Interleukin-36 potently stimulates human M2 macrophages, Langerhans cells and keratinocytes to produce pro-inflammatory cytokines.

Interleukin (IL)-36 cytokines belong to the IL-1 family and include three agonists, IL-36 α, ß and γ and one inhibitor, IL-36 receptor antagonist (IL-36Ra). IL-36 and IL-1 (α and ß) activate similar intracellular pathways via their related heterodimeric receptors, IL-36R/IL-1RAcP and IL-1R1/IL-1RAcP, respectively. However, excessive IL-36 versus IL-1 signaling induces different phenotypes in humans, which may be related to differential expression of their respective receptors. We examined the expression of IL-36R, IL-1R1 and IL-1RAcP mRNA in human peripheral blood, tonsil and skin immune cells by RT-qPCR. Monocyte-derived dendritic cells (MDDC), M0, M1 or M2-polarized macrophages, primary keratinocytes, dermal macrophages and Langerhans cells (LC) were stimulated with IL-1ß or IL-36ß. Cytokine production was assessed by RT-qPCR and immunoassays. The highest levels of IL-36R mRNA were found in skin-derived keratinocytes, LC, dermal macrophages and dermal CD1a(+) DC. In the blood and in tonsils, IL-36R mRNA was predominantly found in myeloid cells. By contrast, IL-1R1 mRNA was detected in almost all cell types with higher levels in tonsil and skin compared to peripheral blood immune cells. IL-36ß was as potent as IL-1ß in stimulating M2 macrophages, keratinocytes and LC, less potent than IL-1ß in stimulating M0 macrophages and MDDC, and exerted no effects in M1 and dermal macrophages. Levels of IL-1Ra diminished the ability of M2 macrophages to respond to IL-1. Taken together, these data are consistent with the association of excessive IL-36 signaling with an inflammatory skin phenotype and identify human LC and M2 macrophages as new IL-36 target cells.

Sex ratio variation shapes the ecological effects of a globally introduced freshwater fish.

Sex ratio and sexual dimorphism have long been of interest in population and evolutionary ecology, but consequences for communities and ecosystems remain untested. Sex ratio could influence ecological conditions whenever sexual dimorphism is associated with ecological dimorphism in species with strong ecological interactions. We tested for ecological implications of sex ratio variation in the sexually dimorphic western mosquitofish, Gambusia affinis. This species causes strong pelagic trophic cascades and exhibits substantial variation in adult sex ratios. We found that female-biased populations induced stronger pelagic trophic cascades compared with male-biased populations, causing larger changes to key community and ecosystem responses, including zooplankton abundance, phytoplankton abundance, productivity, pH and temperature. The magnitude of such effects indicates that sex ratio is important for mediating the ecological role of mosquitofish. Because both sex ratio variation and sexual dimorphism are common features of natural populations, our findings should encourage broader consideration of the ecological significance of sex ratio variation in nature, including the relative contributions of various sexually dimorphic traits to these effects.

Butyrophilin-like 2 modulates B7 costimulation to induce Foxp3 expression and regulatory T cell development in mature T cells.

Naive T cell activation involves at least two signals from an APC, one through the TCR via interaction with peptide-MHC complexes and a second through ligation of CD28 with B7 ligands. Following activation, T cells upregulate a host of other membrane-bound costimulatory molecules that can either promote or inhibit further T cell maturation and proliferation. In some cases, it is necessary to attenuate T cell activation to prevent deleterious inflammation, and inhibitory members of the B7/butyrophilin family of ligands have evolved to balance the strong stimuli the activating B7 ligands confer. Human genetic association and in vitro studies have implicated one such ligand, BTNL2, in controlling inflammation at mucosal surfaces. In this study, we show that recombinant mouse BTNL2 modifies B7/CD28 signaling to promote expression of Foxp3, a transcription factor necessary for regulatory T cell (Treg) development and function. BTNL2 blocks Akt-mediated inactivation of Foxo1, a transcription factor necessary for Foxp3 expression. Immunophenotyping and gene profiling reveal that BTNL2-induced Treg share many properties with natural Treg, and in vivo they suppress enteritis induced by mouse effector T cells. These findings describe a mechanism by which environmental Ag-specific Tregs may be induced by APC expressing specific modulators of costimulatory signals.

