RESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-ß signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-ß signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1ß as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Dependovirus , Fibrose Pulmonar Idiopática , Lisofosfolipídeos , Esfingosina/análogos & derivados , Humanos , Camundongos , Animais , Dependovirus/genética , Pulmão/metabolismo , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/terapia , Fibrose Pulmonar Idiopática/metabolismo , Bleomicina , Modelos Animais , Terapia Genética , Aldeído Liases/genética , Aldeído Liases/metabolismoRESUMO
GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal ß-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual ß-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack ß-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3's role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3's differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients.
Assuntos
Gangliosidose GM1 , Animais , Humanos , Camundongos , beta-Galactosidase/genética , beta-Galactosidase/metabolismo , beta-Galactosidase/uso terapêutico , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M1)/uso terapêutico , Gangliosidose GM1/genética , Glicolipídeos , Neuraminidase/genética , Neuraminidase/uso terapêuticoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to multiple organ systems, including the brain. SARS-CoV-2's neuropathological mechanisms may include systemic inflammation and hypoxia, as well as direct cell damage resulting from viral infections of neurons and glia. How the virus directly causes injury to brain cells, acutely and over the long term, is not well understood. In order to gain insight into this process, we studied the neuropathological effects of open reading frame 3a (ORF3a), a SARS-CoV-2 accessory protein that is a key pathological factor of the virus. Forced ORF3a brain expression in mice caused the rapid onset of neurological impairment, neurodegeneration, and neuroinflammation-key neuropathological features found in coronavirus disease (COVID-19, which is caused by SARS-CoV-2 infection). Furthermore, ORF3a expression blocked autophagy progression in the brain and caused the neuronal accumulation of α-synuclein and glycosphingolipids, all of which are linked to neurodegenerative disease. Studies with ORF3-expressing HeLa cells confirmed that ORF3a disrupted the autophagy-lysosomal pathway and blocked glycosphingolipid degradation, resulting in their accumulation. These findings indicate that, in the event of neuroinvasion by SARS-CoV-2, ORF3a expression in brain cells may drive neuropathogenesis and be an important mediator of both short- and long-term neurological manifestations of COVID-19.
Assuntos
COVID-19 , Doenças Neurodegenerativas , Animais , Humanos , Camundongos , Autofagia , Encéfalo/patologia , COVID-19/patologia , Células HeLa , Homeostase , Lisossomos , Doenças Neurodegenerativas/patologia , Fases de Leitura Aberta , SARS-CoV-2 , EsfingolipídeosRESUMO
Sphingolipids, which function as plasma membrane lipids and signaling molecules, are highly enriched in neuronal and myelin membranes in the nervous system. They are degraded in lysosomes by a defined sequence of enzymatic steps. In the related group of disorders, the sphingolipidoses, mutations in the genes that encode the individual degradative enzymes cause lysosomal accumulation of sphingolipids and often result in severe neurodegenerative disease. Here we review the information indicating that microglia, which actively clear sphingolipid-rich membranes in the brain during development and homeostasis, are directly affected by these mutations and promote neurodegeneration in the sphingolipidoses. We also identify parallels between the sphingolipidoses and more common forms of neurodegeneration, which both exhibit evidence of defective sphingolipid clearance in the nervous system.
Assuntos
Microglia/metabolismo , Mutação , Doenças Neurodegenerativas , Transdução de Sinais , Esfingolipidoses , Esfingolipídeos , Animais , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Esfingolipidoses/genética , Esfingolipidoses/metabolismo , Esfingolipídeos/genética , Esfingolipídeos/metabolismoRESUMO
Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.
