Sphingosine-1-phosphate receptors and innate immunity

Summary

Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates many cellular processes in mammals. One well-studied role of S1P signaling is to modulate T- cell trafficking, which has a major impact on adaptive immunity. Compounds that target S1P signaling pathways are of interest for immune system modulation. Recent studies suggest that S1P signaling regulates many more cell types and processes than previously appreciated. This review will summarize current understanding of S1P signaling, focusing on recent novel findings in the roles of S1P receptors in innate immunity.

Introduction

Sphingosine-1-phosphate (S1P) is a signaling lipid which is an important regulator in inflammation, angiogenesis, vascular permeability, brain and cardiac development and cancer growth and metastasis (Reviewed in: N. J. Pyne et al., 2012, 2016; N. Pyne & Pyne, 2017; S. Pyne, Adams, & Pyne, 2016). S1P is able to affect many processes by signaling extracellularly through S1P receptors (S1PRs). One well studied role of S1P receptor signaling is the modulation of T- cell trafficking, which has a major impact on adaptive immunity (Mandala et al., 2002; Matloubian et al., 2004). Compounds that target S1P receptors are of interest for treatment of autoimmune diseases, the first of these compounds being FTY720 (fingolimod), which was approved in 2010 as a first line treatment for relapsing forms of multiple sclerosis (Brinkmann et al., 2010). Recent studies suggest that S1P signaling regulates many more cell types and processes than previously appreciated, including cells of the innate immune system (Reviewed in: Blaho & Hla, 2017; Garris, Blaho, Hla, & Han, 2014). The innate immune system is the first line of defense against infectious diseases. Understanding the interplay between S1P receptor signaling and the innate immune response is essential, not only to our understanding of S1P biology, but also to design better therapeutics. This review will briefly summarize current understanding of S1P signaling, focusing on recent novel findings in the roles of S1P receptors in innate immunity.

S1P Pathway Overview

The S1P pathway in mammalian systems has been studied in depth (Reviewed in: Mendelson, Evans, & Hla, 2014; N. Pyne & Pyne, 2017; Rosen & Goetzl, 2005; Strub, Maceyka, Hait, Milstien, & Spiegel, 2010) (Figure 1). S1P is produced by the phosphorylation of sphingosine by one of two sphingosine kinases (SK1 and SK2) (Wattenberg, Pitson, & Raben, 2006). S1P levels are regulated by S1P phosphatases which remove the phosphate group or S1P lyase which irreversibly degrades S1P, the final step for sphingolipid degradation (Johnson et al., 2003; Schwab et al., 2005; Reviewed in: Serra & Saba, 2010). Once phosphorylated, S1P is recognized by a family of G-protein coupled receptors (S1PR1–5), that affect downstream effectors (discussed in later sections) (Rivera, Proia, & Olivera, 2008). The distribution of the receptors on different cell types and the coupling of receptors to different G alpha subunits allow S1P to differentially exert its influence in numerous pathways (Figure 1, Table 1) (Reviewed in: Blaho & Hla, 2017). S1P can also signal intracellularly both dependent and independent of extracellular S1P receptors utilizing several different proposed mechanisms (M. M. Adada et al., 2015; Canals et al., 2010; Park et al., 2016; Usatyuk et al., 2011).

Figure 1. Sphingosine-1-Phosphate Signaling Pathways.

Sphingosine is produced from the sphingolipid pathway by the action of ceramidase on ceramide. Sphingosine-1-phosphate (S1P) is produced by phosphorylation of sphingosine by Sphingosine Kinases 1 or 2. S1P can be converted back to sphingosine by S1P phosphatases or irreversibly degraded by S1P lyase. S1P binds to S1P receptors 1–5 (S1PR1–5), which are coupled to G proteins that affect cellular pathways. Shown in this figure are proposed downstream pathways of the G alpha subunits G_i, G_12/13, and G_q which interact with the S1PRs. These pathways may not be active in all cells that express any given S1PR at the same time, but is shown for illustration purposes. New potential mechanisms discussed in this review are outlined in red.

