Sphingosine 1-phosphate receptor-targeted therapeutics in rheumatic diseases

Abstract

Sphingosine 1-phosphate (S1P), which acts via G protein-coupled S1P receptors (S1PRs), is a bioactive lipid essential for vascular integrity and lymphocyte trafficking. The S1P–S1PR signalling axis is a key component of the inflammatory response in autoimmune rheumatic diseases. Several drugs that target S1PRs have been approved for the treatment of multiple sclerosis and inflammatory bowel disease and are under clinical testing for patients with systemic lupus erythematosus (SLE). Preclinical studies support the hypothesis that targeting the S1P–S1PR axis would be beneficial to patients with SLE, rheumatoid arthritis (RA) and systemic sclerosis (SSc) by reducing pathological inflammation. Whereas most preclinical research and development efforts are focused on reducing lymphocyte trafficking, protective effects of circulating S1P on endothelial S1PRs, which maintain the vascular barrier and enable blood circulation while dampening leukocyte extravasation, have been largely overlooked. In this Review, we take a holistic view of S1P–S1PR signalling in lymphocyte and vascular pathobiology. We focus on the potential of S1PR modulators for the treatment of SLE, RA and SSc and summarize the rationale, pathobiology and evidence from preclinical models and clinical studies. Improved understanding of S1P pathobiology in autoimmune rheumatic diseases and S1PR therapeutic modulation is anticipated to lead to efficacious and safer management of these diseases.

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule secreted into the circulation mainly by erythrocytes and endothelial cells. It binds to five high affinity G protein coupled receptors (GPCRs), termed sphingosine 1-phosphate receptors (S1PRs), which are widely expressed and affect cell proliferation, survival and migration both positively and negatively. S1P receptor 1 (S1PR1, also known as S1P1, encoded by S1PR1, originally cloned as endothelial differentiation gene 1 (EDG1)), is highly expressed on endothelial cells (ECs) and increases vascular barrier function after coupling to guanine nucleotide-binding protein (G protein) Gα_i by facilitating the translocation of vascular endothelial (VE)-cadherin (also known as cadherin 5), an EC-specific adhesion molecule, to inter-cellular borders and the assembly of adherens junctions¹. EC S1PR1 signalling also has anti-inflammatory effects by curbing the expression of pro-inflammatory adhesion molecules required for leukocyte transmigration^2-4 and by limiting the production of the cytokines that contribute to the ‘cytokine storm’ in response to viral infection⁵. S1PR1 is also expressed on lymphocytes and is a crucial mediator of lymphocyte trafficking. Lymphocytes migrate out of lymph nodes (LNs), in which S1P levels are kept low by degradative enzymes such as phospholipid phosphatase 3 (LPP3) (also known as lipid phosphate phosphohydrolase) and S1P lyase 1 (SGPL1)^6,7 towards high concentrations of S1P in lymph and blood, using S1PR1 as an egress receptor^8,9.

Whereas S1PR1 functions in vascular and immune systems have been studied extensively, other S1PR isoforms also have important pathophysiological functions^10,11. For example, S1PR2 is induced in ECs during pathological conditions and opposes the actions of S1PR1, by enhancing vascular permeability and inducing inflammatory responses^12-14. In the immune system, S1PR2 restrains the movement of cells (myeloid cells, germinal centre B cells and γδ T cells) by inhibiting chemotactic receptors^15,16. Loss of function of S1PR2 is associated with diffuse large B cell lymphoma severity in humans¹⁷. S1PR3 is expressed in pericytes and fibroblasts, and at low levels in ECs, where it can be upregulated in the setting of lung injury¹⁸. EC S1PR3 mediates S1P-induced vasoconstriction¹⁹, and seems to be crucial for inflammatory and fibrotic responses^20,21. S1PR4 polymorphisms are associated with circulating neutrophil numbers in humans²². S1PR5 is the main S1PR facilitating natural killer cell trafficking from bone marrow and secondary lymphoid organs (SLOs)²³. Because S1PR1 is essential for lymphocyte trafficking, the major EC S1PR, and the target of newer S1PR modulators used in preclinical studies and clinical trials of rheumatic autoimmune diseases, this Review focuses on S1P–S1PR1 biology.

Four S1PR1 modulators have been FDA approved for the treatment of autoimmune inflammation in multiple sclerosis and one has been FDA approved for ulcerative colitis. Fingolimod, approved in 2010, is a sphingosine analogue that is phosphorylated by sphingosine kinase 2 (SPHK2)²⁴ and binds to S1PR1, S1PR3, S1PR4 and S1PR5 (REF.²⁵). Fingolimod has several known adverse effects, including bradycardia, suppression of pulmonary function, hepatocyte damage and macular oedema. Bradycardia occurs secondary to fingolimod engagement of S1PR1-dependent inward-rectifying potassium channels in human cardiomyocytes²⁶. In most individuals, adverse events are mild or moderate, and bradycardia usually resolves within 4 weeks²⁷. The more recently approved drugs, namely, ozanimod, siponimod and ponesimod, are selective for S1PR1 and S1PR5 (REF.²⁸). As some of the safety issues associated with the use of fingolimod could be related to its engagement of other S1PR isoforms, the newer S1PR modulators with greater specificity for S1PR1 are expected to have fewer adverse effects. The active forms of all four of these drugs bind to S1PR1 on the surface of lymphocytes and induce receptor internalization and degradation, thereby acting as functional antagonists and blocking the ability of autoreactive T cells and B cells to enter tissues (see section S1PR1 modulation of lymphocyte trafficking). However, because S1PR1 is highly expressed on ECs and mediates barrier function of blood vessels, the use of these functional antagonists could lead to vascular leakage²⁹ and downstream inflammatory injury³⁰. Thus, in considering the use of S1PR1 modulators in autoimmune rheumatic disease, one must be cognizant of the potential risks of EC S1PR1 antagonism, particularly in patients with clinical and subclinical pulmonary involvement of their rheumatic disease and inflammatory eye diseases, because S1PR1 is highly expressed in lung and retinal vessels^27,31,32. In this Review, we give an overview of S1P–S1PR1 biology, discuss how modulation of the S1P–S1PR1 axis could benefit patients with autoimmune rheumatic diseases and provide a critical assessment of the preclinical and clinical data that support the use of S1PR1 modulators for systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic sclerosis (SSc).

Basic biology of S1P

S1P production and export

S1P is formed by metabolic breakdown of membrane-derived sphingomyelin (FIG. 1). Hydrolysis of sphingomyelin forms ceramide, which is converted to sphingosine by ceramidase; intracellular sphingosine is phosphorylated into S1P by sphingosine kinase 1 (SPHK1) or SPHK2 and degraded by SGPL1 and S1P phosphatase 1 (Spp1) and Spp2¹¹ (FIG. 1). Although the enzymatic properties of SPHKs are similar, differential subcellular localization and tissue specificity of expression suggest some isoform-specific functions. SPHK1 resides predominantly in the cytosol, undergoes translocation to the plasma membrane following phosphorylation and can also be transported outside of the cell³³, whereas SPHK2 is localized in the endoplasmic reticulum, nucleus and mitochondria³⁴ (FIG. 1). Sphk1 knockout (KO) mice demonstrate a 65% reduction in circulating S1P levels but have normal tissue S1P levels and do not exhibit dysregulated lymphocyte trafficking or vascular developmental defects³⁵, whereas Sphk2 KO mice have either normal³⁶ or elevated³⁷ levels of circulating S1P, a finding that has been attributed to the ability of Sphk2 to recycle S1P intracellularly³⁸. Double Sphk1–Sphk2 KO embryos show evidence of both vascular and neural defects and die in utero³⁹, whereas mice with postnatally inducible double Sphk1–Sphk2 KO are viable but demonstrate vascular leakage and increased mortality when challenged with inflammatory mediators⁴⁰.

Fig. 1 ∣. Overview of S1P production, export and chaperones.

Sphingosine 1-phosphate (S1P) is produced intracellularly by sphingosine kinases (SPHK1 and SPHK2) and is degraded by S1P lyases and phosphatases. Extracellular transport of S1P is mediated by spinster homologue 2 (SPNS2) on endothelial cells or major facilitator superfamily domain-containing protein 2B (MFSD2B) on red blood cells and platelets. Extracellular S1P binds to apolipoprotein M (ApoM) in HDL or albumin in the circulation and binds S1P receptor 1 (S1PR1), which is expressed on endothelial cells, lymphocytes and many other cell types.

