RESUMEN
The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019.
Asunto(s)
Ambroxol/farmacología , Antivirales/farmacología , SARS-CoV-2/efectos de los fármacos , Esfingomielina Fosfodiesterasa/genética , Vesiculovirus/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Administración por Inhalación , Animales , Transporte Biológico , Ceramidas/metabolismo , Chlorocebus aethiops , Reposicionamiento de Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/virología , Expectorantes , Expresión Génica , Humanos , Cultivo Primario de Células , Virus Reordenados/efectos de los fármacos , Virus Reordenados/fisiología , SARS-CoV-2/fisiología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Vesiculovirus/fisiologíaRESUMEN
Most patients with cystic fibrosis (CF) suffer from acute and chronic pulmonary infections with bacterial pathogens, which often determine their life quality and expectancy. Previous studies have demonstrated a downregulation of the acid ceramidase in CF epithelial cells resulting in an increase of ceramide and a decrease of sphingosine. Sphingosine kills many bacterial pathogens, and the downregulation of sphingosine seems to determine the infection susceptibility of cystic fibrosis mice and patients. It is presently unknown how deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) connects to a marked downregulation of the acid ceramidase in human and murine CF epithelial cells. Here, we employed quantitative PCR, western blot analysis, and enzyme activity measurements to study the role of IRF8 for acid ceramidase regulation. We report that genetic deficiency or functional inhibition of CFTR/Cftr results in an upregulation of interferon regulatory factor 8 (IRF8) and a concomitant downregulation of acid ceramidase expression with CF and an increase of ceramide and a reduction of sphingosine levels in tracheal and bronchial epithelial cells from both human individuals or mice. CRISPR/Cas9- or siRNA-mediated downregulation of IRF8 prevented changes of acid ceramidase, ceramide, and sphingosine in CF epithelial cells and restored resistance to Pseudomonas aeruginosa infections, which is one of the most important and common pathogens in lung infection of patients with CF. These studies indicate that CFTR deficiency causes a downregulation of acid ceramidase via upregulation of IRF8, which is a central pathway to control infection susceptibility of CF cells.
Asunto(s)
Ceramidasa Ácida/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Células Epiteliales/microbiología , Factores Reguladores del Interferón/metabolismo , Pulmón/microbiología , Infecciones por Pseudomonas/microbiología , Ceramidasa Ácida/genética , Animales , Ceramidas/metabolismo , Fibrosis Quística/inmunología , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Factores Reguladores del Interferón/genética , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Noqueados , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/aislamiento & purificación , Esfingosina/metabolismoRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world's population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers (i.e., IL-6, TNFα, CCL2 and CXCL10). It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Animales , Ratones , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Ceramidas , Modelos Animales de Enfermedad , Fluoxetina/farmacología , Fluoxetina/uso terapéuticoRESUMEN
Sphingosine is a long-chain sphingoid base that has been shown to have bactericidal activity against many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli We have previously demonstrated that sphingosine is present in nasal, tracheal, and bronchial epithelial cells and constitutes a central element of the defense of the airways against bacterial pathogens. Here, using assorted lipid-binding and cell biology assays, we demonstrate that exposing P. aeruginosa and S. aureus cells to sphingosine results in a very rapid, i.e. within minutes, permeabilization of the bacterial plasma membrane, resulting in leakiness of the bacterial cells, loss of ATP, and loss of bacterial metabolic activity. These alterations rapidly induced bacterial death. Mechanistically, we demonstrate that the presence of the protonated NH2 group in sphingosine, which is an amino-alcohol, is required for sphingosine's bactericidal activity. We also show that the protonated NH2 group of sphingosine binds to the highly negatively-charged lipid cardiolipin in bacterial plasma membranes. Of note, this binding was required for bacterial killing by sphingosine, as revealed by genetic experiments indicating that E. coli or P. aeruginosa strains that lack cardiolipin synthase are resistant to sphingosine, both in vitro and in vivo We propose that binding of sphingosine to cardiolipin clusters cardiolipin molecules in the plasma membrane of bacteria. This clustering results in the formation of gel-like or even crystal-like structures in the bacterial plasma membrane and thereby promotes rapid permeabilization of the plasma membrane and bacterial cell death.
