Finding a Way Out: S1P Signaling and Immune Cell Migration.

The signaling lipid sphingosine 1-phosphate (S1P) plays critical roles in an immune response. Drugs targeting S1P signaling have been remarkably successful in treatment of multiple sclerosis, and they have shown promise in clinical trials for colitis and psoriasis. One mechanism of these drugs is to block lymphocyte exit from lymph nodes, where lymphocytes are initially activated, into circulation, from which lymphocytes can reach sites of inflammation. Indeed, S1P can be considered a circulation marker, signaling to immune cells to help them find blood and lymphatic vessels, and to endothelial cells to stabilize the vasculature. That said, S1P plays pleiotropic roles in the immune response, and it will be important to build an integrated view of how S1P shapes inflammation. S1P can function so effectively because its distribution is exquisitely tightly controlled. Here we review how S1P gradients regulate immune cell exit from tissues, with particular attention to key outstanding questions in the field.

Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs.

Much has been learned about how cells enter lymphoid tissues. But how do they leave? Sphingosine-1-phosphate (S1P) has emerged over the past decade as a central mediator of lymphocyte egress. In this review, we summarize the current understanding of how S1P promotes exit from the secondary lymphoid organs and thymus. We review what is known about additional requirements for emigration and summarize the mostly distinct requirements for exit from the bone marrow. Egress from lymphoid organs is limited during immune responses, and we examine how this regulation works. There is accumulating evidence for roles of S1P in directing immune cell behavior within lymphoid tissues. How such actions can fit together with the egress-promoting role of S1P is discussed. Finally, we examine current understanding of how FTY720, a drug that targets S1P receptors and is approved for the treatment of multiple sclerosis, causes immune suppression.

The Bone Marrow Protects and Optimizes Immunological Memory during Dietary Restriction.

Mammals evolved in the face of fluctuating food availability. How the immune system adapts to transient nutritional stress remains poorly understood. Here, we show that memory T cells collapsed in secondary lymphoid organs in the context of dietary restriction (DR) but dramatically accumulated within the bone marrow (BM), where they adopted a state associated with energy conservation. This response was coordinated by glucocorticoids and associated with a profound remodeling of the BM compartment, which included an increase in T cell homing factors, erythropoiesis, and adipogenesis. Adipocytes, as well as CXCR4-CXCL12 and S1P-S1P1R interactions, contributed to enhanced T cell accumulation in BM during DR. Memory T cell homing to BM during DR was associated with enhanced protection against infections and tumors. Together, this work uncovers a fundamental host strategy to sustain and optimize immunological memory during nutritional challenges that involved a temporal and spatial reorganization of the memory pool within "safe haven" compartments.

Gradients of the signaling lipid S1P in lymph nodes position natural killer cells and regulate their interferon-γ response.

The lymph node periphery is an important site for many immunological functions, from pathogen containment to the differentiation of helper T cells, yet the cues that position cells in this region are largely undefined. Here, through the use of a reporter for the signaling lipid S1P (sphingosine 1-phosphate), we found that cells sensed higher concentrations of S1P in the medullary cords than in the T cell zone and that the S1P transporter SPNS2 on lymphatic endothelial cells generated this gradient. Natural killer (NK) cells are located at the periphery of the lymph node, predominantly in the medulla, and we found that expression of SPNS2, expression of the S1P receptor S1PR5 on NK cells, and expression of the chemokine receptor CXCR4 were all required for NK cell localization during homeostasis and rapid production of interferon-γ by NK cells after challenge. Our findings elucidate the spatial cues for NK cell organization and reveal a previously unknown role for S1P in positioning cells within the medulla.

A map of the distribution of sphingosine 1-phosphate in the spleen.

Despite the importance of signaling lipids, many questions remain about their function because few tools are available for charting lipid gradients in vivo. Here we generated a sphingosine 1-phosphate (S1P) reporter mouse and used this mouse to define the distribution of S1P in the spleen. Unexpectedly, the presence of blood did not serve as a predictor of the concentration of signaling-available S1P. Large areas of the red pulp had low concentrations of S1P, while S1P was sensed by cells inside the white pulp near the marginal sinus. The lipid phosphate phosphatase LPP3 maintained low S1P concentrations in the spleen and enabled efficient shuttling of marginal zone B cells. The exquisitely tight regulation of S1P availability might explain how a single lipid can simultaneously orchestrate the movements of many cells of the immune system.

Have Cytokines, Will Travel.

In many contexts, innate lymphoid cells (ILCs) are primarily tissue resident. By contrast, in a recent issue of Science, Huang et al. (2018) show that inflammatory type 2 ILCs migrate from the intestine to the lungs and that this movement is guided by sphingosine-1-phosphate receptors.

Monocyte-derived S1P in the lymph node regulates immune responses.