Extracellular S1P is regulated by the LPP3 enzyme; the active site of this key enzyme is located on the extracellular surface and could be involved in sphingolipid uptake⁴¹. The polymorphism of PLPP3 (encoding LPP3) is associated with cardiovascular disease in humans, suggesting its importance in vascular function⁴². LPP3 is highly expressed on ECs and helps to maintain the vascular barrier, as mice with an EC-specific Plpp3 KO have evidence of vascular leakage in vivo⁴³, and silencing of Lpp3 in ECs decreases vascular resistance in vitro⁴⁴. In the immune system, this enzyme regulates the S1P-dependent trafficking of lymphocytes in the thymus⁴⁵. As LPP3 is active towards multiple lipid phosphates in the extracellular environment, the role of this protein in autoimmune rheumatic diseases warrants further research.

S1P in lymph and plasma originates from different sources. In lymph, the primary source of S1P is the lymphatic endothelial cell^8,46. Lymphatic endothelial cell-specific Sphk1 KO mice have altered lymphatic vascular junctions and decreased egress of lymphocytes to LNs. By contrast, in plasma, erythrocytes and blood vessels ECs are the major sources of S1P⁴⁷. Erythrocytes use Sphk1 to generate S1P and contribute to circulating levels, and EC-specific double Sphk1–Sphk2 KO mice show a ~30% reduction in plasma S1P levels⁴⁸.

Although platelets contain high levels of S1P that is produced by Sphk2 (REF.⁴⁹), they do not contribute to circulating S1P during homeostasis^49,50. However, upon activation with agonists, such as thrombin or collagen, platelets release S1P, which, in turn, induces in platelets a change in shape and the release of arachidonic acid and thromboxane^49,51, thereby fine tuning thrombotic mechanisms. Platelet-released S1P increases endothelial barrier function^52,53, which could limit vascular leakage during thrombosis or inflammatory injury. Moreover, if the level of S1P generated by erythrocytes and ECs becomes insufficient, platelet-derived S1P compensates for essential functions such as embryonic viability⁵². Platelet-derived S1P has a crucial role in the barrier function of high endothelial venules (HEVs), the vessels that enable trafficking of lymphocytes to LNs. Fibroblastic reticular cells surround HEVs and express podoplanin, which activates platelets, thereby inducing the release of S1P, which in turn maintains HEV barrier integrity⁵⁴.

Intracellular S1P is transported to the extracellular space by the transporter spinster homologue 2 (SPNS2)⁵⁵ in ECs and by the major facilitator superfamily domain-containing protein 2B (MFSD2B) in erythrocytes and platelets⁵⁶ (FIG. 1). Spns2 KO mice (either global gene deletion or endothelial-specific deletion) show reduced plasma S1P level and dramatically reduced T cell and immature B cell egress from the thymus and bone marrow, respectively, suggesting that EC-derived S1P is required for adaptive immune system homeostasis^57,58.

Although the cellular and enzymatic contributors to circulating S1P have been defined, the precise mechanisms that maintain physiological S1P levels during homeostasis and inflammation have yet to be elucidated. Inflammatory conditions and ageing lead to reduced plasma S1P levels, suggesting that a stable plasma S1P pool contributes to overall health^59-61.

S1P chaperones

Extracellular S1P in the circulation is bound to either albumin, which carries ~30% of plasma S1P, or apolipoprotein M (ApoM), which carries roughly ~65%⁶² (FIG. 1). ApoM, a lipocalin produced mainly in the liver and kidney, is a constituent of HDL particles. Almost all S1P associated with HDL particles is bound to ApoM⁶². ApoM has anti-atherogenic properties owing to its ability to regulate triglyceride metabolism, adipocyte biology^63,64 and endothelial protection⁶². ApoM can bind to oxidized phospholipids under pathological conditions, even though ApoM-containing HDL particles represent a minor proportion of the heterogeneous HDL particles⁶⁵. APOM is located on the MHC class III region of chromosome 6, a region rich in genes involved in innate immunity and inflammation, including TNF (encoding tumour necrosis factor (TNF)), LTA and LTB (encoding lymphotoxin-α and lymphotoxin-β proteins, respectively)⁶⁶. ApoM KO mice have ~45% reduction in plasma S1P level and are more vulnerable to vascular leakage than wild-type mice but do not show defects in lymphocyte trafficking. However, such mice show increased proliferation of haematopoietic progenitor cells and common lymphoid progenitors in the bone marrow and a heightened adaptive immunity, making them more susceptible to neuroinflammatory insults⁶⁷. The chaperone (albumin or ApoM) presenting S1P to S1PR1 mediates ‘biased’ signalling, as S1P presented in the context of ApoM in HDL particles shows sustained signalling and enhanced endothelial barrier function compared with albumin-bound S1P^62,68-70.

S1PR1 modulation of lymphocyte trafficking

T cells and B cells lacking S1PR1 are trapped in the thymus and bone marrow, respectively, owing to their inability to sense S1P^71-73. During homeostasis, naive lymphocytes egress towards the lymph out of LNs, where S1P levels are kept very low by SGPL1 and LPP3 (REF.⁶). Lymphocyte cell-surface expression of S1PR1 correlates inversely with extracellular S1P concentration to which the receptor is exposed; thus, it is high in the LNs where lymphocytes are poised for egress⁸ (FIG. 2). Lymphatic EC production⁴⁶ and their release of S1P via Spns2 are involved in the egress process^46,58,74. In contrast to the lymphocyte expression of S1PR1, the expression CC-chemokine receptor 7 (CCR7), the principal chemokine receptor for CC-chemokine ligand 21 (CCL21) mediating lymphocyte entry and retention in LNs, is downregulated in LNs but high in blood and lymph, facilitating lymphocyte entrance to and transient arrest in SLOs⁷⁵.

Fig. 2 ∣. Major physiological roles of S1PR1 signalling.

Sphingosine 1-phosphate (S1P) receptor 1 (S1PR1) signalling has roles in lymphocyte trafficking and differentiation and endothelial cell barrier protection. Lymphocytes sense circulating S1P via S1PR1 in lymph nodes where the S1P lyase and phosphatase activities are high, S1PR1 expression is maximal, and lymphocytes are poised to egress. In blood and lymph, high S1P levels induce internalization of S1PR1. Beyond trafficking, S1PR1 signalling restrains CD4⁺T cell differentiation into regulatory T (T_reg) cells and increases their differentiation into T helper 17 (T_H17) cells.Thus, functional antagonists of S1P–S1PR1 signalling increase differentiation into T_reg cells and decrease differentiation into T_H17 cells. On endothelial cells, S1PR1 signalling leads to increased intercellular contacts via adherens junctions mediated, in part, by VE-cadherin and limits vascular leakage and leukocyte transmigration. S1PR1 signalling also limits expression of ICAM1 and VCAM1 in response to inflammatory mediators (such as tumour necrosis factor (TNF)) by blocking NF-κB activation.

Much of the biology of S1P–S1PR1 signalling and its effect on lymphocyte trafficking has been learned from preclinical and clinical experience with fingolimod. Whereas S1P binding to S1PR1 causes internalization of the receptor followed by recycling to the membrane where it can engage extracellular S1P again, fingolimod-P induces S1PR1 internalization, ubiquitination and degradation²⁹, processes that require recruitment of β-arrestin⁷⁶. This difference occurs because fingolimod-P is metabolically more stable, more poorly dissociated from S1PR1 and more efficient at recruiting β-arrestin than S1P⁷⁷. The net result is that lymphocytes lose the sensor (that is, S1PR1) required to exit SLOs. Fingolimod can also reduce SphK1 activity and induce its proteasomal degradation, at least in vitro⁷⁸, which could potentially also contribute to altered lymphocyte trafficking. The lymphocytes affected by fingolimod, namely, naive T cells and central memory T cells, express CCR7 and circulate continuously from the blood to LNs. By contrast, effector memory T cells and CD45RA (also known as receptor-type tyrosine-protein phosphatase C)-expressing effector memory T cells lack expression of CCR7, do not recirculate regularly through LNs and are not affected by fingolimod⁷⁹. Similar T cell subsets are affected in patients taking ozanimod⁸⁰ (see section S1PR1 signalling effects on T_reg cells). Effector memory T cells present in the circulation remain functionally responsive without impairment of defence against pathogens, consistent with the observation that patients taking fingolimod have a low, albeit increased, risk of infectious complications^81,82.