Asunto(s)
Antibacterianos/farmacología , Cardiolipinas/metabolismo , Membrana Celular/metabolismo , Escherichia coli/crecimiento & desarrollo , Pseudomonas aeruginosa/crecimiento & desarrollo , Esfingosina/farmacología , Staphylococcus aureus/crecimiento & desarrollo , Cardiolipinas/genética , Membrana Celular/genética , Escherichia coli/genética , Pseudomonas aeruginosa/genética , Staphylococcus aureus/genéticaRESUMEN
Sphingosine has been shown to prevent and eliminate bacterial infections of the respiratory tract, but it is unknown whether sphingosine can be also employed to prevent viral infections. To test this hypothesis, we analyzed whether sphingosine regulates the infection of cultured and freshly isolated ex vivo human epithelial cells with pseudoviral particles expressing SARS-CoV-2 spike (pp-VSV-SARS-CoV-2 spike) that served as a bona fide system mimicking SARS-CoV-2 infection. We demonstrate that exogenously applied sphingosine suspended in 0.9% NaCl prevents cellular infection with pp-SARS-CoV-2 spike. Pretreatment of cultured Vero epithelial cells or freshly isolated human nasal epithelial cells with low concentrations of sphingosine prevented adhesion of and infection with pp-VSV-SARS-CoV-2 spike. Mechanistically, we demonstrate that sphingosine binds to ACE2, the cellular receptor of SARS-CoV-2, and prevents the interaction of the receptor-binding domain of the viral spike protein with ACE2. These data indicate that sphingosine prevents at least some viral infections by interfering with the interaction of the virus with its receptor. Our data also suggest that further preclinical and finally clinical examination of sphingosine is warranted for potential use as a prophylactic or early treatment for coronavirus disease-19.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Esfingosina/farmacología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Humanos , Mucosa Nasal/metabolismo , Mucosa Nasal/virología , Unión Proteica , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Células Vero , Internalización del Virus/efectos de los fármacosRESUMEN
Previous studies have shown that sphingosine kills a variety of pathogenic bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus Sphingosine concentrations are decreased in airway epithelial cells of cystic fibrosis (CF) mice, and this defect has been linked to the infection susceptibility of these mice. Here, we tested whether the genetic overexpression of acid ceramidase rescues cystic fibrosis mice from pulmonary infections with P. aeruginosa We demonstrate that the transgenic overexpression of acid ceramidase in CF mice corresponds to the overexpression of acid ceramidase in bronchial and tracheal epithelial cells and normalizes ceramide and sphingosine levels in bronchial and tracheal epithelial cells. In addition, the expression of ß1-integrin, which is ectopically expressed on the luminal surface of airway epithelial cells in cystic fibrosis mice, an alteration that is very important for mediating pulmonary P. aeruginosa infections in cystic fibrosis, is normalized in cystic fibrosis airways upon the overexpression of acid ceramidase. Most importantly, the overexpression of acid ceramidase protects cystic fibrosis mice from pulmonary P. aeruginosa infections. Infection of CF mice or CF mice that inhaled sphingosine with P. aeruginosa or a P. aeruginosa mutant that is resistant to sphingosine indicates that sphingosine and not a metabolite kills P. aeruginosa upon pulmonary infection. These studies further support the use of acid ceramidase and its metabolite sphingosine as potential treatments of cystic fibrosis.