Abbreviations: Ras: Ras family small GTPase, ERK: Extracellular Receptor Kinase, PI3K: PI3-kinase, Akt: protein kinase B, eNOS: Endothelial Nitric Oxide Synthase, PLC: Phospholipase C, Rac: Rac family small GTPase, Rho: Rho family of small GTPases, ERM: Ezrin-Radixin-Moesin, Nox2: NADPH oxidase, PTEN: Phosphatase and tensin homolog

Table 1.

Sphingosine-1-phosphate Receptor Summary

Receptor	Expression on immune cells	G alpha subunit	Downstream Pathways	Compounds	References
S1PR1 (EDG1)	T cell, B cell, NK cell, Macrophage, Monocyte, Neutrophil, Eosinophil/Mast cell, Dendritic Cell	Gi	Adenylyl cyclase (inhibitory), Ras/ERK, PI3K/Akt/eNOS, PLC/Ca2+, Rac, Migration	FTY720 (fingolimod)* BAF312 (siponimod)* RCP1063 (ozanimod)* VPC23019* SEW2871 VPC44116* ONO-W061 (ceralifimod)	(Gonzalez et al., 2017; Obinata & Hla, 2012; N. Pyne & Pyne, 2017)
S1PR2 (EDG5)	B cell, Macrophage, Monocyte, Eosinophil/Mast Cell	Gi, G12/13, Gq	Adenylyl cyclase (inhibitory), Ras/ERK, PI3K/Akt/eNOS, PLC/Ca2+, Rac (activated by PI3K, opposed by Rho), Rho/Rho Kinase, ERM phosphorylation	JTE013	(M. Adada et al., 2013; M. M. Adada et al., 2015; Hou et al., 2015; McQuiston et al., 2011)
S1PR3 (EDG3)	B cell, Macrophage, Monocyte, Neutrophil, Eosinophils/Mast Cell, Dendritic Cell	Gi, G12/13, Gq	Adenylyl cyclase (inhibitory), Ras/ERK, PI3K/Akt/eNOS, PLC/Ca2+, Rac (activated by PI3K, opposed by Rho), Rho/Rho Kinase, PI3K/Nox2, PLC/Ca2/P-selectin	FTY720 (fingolimod)* VPC23019* VPC44116* CYM-5541 SPM-242	(Amandeep Bajwa et al., 2016; Blaho & Hla, 2014; Hou et al., 2017; Nussbaum et al., 2015)
S1PR4 (EDG6)	T cell, B cell, Macrophage, Monocyte, Neutrophils, Eosinophil/Mast Cell, Dendritic Cell	Gi, G12/13	Adenylyl cyclase (inhibitory), Ras/ERK, PI3K/Akt/eNOS, PLC/Ca2+, Rac, dendritic cell activation, Rho/PTEN/Akt (inhibitory)	FTY720 (fingolimod)* BAF312 (siponimod)* Cym50138 Cym50358	(Allende et al., 2011; Cencetti et al., 2013; Dillmann et al., 2016; Olesch et al., 2017; Pankratz et al., 2016)
S1PR5 (EDG8)	NK Cell, Eosinophil/Mast Cell, patrolling monocytes	Gi, G12/13	Adenylyl cyclase (inhibitory), Ras/ERK, PI3K/Akt/eNOS, PLC/Ca2+, Rac, Rho, Migration	FTY720 (fingolimod)* BAF312 (siponimod)* RPC1063 (ozanimod)*	(Blaho & Hla, 2014; Debien et al., 2013; Mayol et al., 2011; Scott et al., 2016)

^*=shown to have activity on multiple receptors

Abbreviations:

Ras: Ras family small GTPase, ERK: Extracellular Receptor Kinase, PI3K: PI3-kinase, Akt: protein kinase B, eNOS: Endothelial Nitric Oxide Synthase, PLC: Phospholipase C, Rac: Rac family small GTPase, Rho: Rho family of small GTPases, ERM: Ezrin-Radixin-Moesin, Nox2: NADPH oxidase, PTEN: Phosphatase and tensin homolog

S1PRs and compounds that target them

S1P receptors are a family of seven helix transmembrane G protein coupled receptors that recognize and bind extracellular S1P to affect cellular processes. Sphingosine-1-phosphate receptor 1 (S1PR1) was the first of this family to be discovered during a screen for immediate early endothelial differentiation genes (EDG1) in 1990 (Hla & Maciag, 1990). It was later found that S1P bound specifically to this receptor (Lee et al., 1998). From 1990–2000 four other specific high affinity receptors for S1P were discovered, S1PR2 (EDG5) (Okazaki et al., 1993), S1PR3 (EDG3) (Yamaguchi, Tokuda, Hatase, & Brenner, 1996), S1PR4 (EDG6) (Gräler, Bernhardt, & Lipp, 1998), and S1PR5 (EDG8) (Im et al., 2000). Each member of this family of proteins was found to bind extracellular S1P with high affinity (Spiegel, 2006). To better understand these receptors and to exploit their therapeutic potential, there have been concerted efforts towards the design of compounds that specifically target S1P receptors.