When T cells encounter antigen in the context of dendritic cells (DCs) and become activated, they downregulate S1PR1 transcription and are retained in SLOs via CCR7 retention signals; however, daughter cells begin to recover S1PR1 expression after several divisions and are able to egress in response to S1P⁷⁵. In the presence of inflammatory signals (such as type I interferon and TNF), lymphocytes express early activation antigen CD69, a negative regulator of S1PR1, which, like CCR7, facilitates lymphocyte arrest in SLOs⁸³. A study published in 2021 has shown that during viral infection or neuroinflammation, S1P levels increase in LNs, thereby hindering T cell egress. Unexpectedly, the source of elevated S1P in LNs was activated CD69⁺ monocytes, which helped to differentiate T follicular helper cells and T helper 17 (T_H17) cells. The precise mechanisms by which monocyte-derived S1P affects T cell differentiation have yet to be delineated⁸⁴, but T cells with increased retention time in LNs might have enhanced maturation and antigen specificity.

Although fingolimod treatment leads to sequestration of lymphocytes in LNs, FTY720-treated mice do not exhibit hypertrophic SLOs^85-87. One possibility is that FTY720 induces lymphocyte apoptosis, as several studies have shown^88-91. Another possibility is that FTY720 hinders DC migration to LNs, thereby reducing antigen presentation and T cell and B cell activation, differentiation and proliferation, as has been suggested by some studies^92,93. FTY720 may also limit numbers of activated T_H cells in the lymphoid tissues, which would result in smaller germinal centers⁹⁴, impaired affinity maturation, and diminished class-switching recombination, which could also contribute to smaller SLOs.

S1P and adaptive immunity

S1PR1 signalling effects on T_H17 cells

Fingolimod is beneficial in multiple sclerosis, at least in part, by keeping auto-aggressive central memory T cells out of the central nervous system (CNS). The central memory T cells subset targeted by fingolimod includes T_H17 cells, which produce the inflammatory cytokine IL-17 and are implicated in multiple sclerosis pathogenesis⁹⁵. Mice with a T_H17 cell-specific deletion of S1pr1 (T_H17 S1pr1 KO) are completely resistant to the development of experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG_35–55)⁹⁶. Congruent with the concept that loss of S1PR1 signalling limits trafficking of auto-aggressive T cells, T_H17 S1pr1 KO mice with experimental autoimmune encephalomyelitis had significantly fewer infiltrating T_H17 and T_H1 cells in the CNS than controls. However, contrary to the expectation that T_H17 cells would be trapped in LNs, the percentage of T_H17 cells in the LNs was decreased in KO mice, suggesting an additional role for S1PR1 signalling in the generation and/or survival of T_H17 cells. These data are supported by earlier studies demonstrating that S1PR1 signalling increases T_H17 cell differentiation from CD4⁺ T cells⁹⁷ (FIG. 2). Both S1P and SEW-2871, a S1PR1-specific agonist, increased IL-17 production by T cells⁹⁷. Suppression of T_H1 and T_H2 cell cytokine production occurred concurrently with the development of T_H17 cells⁹⁷. In another study, mice harbouring a variant of S1pr1 encoding a protein with increased signalling capacity had increased production of IL-17, and splenocytes from mice treated with a SGPL1 inhibitor to increase S1P and S1pr1 signalling also demonstrated enhanced IL-17 production and increased susceptibility to experimental autoimmune encephalomyelitis⁹⁸. Taken together, these studies provide strong evidence that S1PR1 signalling augments T_H17 cell differentiation in appropriately primed CD4⁺ T cells and that FTY720, by inducing internalization and degradation of S1PR1, attenuates T_H17 cell differentiation.

S1PR1 signalling effects on T_reg cells

Regulatory T (T_reg) cells inhibit the activation and expansion of CD4⁺ T_H cells, suppress differentiation of cytotoxic CD8⁺ T cells and limit B cell activation. Mice lacking forkhead box protein P3 (Foxp3), the transcription factor required for T_reg cell differentiation and maintenance, develop autoimmunity, and inactivating mutations in FOXP3 in humans are associated with severe autoimmunity^99-101. Increasing T_reg cell numbers and/or function could be beneficial in autoimmune rheumatic disease¹⁰². In NZB/NZW lupus-prone mice, adoptive transfer of T_reg cells before the onset of clinical disease resulted in decreased progression of kidney injury and mortality¹⁰³.

S1P–S1PR1 signalling suppresses T_reg cell numbers and functions (FIG. 2). CD4⁺T-specific S1pr1 KO mice have increased numbers of T_reg cells that have greater suppressive capacity than those in controls¹⁰⁴. Furthermore, mice with CD4⁺T cells overexpressing S1pr1 have impaired suppressive capacity¹⁰⁴. However, a more recent study published in 2017 has added complexity to the paradigm that S1PR1 signalling in T cells negatively affects T_reg cells and autoimmunity. When S1pr1 is knocked out in Foxp3⁺ T cells rather than all CD4⁺ T cells, T_reg cells are trapped in LNs and numbers of tissue-surveilling T_reg cells are decreased, resulting in autoimmunity. Moreover, inducible deletion of S1pr1 in Foxp3⁺ T cells rendered mice more susceptible to experimental autoimmune encephalomyelitis, presumably because of decreased thymic and LN release of T_reg cells to the CNS⁹⁶. In support of the hypothesis that T_reg cells use S1PR1 to exit LNs and enter tissues, KO of S1pr1 in CD4⁺ T cells resulted in T_reg cell trapping in LNs, leading to reduced numbers of T_reg cells in tumours and restrained tumour growth¹⁰⁵. Thus, it is unclear whether the net effect of S1PR1 signalling in lymphocytes is detrimental (by curbing the production of T_reg cells from CD4⁺ T cells) or protective (by enabling T_reg cells to egress from LNs to tissues) against autoimmunity. Some insight can be gleaned from mice and patients taking FTY720/fingolimod. FTY720/fingolimod, acting as a functional antagonist on lymphocytes, has been shown to increase the suppressive capacity of T_reg cells in both mice (FTY720)^106-108 and humans (fingolimod)^109-112. Although the numbers of T_reg cells are expected to fall during treatment with FTY720/fingolimod secondary to trapping in LNs, they diminish to a lesser extent than those of other T cell populations, resulting in the relative preservation of T_reg cells with a memory phenotype^110,113,114. Relative preservation of T_reg cells compared with other T cell subsets was also observed in patients taking ozanimod⁸⁰. This finding could be attributed to reduced expression of S1PR1 in T_reg cells, the altered chemotaxis to S1P of these cells compared with other T cells, and the ability of T_reg cells to use other S1PRs to egress from LNs¹⁰⁶.

Because in SLE, RA and SSc there is evidence of an imbalance of T_H17 cell–T_reg cell populations, S1PR1 modulators, acting as functional antagonists, could help to restore this imbalance and immune tolerance. The ongoing clinical studies using S1PR1 modulators in SLE (NCT03742037) could elucidate whether T_H17 cells and/or T_reg cells trafficking and/or function are positively affected during treatment.

S1P and vascular homeostasis

S1PR1 signalling effect on vascular barrier

S1PR1 is a crucial mediator of vascular development and function; S1pr1 KO mice are not viable as a result of vascular fragility during embryonic development¹¹⁵. S1PR1 inhibits angiogenic sprouting during development^116-118 and stabilizes blood vessels of tumours, which are immature and prone to leakage¹¹⁹. EC S1PR1 is a key mediator of vascular barrier function by facilitating the assembly of VE-cadherin molecules to intercellular borders where they form zipper-like structures that help to maintain the vascular barrier¹ (FIG. 2). Mice with an endothelial-specific¹²⁰ knockdown of S1pr1 or treated with S1PR1 antagonists show evidence of vascular leakage in the lungs^67,121 and brain¹²². Reciprocally, S1PR1 agonists rapidly attenuate vascular leakage, production of inflammatory cytokines and neutrophil infiltration in response to lung injury^120,123, and immune complex mediated vasculopathy¹²¹. However, significant endocytosis of the endothelial S1PR1 after binding to agonists leads to functional antagonism and cessation of the vascular protective response and could even lead to exacerbation of inflammatory injury and parenchymal fibrosis (see section S1PR1 antagonists in preclinical RA)¹²⁴.

