RESUMEN
Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite that regulates numerous processes important for immune responses. S1P is made within cells and must be transported out of cells to exert its effects through activation of 5 specific cell surface GPCRs in an autocrine or paracrine fashion. Spinster 2 (Spns2) transports S1P out of cells, and its deletion in mice reduces circulating levels of S1P, alters immune cell trafficking, and induces lymphopenia. Here we examined the effects of Spns2 deletion on adaptive immune responses and in autoimmune disease models. Airway inflammation and hypersensitivity as well as delayed-type contact hypersensitivity were attenuated in Spns2(-/-) mice. Similarly, Spns2 deletion reduced dextran sodium sulfate- and oxazolone-induced colitis. Intriguingly, Spns2(-/-) mice were protected from the development of experimental autoimmune encephalopathy, a model of the autoimmune disease multiple sclerosis. Deletion of Spns2 also strongly alleviated disease development in collagen-induced arthritis. These results point to a broad role for Spns2-mediated S1P transport in the initiation and development of adaptive immune related disorders.
Asunto(s)
Proteínas de Transporte de Anión/fisiología , Enfermedades Autoinmunes/fisiopatología , Inflamación/fisiopatología , Animales , Proteínas de Transporte de Anión/genética , Modelos Animales de Enfermedad , Ratones , Ratones NoqueadosRESUMEN
Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.
Asunto(s)
Aldehído-Liasas/antagonistas & inhibidores , Antirreumáticos/síntesis química , Imidazoles/síntesis química , Isoxazoles/síntesis química , Oximas/síntesis química , Aldehído-Liasas/genética , Animales , Antirreumáticos/farmacocinética , Antirreumáticos/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Experimental/patología , Presión Sanguínea/efectos de los fármacos , Movimiento Celular , Perros , Frecuencia Cardíaca/efectos de los fármacos , Imidazoles/farmacocinética , Imidazoles/farmacología , Isoxazoles/farmacocinética , Isoxazoles/farmacología , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Oximas/farmacocinética , Oximas/farmacología , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: S1PL is an aldehyde-lyase that irreversibly cleaves sphingosine 1-phosphate (S1P) in the terminal step of sphingolipid catabolism. Because S1P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments. METHODOLOGY: In order to better understand the function of S1PL in this regulatory pathway, we assessed the in vivo effects of different levels of S1PL activity using knockout (KO) and humanized mouse models. PRINCIPAL FINDINGS: Our analysis showed that all S1PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S1PL KO mice (S1PL(-/-)) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S1PL(H/-)) or two alleles (S1PL(H/H)) of human S1PL expressed less than 10 and 20% of normal S1PL activity, respectively. This partial restoration of S1PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S1PL(-/-) mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S1PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S1PL activity affected T-cell egress more than differentiation. SIGNIFICANCE: These findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S1PL activity and suggest that there is a window in which partial inhibition of S1PL could produce therapeutic levels of immunosuppression without causing clinically significant S1P-related lesions in non-lymphoid target organs.
Asunto(s)
Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/metabolismo , Sistema Inmunológico/fisiología , Aldehído-Liasas/genética , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Hematopoyesis/fisiología , Humanos , Linfopenia/inmunología , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Bazo/citología , Bazo/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Timo/citología , Timo/inmunologíaRESUMEN
During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.
Asunto(s)
Aldehído-Liasas/antagonistas & inhibidores , Enfermedades Autoinmunes/tratamiento farmacológico , Imidazoles/farmacología , Administración Oral , Animales , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Imidazoles/administración & dosificación , Imidazoles/uso terapéutico , Recuento de Linfocitos , Ratones , Relación Estructura-ActividadRESUMEN
We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.