Asunto(s)
Ceramidasa Ácida/genética , Ceramidasa Ácida/farmacología , Ceramidasa Ácida/uso terapéutico , Fibrosis Quística/complicaciones , Fibrosis Quística/tratamiento farmacológico , Infecciones por Pseudomonas/etiología , Infecciones por Pseudomonas/prevención & control , Animales , Fibrosis Quística/fisiopatología , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones , Modelos Animales , Pseudomonas aeruginosa/efectos de los fármacos , Virulencia/genéticaRESUMEN
Ceramide is a lipid messenger at the heart of sphingolipid metabolism. In concert with its metabolizing enzymes, particularly sphingomyelinases, it has key roles in regulating the physical properties of biological membranes, including the formation of membrane microdomains. Thus, ceramide and its related molecules have been attributed significant roles in nearly all steps of the viral life cycle: they may serve directly as receptors or co-receptors for viral entry, form microdomains that cluster entry receptors and/or enable them to adopt the required conformation or regulate their cell surface expression. Sphingolipids can regulate all forms of viral uptake, often through sphingomyelinase activation, and mediate endosomal escape and intracellular trafficking. Ceramide can be key for the formation of viral replication sites. Sphingomyelinases often mediate the release of new virions from infected cells. Moreover, sphingolipids can contribute to viral-induced apoptosis and morbidity in viral diseases, as well as virus immune evasion. Alpha-galactosylceramide, in particular, also plays a significant role in immune modulation in response to viral infections. This review will discuss the roles of ceramide and its related molecules in the different steps of the viral life cycle. We will also discuss how novel strategies could exploit these for therapeutic benefit.
Asunto(s)
Ceramidas/metabolismo , VIH-1/metabolismo , Virus de la Influenza A/metabolismo , SARS-CoV-2/metabolismo , Virosis/metabolismo , Virosis/virología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Ceramidas/química , Regulación Viral de la Expresión Génica , VIH-1/patogenicidad , Humanos , Inmunomodulación , Virus de la Influenza A/patogenicidad , SARS-CoV-2/patogenicidad , Virión/crecimiento & desarrollo , Virosis/inmunología , Internalización del Virus , Replicación Viral/efectos de los fármacos , Replicación Viral/inmunologíaRESUMEN
Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Línea Celular , Humanos , Lisosomas/metabolismoRESUMEN
BACKGROUND: Allergic diseases and especially allergic asthma are widespread diseases with high prevalence in childhood, but also in adults. Acid sphingomyelinase (ASM) is a key regulator of the sphingolipid pathway. Previous studies defined the association of ASM with the pathogenesis of TH 1-directed lung diseases like cystic fibrosis and acute lung injury. Here, we define the role of ASM in TH 2-regulated allergic bronchial asthma. METHODS: To determine the role of Asm under baseline conditions, wild-type (WT) and Asm-/- mice were ventilated with a flexiVent setup and bronchial hyperresponsiveness was determined using acetylcholine. Flow cytometry and cytokine measurements in bronchoalveolar lavage fluid and lung tissue were followed by in vitro TH 2 differentiations with cells from WT and Asm-/- mice and blockade of Asm with amitriptyline. As proof of principle, we conducted an ovalbumin-induced model of asthma in WT- and Asm-/- mice. RESULTS: At baseline, Asm-/- mice showed better lung mechanics, but unaltered bronchial hyperresponsiveness. Higher numbers of Asm-/- T cells in bronchoalveolar lavage fluid released lower levels of IL-4 and IL-5, and these results were paralleled by decreased production of typical TH 2 cytokines in Asm-/- T lymphocytes in vitro. This phenotype could be imitated by incubation of T cells with amitriptyline. In the ovalbumin asthma model, Asm-/- animals were protected from high disease activity and showed better lung functions and lower levels of eosinophils and TH 2 cytokines. CONCLUSION: Asm deficiency could induce higher numbers of TH 2 cells in the lung, but those cells release decreased TH 2 cytokine levels. Hereby, Asm-/- animals are protected from bronchial asthma, which possibly offers novel therapeutic strategies, for example, with ASM blockade.
