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Apolipoprotein-M-bound sphingosine-1-phosphate (S1P) is found to restrain the generation of new lymphocytes—and, consequently, adaptive immune responses—by activating the S1P 1 receptor on bone marrow lymphocyte progenitors in mice. Sphingolipid suppression of lymphopoiesis Sphingosine-1-phosphate (S1P) is a lipid mediator concentrated in the blood and lymph and is a signal that regulates a variety of physiological functions. It acts through G-protein-coupled receptors to regulate immune cell trafficking. This study shows that S1P bound to its chaperone apolipoprotein M (ApoM) restrains the generation of new lymphocytes — and, consequently, adaptive immune responses — by activating the S1P 1 receptor on bone marrow lymphocyte progenitors in mice. ApoM–S1P is not required for lymphocyte trafficking however, a function fulfilled by plasma S1P that is a target of fingolimod, an immunomodulating drug recently approved for treating multiple sclerosis. Lipid mediators influence immunity in myriad ways. For example, circulating sphingosine-1-phosphate (S1P) is a key regulator of lymphocyte egress 1 , 2 . Although the majority of plasma S1P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle 3 , the immunological functions of the ApoM–S1P complex are unknown. Here we show that ApoM–S1P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the S1P 1 receptor on bone marrow lymphocyte progenitors. Mice that lacked ApoM ( Apom −/− ) had increased proliferation of Lin − Sca-1 + cKit + haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow. Pharmacological activation or genetic overexpression of S1P 1 suppressed LSK and CLP cell proliferation in vivo . ApoM was stably associated with bone marrow CLPs, which showed active S1P 1 signalling in vivo 4 . Moreover, ApoM-bound S1P, but not albumin-bound S1P, inhibited lymphopoiesis in vitro. Upon immune stimulation, Apom −/− mice developed more severe experimental autoimmune encephalomyelitis 5 , characterized by increased lymphocytes in the central nervous system and breakdown of the blood–brain barrier. Thus, the ApoM–S1P–S1P 1 signalling axis restrains the lymphocyte compartment and, subsequently, adaptive immune responses. Unique biological functions imparted by specific S1P chaperones could be exploited for novel therapeutic opportunities.

Background Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. Methods mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography–tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. Results GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. Conclusions Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.

Membrane sphingolipids are metabolized to sphingosine-1-phosphate (S1P), a bioactive lipid mediator that regulates many processes in vertebrate development, physiology, and pathology. Once exported out of cells by cell-specific transporters, chaperone-bound S1P is spatially compartmentalized in the circulatory system. Extracellular S1P interacts with five GPCRs that are widely expressed and transduce intracellular signals to regulate cellular behavior, such as migration, adhesion, survival, and proliferation. While many organ systems are affected, S1P signaling is essential for vascular development, neurogenesis, and lymphocyte trafficking. Recently, a pharmacological S1P receptor antagonist has won approval to control autoimmune neuroinflammation in multiple sclerosis. The availability of pharmacological tools as well as mouse genetic models has revealed several physiological actions of S1P and begun to shed light on its pathological roles. The unique mode of signaling of this lysophospholipid mediator is providing novel opportunities for therapeutic intervention, with possibilities to target not only GPCRs but also transporters, metabolic enzymes, and chaperones.

Sortilin has been positively correlated with vascular disorders in humans. No study has yet evaluated the possible direct effect of sortilin on vascular function. We used pharmacological and genetic approaches coupled with study of murine and human samples to unravel the mechanisms recruited by sortilin in the vascular system. Sortilin induced endothelial dysfunction of mesenteric arteries through NADPH oxidase 2 (NOX2) isoform activation, dysfunction that was prevented by knockdown of acid sphingomyelinase (ASMase) or sphingosine kinase 1. In vivo, recombinant sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of sphingosine-1-phosphate receptor 3 (S1P3) and gp91phox/NOX2 resulted in preservation of endothelial function and blood pressure homeostasis after 14 days of systemic sortilin administration. Translating these research findings into the clinical setting, we detected elevated sortilin levels in hypertensive patients with endothelial dysfunction. Furthermore, in a population-based cohort of 270 subjects, we showed increased plasma ASMase activity and increased plasma levels of sortilin, S1P, and soluble NOX2-derived peptide (sNOX2-dp) in hypertensive subjects, and the increase was more pronounced in hypertensive subjects with uncontrolled blood pressure. Our studies reveal what we believe is a previously unrecognized role of sortilin in the impairment of vascular function and in blood pressure homeostasis and suggest the potential of sortilin and its mediators as biomarkers for the prediction of vascular dysfunction and high blood pressure.

Lymphocytes require sphingosine-1-phosphate (S1P) receptor-1 to exit lymphoid organs, but the source(s) of extracellular S1P and whether S1P directly promotes egress are unknown. By using mice in which the two kinases that generate S1P were conditionally ablated, we find that plasma S1P is mainly hematopoietic in origin, with erythrocytes a major contributor, whereas lymph S1P is from a distinct radiation-resistant source. Lymphocyte egress from thymus and secondary lymphoid organs was markedly reduced in kinase-deficient mice. Restoration of S1P to plasma rescued egress to blood but not lymph, and the rescue required lymphocyte expression of S1P-receptor-1. Thus, separate sources provide S1P to plasma and lymph to help lymphocytes exit the low-S1P environment of lymphoid organs. Disruption of compartmentalized S1P signaling is a plausible mechanism by which S1P-receptor-1 agonists function as immunosuppressives.