FTY720 was the first compound discovered that targets S1P receptors. FTY720 is an analog of sphingosine that was originally derived from myriocin in 1995 and was found to have potent immunosuppressant activity (Adachi et al., 1995). This compound, like its analog sphingosine, is phosphorylated in vivo by sphingosine kinases, particularly by SK2 (Allende et al., 2004; Kharel et al., 2005). Phosphorylated FTY720 binds S1PR1, 3, 4, and 5 (Rainer Albert et al., 2005). Treatment with FTY720 causes lymphopenia in the blood, and sequestration of lymphocytes in lymph nodes (Brinkmann et al., 2002; Mandala et al., 2002). Sequestration of lymphocytes is mediated by the agonism of S1PR1 by phosphorylated FTY720 which leads to a functional antagonism once the receptor has been internalized and degraded (Oo et al., 2007). This is a simplified explanation of the effect of phosphorylated FTY720 on lymphocyte egress, as this effect is appears to be the result of multiple factors on multiple cell types and is outside the scope of this review (Reviewed in: Cyster & Schwab, 2012; Garris et al., 2014).

FTY720, under the name Gilenya ® (fingolimod) (Novartis AG, Basel Switzerland), is approved by the United States Food and Drug administration as a first line treatment for relapsing remitting multiple sclerosis. Additionally, BAF312 (siponimod), and RPC1063 (ozanimod), derivatives of FTY720, recently met their primary endpoint in phase III clinical trials as treatments for secondary progressive multiple sclerosis and relapsing multiple sclerosis respectively (Althoff & Fiorin, 2016; Catherine Cantone, 2017). Many studies have put forth S1P receptor targeted compounds as potential therapeutics for a variety of immune related, and infectious diseases (Reviewed in: Arish et al., 2016)). A host of small molecules that target specific receptors have been produced from the broad-spectrum parent compound FTY720 (Table 1). These compounds are excellent tools that are used in conjunction with genetic models to learn more about the importance of specific receptors and have led to novel findings across many medically related fields. The following sections will highlight findings with implications for innate immune cells (Figure 2).

Figure 2. Sphingosine-1-Phosphate Receptors on Innate Immune Cells.

S1P receptors 1–5 (S1PR1–5) have been reported to be expressed on various innate immune cell subtypes. S1PR1 is ubiquitously expressed, S1PR2 is expressed on macrophages, monocytes, and eosinophils and mast cells. S1PR3 is expressed on macrophages, neutrophils (during inflammation), dendritic cells, monocytes, eosinophils and mast cells. S1PR4 is expressed on macrophages, neutrophils, dendritic cells, monocytes, eosinophils and mast cells. S1PR5 is expressed on patrolling monocytes and natural killer cells. The major biological outcomes associated with each receptor on each cell type as discussed in this review are shown.

S1PR1

S1PR1 is one of the most widely studied receptors of S1P (Blaho & Hla, 2014; Garris et al., 2014; N. Pyne & Pyne, 2017). Recent evidence suggests that signaling through S1PR1 occurs via the binding of G_i and β-arrestin together to allow for binding of Src leading to receptor internalization mediated by clathrin and dynamin-2 and activation of downstream pathways (N. Pyne & Pyne, 2017).