S1PR1 signalling also induces vasorelaxation by activating endothelial nitric oxide synthase (eNOS)^19,125; mice with EC-specific S1pr1 KO have significantly higher blood pressure than littermate controls¹⁹. However, in the afferent arterioles of the kidney, S1PR1 agonist SEW-2871 had a vasoconstrictive effect, even at doses as low as 10 nM¹²⁶. Because afferent arterioles are of major importance in regulating glomerular filtration rate, the vasoconstrictor effect of S1P selectively in afferent but not efferent arterioles strongly implicates S1PR1 signalling in controlling glomerular filtration rate¹²⁷. How EC S1PR1 signalling affects the pathophysiology of lupus nephritis has yet to be elucidated.

Circulating S1P–ApoM and vascular health

ApoM on HDL particles delivers S1P to S1PR1 and has anti-inflammatory effects by limiting expression of pro-inflammatory adhesion molecules such as ICAM1 and VCAM1, which are required for leukocyte transmigration into tissues^2,3 (FIG. 2). As an example of ’biased’ signalling, S1P–ApoM is more effective than S1P–albumin at preventing TNF-induced ICAM1 expression in ECs; this enhanced signalling is secondary to the ability of S1P–ApoM to attenuate NF-κB phosphorylation², which is essential for TNF-mediated inflammatory responses.

Many lines of evidence suggest that ApoM delivery of S1P to ECs also protects against vascular injury and inflammation, which are important pathogenetic mechanisms in cardiovascular disease. ApoM has been shown to mediate cholesterol efflux and susceptibility to atherosclerosis¹²⁸. Furthermore, the cardiovascular protective effects of S1P–ApoM were demonstrated in mouse models of myocardial infarction and stroke, in which an engineered S1P chaperone ApoM–S1P Fc attenuated inflammatory damage in response to ischaemic injury⁶⁹.

ApoM also has protective effects on blood pressure: Apom KO mice showed significantly increased resting blood pressure compared with wild-type mice, and ApoM–S1P Fc increased circulating nitrite levels and protected against angiotensin-induced hypertension, suggesting that ApoM activates the eNOS⁶⁹.

S1P–S1PR1 modulators in rheumatic disease

SLE, RA and SSc are complex and heterogeneous diseases that share some pathological features, including autoreactive lymphocytes, dysregulated T_H17 cells and T_reg cells that are not effective enough to quell autoimmune-mediated damage. As detailed above, there is strong evidence that these pathological features could be ameliorated by S1PR1 modulation. Moreover, cytokine-induced vascular permeability in each of these diseases facilitates extravasation of inflammatory mediators^129,130, which could be attenuated by EC S1PR1 agonism. The clinical efficacy of fingolimod for the treatment of multiple sclerosis has encouraged the development of several S1PR1-specific agonists and antagonists for the treatment of other autoimmune diseases. In the following sections, we review the evidence that the S1P–S1PR axis is dysfunctional in SLE, RA and SSc and the preclinical and clinical studies supporting the use of S1PR modulators in each of these diseases. We take a holistic view that therapeutic modulation of the S1P signalling axis should consider not only the immune system but also the vascular system and connective tissue cells. Reviews on autoimmune pathophysiology are available^131,132.

Systemic lupus erythematosus

SLE is the prototypic systemic autoimmune disease, with all components of the immune system contributing to autoimmunity against nucleic acid-containing cellular elements. The innate immune system is activated, in part, by cellular nucleic acid sensors and neutrophil extracellular traps (NETs), which stimulate production of type I interferon, a key contributor to organ damage. Adaptive immune responses are also altered¹³³. T cell dysregulation is evidenced by altered signalling, production of cytokines and proliferation and function of T cell subsets¹³⁴. In SLE, expansion of follicular T_H cells promotes differentiation of autoantigen-specific B cells, whereas depletion of T_reg cells is associated with impaired capacity to suppress immune responses and T_H17 cells¹³⁵. B cell function is also disturbed, with increased cytokine production and augmented presentation of antigens to T cells. Consequent expansion of autoreactive B cells and production of autoantibodies leads to intravascular immune complex formation. SLE ECs display an activated, inflamed phenotype, with increased expression of adhesion molecules related to exposure to cytokines (that is, type I interferons), complement activation fragments and products of stimulated innate immune cells¹³⁶. Immune complexes escape through permeant blood vessels, activate complement, neutrophils and monocytes, and thereby serve as the primary drivers of inflammatory-mediated organ damage¹³⁷.

All-cause SLE mortality has not improved in the past two decades and remains twofold higher than that in the general population^138,139, with infection and cardiovascular disease now the most common causes of mortality^140,141. Novel therapies are needed to decrease the use of corticosteroids, induce remission and mitigate end organ damage. S1PR1 modulators could be of particular benefit in SLE because, in addition to their effect on lymphocyte trafficking, they protect the vasculature and reduce the production of type I interferons (see section IFNα production in SLE as target of S1P–S1PR1 modulators).

Evidence that S1P levels are dysregulated in SLE.

S1P levels in SLE have been assessed in small studies^142-144, with two showing that patients had elevated S1P that did not correlate with disease activity and another showing that the ceramide:S1P ratios significantly decreased after treatment (n = 22)¹⁴⁴, suggesting that a drop in ceramide and/or an increase in S1P was associated with an improvement in SLE. The differing results of these studies could be the result of technical differences in blood drawing and handling, as platelet activation and haemolysis contribute to S1P levels. S1P in serum is higher than in plasma because of the contribution of platelet and erythrocyte S1P; thus, sera levels might not accurately reflect circulating S1P levels. Larger longitudinal studies using plasma rather than serum from patients before and after successful treatment could help to determine whether S1P is a useful biomarker in SLE. It is also unknown whether dysregulated S1P levels contribute to aberrant lymphocyte trafficking and lymphopenia in SLE.

Evidence that ApoM is dysregulated in SLE.

Circulating ApoM levels were reduced in patients with SLE and were inversely correlated with SLE disease activity index (SLEDAI) scores in two studies^145,146. In the first study, two groups of patient samples were assessed. In group I (n = 84), bio-banked samples and the corresponding SLEDAI-2K score obtained on the day of collection were evaluated. Group II (n = 140) consisted of patients with SLE participating in studies related to cardiovascular disease over a 2-year period. In group I, ApoM concentrations were significantly lower in patients with active renal disease, skin involvement, leukopenia or anti-dsDNA antibodies than in patients with SLE without these disease manifestations. Endothelial function using a finger plethysmograph (a device measuring arterial blood flow) (EndoPAT, Itamar Medical, Israel) was assessed in patient group II. A low Reactive Hyperemia Index value on EndoPAT indicates endothelial dysfunction¹⁴⁷. In support of a vascular protective effect of ApoM, there was an association between Reactive Hyperemia Index and ApoM levels in patients with SLE of 20–45 years of age (n = 60) that remained after adjustment for cardiovascular disease risk factors, medications and SLE disease activity.

In the second study, ApoM levels were compared between individuals newly diagnosed with SLE and age-matched and sex-matched controls (n = 52 per group)¹⁴⁵. Because ApoM is produced mainly in the liver and kidney, patients with liver or kidney disease were excluded, as were patients taking steroids or other immunosuppressive agents. ApoM levels were significantly lower in patients with SLE than those in healthy controls and correlated negatively with the levels of SLEDAI scores and the presence of anti-dsDNA antibodies.

Why ApoM levels would drop during periods of increased disease activity is not clear, but data from both humans and mice demonstrate that ApoM expression is downregulated during the acute phase response induced by infection and inflammation¹⁴⁸. An interesting and yet unanswered question is whether ApoM undergoes post-translational modifications during chronic inflammation that impair its vascular protective effects. Preclinical studies are needed to determine whether S1P–ApoM delivery attenuates disease activity in mouse models of SLE.