Protein tyrosine phosphatase expression profile of rheumatoid arthritis fibroblast-like synoviocytes: a novel role of SH2 domain-containing phosphatase 2 as a modulator of invasion and survival.

OBJECTIVE: The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS. METHODS: A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain-containing phosphatase 2 (SHP-2), a PTP that was up-regulated in RA, was then analyzed by knockdown using cell-permeable antisense oligonucleotides. RESULTS: PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP-2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP-2 knockdown. RA FLS that were deficient in SHP-2 exhibited decreased activation of focal adhesion kinase and mitogen-activated protein kinases. CONCLUSION: These findings indicate that SHP-2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP-2 to be a candidate therapeutic target for RA.

Quinolinone-based agonists of S1P1: use of a N-scan SAR strategy to optimize in vitro and in vivo activity.

We reveal how a N-scan SAR strategy (systematic substitution of each CH group with a N atom) was employed for quinolinone-based S1P(1) agonist 5 to modulate physicochemical properties and optimize in vitro and in vivo activity. The diaza-analog 17 displays improved potency (hS1P(1) RI; 17: EC(50)=0.020 µM, 120% efficacy; 5: EC(50)=0.070 µM, 110% efficacy) and selectivity (hS1P(3) Ca(2+) flux; 17: EC(50) >25 µM; 5: EC(50)=1.5 µM, 92% efficacy), as well as enhanced pharmacokinetics (17: CL=0.15 L/h/kg, V(dss)=5.1L/kg, T(1/2)=24h, %F=110; 5: CL=0.93L/h/kg, V(dss)=11L/kg, T(1/2)=15 h, %F=60) and pharmacodynamics (17: 1.0mg/kg po, 24h PLC POC=-67%; 5: 3mg/kg po, 24h PLC POC=-51%) in rat.

Diverse functions of reactive cysteines facilitate unique biosynthetic processes of aggregate-prone interleukin-31.

Interleukin-31 (IL-31) is a member of the four helical-bundle gp130/IL-6 cytokine family. Despite its implicated roles in inflammatory diseases, the biosynthetic processes of IL-31 have been poorly investigated. A detailed understanding of IL-31 biosynthesis and the nature of ligand-receptor interactions can provide insights into effective strategies for the design of therapeutic approaches. By using various heterologous protein expression systems, we demonstrated that murine IL-31 was secreted as inter-molecularly disulfide-bonded covalent aggregates. Covalently aggregated IL-31 appeared while trafficking in the secretory pathway, but was not actively retained in the ER. The aggregate formation was not caused by a dysfunctional ER quality control mechanism or an intrinsic limitation in protein folding capacity. Furthermore, secreted IL-31 aggregates were part of a large complex composed of various pleiotropic secretory factors and immune-stimulators. The extent and the heterogeneous nature of aggregates may imply that IL-31 was erroneously folded, but it was capable of signaling through cognate receptors. Mutagenesis revealed the promiscuity of all five cysteines in inter-molecular disulfide formation with components of the hetero-aggregates, but no cysteine was required for IL-31 secretion itself. Our present study not only illustrated various functions that cysteines perform during IL-31 biosynthesis and secretion, but also highlighted their potential roles in cytokine effector functions.

Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during experimental autoimmune encephalomyelitis.

BACKGROUND: Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays. RESULTS: Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals. CONCLUSIONS: These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.

Gatekeepers of intestinal inflammation.

The intestine is subjected to a barrage of insults from food, bacterial flora, and pathogens. Despite this constant antigenic challenge, the mucosal tissues lining the intestinal tract remain largely under control. The mechanisms regulating the homeostatic balance in the gut have been investigated for many years by many groups, but the precise nature of the regulatory control remains elusive. In this review, we provide an overview of pathways proposed to be involved in dampening the inflammatory response and maintaining the homeostatic balance in the intestine, and how these pathways may be disrupted in ulcerative colitis and Crohn's disease.

Mechanisms of oncostatin M-induced pulmonary inflammation and fibrosis.