In innate immunity, S1PR1 is ubiquitously expressed and has been found to mediate functions in most innate immune cells. Macrophages express S1PR1 and migration toward S1P was shown to be dependent on expression of S1PR1 in studies which utilized an S1PR1/3 antagonist, VPC23019, and S1PR1 deficient mice (Weichand et al., 2013). Other work utilized S1PR1 specific agonist, SEW2871, and S1PR1 antagonist, VPC44116, to show that stimulation of S1PR1 can induce an antiinflammatory phenotype (Hughes et al., 2008). Additionally, recent work in myeloid specific S1PR1 deficient mice, showed enhanced protection of macrophages from apoptosis both in vitro and in vivo (Gonzalez, Qian, Tahir, Yu, & Trigatti, 2017). S1PR1 has also been linked to neutrophil migration. In a rat model of hyperalgesia S1PR1 was found to be necessary for neutrophil recruitment (Finley et al., 2013). In a model of Candida albicans water-soluble fraction induced vasculitis, treatment with ONO-W061, a S1PR1 agonist, decreased neutrophil recruitment (Miyabe et al., 2017). In mast cells, S1PR1 is reported to induce migration similar to what has been observed in lymphocytes (Oskeritzian et al., 2010). In eosinophils, S1PR1 inhibition by FTY720 or SEW2871, an S1PR1 agonist, lead to reduced recruitment of eosinophils during hapten application-induced cutaneous responses in mice and reduced chemotaxis in vitro (Sugita et al., 2010). Additionally, S1PR1 has been implicated in plasmacytoid dendritic cell signaling, by degrading interferon alpha receptor 1 to inhibit aberrant interferon alpha production during viral infection (Teijaro et al., 2016). The role of S1PR1 in innate cell migration can also be observed in a model of systemic Yersinia pestis, where it was found that S1PR1 specific agonist, SEW2871, and S1PR1 conditional deletion in mononuclear phagocytes reduced trafficking of infected dendritic cells and monocytes and prevented disease progression (St John et al., 2014). S1PR1 was also found to contribute to natural killer cell trafficking, but it’s role is purported to be minor due to a dependence on CD69 regulation (Jenne et al., 2009; Shiow et al., 2006). S1PR1 exerts its influence in many different pathways and is a major regulator of immunity in both adaptive and innate immune response. Although many findings have been geared towards defining its role in autoimmune responses, they also support the idea that S1PR1 plays a role in immune responses to infectious diseases by affecting recruitment and trafficking of innate immune cells, macrophage polarization, and plasmacitoid dendritic cell functions.

S1PR2

Unlike S1PR1, S1PR2 has been reported to be able to signal through several different G alpha subunits, G_i, G_12/13 and G_q. Each of these subunits can activate an array of downstream pathways, depending on the stimulus and cell type (M. Adada, Canals, Hannun, & Obeid, 2013). One recognized role of S1PR2 is to oppose the activity of S1PR1 by repelling rather than attracting cells in response to S1P. It has been suggested that induction of G_12/13 opposes the actions of G_i by inhibiting Rac and Akt (Green et al., 2011; Sanchez et al., 2007; Takashima et al., 2008).

In innate cells, S1PR2 is present on macrophages, monocytes and granulocytes (Blaho & Hla, 2014). S1PR2 was found to mediate S1P dependent enhancement of phagocytosis of the fungal pathogen Cryptococcus neoformans by utilizing both S1PR2 knockout alveolar macrophages and macrophages treated with S1PR2 antagonist JTE-013. S1PR2 knockout macrophages were found to have significantly lower expression levels of phagocytic receptors FcγR I, II, and III (McQuiston, Luberto, & Del Poeta, 2011). Another study found that S1PR2 knockout macrophages exhibited enhanced opsonin-independent phagocytosis of Escherichia coli due to S1PR2 signaling causing inhibition of RhoA-dependent cell contraction and IQGAP1-Rac1-dependent lamellipodial protrusion (Hou et al., 2015). The difference between these two observations lies in the different pathogens and pathways used for phagocytosis. In vitro, C. neoformans requires opsonization in order for phagocytosis to take place; whereas, phagocytosis of E. coli occurs independently of opsonin. The enhanced phagocytosis described by McQuiston was only observed in response to antibody opsonized phagocytosis, not complement mediated, showing that the effect of S1P mediated enhancement via S1PR2 was specific to FcyR mediated phagocytosis and did not affect other phagocytic pathways, making these two observations not mutually exclusive. S1PR2, via use of antagonist JTE-013, has also been linked to mast cell triggering and release of antimicrobial peptides during vaccinia virus infection (Wang et al., 2012). Additionally a new pathway has been discovered in which intracellular S1P participates in S1PR2 activation to cause phosphorylation of Ezrin-Radixin-Moesin (ERM) proteins, which had been previously unreported (M. M. Adada et al., 2015). ERM proteins play an important role in phagocytic cell function, and this mechanism could possibly be involved in phagosome maturation (Erwig et al., 2006). These findings and others point to an important role for S1PR2 in innate immune cells by increasing antibody mediated phagocytosis of fungi and inhibiting phagocytosis of bacteria in alveolar macrophages, affecting mast cell triggering during viral infection, and affecting ERM phosphorylation.