Assuntos
Proteínas de Membrana/genética , Bainha de Mielina/genética , Esfingolipídeos/genética , Animais , Ceramidas/genética , Células HeLa , Humanos , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Transdução de Sinais/genética , Esfingolipídeos/biossínteseRESUMO
GM1 gangliosidosis is an autosomal recessive neurodegenerative disorder caused by the deficiency of lysosomal ß-galactosidase (ß-gal) and resulting in accumulation of GM1 ganglioside. The disease spectrum ranges from infantile to late onset and is uniformly fatal, with no effective therapy currently available. Although animal models have been useful for understanding disease pathogenesis and exploring therapeutic targets, no relevant human central nervous system (CNS) model system has been available to study its early pathogenic events or test therapies. To develop a model of human GM1 gangliosidosis in the CNS, we employed CRISPR/Cas9 genome editing to target GLB1 exons 2 and 6, common sites for mutations in patients, to create isogenic induced pluripotent stem (iPS) cell lines with lysosomal ß-gal deficiency. We screened for clones with <5% of parental cell line ß-gal enzyme activity and confirmed GLB1 knockout clones using DNA sequencing. We then generated GLB1 knockout cerebral organoids from one of these GLB1 knockout iPS cell clones. Analysis of GLB1 knockout organoids in culture revealed progressive accumulation of GM1 ganglioside. GLB1 knockout organoids microinjected with AAV9-GLB1 vector showed a significant increase in ß-gal activity and a significant reduction in GM1 ganglioside content compared with AAV9-GFP-injected organoids, demonstrating the efficacy of an AAV9 gene therapy-based approach in GM1 gangliosidosis. This proof-of-concept in a human cerebral organoid model completes the pre-clinical studies to advance to clinical trials using the AAV9-GLB1 vector.
RESUMO
The bioactive lipid mediator sphingosine 1-phosphate (S1P) was recently assigned critical roles in platelet biology: whereas S1P1 receptor-mediated S1P gradient sensing was reported to be essential for directing proplatelet extensions from megakaryocytes (MKs) toward bone marrow sinusoids, MK sphingosine kinase 2 (Sphk2)-derived S1P was reported to further promote platelet shedding through receptor-independent intracellular actions, and platelet aggregation through S1P1 Yet clinical use of S1P pathway modulators including fingolimod has not been associated with risk of bleeding or thrombosis. We therefore revisited the role of S1P in platelet biology in mice. Surprisingly, no reduction in platelet counts was observed when the vascular S1P gradient was ablated by impairing S1P provision to plasma or S1P degradation in interstitial fluids, nor when gradient sensing was impaired by S1pr1 deletion selectively in MKs. Moreover, S1P1 expression and signaling were both undetectable in mature MKs in situ, and MK S1pr1 deletion did not affect platelet aggregation or spreading. When S1pr1 deletion was induced in hematopoietic progenitor cells, platelet counts were instead significantly elevated. Isolated global Sphk2 deficiency was associated with thrombocytopenia, but this was not replicated by MK-restricted Sphk2 deletion and was reversed by compound deletion of either Sphk1 or S1pr2, suggesting that this phenotype arises from increased S1P export and S1P2 activation secondary to redistribution of sphingosine to Sphk1. Consistent with clinical observations, we thus observe no essential role for S1P1 in facilitating platelet production or activation. Instead, S1P restricts megakaryopoiesis through S1P1, and can further suppress thrombopoiesis through S1P2 when aberrantly secreted in the hematopoietic niche.
Assuntos
Plaquetas/metabolismo , Lisofosfolipídeos/metabolismo , Megacariócitos/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Nicho de Células-Tronco , Trombopoese , Animais , Plaquetas/citologia , Lisofosfolipídeos/genética , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Esfingosina/genética , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMO
Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of ß-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses.