IFNα production in SLE as a target of S1P–S1PR1I modulators.

Dysregulation of type I interferon production is a cornerstone of SLE pathophysiology^149-152, and type I interferons, as well as their major producers, plasmacytoid dendritic cells (pDCs), are now targets for therapy in SLE. Understanding how S1P–S1PR1 signalling affects type I interferon production in response to viral infection could reveal mechanistic insights and targets for the treatment of SLE, as viral nucleic acids and human nucleic acids contained in immune complexes and/or NETs in SLE use common molecular pathways to promote the production of type I interferons.

The S1PR1 and S1PR5 agonist CYM-5442 was shown to attenuate IFNα production in pDCs challenged either with influenza virus or CpG-A (a synthetic nucleotide that activates Toll-like receptor 9 (TLR9)). Mechanistically, CYM-5442 induced co-internalization and degradation of S1PR1 and the type I interferon receptor interferon α/β receptor 1 (IFNAR), thereby impeding the ability of IFNα to turn on an autocrine feedback loop and attenuating production of IFNα¹⁰⁸. CYM-5442 and other S1PR1 agonists have also been shown to attenuate production of type I interferons and mortality in response to viral infection with influenza A H1N1 in vivo^5,153. Interestingly, these studies demonstrated that EC S1PR1 signalling reduces the production of type I interferons and other key mediators of inflammatory injury including CCL2 and CXC-chemokine ligand 10 (CXCL10), implicating ECs as orchestrators of inflammatory injury in this model. Because NETs and immune complexes from patients with SLE have also been demonstrated to induce production of type-I interferons by pDCs^154,155 and ECs in a nucleic acid-dependent and endosomal TLR-dependent manner, studies to test whether S1PR1 agonists attenuate NET-driven type I interferon production would help to elucidate whether S1PR1 agonists have the potential to have immunomodulatory roles beyond lymphocyte trafficking in SLE.

Taken together, these studies suggest that S1PR1 modulators could attenuate type I interferon production by pDCs and/or ECs in SLE (FIG. 3). However, preclinical studies investigating a modulatory role of S1PR1 agonists on IFNα production have yielded mixed results. In the NZBWF1 mouse model of SLE, ozanimod attenuated proteinuria and inflammatory renal injury (as assessed by histology) and reduced mortality but did not affect the interfern signature or decrease IFNAR expression on pDCs¹⁵⁶. By contrast, cenerimod, another S1PR1 and S1PR5 agonist, reduced type I interferon levels in both plasma and brain of MRL/lpr mice¹⁵⁷. It is not known whether distinct signalling properties of these S1PR1 modulators might account for these differences and/or whether one mouse model of SLE is more responsive than others to S1PR1 modulation of IFNα.

Fig. 3 ∣. Potential mechanisms by which S1PR1 modulators could mitigate disease pathogenesis.

Small-molecule sphingosine 1-phosphate receptor 1 (S1PR1) modulators could ameliorate disease in systemic lupus erythematosus (SLE) by decreasing the trafficking of autoreactive lymphocytes, increasing numbers and functions of regulatory T (T_reg) cells, decreasing T helper 17 (T_H17) cell differentiation and decreasing autoantibody production. Moreover, S1PR1 modulators could attenuate disease by increasing endothelial cell barrier function, increasing the blood–brain barrier, and decreasing the expression of inflammatory adhesion molecules, which would contribute to decreased leukocyte transmigration. S1PR1 modulators have also been shown to decrease type I interferons in response to viral infection or CpG oligonucleotides, and have the potential to attenuate type I interferons in SLE. These mechanisms could impart protection against renal, neuropsychiatric and atherosclerotic manifestations of SLE. EC, endothelial cell.

Ameliorative effects of FTY720 in preclinical studies of SLE.

The MRL/lpr model of SLE shares many aspects of human disease, including anti-dsDNA antibodies, renal deposition of IgG antibodies and complement, and proteinuria¹⁵⁸. MRL/lpr mice have a deletion in Fas causing failure of apoptotic cell death in CD4⁺ T cells, leading to the persistence of pathogenetic autoreactive T_H cells and the subsequent production of autoantibodies¹⁵⁹. As a result, these mice develop lymphadenopathy, splenomegaly and spontaneous autoimmune glomerulonephritis. There is evidence that FTY720 treatment markedly attenuates pathogenesis and mortality in the MRL/lpr model of SLE^90,160 (TABLE 1). If the major effect of FTY720 was to halt lymphocyte trafficking out of SLOs, one might expect increased lymphadenopathy and splenomegaly in MPL/lpr mice. However, FTY720-treated MRL/lpr mice showed significantly reduced weights of SLOs and decreased numbers of T cells and B cells in the thymus⁹¹, suggesting that FTY720 induced lymphocyte apoptosis. In support of this hypothesis, FTY720 did induce lymphocyte apoptosis in vitro, and FTY720-treated mice showed increased numbers of apoptotic cells in SLOs⁹⁰, implicating lymphocyte apoptosis as a major mechanism contributing to its beneficial effects. FTY720 also significantly suppressed the production of anti-dsDNA antibodies and reduced the deposition of IgG in glomeruli in mice that had already developed renal disease, indicating its therapeutic potential in SLE¹⁶⁰.

Table 1 ∣.

Preclinical studies using S1PR1 modulators or SPHK inhibitors in mouse models of SLE

SLE model	S1PR modulator	Target	Outcome	Interferon signature	Refs
MRL-lpr	FTY720	S1PR1, S1PR3, S1PR4 and S1PR5	↓Glomerulonephritis and mortality ↓Neuropsychiatric disease, blood–brain barrier permeability, brain tissue cytokines	NA	85,90,161
	Amiselimod	S1PR1, S1PR4 and S1PR5	↓Glomerulonephritis	NA	87
	Cenerimod	S1PR1 and S1PR5	↓Glomerulonephritis, ↓brain tissue cytokines and mortality	Reduced	157
	KRP-203	S1PR1 and S1PR4	↓Glomerulonephritis and mortality	NA	86
NZBWF1	Ozanimod	S1PR1 and S1PR5	↓Glomerulonephritis and mortality	Unchanged	156
	Amiselimod	S1PR1, S1PR4 and S1PR5	↓Glomerulonephritis	NA	87
BXSB	FTY720	S1PR1, S1PR3, S1PR4 and S1PR5	↓Glomerulonephritis and mortality	NA	163

NA, not assessed; S1P, sphingosine 1-phosphate; S1PR, S1P receptor; SLE, systemic lupus erythematosus.

Neuropsychiatric features in MRL/lpr mice^85,161 have some similarities with human disease, and FTY720 has been shown to decrease depression-like behaviour, spatial and recognition memory deficits, and leukocyte infiltration of the choroid plexus⁸⁵. Mechanistically, FTY720 reduced neuron damage, attenuated the production of inflammatory cytokines in the brain and protected the blood–brain barrier by directly affecting brain microvascular ECs. The brain EC transcriptome showed marked decreases in the expression of genes involved in cellular adhesion and in the interferon response⁸⁵. As in the aforementioned studies, FTY720 treatment was associated with reduced splenomegaly and lymphadenopathy⁸⁵. These studies support the need for further work to evaluate the role of FTY720 and/or other S1PR1 modulators as potential therapies for neuropsychiatric SLE.

Finally, in another model of SLE, male mice carry the Yaa mutation inducing overexpression of TLR7, resulting in accelerated autoimmunity and glomerulonephritis¹⁶²; in this model, FTY720 protected against proteinuria, which occurred in 20% of treated animals compared with 100% of untreated controls, and lowered mortality¹⁶³.

Taken together, these data support the idea that fingolimod could benefit patients with SLE, particularly those with glomerulonephritis. However, FTY720 has several known adverse effects, including bradycardia, bronchoconstriction and macular oedema. As some of the safety issues related to use of FTY720 are associated with its engagement of S1PR3, newer S1PR modulators with increased specificity for S1PR1 are expected to have fewer adverse effects¹⁶⁴.

Newer S1PR modulators in preclinical SLE.

