The outs and the ins of sphingosine-1-phosphate in immunity

Abstract

The potent lipid mediator sphingosine-1-phosphate (S1P) is produced inside cells by two closely related kinases, sphingosine kinase 1 (SPHK1) and SPHK2, and has emerged as a crucial regulator of immunity. Many of the actions of S1P in innate and adaptive immunity are mediated by its binding to five G protein-coupled receptors, designated S1PR1-5, but recent findings have also identified important roles for S1P as a second messenger during inflammation. In this Review, we discuss recent advances in our understanding of the roles of S1P receptors and describe the newly identified intracellular targets of S1P that are crucial for immune responses. Finally, we discuss the therapeutic potential of new drugs that target S1P signalling and functions.

Figure 1. A simplified scheme of S1P synthesis and metabolism and inside-out signalling

Sphingosine-1-phosphate (S1P) is synthesized by phosphorylation of sphingosine in a reaction that is catalysed by sphingosine kinase 1 (SPHK1) at the plasma membrane (a) and by SPHK2 at the endoplasmic reticulum (ER), mitochondria and nucleus (b). At the ER, S1P is irreversibly degraded by S1P lyase or dephosphorylated to sphingosine by an S1P phosphatase (S1Pase). S1P produced at the plasma membrane in response to stimuli is released by specific transporters and regulates immune functions by binding to specific S1P receptors (S1PRs) and initiating downstream signalling pathways (inside-out signalling). S1P produced in the mitochondria and nucleus by SPHK2 has direct intracellular targets, and S1P generated by SPHK1 at the plasma membrane can also function intracellularly. In the blood, S1P is produced mainly by erythrocytes, is bound to albumin and high-density lipoprotein (HDL) and can activate S1PRs. ACDase, acid ceramidase; ASMase, acid sphingomyelinase.

Figure 2. Regulation of T cell egress by S1PR1

When mature single-positive (SP) thymocytes are ready to exit the thymus (a), they upregulate Krüppel-like factor 2 (KLF2) and its target sphingosine-1-phosphate receptor 1 (S1PR1). The re-expression of S1PR1 enables thymocytes to exit the thymus in response to S1P locally supplied by perivascular cells before encountering the endothelium and then the high levels of S1P in blood vessels. S1PR1 on circulating T cells is internalized owing to high levels of S1P and reappears when the T cells enter non-inflamed lymphoid tissues (b) that contain low levels of S1P. However, in inflamed lymphoid tissues (c), CD69 is expressed on lymphocytes and causes internalization and degradation of S1PR1 to delay exit. After undergoing several rounds of division, the newly generated effector T cells upregulate S1PR1, lose CC-chemokine receptor 7 (CCR7) expression and exit into the circulation. TCR, T cell receptor.

Figure 3. FHL2-mediated repression of S1pr1 expression and regulation of DC migration

Ligation of sphingosine-1-phosphate receptor 2 (S1PR2) on immature dendritic cells (DCs) (a) activates the small GTPase RHO, leading to the translocation of four and a half LIM domains protein 2 (FHL2) to the nucleus, where it represses S1pr1 expression. In addition, immature DCs express only low levels of CC-chemokine receptor 7 (CCR7) and this ensures that RAC activation is limited. Such repression of S1PR1 and RAC ensures that migratory responses of immature DCs are restricted. During maturation (b), DCs upregulate CCR7 and downregulate S1PR2; this results in increasedS1pr1 expression and RAC activation, leading to enhanced migration of mature DCs. CCL19, CC-chemokine ligand 19.

Figure 4. The dual role of PAR1 and inside-out signalling by S1P in the regulation of endothelial barrier function

Under physiological conditions, plasma sphingosine-1-phosphate (S1P) is produced and released by erythrocytes to maintain barrier integrity. Protease-activated receptor 1 (PAR1) activation by thrombin (the levels of which are elevated during pathology) increases endothelial permeability; this process may also be dependent on sphingosine kinase 1 (SPHK1) and S1P receptor 3 (S1PR3). Permeability is gradually counteracted by thrombin-mediated cleavage of protein C to activated protein C (APC), which then binds to the endothelial protein C receptor (EPCR), stimulating SPHK1 expression and signalling through the S1P–S1PR1 axis that reseals the endothelial cell barrier. S1PR1 or S1PR3 induce RAC- or RHO-dependent cytoskeletal rearrangements to promote or disrupt adherens junctions, respectively. Sph, sphingosine.

