR1 signaling exerts an endothelial-protective function under normal conditions as the normal concentrations of S1P in the plasma are high enough to fully activate S1PR1

Down-regulation/inactivation could sequestrate lymphocytes in the peripheral lymph node to mitigate autoimmune responses Down-regulation/inactivation could decrease IL-17 secretion and TH17 polarization and mitigate brain damage Down-regulation/inactivation could reduce microglia-associated inflammation

Potential interventions and possible benefits

M. Han, et al.

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Fig. 3. The interaction between SphK1 and HIF. When oxygen supply is sufficient; PHDs bind with the α-subunit of HIF, enabling it to be recognized by pVHL, which forms an E3 ubiquitin ligase complex with other proteins, leading to HIF proteasomal degradation. However, when oxygen is shorted, PHDs will become inactivated and leads to the accumulation of HIF-α. Then, HIF-2α bind with HREs in SphK1 promotor regions to facilitate its transcription, and SphK1 could stabilize HIF-1α through the Akt/GSK3β pathway and thus prevents its pVHL-dependent proteasomal degradation. HIF (hypoxia inducible factor); PHDs (prolyl-hydroxylases); pVHL (von Hippel-Lindau tumor suppressor protein); GSK3β (glycogen synthase kinase-3β); Akt (protein kinase B); PI3K (phosphatidylinositol 3-kinase); NF-κB (nuclear factor-kappa B); SphK1 (sphingosine kinase 1); CBP (CREB binding protein); HRE (hypoxia response consensus sequence).

promoted by hypoxia-inducible factor (HIF): a transcription factor comprising an unstable α subunit (HIF-1α or HIF-2α) and a constitutively expressed nuclear β subunit (HIF-β). Under normoxic conditions, the HIF-α subunit is hydroxylated by iron-dependent prolylhydroxylases (PHDs), enabling it to be recognized by the von HippelLindau tumor suppressor protein (pVHL). Together with other proteins, pVHL forms an E3 ubiquitin ligase complex, targeting HIF-α for proteasomal degradation. The interruption of oxygen supply inactivates PHDs and leads to the accumulation of HIF-α, which acts as a transcriptional regulator that bind with genes whose promoters contain hypoxia response consensus sequences (HREs). In this case, the SphK1 5′ flanking region possesses multiple HRE sites to which HIF-2α, not HIF-1α, can directly bind to recruit a number of coactivators. SphK1 transcription and concentrations as well as S1P production are thereby enhanced [114]. Contrastingly, SphK1 stabilizes HIF-1α through the Akt/GSK3β pathway and thus prevents its pVHL-dependent proteasomal degradation [129] (Fig. 3). Because SphK1 and HIF can both be activated by the same molecules, including TNF-α, IL-17, TLR2, and regulate the NF-κB pathway [130], SphK1 could mediate hypoxia–inflammation signaling crosstalk and thus act as a proinflammatory molecule. In mouse models of

polymicrobial peritonitis and sepsis, elevated cytosolic SphK1 activity leads to aberrant inflammatory responses. In these same models, SphK1 inhibition decreases mortality and reduces the plasma concentrations of TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1, and high-mobility group protein B1 [131], indicating the critical role of SphK1 in inflammation. In microglia, TNF-α could activate SphK1, which phosphorylates sphingosine to S1P. S1P subsequently binds to TRAF2, thus fulfilling an important role in NF-κB activation [126,132]. TNF-α also stimulates the accumulation of ubiquitinated HIF via the NF-κB–dependent pathway, which directly induces HIF-1α mRNA transcription [133]. HIF could then activate the NF-κB pathway, forming a positive feedback loop [130]. The increased NF-kB activity in phagocytes could prolong their survival and induce sustained inflammation. As aforementioned, TLR2 upregulation in microglial cells following ischemia could activate SphK1 and sequentially elevate pro-inflammatory cytokines, contributing to the differentiation of microglia into M1 phenotypes [127]. TLR ligation also stabilizes HIF-1α in dendritic cells, which might be involved in promoted antigen presentation. Furthermore, HIF1α in myeloid cells could increase the transcription of key glycolytic enzymes and thereby elevate the glycolytic rate, which is beneficial to M1 macrophage polarization because it relies on glycolysis as an energy 9