Assuntos
Diferenciação Celular , Córtex Cerebral/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios/patologia , Organoides/patologia , Doença de Sandhoff/patologia , Proliferação de Células , Células Cultivadas , Humanos , Lisossomos/metabolismo , beta-N-Acetil-Hexosaminidases/deficiência , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Sphingolipids are a diverse class of essential cellular lipids that function as structural membrane components and as signaling molecules. Cells acquire sphingolipids by both de novo biosynthesis and recycling of exogenous sphingolipids. The individual importance of these pathways for the generation of essential sphingolipids in differentiated cells is not well understood. To investigate the requirement for de novo sphingolipid biosynthesis in adipocytes, a cell type with highly regulated lipid metabolism, we generated mice with an adipocyte-specific deletion of Sptlc1 Sptlc1 is an obligate subunit of serine palmitoyltransferase, the enzyme responsible for the first and rate-limiting step of de novo sphingolipid biosynthesis. These mice, which initially developed adipose tissue, exhibited a striking age-dependent loss of adipose tissue accompanied by evidence of adipocyte death, increased macrophage infiltration, and tissue fibrosis. Adipocyte differentiation was not affected by the Sptlc1 deletion. The mice also had elevated fasting blood glucose, fatty liver, and insulin resistance. Collectively, these data indicate that de novo sphingolipid biosynthesis is required for adipocyte cell viability and normal metabolic function and that reduced de novo sphingolipid biosynthesis within adipocytes is associated with adipocyte death, adipose tissue remodeling, and metabolic dysfunction.
Assuntos
Adipócitos/citologia , Homeostase , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/biossíntese , Adiposidade , Animais , Diferenciação Celular , Sobrevivência Celular , Deleção de Genes , Inflamação , Resistência à Insulina , Metabolismo dos Lipídeos , Lipogênese , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Serina C-Palmitoiltransferase/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1(-/-) mice, Sgpp2(-/-) mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2(-/-) mice had normal pancreatic islet size; however, they exhibited defective adaptive ß-cell proliferation that was demonstrated after treatment with either a high-fat diet or the ß-cell-specific toxin, streptozotocin. Importantly, ß-cells from untreated Sgpp2(-/-) mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with ß-cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes ß-cell endoplasmic reticulum stress, which is a known cause of ß-cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes.
Assuntos
Proliferação de Células/genética , Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/genética , Monoéster Fosfórico Hidrolases/genética , Animais , Dieta Hiperlipídica , Chaperona BiP do Retículo Endoplasmático , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico , Humanos , Imuno-Histoquímica , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoéster Fosfórico Hidrolases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingolipídeos/metabolismo , Estreptozocina/farmacologiaRESUMO
Both genes and the environment are determinants in the susceptibility to allergies and may alter the severity of the disease. We explored whether an increase in the levels of the lipid mediator S1P in vivo, a condition found during allergic asthma, could affect the sensitivity or the response of MCs to IgE/Ag and the onset of allergic disease. We found that increasing S1P levels by genetic deletion of S1P lyase, the enzyme catabolizing S1P, led to elevated activity of circulating tryptase. Accordingly, MCs of S1P lyase-deficient mice were mostly degranulated in the tissues and showed enhanced calcium levels, degranulation, and cytokine production in response to IgE/Ag in vitro. Th 1-skewed mice (C57BL/6) had lower levels of S1P in circulation and histamine responses than did Th 2-skewed (129/Sv) mice. However, when S1P levels were increased by pharmacologic inhibition of S1P lyase, the C57BL/6 mice showed increased histamine release into the circulation and anaphylactic responses similar to those in the 129/Sv mice. Culturing of MCs in the presence of S1P enhanced their degranulation responses, and when the S1P-treated MCs were used to reconstitute MC-deficient (Kit(W-sh)) mice, they caused enhanced anaphylaxis. Gene expression arrays in S1P lyase-deficient MCs and MCs treated with S1P continuously revealed increased expression of numerous genes, including the adhesion molecule CNTN4,which contributed to the enhanced responses. Our findings argue that dysregulation in the metabolism of S1P is a contributing factor in modulating MC responsiveness and the allergic response.