Asunto(s)
Asma , Hiperreactividad Bronquial , Animales , Líquido del Lavado Bronquioalveolar , Citocinas , Modelos Animales de Enfermedad , Pulmón , Ratones , Ratones Endogámicos BALB C , Ovalbúmina , Esfingomielina Fosfodiesterasa/genética , Células Th2RESUMEN
BACKGROUND/AIMS: Recent studies indicated that an inhalation treatment of cystic fibrosis mice with acid ceramidase prevents and eliminates infections with Pseudomonas aeruginosa and Stapyhlococcus aureus. Inhalation of acid ceramidase facilitated the elimination of P. aeruginosa in acutely- or chronically-infected mice with cystic fibrosis. Thus, inhalation of acid ceramidase might be a preventive and/or curative treatment for patients with cystic fibrosis suffering from pneumonia. METHODS: We treated cultured epithelial cells or leukemic T-lymphocytes (Jurkat cells) with purified acid ceramidase and determined intracellular signalling events, proliferation and cell survival. Specifically, we measured the activity of AKT, p38-kinase and p70S6-kinase using activation-specific phospho-antibodies in western blot studies. Trypan Blue staining served to analyze proliferation and cell survival. RESULTS: Our studies indicate that treatment of Chang epithelial cells or Jurkat T lymphocytes with purified acid ceramidase results in a dose dependent activation of AKT, p38-kinase and p70S6-kinase, while tyrosine phosphorylation of intracellular proteins remains largely unchanged. Acid ceramidase treatment did not change expression of tight junction proteins such as ZO-1, ZO-2 and occludin. Cellular viability and proliferation were not affected by acid ceramidase treatment. CONCLUSION: Our data suggest that treatment of epithelial cells and lymphocytes with acid ceramidase results in activation of distinct pathways, in particular AKT- and p38K-dependent pathways, while no global activation or cell death was observed.
Asunto(s)
Ceramidasa Ácida/farmacología , Células Epiteliales/metabolismo , Leucemia/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Células Epiteliales/patología , Humanos , Células Jurkat , Leucemia/patologíaRESUMEN
BACKGROUND/AIMS: We have previously shown that inhibition of the mitochondrial Kv1.3 channel results in an initial mitochondrial hyperpolarization and a release of oxygen radicals that mediate mitochondrial depolarization, cytochrome c release and death. Here, we investigated whether inhibition of Kv1.3 channels can also induce cellular resistance mechanisms that counteract the induction of cell death under certain conditions. METHODS: We treated leukemic T cells with the mitochondria-targeted Kv1.3 inhibitor PCARBTP and determined the activity of different kinases associated with cell survival including ZAP70, PI-3-K, AKT, JNK and ERK by measuring the activation-associated phosphorylation of these proteins. Furthermore, we inhibited AKT and JNK and determined the effect of PCARBTP-induced tumor cell death. RESULTS: We demonstrate that treatment of Jurkat T leukemia cells with low doses of the mitochondria-targeted inhibitor of Kv1.3 PCARBTP (0.25 µM or 1 µM) for 10 minutes induced a constitutive phosphorylation/activation of the pro-survival signaling molecules ZAP70, PI-3-K, AKT and JNK, while the phosphorylation/activation of ERK was not affected. Stimulation of Jurkat cells via the TCR/CD3 complex induced an additional activation of a similar pattern of signaling events. Higher doses of the Kv1.3 inhibitor, i.e. 10 µM PCARBTP, reduced the basal phosphorylation/activation of these signaling molecules and also impaired their activation upon stimulation via the TCR/CD3 complex. A low dose of PCARBTP, i.e. 0.25 µM PCARBTP, was almost without any effect on cell death. In contrast, concomitant inhibition of PI-3-K or AKT greatly sensitized Jurkat leukemia cells to the Kv1.3 inhibitor PCARBTP and allowed induction of cell death already at 0.25 µM PCARBTP. CONCLUSION: These studies indicate that Jurkat leukemia cells respond to low doses of the mitochondria-targeted Kv1.3 inhibitor PCARBTP with an activation of survival signals counteracting cell death. Inhibition of these T cell survival signals sensitizes leukemia cells to death induced by mitochondria-targeted Kv1.3 inhibitors. High doses of the Kv1.3 inhibitor inactivate these signals directly permitting death.
Asunto(s)
Apoptosis/efectos de los fármacos , Cumarinas/farmacología , Compuestos Organofosforados/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células Jurkat , Leucemia/metabolismo , Leucemia/patología , Mitocondrias/metabolismo , Fosfatidilinositol 3-Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteína Tirosina Quinasa ZAP-70/antagonistas & inhibidores , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
BACKGROUND/AIMS: Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required. METHODS: Here, we inhaled mini pigs with increasing doses of sphingosine for 10 days and analyzed the uptake of sphingosine into epithelial cells of bronchi as well as into the trachea and lung and the systemic circulation. Moreover, we measured the generation of ceramide and sphingosine 1-phosphate that potentially mediate inflammation, the influx of leukocytes, epithelial cell death and disruption of the epithelial cell barrier. RESULTS: We demonstrate that inhalation of sphingosine results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea, but not in systemic accumulation. Inhaled sphingosine had no side effects up to very high doses. CONCLUSION: In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.