S1PR3

S1PR3, like S1PR2, has been reported to couple with G alpha subunits, G_i, G_12/13, and G_q. In the past, S1PR3 has been studied in conjunction with the other receptors, but recent availability of genetic models (Kono et al., 2004) and S1PR3 allosteric agonist, CYM-5541 and antagonist SPM-242 (Jo et al., 2012) allow for more thorough study of this receptor.

In innate immunity, S1PR3 was found to mediate S1P induced increase in mature dendritic cell migration and endocytosis (Maeda et al., 2007). More recently, it has been found to also be involved in dendritic cell maturation and promotion of a Th1 response and dendritic cell suppression of T cell regulatory responses in an ischemic reperfusion injury murine model (Bajwa et al., 2012; Bajwa et al., 2016). S1PR3 has been shown to mediate chemotaxis of macrophages in vitro and has been linked to macrophage and monocyte recruitment to plaques in atherosclerosis models (Keul et al., 2011). A recent publication showed that S1PR3 drives bactericidal killing in macrophages by promoting reactive oxygen species generation by NOX2 and promoting phagosome maturation in a murine sepsis model (Hou et al., 2017). S1PR3 was found to be elevated in septic patients and was associated with increased bacterial clearance, better immune status and preferable outcomes (Hou et al., 2017). In neutrophils, S1PR3 is upregulated when isolated from patients with pneumonia and the S1PR3 expressing neutrophils show enhanced chemotaxis in response to S1P (Rahaman, Costello, Belmonte, Gendy, & Walsh, 2006). Studies in S1PR3 knock out mice show reduced inflammation during bleomycin induced lung injury, this observed inflammation may be mediated in part by S1PR3 expressing neutrophils (Murakami et al., 2014). Similarly, S1PR3 is also found to be highly expressed on eosinophils during hapten application-induced cutaneous responses and may play a role in mediating recruitment (Sugita et al., 2010). Studies have also linked S1PR3 to other leukocyte functions. Expression of S1PR3 in endothelial cells drives leukocyte rolling by upregulating P-selectin in a G_q, PLC dependent fashion. S1P released by mast cells acts on S1PR3 to drive leukocyte rolling and recruitment to sites of inflammation (Nussbaum et al., 2015). Altogether, these studies present compelling evidence that S1PR3 plays a multifactorial role in immunity by affecting dendritic cell maturation, macrophage chemotaxis and killing, and neutrophil and eosinophil recruitment, while also promoting immune cell recruitment by driving leukocyte rolling on endothelial cells.

S1PR4

S1PR4, although less studied than other receptors, is abundant on immune cells (Olesch, Ringel, Brüne, & Weigert, 2017). S1PR4 signals through G alpha subunits G_i and G_12/13 (Gräler et al., 2003). It has been suggested that one of the major roles of S1PR4 is to activate Rho kinase downstream of G_12/13 and affect cytoskeletal rearrangement (Sit & Manser, 2011). Another proposed downstream pathway is that Rho kinase activates PTEN which in turn opposes Akt signaling (as in the S1PR2 pathway) to promote apoptosis in myoblasts, this mechanism could also be present in other cell types but could have additional affects since Akt also regulates the cell cycle, metabolism, and autophagy (Cencetti et al., 2013; Manning & Toker, 2017).