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source [130]. Hence, a TLR/HIF-1α/SphK1 axis that contributes to microphage M1 differentiation during IR injury may exist. Moreover, the SphK1-S1P axis contributes to post-stroke TH17 polarization through IL-17 production [125]. HIF-1α concurrently enhances TH17 development through direct transcriptional activation and attenuates Treg development by binding to Foxp3 and thus labelling it for proteasomal degradation. Mice with HIF-1α-deficient T cells are resistant to the induction of TH17-dependent EAE, which is manifested as immunosuppression due to the increase in the concentration of Treg cells [134]. Hence, SphK1, HIF, and related cytokines may form a complicated inflammatory network, in which they interdependently amplify stroke-induced neural damage.

ischemia, SphK2 is considered a protective factor. However, this view is rotted in a relatively limited body of research and may require further experimental validation. Hypoxic preconditioning (HPC) is a well-established approach for inducing tolerance to focal and global cerebral ischemia in both neonates and adults, as it reduces infarct volume and neurological deficits as well as attenuates the reduction in ipsilateral edema [145]. The attribution of these effects to the rapid HPC-mediated increase in microvascular SphK2 protein expression and activity, which peak at 2 h [146], indicates that SphK2 is strongly activated during ischemia. Hypoxia preconditioning significantly reduces infarct volume and improves neurological outcome in wild-type and SphK1−/− mice, but not in SphK2−/− mice [147,148], suggesting that SphK2 is involved in the induction of hypoxic tolerance. Pfeilschifter et al. found that the genetic deletion of SphK2 increases the size of ischemic lesion size and worsens neurological function after the induction of tMCAO [149]. Further study demonstrates that Sphk2 is induced to mediate ischemic tolerance by microvascular and neuronal mechanisms. SphK2 activity is critical to the pre-stroke strengthening of the BBB afforded by HPC, the adherens junction protein VE-cadherin, and the tight junction proteins claudin-5, occludin, and ZO-1; none of these proteins are upregulated in SphK2−/− mice [147]. HIF accumulation is observed following HPC, which causes a downstream increase in SphK2 expression. S1P subsequently acts through the S1PR1 receptor to increase CCL2 expression [145]. The binding of CCL2 with CCLR in astrocytes, microglia, endothelial cells, neurons, and immune cells could regulate the genomic reprogramming required for ischemic tolerance, culminating in the establishment of an ischemia-tolerant phenotype [150]. Sheng et al. found that SphK2 knockdown prevented preconditioning-induced autophagy in primary mouse cortical neurons. Further, S1P and FTY720 did not protect neurons against oxygen glucose deprivation and did not alter the expression of LC3 and p62 [151], suggesting that SphK2mediated autophagy and protection are not S1P-dependent. Both coimmunoprecipitation and GST pulldown analysis indicated that SphK2 directly interacts with Bcl-2 via its BH3 domain, thereby dissociating it from Beclin-1 and activating autophagy in neurons [152]. Krüppel-like factor 4 (KLF4) is a transcription factor that binds with the promoter region of Agr1, accelerating its transcription and contributing to microglial M2 polarization. Recent study has also found that the intranuclear SphK2-S1P axis inhibits KLF4 from interacting with HDAC1 and suppressing KLF4 deacetylation; the transformation of microglial polarization from the M1 to the M2 phenotype is thereby facilitated, and cerebral IR injury is attenuated [153].