Asunto(s)
Antibacterianos/metabolismo , Esfingosina/metabolismo , Administración por Inhalación , Animales , Antibacterianos/farmacología , Bronquios/metabolismo , Bronquios/patología , Ceramidas/análisis , Humanos , Pulmón/patología , Lisofosfolípidos/análisis , Espectrometría de Masas , Pseudomonas aeruginosa/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/análisis , Esfingosina/farmacología , Staphylococcus aureus/efectos de los fármacos , Porcinos , Porcinos Enanos , Tráquea/metabolismo , Tráquea/patologíaRESUMEN
BACKGROUND/AIMS: Multiple sclerosis (MS) is one of the most common autoimmune disorders of the central nervous system (CNS) and the leading cause of neurological disability among young adults in the Western world. We have previously shown that the acid sphingomyelinase plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: We induced adoptively transferred EAE in wildtype and acid sphingomyelinase-deficient mice. In addition, we immunized mice with MOGaa35-55 to induce active EAE and treated the mice with amitriptyline, a functional inhibitor of the acid sphingomyelinase. We investigated symptoms of EAE, blood-brain barrier integrity and neuroinflammation. RESULTS: In the model of adoptively transferred EAE we demonstrate that expression of acid sphingomyelinase in the recipients rather than on transferred encephalitogenic T cells contributes to the clinical development of EAE symptoms. To test if pharmacological targeting of acid sphingomyelinase can be explored for the development of novel therapies for MS, we inhibited acid sphingomyelinase with amitriptyline in mice in which EAE was induced by active immunization. We demonstrate that pharmacological inhibition of acid sphingomyelinase using amitriptyline protects against the development of EAE and markedly attenuates the characteristic detrimental neuroinflammatory response. CONCLUSION: The studies identify the acid sphingomyelinase as a novel therapeutic target for treating MS patients.
Asunto(s)
Amitriptilina/uso terapéutico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/enzimología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/deficiencia , Inhibidores de Captación Adrenérgica/farmacología , Inhibidores de Captación Adrenérgica/uso terapéutico , Amitriptilina/farmacología , Animales , Encefalomielitis Autoinmune Experimental/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esfingomielina Fosfodiesterasa/genéticaRESUMEN
Major depressive disorder (MDD) is a common and severe disease characterized by mood changes, somatic alterations, and often suicide. MDD is treated with antidepressants, but the molecular mechanism of their action is unknown. We found that widely used antidepressants such as amitriptyline and fluoxetine induce autophagy in hippocampal neurons via the slow accumulation of sphingomyelin in lysosomes and Golgi membranes and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A and thereby the autophagy proteins Ulk, Beclin, Vps34/Phosphatidylinositol 3-kinase, p62, and Lc3B. Although treatment with amitriptyline or fluoxetine requires at least 12 days to achieve sphingomyelin accumulation and the subsequent biochemical and cellular changes, direct inhibition of sphingomyelin synthases with tricyclodecan-9-yl-xanthogenate (D609) results in rapid (within 3 days) accumulation of ceramide in the ER, activation of autophagy, and reversal of biochemical and behavioral signs of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioral changes typical of MDD. These findings identify sphingolipid-controlled autophagy as an important target for antidepressive treatment methods and provide a rationale for the development of novel antidepressants that act within a few days.