Oncostatin M (OSM), an IL-6 family cytokine, has been implicated in a number of biological processes including the induction of inflammation and the modulation of extracellular matrix. In this study, we demonstrate that OSM is up-regulated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and scleroderma, and investigate the pathological consequences of excess OSM in the lungs. Delivery of OSM to the lungs of mice results in a significant recruitment of inflammatory cells, as well as a dose-dependent increase in collagen deposition in the lungs, with pathological correlates to characteristic human interstitial lung disease. To better understand the relationship between OSM-induced inflammation and OSM-induced fibrosis, we used genetically modified mice and show that the fibrotic response is largely independent of B and T lymphocytes, eosinophils, and mast cells. We further explored the mechanisms of OSM-induced inflammation and fibrosis using both protein and genomic array approaches, generating a "fibrotic footprint" for OSM that shows modulation of various matrix metalloproteinases, extracellular matrix components, and cytokines previously implicated in fibrosis. In particular, although the IL-4/IL-13 and TGF-beta pathways have been shown to be important and intertwined of fibrosis, we show that OSM is capable of inducing lung fibrosis independently of these pathways. The demonstration that OSM is a potent mediator of lung inflammation and extracellular matrix accumulation, combined with the up-regulation observed in patients with pulmonary fibrosis, may provide a rationale for therapeutically targeting OSM in human disease.

Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis.

We provide a single-cell atlas of idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease, by profiling 312,928 cells from 32 IPF, 28 smoker and nonsmoker controls, and 18 chronic obstructive pulmonary disease (COPD) lungs. Among epithelial cells enriched in IPF, we identify a previously unidentified population of aberrant basaloid cells that coexpress basal epithelial, mesenchymal, senescence, and developmental markers and are located at the edge of myofibroblast foci in the IPF lung. Among vascular endothelial cells, we identify an ectopically expanded cell population transcriptomically identical to bronchial restricted vascular endothelial cells in IPF. We confirm the presence of both populations by immunohistochemistry and independent datasets. Among stromal cells, we identify IPF myofibroblasts and invasive fibroblasts with partially overlapping cells in control and COPD lungs. Last, we confirm previous findings of profibrotic macrophage populations in the IPF lung. Our comprehensive catalog reveals the complexity and diversity of aberrant cellular populations in IPF.

Regulation of costimulation in the era of butyrophilins.

The butyrophilin and butyrophilin-like superfamily of molecules has garnered attention in the immunology world in the past few years as a result of the observation that the butyrophilin-like 2 molecule, BTNL2, can alter T cell responsiveness. Additional interest in this superfamily solidified following the discovery that genetic polymorphisms in BTNL2 are associated with predisposition to many human diseases. In this review, we will provide an overview of the members comprising the butyrophilin superfamily of molecules. We will then discuss BTNL2 immunomodulatory function, and BTNL2 structural associations with other costimulatory molecules. We will then draw your attention to some of the lesser-known butyrophilin superfamily members by describing the expression patterns of these molecules in human tissues and cells. And we will finish by hypothesizing on the potential influence on general immune homeostasis that might be mediated by this, thus-far little-studied, family of molecules.

Limited differential expression of miRNAs and other small RNAs in LPS-stimulated human monocytes.

Monocytes are a distinct subset of myeloid cells with diverse functions in early inflammatory immune modulation. While previous studies have surveyed the role of miRNA regulation on different myeloid cell lines and primary cultures, the time-dependent kinetics of inflammatory stimulation on miRNA expression and the relationship between miRNA-to-target RNA expression have not been comprehensively profiled in monocytes. In this study, we use next-generation sequencing and RT-PCR assays to analyze the non-coding small RNA transcriptome of unstimulated and lipopolysaccharide (LPS)-stimulated monocytes at 6 and 24 hours. We identified a miRNA signature consisting of five mature miRNAs (hsa-mir-146a, hsa-mir-155, hsa-mir-9, hsa-mir-147b, and hsa-mir-193a) upregulated by LPS-stimulated monocytes after 6 hours and found that most miRNAs were also upregulated after 24 hours of stimulation. Only one miRNA gene was down-regulated and no other small RNAs were found dysregulated in monocytes after LPS treatment. In addition, novel tRNA-derived fragments were also discovered in monocytes although none showed significant changes upon LPS stimulation. Interrogation of validated miRNA targets by transcriptomic analysis revealed that absolute expression of most miRNA targets implicating in innate immune response decreased over time in LPS-stimulated monocytes although their expression patterns along the treatment were heterogeneous. Our findings reveal a potential role by which selective miRNA upregulation and stable expression of other small RNAs enable monocytes to develop finely tuned cellular responses during acute inflammation.