KRP-203 is an S1PR modulator with specificity to S1PR1 and S1PR4. MRL/lpr mice preventively and therapeutically treated with KRP-203 had increased survival⁸⁶. KRP-203 treatment resulted in a marked decrease in both peripheral lymphocytes and monocytes, attenuated glomerulonephritis, reduced interstitial and perivascular infiltrates, and decreased proteinuria. However, IgG and complement deposition were not decreased. Despite initial adenopathy in MRL/lpr mice treated with KRP-203, there was ultimately a reduction in SLO weights as well as a decrease in the absolute number of lymphocytes in these organs. Again, lymphocyte apoptosis was deemed most likely to account for these findings⁸⁶.

Amiselimod, a S1PR modulator with specificity to S1PR1, was shown to decrease anti-dsDNA antibody titres, the number of plasma cells and the development of glomerulonephritis, splenomegaly and lymphadenopathy in the MRL/lpr model when given preventively and therapeutically⁸⁷. Amiselimod also prevented progression of nephritis in NZBWF1 mice. As expected, the treatment was shown to induce lymphopenia⁸⁷.

Cenerimod, a S1PR modulator with specificity to S1PR1 and S1PR5, leads to a 500-fold decrease in potency in Ca⁺⁺ signalling (mediated by Gi–phospholipase C–inositol trisphosphate) compared with S1P, FTY720 and amiselimod, thereby reducing its ability to induce bronchoconstriction ex vivo¹⁶⁵. Cenerimod attenuated renal disease and prolonged survival in the MRL/lpr model; treated animals showed decreased circulating blood CD19⁺ B cells and CD4⁺ and CD8⁺ T cells; reduced plasma anti-dsDNA antibodies, proteinuria and brain inflammation; and increased survival. Plasma and tissue levels of IFNα, and other known mediators of SLE, including TNF, IL-6, BAFF (also known as tumour necrosis factor ligand superfamily member 13B) and IL-10, were also reduced¹⁵⁷.

Ozanimod was approved by the US FDA for the treatment of multiple sclerosis in 2020 and ulcerative colitis in 2021. It has specificity for S1PR1 and S1PR5, and its active metabolite, RP-101075, decreased proteinuria, renal histological scores and mortality in NZBWF1 mice. RP-101075 treatment suppressed expression of profibrotic and inflammatory genes but did not significantly alter the expression of interferon-inducible genes¹⁵⁶. Both ozanimod and RP-101075 significantly reduced peripheral and splenic B cell subsets, but neither treatment was associated with a sustained drop in anti-dsDNA antibodies¹⁵⁶.

In summary, multiple S1PR modulators have been shown to attenuate experimental SLE. Cenerimod is particularly promising because of its defined ability to attenuate type I interferon production as well as its unique signalling profile that could mitigate the risk of bronchoconstriction. As discussed in the next section, cenerimod is currently being evaluated in a phase II clinical trial for SLE.

Clinical trials of S1PR1 modulators in human SLE.

Amiselimod has a more favourable cardiac safely profile than fingolimod¹⁶⁶. In an open label, non-randomized multicentre phase Ib safety trial, during which immunosuppressive and antimalarial drugs were prohibited, patients were treated with either 0.2 or 0.4 mg of amiselimod for 24 weeks¹⁶⁷. In a second part of the trial, low-dose 0.2-mg amiselimod was administered with concomitant submaximal doses of immunosuppressive agents. Individuals with severe active lupus nephritis, CNS lupus, thrombosis, lymphopenia or evidence of cardiovascular disease were excluded. A decrease in peripheral lymphocyte count was observed in all 17 patients treated with active drug without a dose-dependent response, but no severe infections occurred, and there were no serious adverse events. Of the 9 out of 17 individuals (53%) who were positive for anti-dsDNA antibodies at baseline, 8 (89%) had decreased antibody levels by week 24. Although this exploratory study showed that amiselimod was well tolerated, its half-life was determined to be 20–23 days, which could limit its clinical use.

A multicentre, double-blind, placebo-controlled study was conducted to test the safety of cenerimod: patients with SLE were randomized to receive oral cenerimod (0.5, 1, 2 mg or placebo) for 12 weeks¹⁵⁷. After an interim safety review, additional individuals were randomized to receive either 4 mg cenerimod or placebo. End points included changes in total lymphocyte count, SLEDAI-2K (modified to exclude lymphopenia) and anti-dsDNA antibodies, pharmacokinetic assessments and adverse events. 1- and 2-mg doses induced a drop in the levels of CD19⁺ B cells and CD4⁺ and CD8⁺ T cells. Cenerimod attenuated plasma IFNα and downstream mediators CXCL9, CXCL10 and galectin 9 in humans with SLE after 12 weeks of treatment¹⁵⁷.

An unbiased analysis of circulating B cells revealed two specific cell clusters that were reduced after treatment with cenerimod. Interestingly, these clusters demonstrated elevated expression of CD27 antigen and CD38 antigen (also known as ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1), resembling antibody-secreting cells, which have previously been identified as key mediators of autoimmunity¹⁵⁷. Plasma interferon-associated biomarkers were decreased in the majority of patients taking cenerimod. A larger study of cenerimod treatment for up to 12 months is currently ongoing (NCT03742037).

Rheumatoid arthritis

RA is the most prevalent systemic autoimmune rheumatic disease, affecting 1.5 million individuals in the USA, characterized by inflammatory and often erosive arthritis and the presence of autoantibodies against IgG (rheumatoid factor) and anti-citrullinated peptides antibodies¹⁶⁸. Despite multiple FDA-approved medications for the treatment of RA, treatment-resistant RA occurs in 6–17% of patients¹⁶⁹, and many patients do not have sustained remission. RA-related inflammation increases the risk of cardiovascular disease^170,171, with patients having a 2–3-fold higher risk of myocardial infarction and a 50% higher risk of stroke than healthy controls^172,173. Thus, there is a real medical need for novel therapies that can be used safely and concomitantly with approved medications to ameliorate the quality of life and lower mortality in patients with RA. The mechanisms by which S1PR1 modulators could ameliorate RA include blockade of auto-aggressive T cells to synovial tissues, restoration of the T_reg–T_H17 cell imbalance and bolstering of the vascular barrier to attenuate both microvascular¹²¹ and atherosclerotic disease^174,175.

Evidence that S1P levels are dysregulated in human RA and experimental arthritis.

S1P levels have been shown to be elevated in RA synovial fluids compared with osteoarthritis synovial fluids¹⁷⁶, which could result from S1P release from activated platelets, fibroblasts and leukocytes^176-179 (FIG. 4). S1P has many pro-inflammatory effects in RA, including its ability to induce synoviocyte proliferation¹⁸⁰ and synoviocyte production of prostaglandin E2 (PGE₂) and cyclooxygenase 2 (COX2, also known as prostaglandin G/H synthase 2) in response to inflammatory cytokines in vitro¹⁷⁶. S1P hinders apoptosis of fibroblast-like synoviocytes via S1PR1 (REF.¹⁸¹) and increases receptor activator of nuclear factor κB ligand (RANKL, also known as tumour necrosis factor ligand superfamily member 11) expression on synoviocytes and CD4⁺ T cells, thereby promoting osteoclastogenesis¹⁸¹, which could contribute to bone erosions. RANKL signalling downregulates the expression of S1PR1 on osteoclast precursors, inhibiting their re-exit into the circulation, and enhances osteoclast maturation and bone resorption¹⁸². Because lymphocyte S1PR1 expression and the local S1P level are inversely related, elevated S1P levels in synovial tissues decrease T cell expression of S1PR1, thereby increasing T cell retention time in tissues^183,184, potentially exacerbating disease.

Fig. 4 ∣. Contribution of S1P to inflammatory injury and joint destruction in RA.

Elevated sphingosine 1-phosphate (S1P) levels in rheumatoid arthritis (RA) synovium (from multiple sources, including platelets) could contribute to disease pathogenesis by increasing osteoclast precursor retention and bone resorption, and increasing synoviocyte proliferation and production of prostaglandin E2 (PGE₂), tumour necrosis factor (TNF) and IL-1β.The effect of increased S1P receptor 1 (S1PR1) signalling on angiogenesis in the RA synovium remains to be elucidated.