Assuntos
Contactinas/fisiologia , Hipersensibilidade/etiologia , Lisofosfolipídeos/fisiologia , Mastócitos/fisiologia , Esfingosina/análogos & derivados , Animais , Cálcio/metabolismo , Liberação de Histamina , Camundongos , Camundongos Endogâmicos C57BL , Esfingosina/fisiologia , TranscriptomaRESUMO
Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphorylated by the actions of two S1P-specific phosphatases, sphingosine-1-phosphate phosphatases 1 and 2. To identify the physiological functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1(-/-) mice appeared normal at birth, but during the 1st week of life they exhibited stunted growth and suffered desquamation, with most dying before weaning. Both Sgpp1(-/-) pups and surviving adults exhibited multiple epidermal abnormalities. Interestingly, the epidermal permeability barrier developed normally during embryogenesis in Sgpp1(-/-) mice. Keratinocytes isolated from the skin of Sgpp1(-/-) pups had increased intracellular S1P levels and displayed a gene expression profile that indicated overexpression of genes associated with keratinocyte differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis.
Assuntos
Diferenciação Celular/genética , Epiderme/metabolismo , Queratinócitos/metabolismo , Proteínas de Membrana/genética , Monoéster Fosfórico Hidrolases/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Análise por Conglomerados , Epiderme/embriologia , Epiderme/crescimento & desenvolvimento , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/genética , Queratinócitos/citologia , Lisofosfolipídeos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Monoéster Fosfórico Hidrolases/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/embriologia , Pele/crescimento & desenvolvimento , Pele/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de TempoRESUMO
Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.
Assuntos
Aldeído Liases , Movimento Celular/imunologia , Lisofosfolipídeos/metabolismo , Neutrófilos , Transdução de Sinais/imunologia , Esfingosina/análogos & derivados , Proteínas de Fase Aguda/imunologia , Aldeído Liases/genética , Aldeído Liases/imunologia , Aldeído Liases/metabolismo , Animais , Biomarcadores/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Hematopoese/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Lisofosfolipídeos/imunologia , Camundongos , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Esfingosina/imunologia , Esfingosina/metabolismoRESUMO
S1P1 receptor expression is required for the egress of newly formed T cells from the thymus and exit of mature T and B cells from secondary lymphoid organs. In this study, we deleted the expression of the S1P1 receptor gene (S1pr1) in developing B cells in the bone marrow. Although B cell maturation within the bone marrow was largely normal in the B cell-specific S1pr1 knockout (B-S1pr1KO) mice, their newly generated immature B cells appeared in the blood at abnormally low numbers as compared with control mice. In the bone marrow of B-S1pr1KO mice, immature B cells in contact with the vascular compartment displayed increased apoptosis as compared with control mice. Forced expression of CD69, a negative regulator of S1P1 receptor expression, in developing bone marrow B cells also reduced the number of immature B cells in the blood. Attenuation of CXCR4 signaling, which is required for the proper retention of developing B cells in bone marrow, did not release immature B cells into the blood of B-S1pr1KO mice as effectively as in control mice. Our results indicate that the S1P1 receptor provides a signal necessary for the efficient transfer of newly generated immature B cells from the bone marrow to the blood.
Assuntos
Medula Óssea/imunologia , Células Precursoras de Linfócitos B/imunologia , Receptores de Lisoesfingolipídeo/genética , Animais , Apoptose , Deleção de Genes , Regulação da Expressão Gênica , Homeostase , Integrases/genética , Camundongos , Camundongos Knockout , Células Precursoras de Linfócitos B/fisiologia , Receptores de Lisoesfingolipídeo/deficiência , Receptores de Lisoesfingolipídeo/fisiologia , Receptores de Esfingosina-1-Fosfato , Baço/imunologia , Linfócitos T/imunologia , Timo/imunologiaRESUMO
Although much is known about the migration of T cells from blood to lymph nodes, less is known about the mechanisms regulating the migration of T cells from tissues into lymph nodes through afferent lymphatics. Here we investigated T cell egress from nonlymphoid tissues into afferent lymph in vivo and developed an experimental model to recapitulate this process in vitro. Agonism of sphingosine 1-phosphate receptor 1 inhibited the entry of tissue T cells into afferent lymphatics in homeostatic and inflammatory conditions and caused the arrest, mediated at least partially by interactions of the integrin LFA-1 with its ligand ICAM-1 and of the integrin VLA-4 with its ligand VCAM-1, of polarized T cells at the basal surface of lymphatic but not blood vessel endothelium. Thus, the increased sphingosine 1-phosphate present in inflamed peripheral tissues may induce T cell retention and suppress T cell egress.