Lymphotoxin beta receptor (Lt betaR): dual roles in demyelination and remyelination and successful therapeutic intervention using Lt betaR-Ig protein.

Inflammation mediated by macrophages is increasingly found to play a central role in diseases and disorders that affect a myriad of organs, prominent among these are diseases of the CNS. The neurotoxicant-induced, cuprizone model of demyelination is ideally suited for the analysis of inflammatory events. Demyelination on exposure to cuprizone is accompanied by predictable microglial activation and astrogliosis, and, after cuprizone withdrawal, this activation reproducibly diminishes during remyelination. This study demonstrates enhanced expression of lymphotoxin beta receptor (Lt betaR) during the demyelination phase of this model, and Lt betaR is found in areas enriched with microglial and astroglial cells. Deletion of the Lt betaR gene (Lt betaR-/-) resulted in a significant delay in demyelination but also a slight delay in remyelination. Inhibition of Lt betaR signaling by an Lt betaR-Ig fusion decoy protein successfully delayed demyelination in wild-type mice. Unexpectedly, this Lt betaR-Ig decoy protein dramatically accelerated the rate of remyelination, even after the maximal pathological disease state had been reached. This strongly indicates the beneficial role of Lt betaR-Ig in the delay of demyelination and the acceleration of remyelination. The discrepancy between remyelination rates in these systems could be attributed to developmental abnormalities in the immune systems of Lt betaR-/- mice. These findings bode well for the use of an inhibitory Lt betaR-Ig as a candidate biological therapy in demyelinating disorders, because it is beneficial during both demyelination and remyelination.

Considerations for the sensible use of rodent models of inflammatory disease in predicting efficacy of new biological therapeutics in the clinic.

Successful therapeutics for treating autoimmune and inflammatory diseases must be able to significantly dampen, and ideally reverse, the complex processes involved in the manifestation of inflammatory pathology in intact tissues and organs. Studies on human cells and tissues - both normal and diseased - are obviously critical for moving forward with a particular therapeutic strategy, but these types of studies are oftentimes limited in their complexity and usually fail to fully replicate the biology of the intact inflammatory environment and disease process. It is for this reason that development of a new drug generally relies on data generated from in vivo animal models that have been created to mimic aspects of the complex disease process in whole organs and whole animals. Although the intact animal model of disease provides the opportunity for key elements involved in inflammatory processes to be investigated in natural surroundings, the primary trigger for inflammatory activation in animal models is, by necessity, artificial and, of course, differs from the natural pathogenesis driving disease in humans. Despite the artificial way of inducing inflammatory responses, animal models of disease have proven invaluable for providing insight into the potential efficacy of new drugs, particularly when careful consideration has been given to ensure that the model system under study resembles the inflammatory pathway expected in human disease. The most common artificial approaches for stimulating inflammatory diseases in mice are quite varied, and range from overexpression or targeted deletion of genes in transgenic or knockout animals, immunization of animals with putative autoantigens, all the way to synthetic, chemical challenges. None of these artificial systems or triggers is wholly perfect at mimicking the complexity of human autoimmune and inflammatory diseases, but animal disease model data is an important, and very necessary, step in the path of drug development. This review will focus on the critical aspects of disease modeling in animals that should be considered when embarking on drug discovery programs, with particular attention on three of the major inflammatory diseases - rheumatoid arthritis, multiple sclerosis and asthma. We will discuss the use of rodent models in predicting the outcomes of currently approved medicines with a focus on biological therapeutics, and will highlight ongoing clinical trials where there appears to be strong correlation between animal models and the initial indication of clinical efficacy.

Predator-induced phenotypic plasticity of shape and behavior: parallel and unique patterns across sexes and species.