S1PR4, like the other receptors, has been proposed to play an important role in innate immune cells. Recent published work showed that S1PR4 knockout in mice lead to a decrease in plasmacytoid dendritic cell differentiation (Dillmann, Mora, Olesch, Brüne, & Weigert, 2015). Additionally, agonist Cym50138 which targets S1PR4, but not other compounds which target S1PR1–3, blocked human plasmacytoid activation and restricted production of interferon alpha in response to CpG oligodeoxynucleotides or tick-borne encephalitis vaccine, whereas S1P4 antagonist Cym50358 prevented the S1P triggered decrease in interferon alpha (Dillmann et al., 2016). S1PR4 has also been linked to roles in neutrophil recruitment. In mice that are deficient in S1P lyase, S1P accumulates and causes neutrophilia, which is partially rescued when S1PR4 is knocked out (Allende et al., 2011). Neutrophils upregulate S1PR4 upon stimulation and FTY720 abrogates neutrophil homing to lymph nodes, further suggesting that S1PR4 is important for neutrophil migration (Gorlino et al., 2014). Additionally, a recent meta-analysis of human genetic variants revealed an association between S1PR4 missense variant and lowered circulating neutrophil counts and confirmed this phenotype in S1PR4 knockout mice and zebrafish (Pankratz et al., 2016). Recent work showed that S1PR4 is the most highly expressed receptor on human alveolar macrophages and that expression of S1PR2 and S1PR4 were highly correlated (Barnawi et al., 2015). In a comparison between M1 and M2 activated macrophages isolated from mouse bone marrow, S1PR4 was found to be the only receptor with a significantly different expression profile when comparing the two conditions and the observed down regulation on M1 polarized macrophages could be responsible for differing responses to S1P, such as differences in migration, and cytokine production (Müller, von Bernstorff, Heidecke, & Schulze, 2017). S1PR4 is widely expressed on immune cells, and current findings suggest a role in plasmacitoid dendritic cell differentiation and activation, neutrophil recruitment, and also point to roles in macrophages as well. Altogether these findings warrant further investigation into the potential roles of S1PR4 in innate immune cells during infection.

S1PR5

Like S1PR4, S1PR5 signals through G alpha subunits G_i and G_12/13. Historically, S1PR5 was thought to be restricted to oligodendrocytes, but has recently been shown to be important for natural killer cells and patrolling monocytes. S1PR5 deficient mice have defective natural killer cell homing and S1PR5 is required for recruitment into inflamed organs (Walzer et al., 2007). The coordinated expression of CXCR4 and S1PR5 allows for mature natural killer cells to leave lymph nodes as expression levels of these proteins change during cell development (Mayol, Biajoux, Marvel, Balabanian, & Walzer, 2011). Patrolling monocytes require S1PR5 to egress from the bone marrow but do not require S1P gradients for their trafficking, unlike the relationship between T cells and S1P, suggesting that S1PR5 mediated trafficking may occur by a different mechanism (Debien et al., 2013). The mechanism by which S1PR5 influences trafficking warrants further study, and there may be other cell types that rely on this receptor for trafficking or other downstream pathways, that have yet to be investigated.

Conclusions and Final Thoughts

Great advances have been made toward understanding the biology of S1P signaling. S1P receptors are attractive targets for design of small molecules and this allows for advanced study of their roles in a variety of biological processes. In the field of immunity, there have been many studies which point to the role of S1PR signaling in trafficking, differentiation, and activation of immune cell effector functions, but there remain many unanswered questions when it comes to defining the specific mechanisms of these effects and how these receptors may affect responses to infectious diseases in innate immune cells. These unknowns need to be addressed, especially as S1PR agonists and antagonists are being used and proposed as treatments for disorders. Recently in the post marketing setting, treatment with fingolimod has been linked to several cases of infection with the opportunistic fungal pathogen C. neoformans, which causes a severe fungal meningitis if left untreated (Achtnichts, Obreja, Conen, Fux, & Nedeltchev, 2015; Commissioner, n.d.; Grebenciucova, Reder, & Bernard, 2016; Huang, 2015; Ward, Jones, & Goldman, 2016). Previous research showed that S1P is important for maintaining C. neoformans in granulomas in the lung and in neutrophil killing (Farnoud, Bryan, Kechichian, Luberto, & Del Poeta, 2015; McQuiston, Luberto, & Del Poeta, 2010). The influence of S1P receptors on innate immunity could be one of the reasons for this increased susceptibility. The innate immune system is the first line defense against pathogens, and understanding the implications of S1P receptor signaling in innate immunity is valuable when proposing the use of these inhibitors in patients.