Type 1 innate lymphoid cells (ILC1s) are a class of tissue-resident cells with antitumor activity, suggesting its possible role in solid tumor immune surveillance, but it is not clear whether manipulating ILC1s can induce potent antitumor immune responses. Here, we found that G-protein-coupled receptor 34 (GPR34), a receptor for lysophosphatidylserine (LysoPS), was highly expressed on ILC1s but not on conventional natural killer cells in the tumor microenvironment. LysoPS was enriched in the tumor microenvironment and could inhibit ILC1 activation via GPR34. Genetic deletion of LysoPS synthase Abhd16a expression in tumors or Gpr34 expression in ILC1s or antagonizing GPR34 enhanced ILC1 antitumor activity. In individuals with cancer, ABHD16A expression in tumors or GPR34 expression in ILC1s was inversely correlated with the antitumor activity of ILC1s or ILC1-like cells. Thus, our results demonstrate that manipulating ILC1s can induce potent antitumor immunity, and GPR34 is a metabolic immune checkpoint that can be targeted to develop ILC1-based immunotherapy. Zhou and colleagues find that GPR34 is a metabolic immune checkpoint for ILC1-mediated antitumor immunity.

A cofactor for the E3 ubiquitin ligase TRAF2 TRAF2 (tumour-necrosis factor receptor-associated factor 2), a component of the NF-κB activation pathway important in inflammatory, anti-apoptotic, and immune processes, is shown to be a target of sphingosine kinase 1, one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate inside cells. In addition, sphingosine kinase 1 is revealed as the 'missing' cofactor for TRAF2 E3 ubiquitin ligase activity. These findings provide a mechanistic explanation for the numerous observations of the importance of SphK1 in inflammatory, anti-apoptotic and immune processes. Engagement of the tumour-necrosis factor (TNF) receptor results in the assembly of multi-component signalling complexes by adaptor proteins that include TNF receptor-associated factor 2 (TRAF2). Genetic evidence indicates that TRAF2 is needed for the polyubiquitination of receptor interacting protein 1 (RIP1), but direct evidence has been lacking. Here it is shown that the lipid sphingosine-1-phosphate is a co-factor needed for this ubiquitination activity of TRAF2. Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-κB signalling triggered by TNF-α 1 , 2 . Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1) 3 that then serves as a platform for recruitment and stimulation of IκB kinase, leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1) 4 , one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and the production of S1P is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of IκB kinase and IκBα, and IκBα degradation, leading to NF-κB activation. These responses were mediated by intracellular S1P independently of its cell surface G-protein-coupled receptors. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signalling and the canonical NF-κB activation pathway important in inflammatory, antiapoptotic and immune processes.

Cauda equina compression (CEC) is a major cause of neurogenic claudication and progresses to neuropathic pain (NP). A lipid mediator, lysophosphatidic acid (LPA), is known to induce NP via the LPA 1 receptor. To know a possible mechanism of LPA production in neurogenic claudication, we determined the levels of LPA, lysophosphatidylcholine (LPC) and LPA-producing enzyme autotaxin (ATX), in the cerebrospinal fluid (CSF) and spinal cord (SC) using a CEC as a possible model of neurogenic claudication. Using silicon blocks within the lumbar epidural space, we developed a CEC model in rats with motor dysfunction. LPC and LPA levels in the CSF were significantly increased from day 1. Importantly, specific LPA species (16:0, 18:2, 20:4) were upregulated, which have been shown to produce by ATX detected in the CSF, without changes on its level. In SC, the LPC and LPA levels did not change, but mass spectrometry imaging analysis revealed that LPC was present in a region where the silicon blocks were inserted. These results propose a model for LPA production in SC and CSF upon neurogenic claudication that LPC produced locally by tissue damages is converted to LPA by ATX, which then leak out into the CSF.

GPR55 is an orphan G protein-coupled receptor (GPCR) and represents a promising drug target for cancer, inflammation, and metabolic diseases. The endogenous activation of lipid GPCRs can be solely mediated by membrane components and different lipids have been proposed as endogenous activators of GPR55, such as cannabinoids and lysophosphatidylinositols. Here, we determine high-resolution cryo-electron microscopy structures of the activated GPR55 in complex with heterotrimeric G 13 and two structurally diverse ligands: the putative endogenous agonist 1-palmitoyl-2-lysophosphatidylinositol (LPI) and the synthetic agonist ML184. These results reveal insights into ligand recognition at GPR55, G protein coupling and receptor activation. Notably, an orthosteric binding site opening towards the membrane is observed in both structures, enabling direct interaction of the agonists with membrane lipids. The structural observations are supported by mutagenesis and functional experiments employing G protein dissociation assays. These findings will be of importance for the structure-based development of drugs targeting GPR55. Endogenous activation of lipid GPCRs can be mediated by membrane components. Here, authors show an orthosteric binding site opens towards the membrane, setting the foundation for structure-based development of drugs targeting GPR55.

The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.