Asunto(s)
Antidepresivos/farmacología , Trastorno Depresivo Mayor/tratamiento farmacológico , Esfingomielina Fosfodiesterasa/genética , Animales , Antidepresivos/metabolismo , Autofagia/efectos de los fármacos , Hidrocarburos Aromáticos con Puentes/farmacología , Ceramidas/metabolismo , Ceramidas/farmacología , Corticosterona/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Femenino , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Norbornanos , Proteína Fosfatasa 2/efectos de los fármacos , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Tiocarbamatos , Tionas/farmacologíaRESUMEN
Farber disease is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments for Farber disease are clinically available, and affected patients have a severely shortened lifespan. We have recently reported a novel acid ceramidase deficiency model that mirrors the human disease closely. Acid sphingomyelinase is the enzyme that generates ceramide upstream of acid ceramidase in the lysosomes. Using our acid ceramidase deficiency model, we tested if acid sphingomyelinase could be a potential novel therapeutic target for the treatment of Farber disease. A number of functional acid sphingomyelinase inhibitors are clinically available and have been used for decades to treat major depression. Using these as a therapeutic for Farber disease, thus, has the potential to improve central nervous symptoms of the disease as well, something all other treatment options for Farber disease can't achieve so far. As a proof-of-concept study, we first cross-bred acid ceramidase deficient mice with acid sphingomyelinase deficient mice in order to prevent ceramide accumulation. Double-deficient mice had reduced ceramide accumulation, fewer disease manifestations, and prolonged survival. We next targeted acid sphingomyelinase pharmacologically, to test if these findings would translate to a setting with clinical applicability. Surprisingly, the treatment of acid ceramidase deficient mice with the acid sphingomyelinase inhibitor amitriptyline was toxic to acid ceramidase deficient mice and killed them within a few days of treatment. In conclusion, our study provides the first proof-of-concept that acid sphingomyelinase could be a potential new therapeutic target for Farber disease to reduce disease manifestations and prolong survival. However, we also identified previously unknown toxicity of the functional acid sphingomyelinase inhibitor amitriptyline in the context of Farber disease, strongly cautioning against the use of this substance class for Farber disease patients.
Asunto(s)
Lipogranulomatosis de Farber/enzimología , Esfingomielina Fosfodiesterasa/deficiencia , Ceramidasa Ácida/metabolismo , Amitriptilina/farmacología , Animales , Ceramidas/metabolismo , Citocinas/metabolismo , Lipogranulomatosis de Farber/patología , Ratones Endogámicos C57BL , Esfingomielina Fosfodiesterasa/metabolismo , Análisis de Supervivencia , Aumento de Peso/efectos de los fármacosRESUMEN
Staphylococcus aureus (S. aureus) infections are among the most common and severe infections, garnering notoriety in an era of increasing resistance to antibiotics. It is therefore important to define molecular mechanisms by which this pathogen attacks host cells. Here, we demonstrate that alpha-toxin, one of the major toxins of S. aureus, induces activation of acid sphingomyelinase and concomitant release of ceramide in endothelial cells treated with the toxin. Activation of acid sphingomyelinase by alpha-toxin is mediated via ADAM10. Infection experiments employing alpha-toxin-deficient S. aureus and the corresponding wild-type strain reveal that activation of acid sphingomyelinase in endothelial cells requires alpha-toxin expression by the pathogen. Activation of acid sphingomyelinase is linked to degradation of tight junctions in endothelial cells in vitro, which is blocked by pharmacological inhibition of acid sphingomyelinase. Most importantly, alpha-toxin induces severe degradation of tight junctions in the lung and causes lung edema in vivo, which is prevented by genetic deficiency of acid sphingomyelinase. These data indicate a novel and important role of the acid sphingomyelinase/ceramide system for the endothelial response to toxins and provide a molecular link between alpha-toxin and the degradation of tight junctions. The data also suggest that inhibition of acid sphingomyelinase may provide a novel treatment option to prevent lung edema caused by S. aureus alpha-toxin.