Assuntos
Vasos Linfáticos/imunologia , Lisofosfolipídeos/metabolismo , Modelos Imunológicos , Receptores de Lisoesfingolipídeo/imunologia , Esfingosina/análogos & derivados , Linfócitos T/imunologia , Animais , Movimento Celular , Endotélio Linfático/imunologia , Cloridrato de Fingolimode , Homeostase , Inflamação/imunologia , Integrina alfa4beta1/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Linfonodos/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Propilenoglicóis/farmacologia , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Transdução de Sinais , Esfingosina/metabolismo , Esfingosina/farmacologia , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
The S1P1 receptor, on the surface of lymphocytes and endothelial cells, regulates the unique trafficking behavior of certain lymphocyte populations. We have examined whether the S1P1 receptor also dictates the distinctive tissue distribution of V alpha14-J alpha18 natural killer T (NKT) cells, whose trafficking pattern is not well understood. Mice (TCS1P1 KO) were established with a conditional deletion of the S1P1 receptor in thymocytes that included precursors of NKT cells. Within the thymus, NKT cells were found at normal or increased levels, indicating that S1P1 receptor expression was dispensable for NKT cell development. However, substantially reduced numbers of NKT cells were detected in the peripheral tissues of the TCS1P1 KO mice. Short-term S1P1 deletion after NKT cells had established residence in the periphery did not substantially alter their distribution in tissues, except for a partial decrease in the spleen. FTY720, a S1P1 receptor ligand that has potent effects on the trafficking of conventional T cells, did not alter the preexisting distribution of NKT cells within peripheral tissues of wild-type mice. Our results indicate that the S1P1 receptor expression on NKT cells is dispensable for development within thymus but is essential for the establishment of their tissue residency in the periphery.
Assuntos
Células Matadoras Naturais/citologia , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/fisiologia , Transdução de SinaisRESUMO
After induction in secondary lymphoid organs, a subset of antibody-secreting cells (ASCs) homes to the bone marrow (BM) and contributes to long-term antibody production. The factors determining secondary lymphoid organ residence versus BM tropism have been unclear. Here we demonstrate that in mice treated with FTY720 or that lack sphingosine-1-phosphate (S1P) receptor-1 (S1P1) in B cells, IgG ASCs are induced and localize normally in secondary lymphoid organs but they are reduced in numbers in blood and BM. Many IgG ASCs home to BM on day 3 of the secondary response and day 3 splenic ASCs exhibit S1P responsiveness, whereas the cells remaining at day 5 are unable to respond. S1P1 mRNA abundance is higher in ASCs isolated from blood compared to spleen, whereas CXCR4 expression is lower. Blood ASCs also express higher amounts of Kruppel-like factor (KLF)2, a regulator of S1P1 gene expression. These findings establish an essential role for S1P1 in IgG plasma cell homing and they suggest that differential regulation of S1P1 expression in differentiating plasma cells may determine whether they remain in secondary lymphoid organs or home to BM.