3.2. SphK2 is instrumental in hypoxic preconditioning The role of SphK2 in inflammation is controversial. ABC294640, a pharmacological inhibitor of SphK2, exerts anti-inflammatory effects in both acute and chronic mouse models of ulcerative colitis [135], Crohn's disease [136], and arthritis [137]. These include the attenuation of a broad spectrum of inflammatory mediators, including S1P, NFkB, TNF-α, VCAM-1, ICAM-1, COX-2, IL-1β, and IL-6, implicating SphK2 as an inflammatory mediator. Furthermore, the treatment of a mouse bilateral carotid artery stenosis model of chronic cerebral hypoperfusion with the SphK inhibitor SKI-II, which has slightly higher affinity for SphK2 than SphK1, attenuated white matter lesions [138]. More direct evidence indicates that the deletion of SphK2 exerts a protective effect on the pathogenesis of EAE, leading to a reduction of TNF-α-induced permeability in mouse vascular endothelial cells. This is paralleled by both the enhanced expression of PECAM-1 and the reduced expression of ICAM-1 and VCAM-1 [139]. Moreover, using yeast two-hybrid screening, Yoshimoto et al. associated the mouse SphK2 with the IL-12Rβ1 cytoplasmic region, probably through a proline-rich domain, as well as with the positive modulation of IL-12 signaling, especially IL-12-induced IFN-γ production via the STAT4 pathway [140]. Contrastingly, SphK2 seems to suppress the secretion of inflammatory cytokines in some immune cells. Bajwa et al. found that the deletion or pharmacologic inhibition of SphK2 protects kidneys from fibrosis. This is possibly achieved through the upregulation of IFN-γ, which has anti-fibrotic properties, further suggesting that SphK2 is a negative regulator of IFN-γ; this interpretation conflicts with the findings of Yoshimoto et al. Using splenic T cells from untreated Sphk2−/− mice, Bajwa et al. observed that Sphk2-null T cells were hyperproliferative and produced more IFN-γ than did those of WT or SphK1−/− mice [141]. Similarly, Sphk2−/− CD4+ T cells were also found to exhibit a hyperactivated phenotype with significantly enhanced proliferation and cytokine secretion in response to IL-2, as well as reduced sensitivity to Treg cell-mediated suppression in vitro [142]. Additionally, tumor-associated macrophages from SphK2-deficient tumors evinced an increased expression of pro-inflammatory markers, such as NO, TNF-α, IL-12, and MHCII, and a lower expression of anti-inflammatory IL-10 and CD206 [143]. Using primary human macrophages, a recent investigation showed that SphK2 expression negatively correlated with and was functionally coupled to inflammatory cytokine production in response to LPS. Thus, SphK2 restricts inflammation, and its downregulation is essential to LPS-induced macrophage activation [144]. The role of SphK2 this seems to be anti-inflammatory. However, as aforementioned, the blood and lymphatic fluid of Sphk2−/− mice have been found to feature higher concentrations of S1P and SphK1, both of which are actually inflammatory chemokines. However, determining whether this excessive inflammation is caused by the deletion of SphK2 or increase of SphK1 and S1P remains difficult, and the function of SphK2 in immune cells remains unclear. Future studies using genetic knockout of SphK2 are thus warranted to exclude the influence of S1P levels and SphK1 activity. SphK2's role in cerebral IR injury is well understood. During brain

4. Conclusion and future perspectives While early studies mainly focused on the protective role of S1P in endothelial cells, the role of S1P in the pathology of IR injury has garnered increasing attention, especially after the benefits of FTY720 were elucidated. After stroke, local S1P concentration is augmented due to the rupture of erythrocytes and the thrombosis-mediated stimulation of platelets. This response comprises a self-protective signal to ensure vascular integrity and prevent cell apoptosis. However, the local engulfment of cell debris is limited, and S1P, together with other inflammatory cytokines, sends “find me” and “eat me” signals to promote immune cells in circulation to remove apoptotic cells. S1P, as an “inside-out signaling” molecule, contributes to both the chemotaxis of lymphocytes and the inflammatory responses of local cells, such as microglia, but uncontrolled inflammation might cause irreversible neurological deficit or even death. By registering external stimulation and transducing internal signals, respectively, S1PRs and SphKs cooperate closely and might form a positive feedback loop to aggregate immune attack. Indeed, sphingolipid signaling is very complex. Hannun and Obeid proposed that at least two levels of investigation are required to gain a comprehensive understanding of bioactive sphingolipids: The basic level is the understanding of their various specific metabolistic mechanisms, while at an advanced level, focus should be placed on 10

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lipid-mediated pathways and the metabolic organization of enzymes [154]. Future research based on these principles is needed to decipher the complex interplay transduced by S1P, especially S1P receptors and SphKs.