Asunto(s)
Toxinas Bacterianas/metabolismo , Ceramidas/metabolismo , Células Endoteliales/metabolismo , Proteínas Hemolisinas/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Staphylococcus aureus/metabolismo , Uniones Estrechas/metabolismo , Proteína ADAM10/metabolismo , Animales , Células Cultivadas , Células Endoteliales/virología , Pulmón/metabolismo , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Edema Pulmonar/metabolismo , Edema Pulmonar/virología , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/virología , Uniones Estrechas/virologíaRESUMEN
Pulmonary infections of cystic fibrosis (CF) patients with Staphylococcus aureus (S. aureus) occur very early in the disease. The molecular details that cause infection-susceptibility of CF patients to and mediate infection with S. aureus are poorly characterized. Therefore, we aimed to identify the role of α-toxin, a major S. aureus toxin, for pulmonary infection of CF mice. Infection with S. aureus JE2 resulted in severe pneumonia in CF mice, while wildtype mice were almost unaffected. Deficiency of α-toxin in JE2-Δhla reduced the pathogenicity of S. aureus in CF mice. However, CF mice were still more susceptible to the mutant S. aureus strain than wildtype mice. The S. aureus JE2 induced a marked increase of ceramide and a downregulation of sphingosine and acid ceramidase expression in bronchi of CF mice. Deletion of α-toxin reduced these changes after infection of CF mice. Similar changes were observed in wildtype mice, but at much lower levels. Our data indicate that expression of α-toxin is a major factor causing S. aureus infections in CF mice. Wildtype S. aureus induces a marked increase of ceramide and a reduction of sphingosine and acid ceramidase expression in bronchial epithelial cells of wildtype and CF mice, changes that determine infection susceptibility.
Asunto(s)
Toxinas Bacterianas/metabolismo , Fibrosis Quística/complicaciones , Fibrosis Quística/metabolismo , Proteínas Hemolisinas/metabolismo , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Animales , Fibrosis Quística/microbiología , Femenino , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Infecciones Estafilocócicas/microbiologíaRESUMEN
Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments are clinically available and affected patients have a severely shortened lifespan. Due to the low incidence, the pathogenesis of FD is still poorly understood. Here, we report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. Asah1tmEx1 mice were generated by deletion of the acid ceramidase signal peptide sequence. The effects on lysosomal targeting and activity of the enzyme were assessed. Ceramide and sphingomyelin levels were quantified by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and disease manifestations in several organ systems were analyzed by histology and biochemistry. We show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. The affected mice fail to thrive and die early. Histiocytic infiltrations were observed in many tissues, as well as lung inflammation, liver fibrosis, muscular disease manifestations and mild kidney injury. Our new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. In the future, it may also facilitate the development of urgently needed therapies.
Asunto(s)
Modelos Animales de Enfermedad , Lipogranulomatosis de Farber/patología , Animales , Ceramidas/análisis , Ceramidas/metabolismo , Cromatografía Liquida , Lipogranulomatosis de Farber/metabolismo , Ratones , Ratones Endogámicos C57BL , Esfingomielinas/análisis , Esfingomielinas/metabolismo , Espectrometría de Masas en TándemRESUMEN
CD4+ Foxp3+ regulatory T cells (Tregs) depend on CD28 signaling for their survival and function, a receptor that has been previously shown to activate the acid sphingomyelinase (Asm)/ceramide system. In this article, we show that the basal and CD28-induced Asm activity is higher in Tregs than in conventional CD4+ T cells (Tconvs) of wild-type (wt) mice. In Asm-deficient (Smpd1-/-; Asm-/-) mice, as compared with wt mice, the frequency of Tregs among CD4+ T cells, turnover of the effector molecule CTLA-4, and their suppressive activity in vitro were increased. The biological significance of these findings was confirmed in our Treg-sensitive mouse model of measles virus (MV) CNS infection, in which we observed more infected neurons and less MV-specific CD8+ T cells in brains of Asm-/- mice compared with wt mice. In addition to genetic deficiency, treatment of wt mice with the Asm inhibitor amitriptyline recapitulated the phenotype of Asm-deficient mice because it also increased the frequency of Tregs among CD4+ T cells. Reduced absolute cell numbers of Tconvs after inhibitor treatment in vivo and extensive in vitro experiments revealed that Tregs are more resistant toward Asm inhibitor-induced cell death than Tconvs. Mechanistically, IL-2 was capable of providing crucial survival signals to the Tregs upon inhibitor treatment in vitro, shifting the Treg/Tconv ratio to the Treg side. Thus, our data indicate that Asm-inhibiting drugs should be further evaluated for the therapy of inflammatory and autoimmune disorders.