Assuntos
Células da Medula Óssea/citologia , Movimento Celular/imunologia , Tecido Linfoide/metabolismo , Lisofosfolipídeos/biossíntese , Plasmócitos/metabolismo , Receptores de Lisoesfingolipídeo/biossíntese , Esfingosina/análogos & derivados , Animais , Células Produtoras de Anticorpos/citologia , Células Produtoras de Anticorpos/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Movimento Celular/genética , Imunoglobulina G/biossíntese , Tecido Linfoide/citologia , Lisofosfolipídeos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/citologia , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/fisiologia , Esfingosina/biossíntese , Esfingosina/fisiologiaRESUMO
Complex sphingolipids are abundant as eukaryotic cell membrane components, whereas their metabolites, in particular ceramide, sphingosine, and sphingosine 1-phosphate, are involved in diverse cell signaling processes. In mammals, degradation of ceramide by ceramidase yields sphingosine, which is phosphorylated by the action of sphingosine kinase to generate sphingosine 1-phosphate. Therefore, ceramidases are key enzymes in the regulation of the cellular levels of ceramide, sphingosine, and sphingosine 1-phosphate. To explore the physiological functions of a neutral ceramidase with diverse cellular locations, we disrupted the Asah2 gene in mice. Asah2 null mice have a normal life span and do not show obvious abnormalities or major alterations in total ceramide levels in tissues. The Asah2-encoded neutral ceramidase is highly expressed in the small intestine along the brush border, suggesting that the neutral ceramidase may be involved in a pathway for the digestion of dietary sphingolipids. Indeed, Asah2 null mice were deficient in the intestinal degradation of ceramide. Thus, the results indicate that the Asah2-encoded neutral ceramidase is a key enzyme for the catabolism of dietary sphingolipids and regulates the levels of bioactive sphingolipid metabolites in the intestinal tract.
Assuntos
Amidoidrolases/genética , Amidoidrolases/fisiologia , Alelos , Amidoidrolases/metabolismo , Animais , Northern Blotting , Western Blotting , Membrana Celular/metabolismo , Ceramidases , Ceramidas/química , Ceramidas/metabolismo , Cromatografia Líquida , Vetores Genéticos , Heterozigoto , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Lisofosfolipídeos/química , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Ceramidase Neutra , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray , Esfingolipídeos/química , Esfingolipídeos/metabolismo , Esfingomielinas/química , Esfingosina/análogos & derivados , Esfingosina/química , Fatores de Tempo , Distribuição TecidualRESUMO
Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1-/-tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1-/- mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1-/- mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.
Assuntos
Linfopenia/etiologia , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Propilenoglicóis/toxicidade , Esfingosina/análogos & derivados , Animais , Sequência de Bases , DNA/genética , Cloridrato de Fingolimode , Imunossupressores/metabolismo , Imunossupressores/toxicidade , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , Linfopenia/induzido quimicamente , Linfopenia/enzimologia , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Pró-Fármacos/metabolismo , Pró-Fármacos/toxicidade , Propilenoglicóis/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/metabolismoRESUMO
Angiogenesis, also known as new blood vessel formation, is regulated coordinately with other tissue differentiation events during limb development. Although vascular endothelial cell growth factor (VEGF) is important in the regulation of angiogenesis, chondrogenesis and osteogenesis during limb development, the role of other angiogenic factors is not well understood. Sphingosine 1-phosphate, a platelet-derived lipid mediator, regulates angiogenesis and vascular maturation via its action on the G-protein-coupled receptor S1P(1) (also known as EDG-1). In addition to vascular defects, abnormal limb development was also observed in S1p(1)(-/-) mice. Here we show that strong induction of S1P(1) expression is observed in the blood vessels and the interdigital mesenchymal cells during limb development. Deletion of S1P(1) results in aberrant chondrocyte condensation and defective digit morphogenesis. Interestingly, the vasculature in the S1p(1)(-/-) limbs was hyperplastic and morphologically altered. In addition, the hypoxia inducible factor (HIF)-1 alpha and its response gene VEGF were induced in S1p(1)(-/-) limbs. However, aberrant regulation of HIF-1 alpha and VEGF were not observed in embryonic fibroblasts derived from S1p(1)(-/-) mice, suggesting a non-cell autonomous effect of S1P(1) on VEGF expression. Indeed, similar limb defects were observed in endothelium-specific S1P(1) null mice in vivo. These data suggest that the function of S1P(1) in the developing vasculature is essential for proper limb development.