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Author contribution Mengtao Han: Conceptualization, Writing - Original Draft, Visualization. Tao Sun and Haijun Chen: Investigation, Resources, Validation. Mingzhi Han: Writing - Review & Editing. Donghai Wang: Project administration, Supervision. Formatting of funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of competing interest The authors declare that there are no conflicts of interest. Acknowledgement We are grateful to Dr. Wang Jian for helping with earlier versions of this manuscript and for his comments. References [1] S. Prabhakaran, I. Ruff, R.A. Bernstein, Acute stroke intervention, JAMA 313 (2015) 1451. [2] H.K. Eltzschig, T. Eckle, Ischemia and reperfusion—from mechanism to translation, Nat. Med. 17 (2011) 1391–1401. [3] A. Liesz, E. Suri-Payer, C. Veltkamp, H. Doerr, C. Sommer, S. Rivest, et al., Regulatory T Cells Are Key Cerebroprotective Immunomodulators in Acute Experimental Stroke. 15 (2009) 192–199. [4] Y. Fu, Q. Liu, J. Anrather, F.-D. Shi, Immune interventions in stroke, Nat. Rev. Neurol. 11 (2015) 524–535. [5] C. Romer, O. Engel, K. Winek, S. Hochmeister, T. Zhang, G. Royl, et al., Blocking Stroke-Induced Immunodeficiency Increases CNS Antigen-Specific Autoreactivity but Does Not Worsen Functional Outcome After Experimental Stroke. 35 (2015) 7777–7794. [6] Stys PK, Zamponi GW, Van Minnen J, Geurts JJG. Will the Real Multiple Sclerosis Please Stand Up? 2012;13:507–14. [7] R.E. Cannon, J.C. Peart, B.T. Hawkins, C.R. Campos, D.S. Miller, Targeting bloodbrain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 15930–15935. [8] Y. Fu, N. Zhang, L. Ren, Y. Yan, N. Sun, Y.-J. Li, et al., Impact of an immune modulator fingolimod on acute ischemic stroke, Proc. Natl. Acad. Sci. 111 (2014) 18315–18320. [9] Y. Wei, M. Yemisci, H.-H. Kim, L.M. Yung, H.K. Shin, S.-K. Hwang, et al., Fingolimod Provides Long-Term Protection in Rodent Models of Cerebral Ischemia. 69 (2011) 119–129. [10] A. Huwiler, U. Zangemeister-Wittke, The sphingosine 1-phosphate receptor modulator fingolimod as a therapeutic agent: recent findings and new perspectives, Pharmacol. Ther. 185 (2018) 34–49. [11] N. Sun, R.F. Keep, Y. Hua, G. Xi, Critical role of the sphingolipid pathway in stroke: a review of current utility and potential therapeutic targets, Transl. Stroke Res. 7 (2016) 420–438. [12] S. Spiegel, M.A. Maczis, M. Maceyka, S. Milstien, New insights into functions of the sphingosine-1phosphate transporter SPNS2, J. Lipid Res. 60 (2019). [13] N.C. Hait, J. Allegood, M. Maceyka, G.M. Strub, K.B. Harikumar, S.K. Singh, et al., Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate, Science 325 (2009) 1254–1257. [14] Książek M, Chacińska M, Chabowski A, Baranowski M. Sources, Metabolism, and Regulation of Circulating Sphingosine-1-phosphate. 2015;56:1271–81. [15] Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nature Reviews | Molecular Cell Biology. 2003;4 397–407. [16] C. Christoffersen, L.B. Nielsen, O. Axler, A. Andersson, A.H. Johnsen, B. Dahlbäck, Isolation and characterization of human apolipoprotein M-containing lipoproteins, J. Lipid Res. 47 (2006) 1833–1843. [17] C. Christoffersen, L.B. Nielsen, Apolipoprotein M: bridging HDL and endothelial function, Curr. Opin. Lipidol. 24 (2013) 295–300. [18] C. Christoffersen, H. Obinata, S.B. Kumaraswamy, S. Galvani, J. Ahnstrom, M. Sevvana, et al., Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M, Proc. Natl. Acad. Sci. 108 (2011) 9613–9618.

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