Figure 5. Roles of S1P produced by SPHK1 in TNFR signalling

Engagement of TNF receptor 1 (TNFR1) by tumour necrosis factor (TNF) leads to the recruitment of a signalling complex containing TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), receptor-interacting protein 1 (RIP1; also known as RIPK1), cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2. Interaction of TRAF2 with sphingosine kinase 1 (SPHK1) stimulates it and brings it to the plasma membrane where its substrate, sphingosine, resides. Intracellular sphingosine-1-phosphate (S1P) is a required cofactor for the K63-linked polyubiquitylation of RIP1 by TRAF2, and the ubiquitin chain then acts as a scaffold to recruit and activate TGFβ-activated kinase 1 (TAK1; also known as MAP3K7) and IκB kinase (IKK) complexes. The IKK complex phosphorylates NF-κB inhibitor-α (IκBα), leading to the activation of nuclear factor-κB (NF-κB). Polyubiquitylation of RIP1 also prevents its interaction with pro-caspase 8, and this limits the processing of this caspase precursor and thus the initiation of apoptosis. Intracellular S1P can be exported out of cells and can then activate its cell surface receptors and downstream signalling pathways, culminating in proliferation, survival, motility and the production of cytokines, chemokines and prostaglandins. S1PR, S1P receptor; Sph, sphingosine; TAB2, TAK1-binding protein 2; Ub, ubiquitin.

Figure 6. Roles of S1P produced by SPHK1 in TLR4 signalling

Engagement of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) at the plasma membrane promotes the recruitment of the adaptor myeloid differentiation primary response protein 88 (MYD88) and the assembly of a signalling complex that includes another adaptor called TIR domain-containing adaptor protein (TIRAP), TNFR-associated factor 6 (TRAF6) and the protein kinase TGFβ-activated kinase 1 (TAK1; also known as MAP3K7). Sphingosine-1-phosphate (S1P) generation by sphingosine kinase 1 (SPHK1) mediates the activation of protein kinase Cδ (PKCδ), which phosphorylates an unknown target to promote IκB kinase (IKK) and nuclear factor-κB (NF-κB) activation through an ill-defined mechanism. It is also possible that S1P enhances the autoubiquitylation of TRAF6, which then recruits and activates TAK1. TAK1 phosphorylates the IKK complex, leading to the activation of NF-κB and mitogen-activated protein kinases (MAPKs; namely, extracellular signal-regulated kinase (ERK), JUN N-terminal kinase (JNK) and p38). Dashed arrows indicate pathways in TLR4 signalling that could potentially involve S1P. AP1, activator protein 1; IκBα, NF-κB inhibitor-α; IRAK, IL-1R-associated kinase; IRF3, interferon-regulatory factor 3; MD2, myeloid differentiation factor 2 (also known as LY96); RIP1, receptor-interacting protein 1 (also known as RIPK1); Sph, sphingosine; TAB, TAK1-binding protein; TBK1, TANK-binding kinase 1; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor protein inducing IFNβ; Ub, ubiquitin.

Figure 7. S1P produced by SPHK2 in the nucleus inhibits HDACs and regulates gene transcription

Nuclear sphingosine kinase 2 (SPHK2) is associated with histone deacetylase 1 (HDAC1) and HDAC2 in repressor complexes at the promoters of specific genes (such as CDKN1A, which encodes p21). In response to external stimuli, protein kinase C (PKC) is activated (probably via extracellular signal-regulated kinase 1 (ERK1)) and phosphorylates and activates SPHK2, resulting in increased production of sphingosine-1-phosphate (S1P). S1P binds to and inhibits HDAC1 and HDAC2, leading to increased levels of histone acetylation and gene transcription. Dashed arrows indicate pathways that could potentially activate SPHK2. Ac, acetyl; H3, histone H3.