Consistent with the findings that S1P contributes to RA pathogenesis, decreasing S1P levels by pharmacological or small interfering RNA knockdown of Sphk1 was shown to reduce the production of key mediators of inflammatory arthritis, including TNF and IL-1β, and attenuate collagen-induced arthritis^185,186. Moreover, crossing of TNF transgenic mice, which develop spontaneous arthritis, and Sphk1 KO mice protected offspring from developing the disease¹⁸⁷. Mechanistically, the SphK1 KO mice had reduced levels of articular COX2 and fewer synovial T_H17 cells than did transgenic TNF–SphK1^+/+ littermates. In another study implicating SPHK1 as a contributor to RA pathogenesis, SPHK1-mediated production of S1P inhibited Fas-mediated apoptosis of lymphoblastoid cell lines prepared from peripheral B cells cultured from patients with RA¹⁸⁸. However, in other studies, Sphk1 KO mice did not show decreased severity in collagen-induced arthritis¹⁸⁹. These apparently discrepant results could be secondary to differences in the genetic strains and the models of arthritis utilized for these studies.

Whereas S1P generation by SPHK1 has been linked to inflammatory signalling via activating S1PR1 signalling, the role of SPHK2 is unclear, with conflicting results regarding SPHK2 roles in inflammation. Although the differences in genetic strains and arthritis models used for these studies could account for these discrepant results, added complexity stems from the fact that Sphk1 activity is increased when Sphk2 is knocked out^186,190-193

Evidence that APOM polymorphisms are risk factors for RA.

Polymorphisms in the APOM promoter that result in reduced ApoM levels are associated with an increased risk of RA. Polymorphisms at SNP rs805297 have been shown to decrease APOM transcriptional activity and be significantly associated with RA (OR = 1.56)¹⁹⁴. In a study from Korea, this polymorphism was found to be associated with both RA susceptibility and dyslipidaemia, and the A/A genotype was associated with both decreased levels of HDL particles and ApoM but not disease activity¹⁹⁵. A study from China of patients with RA (n = 520) showed an association of APOM rs805297 TT genotype with an increased risk of developing RA, with an odds ratio of 4.01 among men. In a sub-analysis of 84 patients, the average plasma concentration of ApoM was significantly higher in patients with RA than in age-matched and sex-matched controls¹⁹⁶. Given the protective role of ApoM on the vasculature and the adaptive immune response, the elevated levels in RA are likely to be the consequence of inflammation.

Whether certain APOM polymorphisms predispose to disease or are rather the result of linkage disequilibrium is yet to be elucidated. On the one hand, linkage disequilibrium is supported by the fact that APOM is located in the MHC class III region along with TNF. On the other hand, patients with RA have a more atherogenic lipid profile than healthy controls, even in the preclinical non-inflammatory phase¹⁹⁷, raising the possibility that dyslipidaemia contributes to the risk of developing RA. As stated previously, ApoM has been implicated in adipocyte and triglyceride biology, which could affect autoimmune mechanisms. Indeed, anti-inflammatory roles of S1P–ApoM signalling include its ability to decrease ICAM1, VCAM1 and E-selectin expression on ECs in the presence of inflammatory cytokines^2,3 and to restrain lymphopoiesis and adaptive immune responses, which could predispose to the development of RA⁶⁷.

Ameliorative effects of FTY720 in preclinical studies of RA.

As observed in SLE, there is evidence that fingolimod attenuates disease severity in experimental inflammatory arthritis. Supporting the concept that blocking the egress of autoreactive lymphocytes out of SLOs is a major mechanism by which FTY720 ameliorates disease, the ability of FTY720 to attenuate arthritis in both collagen-induced and adjuvant induced arthritis models was associated with reduced numbers of circulating and synovial CD4⁺ T cells^198-202. SKG mice, another RA model, spontaneously develop T cell-mediated autoimmune arthritis owing to a mutation in Zap70, which impairs negative selection of T cells in the thymus²⁰³. FTY720 treatment in this model resulted in sequestration of CD4⁺ T cells in the thymus, reduced numbers of circulating CD4⁺ T cells and attenuated arthritis²⁰⁴. In addition, FTY720 has been shown to inhibit TNF-induced synoviocyte production of PGE₂ (REFS^108,204), IL-1β, IL-6 and IL-8 (REF.²⁰⁰). Consistent with the aforementioned studies showing that S1pr1 KO in CD4⁺ T cells led to increased numbers and function of T_reg cells, FTY720, acting as a functional antagonist of S1PR1, attenuated collagen induced arthritis by increasing T_reg cell numbers and their ability to induce anergy in collagen-specific T cells¹⁰⁸.

DCs express S1PR1 and are also affected by FTY720 and other S1PR1 modulators²⁰⁵. In a study focused on the role of FTY720 in DC trafficking and function in collagen-induced arthritis, FTY720 attenuated production of several cytokines, including TNF, IL-1β and Il-6, as well as the incidence and severity of arthritis⁹². To determine whether the ability of FTY720 to attenuate arthritis was mediated, at least in part, by an effect on DCs, a DC transfer mouse model of arthritis was used. In this model, murine bone marrow-derived DCs are activated with lipopolysaccharide and collagen in vitro and injected into the paw, causing localized arthritis. Ex vivo treatment of DCs with FTY720 before injection into the paw significantly reduced DC trafficking to draining LNs and attenuated arthritis, demonstrating that DCs are an important target of FTY720 (REF.⁹²).

S1PR1 antagonists in preclinical RA.

Because of their ability to block lymphocyte trafficking, S1PR1 antagonists have been tested for their efficacy in preclinical models of arthritis²⁰⁶. However, although S1PR1 antagonism, induced by either functional or competitive antagonists, mitigates trafficking of central memory T cells and naive T cells to tissues, it also blocks S1PR1 signalling on ECs, leading to vascular leakage. In a study on adjuvant induced arthritis, the S1PR1-specific antagonist NIBR-0213 was effective at preventing arthritis, but it induced diffuse alveolar and interstitial haemorrhage, oedema and inflammation. In mice treated with NIBR-0213 for over 2 weeks, chronic changes were observed, including necrotic and fibrotic foci in both the lung and the heart²⁰⁷. These data suggest that the use of S1PR1 functional antagonists that induce endothelial injury might pose risks for patients with RA, because clinically significant interstitial lung disease occurs in ~10%^208-210 of these patients and subclinical disease occurs in up to 60%, as detected by high-resolution CT²⁰⁹.

Clinical trials targeting S1P–S1PR1 axis in RA.

Although fingolimod has not yet been tested for RA treatment in clinical trials, a different strategy to block S1PR1 signalling and induce lymphopenia has been used in phase I and II clinical trials. Inhibition of SGPL1 resulted in >100-fold accumulation of S1P in thymus and SLOs, thereby eliminating the S1P gradient and inducing lymphopenia⁶. The SGPL1 inhibitor LX2931 (REF.²¹¹) was tested in phase II clinical trials on 208 patients with active RA who had incomplete response to methotrexate. There were no safety issues, but analysis of the results suggested that the doses tested were subtherapeutic. No further studies using this agent have been reported, as the study failed to meet its primary end point of ACR20 response at week 12 (REF.²¹²). This approach as a treatment for RA is not supported by preclinical data, as mice with Sgpl1 KO have neutrophilia and increased levels of inflammatory cytokines, particularly IL-17 (REF.²¹³).

Systemic sclerosis

SSc is a rare complex systemic autoimmune rheumatic disease characterized by vasculopathy and fibrosis, mostly of skin, gastrointestinal tract and lungs. Symptomatic interstitial lung disease is present in >50% of patients with diffuse disease and in up to 35% of individuals with limited cutaneous SSc, and subclinical interstitial lung disease is seen on high-resolution CT in up to 90% of patients with SSc^214,215. Data from the Scleroderma Lung Study II have shown that the mortality from interstitial lung disease is ~20% with a median follow-up of 3.6 years after the diagnosis of interstitial lung disease^216,217. Pulmonary hypertension, either in its primary form or secondary, to severe interstitial lung disease occurs in 8–12% of patients, with 25% mortality at 3 years after pulmonary hypertension diagnosis²¹⁸. Although there has been improvement in mortality over the past few decades^216,219, novel therapies are direly needed to decrease morbidity and mortality in SSc.