The lipid chemoattractant sphingosine 1-phosphate (S1P) guides cells out of tissues, where the concentration of S1P is relatively low, into circulatory fluids, where the concentration of S1P is high1. For example, S1P directs the exit of T cells from lymph nodes, where T cells are initially activated, into lymph, from which T cells reach the blood and ultimately inflamed tissues1. T cells follow S1P gradients primarily using S1P receptor 1 (ref. 1). Recent studies have described how S1P gradients are established at steady state, but little is known about the distribution of S1P in disease or about how changing levels of S1P may affect immune responses. Here we show that the concentration of S1P increases in lymph nodes during an immune response. We found that haematopoietic cells, including inflammatory monocytes, were an important source of this S1P, which was an unexpected finding as endothelial cells provide S1P to lymph1. Inflammatory monocytes required the early activation marker CD69 to supply this S1P, in part because the expression of CD69 was associated with reduced levels of S1pr5 (which encodes S1P receptor 5). CD69 acted as a 'stand-your-ground' signal, keeping immune cells at a site of inflammation by regulating both the receptors and the gradients of S1P. Finally, increased levels of S1P prolonged the residence time of T cells in the lymph nodes and exacerbated the severity of experimental autoimmune encephalomyelitis in mice. This finding suggests that residence time in the lymph nodes might regulate the differentiation of T cells, and points to new uses of drugs that target S1P signalling.

Get me out of here: Sphingosine 1-phosphate signaling and T cell exit from tissues during an immune response.

During an immune response, the duration of T cell residence in lymphoid and non-lymphoid tissues likely affects T cell activation, differentiation, and memory development. The factors that govern T cell transit through inflamed tissues remain incompletely understood, but one important determinant of T cell exit from tissues is sphingosine 1-phosphate (S1P) signaling. In homeostasis, S1P levels are high in blood and lymph compared to lymphoid organs, and lymphocytes follow S1P gradients out of tissues into circulation using varying combinations of five G-protein coupled S1P receptors. During an immune response, both the shape of S1P gradients and the expression of S1P receptors are dynamically regulated. Here we review what is known, and key questions that remain unanswered, about how S1P signaling is regulated in inflammation and in turn how S1P shapes immune responses.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Lymphatic endothelial S1P promotes mitochondrial function and survival in naive T cells.

Effective adaptive immune responses require a large repertoire of naive T cells that migrate throughout the body, rapidly identifying almost any foreign peptide. Because the production of T cells declines with age, naive T cells must be long-lived. However, it remains unclear how naive T cells survive for years while constantly travelling. The chemoattractant sphingosine 1-phosphate (S1P) guides T cell circulation among secondary lymphoid organs, including spleen, lymph nodes and Peyer's patches, where T cells search for antigens. The concentration of S1P is higher in circulatory fluids than in lymphoid organs, and the S1P1 receptor (S1P1R) directs the exit of T cells from the spleen into blood, and from lymph nodes and Peyer's patches into lymph. Here we show that S1P is essential not only for the circulation of naive T cells, but also for their survival. Using transgenic mouse models, we demonstrate that lymphatic endothelial cells support the survival of T cells by secreting S1P via the transporter SPNS2, that this S1P signals through S1P1R on T cells, and that the requirement for S1P1R is independent of the established role of the receptor in guiding exit from lymph nodes. S1P signalling maintains the mitochondrial content of naive T cells, providing cells with the energy to continue their constant migration. The S1P signalling pathway is being targeted therapeutically to inhibit autoreactive T cell trafficking, and these findings suggest that it may be possible simultaneously to target autoreactive or malignant cell survival.

Secrets and lyase: Control of sphingosine 1-phosphate distribution.

The signaling lipid sphingosine 1-phosphate (S1P) plays key roles in many physiological processes. In the immune system, S1P's best-described function is to draw cells out of tissues into circulation. Here, we will review models of S1P distribution in the thymus, lymph nodes, spleen, and nonlymphoid tissues. These models have been challenging to construct, because of the lack of tools to map lipid gradients. Nonetheless, evidence to date suggests that S1P distribution is exquisitely tightly controlled, and that concentrations of signaling-available S1P cannot be predicted by standard rules of thumb. The fine regulation of S1P gradients may explain how S1P can simultaneously direct multiple cell movements both between tissues and circulation and within tissues. It may also make it feasible to develop drugs that enable spatially specific modulation of S1P signaling.

Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells.

The cellular dynamics of the egress of lymphocytes from lymph nodes are poorly defined. Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma. T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses. In some sinuses, T cells became rounded and moved unidirectionally. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph.

Finding a way out: lymphocyte egress from lymphoid organs.

The egress of lymphocytes from the thymus and secondary lymphoid organs into circulatory fluids is essential for normal immune function. The discovery that a small-molecule inhibitor of lymphocyte exit, FTY720, is a ligand for sphingosine 1-phosphate (S1P) receptors led to studies demonstrating that S1P receptor type 1 (S1P1) is needed in T cells and B cells for their egress from lymphoid organs. S1P exists in higher concentrations in blood and lymph than in lymphoid organs, and this differential is also required for lymphocyte exit. Transcriptional and post-translational mechanisms regulate S1P1 and thus the egress of lymphocytes. In this review we discuss the body of evidence supporting a model in which lymphocyte egress is promoted by encounter with S1P at exit sites. We relate this model to work examining the effects of S1P receptor agonists on endothelium.

Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells.

The intestinal microbiota has a critical role in immune system and metabolic homeostasis, but it must be tolerated by the host to avoid inflammatory responses that can damage the epithelial barrier separating the host from the luminal contents. Breakdown of this regulation and the resulting inappropriate immune response to commensals are thought to lead to the development of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. We proposed that the intestinal immune system is instructed by the microbiota to limit responses to luminal antigens. Here we demonstrate in mice that, at steady state, the microbiota inhibits the transport of both commensal and pathogenic bacteria from the lumen to a key immune inductive site, the mesenteric lymph nodes (MLNs). However, in the absence of Myd88 or under conditions of antibiotic-induced dysbiosis, non-invasive bacteria were trafficked to the MLNs in a CCR7-dependent manner, and induced both T-cell responses and IgA production. Trafficking was carried out by CX(3)CR1(hi) mononuclear phagocytes, an intestinal-cell population previously reported to be non-migratory. These findings define a central role for commensals in regulating the migration to the MLNs of CX(3)CR1(hi) mononuclear phagocytes endowed with the ability to capture luminal bacteria, thereby compartmentalizing the intestinal immune response to avoid inflammation.

Exit Strategies: S1P Signaling and T Cell Migration.

Whereas the role of sphingosine 1-phosphate receptor 1 (S1PR1) in T cell egress and the regulation of S1P gradients between lymphoid organs and circulatory fluids in homeostasis are increasingly well understood, much remains to be learned about S1P signaling and distribution during an immune response. Recent data suggest that the role of S1PR1 in directing cells from tissues into circulatory fluids is reprised again and again, particularly in guiding activated T cells from non-lymphoid tissues into lymphatics. Conversely, S1P receptor 2 (S1PR2), which antagonizes migration towards chemokines, confines cells within tissues. Here we review the current understanding of the roles of S1P signaling in activated T cell migration. In this context, we outline open questions, particularly regarding the shape of S1P gradients in different tissues in homeostasis and inflammation, and discuss recent strategies to measure S1P.

S1PR1 inhibition induces proapoptotic signaling in T cells and limits humoral responses within lymph nodes.

Effective immunity requires a large, diverse naive T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we addressed how S1P enables T cell survival and the implications for patients treated with S1PR1 antagonists. We found that S1PR1 limited apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization were required to prevent this proapoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naive T cells limited B cell responses. Our findings highlighted an effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggested both limitations and additional uses of this important class of drugs.

Sphingosine 1-phosphate receptor 1 inhibition induces a pro-apoptotic signaling cascade in T cells.

Effective immunity requires a large, diverse naïve T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we address how S1P enables T cell survival, and the implications for patients treated with S1PR1 antagonists. Contrary to expectations, we found that S1PR1 limits apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization are required to prevent this pro-apoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naïve T cells limited B cell responses. Our findings highlight an unexpected effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggest both limitations and novel uses of this important class of drugs.

Blood-thirsty: S1PR5 and TRM.

In this elegant study, Evrard et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210116) find that sphingosine 1-phosphate receptor 5 (S1PR5) powerfully impairs tissue-resident memory T cell (TRM) formation, and that tissue-derived TGF-ß limits S1pr5 expression by infiltrating T cells.

Sphingosine 1-phosphate signaling in perivascular cells enhances inflammation and fibrosis in the kidney.

Chronic kidney disease (CKD), characterized by sustained inflammation and progressive fibrosis, is highly prevalent and can eventually progress to end-stage kidney disease. However, current treatments to slow CKD progression are limited. Sphingosine 1-phosphate (S1P), a product of sphingolipid catabolism, is a pleiotropic mediator involved in many cellular functions, and drugs targeting S1P signaling have previously been studied particularly for autoimmune diseases. The primary mechanism of most of these drugs is functional antagonism of S1P receptor-1 (S1P1) expressed on lymphocytes and the resultant immunosuppressive effect. Here, we documented the role of local S1P signaling in perivascular cells in the progression of kidney fibrosis using primary kidney perivascular cells and several conditional mouse models. S1P was predominantly produced by sphingosine kinase 2 in kidney perivascular cells and exported via spinster homolog 2 (Spns2). It bound to S1P1 expressed in perivascular cells to enhance production of proinflammatory cytokines/chemokines upon injury, leading to immune cell infiltration and subsequent fibrosis. A small-molecule Spns2 inhibitor blocked S1P transport, resulting in suppression of inflammatory signaling in human and mouse kidney perivascular cells in vitro and amelioration of kidney fibrosis in mice. Our study provides insight into the regulation of inflammation and fibrosis by S1P and demonstrates the potential of Spns2 inhibition as a treatment for CKD and potentially other inflammatory and fibrotic diseases that avoids the adverse events associated with systemic modulation of S1P receptors.