Phenotypic plasticity is often an adaptation of organisms to cope with temporally or spatially heterogenous landscapes. Like other adaptations, one would predict that different species, populations, or sexes might thus show some degree of parallel evolution of plasticity, in the form of parallel reaction norms, when exposed to analogous environmental gradients. Indeed, one might even expect parallelism of plasticity to repeatedly evolve in multiple traits responding to the same gradient, resulting in integrated parallelism of plasticity. In this study, we experimentally tested for parallel patterns of predator-mediated plasticity of size, shape, and behavior of 2 species and sexes of mosquitofish. Examination of behavioral trials indicated that the 2 species showed unique patterns of behavioral plasticity, whereas the 2 sexes in each species showed parallel responses. Fish shape showed parallel patterns of plasticity for both sexes and species, albeit males showed evidence of unique plasticity related to reproductive anatomy. Moreover, patterns of shape plasticity due to predator exposure were broadly parallel to what has been depicted for predator-mediated population divergence in other studies (slender bodies, expanded caudal regions, ventrally located eyes, and reduced male gonopodia). We did not find evidence of phenotypic plasticity in fish size for either species or sex. Hence, our findings support broadly integrated parallelism of plasticity for sexes within species and less integrated parallelism for species. We interpret these findings with respect to their potential broader implications for the interacting roles of adaptation and constraint in the evolutionary origins of parallelism of plasticity in general.

Functional genomic analysis of remyelination reveals importance of inflammation in oligodendrocyte regeneration.

Tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, was shown previously to promote remyelination and oligodendrocyte precursor proliferation in a murine model for demyelination and remyelination. We used Affymetrix microarrays in this study to identify (1) changes in gene expression that accompany demyelination versus remyelination and (2) changes in gene expression during the successful remyelination of wild-type mice versus the unsuccessful attempts in mice lacking TNFalpha. Alterations in inflammatory genes represented the most prominent changes, with major histocompatibility complex (MHC) genes dramatically enhanced in microglia and astrocytes during demyelination, remyelination, and as a consequence of TNFalpha stimulation. Studies to examine the roles of these genes in remyelination were then performed using mice lacking specific genes identified by the microarray. Analysis of MHC-II-null mice showed delayed remyelination and regeneration of oligodendrocytes, whereas removal of MHC-I had little effect. These data point to the induction of MHC-II by TNFalpha as an important regulatory event in remyelination and emphasize the active inflammatory response in regeneration after pathology in the brain.

Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy.

Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.

Immune modulation by butyrophilins.

The B7 family of co-stimulatory molecules has an important role in driving the activation and inhibition of immune cells. Evolving data have shown that a related family of molecules - the butyrophilins - have similar immunomodulatory functions to B7 family members and may represent a novel subset of co-stimulatory molecules. These studies have taken the field by surprise, as the butyrophilins were previously thought to only be important in lactation and milk production. In this Review, we describe the expression patterns of the various members of the butyrophilin family and explore their immunomodulatory functions. In particular, we emphasize the contribution of butyrophilins to immune homeostasis and discuss the potential of targeting these molecules for therapeutic purposes.

Optimization of a Potent, Orally Active S1P1 Agonist Containing a Quinolinone Core.

The optimization of a series of S1P1 agonists with limited activity against S1P3 is reported. A polar headgroup was used to improve the physicochemical and pharmacokinetic parameters of lead quinolinone 6. When dosed orally at 1 and 3 mg/kg, the azahydroxymethyl analogue 22 achieved statistically significant lowering of circulating blood lymphocytes 24 h postdose. In rats, a dose-proportional increase in exposure was measured when 22 was dosed orally at 2 and 100 mg/kg.

4-Methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide: A Potent and Selective Agonist of S1P1.

The sphingosine-1-phosphate-1 receptor (S1P1) and its endogenous ligand sphingosine-1-phosphate (S1P) cooperatively regulate lymphocyte trafficking from the lymphatic system. Herein, we disclose 4-methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide (8), an uncommon example of a synthetic S1P1 agonist lacking a polar headgroup, which is shown to effect dramatic reduction of circulating lymphocytes (POC = -78%) in rat 24 h after a single oral dose (1 mg/kg). The excellent potency that 8 exhibits toward S1P1 (EC50 = 0.035 µM, 96% efficacy) and the >100-fold selectivity that it displays against receptor subtypes S1P2-5 suggest that it may serve as a valuable tool to understand the clinical relevance of selective S1P1 agonism.