The use of S1PR1 modulators could have beneficial effects in SSc. For example, S1PR1 modulators decrease type I interferon production by pDCs²²⁰ and thereby have the potential to decrease the upregulated production of type I interferons in SSc^221-224. IFNα could contribute to SSc pathogenesis by increasing blood vessel permeability via downregulation of FLI1 (encoding Friend leukaemia integration 1 transcription factor) and CDH5 (encoding VE-cadherin)²²⁵ and by contributing to skin fibrosis, as has been shown in mouse studies²²⁶. S1PR1 modulators (acting as functional antagonists) have also been shown to diminish differentiation and function of T_H17 cells, both of which are increased in patients with SSc^227-229 and contribute to fibrosis in animal models of SSc^230,231. However, in terms of restoring the T_H17–T_reg cell imbalance, S1PR1 modulation could be problematic in SSc, as T_reg cells have the potential to exacerbate pulmonary fibrosis by increasing T_H2 cell immune responses²³². Moreover, S1PR1 antagonism, by treatment with either competitive or functional antagonists, can increase the risk of pulmonary vascular leakage.

S1PR1 modulation to target vascular leakage and fibrosis in SSc.

Bolstering EC S1PR1 signalling limits vascular leakage and decreases vascular tone²³³ and therefore could be of particular benefit in SSc, because vascular leakage is a predominant clinical feature^234,235, and almost all patients have Raynaud phenomenon as a result of vascular dysfunction. The vasculopathy of SSc is characterized by increased vascular permeability, decreased VE-cadherin expression and dysregulated angiogenesis²³⁶. These features are prominent in mice with an EC-specific KO of Fli1, a transcription factor that is markedly decreased in SSc skin and is a negative regulator of collagen production²³⁷. Isolated dermal ECs from these mice show markedly decreased expression of S1pr1 and VE-cadherin. Chromatin immunoprecipitation experiments showed that the promoter of S1pr1 is targeted by Fli1 (REF.²³⁷). Taken together, these data support the hypothesis that low levels of Fli1 in SSc ECs lead to decreased transcription of S1PR1 and CDH5 (VE-cadherin), leading to increased vascular instability and leakage.

S1PR1 modulation has also been studied in mouse models of pulmonary fibrosis. Chronic administration of FTY720 or an S1PR1-specific functional antagonist exacerbated vascular leakage and intra-alveolar coagulation in response to bleomycin, leading to increased pulmonary fibrosis and mortality in bleomycin-treated mice¹²⁴. In in vitro studies, short-term (1-h) EC exposure to S1P or S1PR1 agonists decreased permeability and protected against thrombin-mediated barrier disruption, whereas prolonged (24-h) exposure to S1PR1 agonists exerted the opposite effect¹²⁴, illustrating that the dose and the frequency of administration of a S1PR1 modulator will determine whether it acts as an agonist or as an antagonist (FIG. 5). In follow-up studies on pulmonary fibrosis induced by bleomycin and FTY720, fibrosis was attenuated by inhibiting thrombin generation, thereby showing that extravascular coagulation induced by vascular leak is a driver of fibrosis. Mechanistically, thrombin binding to protease activated receptor 1 (PAR1) increased activation of transforming growth factor-β1 (TGFβ1) via avβ6 integrin²³⁸. Importantly, a study supports the hypothesis that EC S1PR1 protects against pulmonary fibrosis by mitigating vascular leakage, leukocyte infiltration, and intravascular coagulation²³⁹. These data support the development of novel EC S1PR1 targeted agonists for the treatment of SSc.

Fig. 5 ∣. S1PR1 modulators act as agonists and/or functional antagonists depending on dose and target cell type.

Sphingosine 1-phosphate (S1P) receptor 1 (S1PR1) modulators have differing effects on endothelial cells (ECs) and lymphocytes. After binding of S1PR modulators to S1PR1, signalling is activated and the S1PR1 modulator–S1PR1 complex can be internalized and degraded in proteasomes and lysosomes. S1PR1-targeted small molecule functional antagonists at therapeutic concentrations induce irreversible internalization and degradation of S1PR1, resulting in lymphopenia. At such doses, endothelial S1PR1 antagonism does not lead to substantial vascular leak, owing to the presence of large receptor reserve. However, at supratherapeutic doses, S1PR1 functional antagonists induce degradation of S1PR1 in both lymphocytes and endothelial cells, leading to lymphopenia as well as vascular leakage.

S1PR modulators in mouse models of SSc.

Cenerimod attenuated the infiltration of immune cells and inflammatory cytokines IL-6, IL-13 and Il-1β in a mouse model of chronic graft-versus-host disease (Scl-cGVHD)²⁴⁰. Consistent with previous data that S1PR1 is a negative regulator of T_reg cell differentiation, cenerimod (functioning as an antagonist on CD4⁺ T cells) increased the percentage of T_reg cells both in the spleen and skin compared with control mice²⁴⁰. Moreover, cenerimod decreased IL-6 and collagen deposition in both skin and lung of the bleomycin-induced fibrosis model²⁴⁰. Experiments to assess vascular permeability were not performed in these studies, but the protective effects of cenerimod were possibly related, at least in part, to agonist signalling on ECs²⁴⁰. In addition, because S1PR5 was implicated in the early fibrotic changes in the skin in the bleomycin model of SSc²⁴¹, one could speculate that cenerimod’s anti-fibrotic effects may have been mediated, at least in part, by modulating S1PR5 signalling. Thus, multiple S1PRs could be involved in the complex pathophysiology of SSc by acting on immune, vascular and connective tissue cells. Such complex biology needs to be understood much better to develop rational therapeutic approaches.

Future directions and challenges

The application of S1PR modulators to inhibit autoreactive lymphocyte trafficking has proven successful in patients with multiple sclerosis and inflammatory bowel disease. However, the complex biology of S1P has hindered the wider application of this therapeutic principle in additional immunological diseases. Further complicating the application of S1PR1 modulators to autoimmune rheumatic diseases, S1PR1 signalling can be enhanced or antagonized by the same drug depending on the concentration of the drug, the duration of treatment and the type of cell targeted (FIG. 5).

It is well established that patients with SLE, RA and SSc show substantial endothelial dysfunction^{169,234,235,242-244}. Although preclinical studies support the use of S1PR1 modulators for SLE, RA and SSc, vascular S1PRs have received scant attention in past therapeutic efforts. The discovery that HDL-bound S1P protects the vascular endothelium during inflammatory insult has provided impetus for therapeutic development. Newer S1PR1 agonists do not induce internalization and degradation of S1PR1 and do not affect lymphocyte percentages in peripheral blood¹⁶¹. We hypothesize that S1PR1 agonists specifically targeting ECs could benefit patients with autoimmune rheumatic disease on standard therapies, because bolstering vascular integrity might synergize with immunosuppressive therapy. A potential benefit of the use of concomitant immunosuppressive and endothelial barrier-targeted drugs is that, although the anti-inflammatory effects of both drugs could be compounded, the immunosuppressive effects are not likely to be, as EC S1PR1-bolstering drugs should attenuate the risk of infection^4,5,153. An optimal approach will be to reset the immune cell repertoire while maintaining optimal EC function. We posit that an in-depth understanding of S1P biology is an essential prerequisite in the rational therapeutic development of novel agents to control SLE, RA and SSc.

Key points.

• Autoimmune rheumatic diseases are complex and heterogeneous diseases that have fundamental pathophysiological pathways in common.

• These dysregulated pathways include lymphocyte autoreactivity, myeloid and endothelial cell activation, and extravasation of inflammatory mediators into tissues.

• Chronic inflammatory damage in autoimmune rheumatic disease leads to end organ damage.

• Sphingosine 1-phosphate receptor 1 (S1PR1) is expressed on leukocytes and endothelial cells and is an important mediator of lymphocyte trafficking, regulatory T/T helper 17 cell homeostasis and vascular permeability.

• Use of S1PR1 modulators in preclinical studies of systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis has shown promise in attenuating inflammatory injury and end organ damage.

• Modulation of S1PR1 signalling in leukocytes and/or endothelial cells warrants further evaluation in clinical studies in autoimmune rheumatic diseases.