IntroductionCOVID-19, caused by SARS-CoV-2, remains a global health challenge despite the availability of vaccines and antiviral treatments. The emergence of immune escape variants and the persistence of long COVID symptoms continue to complicate prevention and therapy, especially in low-resource settings. Functional inhibitors of acid sphingomyelinase (FIASMAs) have shown promise as broad-spectrum antiviral agents.MethodsWe evaluated the antiviral efficacy of amitriptyline, a widely used antidepressant and known FIASMA, against SARS-CoV-2. In vitro neutralization assays using pseudotyped virus-like particles (VLPs) and clinical isolates were performed to assess its ability to inhibit viral entry and replication.ResultsAmitriptyline significantly inhibited infection by SARS-CoV-2 VLPs bearing spike proteins, including those with mutations in the receptor-binding domain. Moreover, it reduced replication of clinical SARS-CoV-2 isolates D614G, Omicron BA.5, and Omicron XBB.1 in a dose-dependent manner at subtoxic concentrations.DiscussionOur findings demonstrate that amitriptyline neutralizes SARS-CoV-2 across multiple variants. These results support the potential of amitriptyline as a repurposed antiviral drug. Further clinical studies are warranted to evaluate its efficacy and safety in treating COVID-19 in humans.

The pandemic caused by SARS-CoV-2 is still a major health problem. Newly emerging variants and long-COVID-19 represent a challenge for the global health system. In particular, individuals in developing countries with insufficient health care need easily accessible, affordable and effective treatments of COVID-19. Previous studies have demonstrated the efficacy of functional inhibitors of acid sphingomyelinase against infections with various viruses, including early variants of SARS-CoV-2. This work investigated whether the acid sphingomyelinase inhibitors fluoxetine and sertraline, usually used as antidepressant molecules in clinical practice, can inhibit the replication of the former and recently emerged SARS-CoV-2 variants in vitro. Fluoxetine and sertraline potently inhibited the infection with pseudotyped virus-like particles and SARS-CoV-2 variants D614G, alpha, delta, omicron BA.1 and omicron BA.5. These results highlight fluoxetine and sertraline as priority candidates for large-scale phase 3 clinical trials at different stages of SARS-CoV-2 infections, either alone or in combination with other medications.

Pancreas ductal adenocarcinoma (PDAC) remains a malignant tumor with very poor prognosis and low 5-year overall survival. Here, we aimed to simultaneously target mitochondria and lysosomes as a new treatment paradigm of malignant pancreas cancer in vitro and in vivo. We demonstrate that the clinically used sphingosine analog FTY-720 together with PAPTP, an inhibitor of mitochondrial Kv1.3, induce death of pancreas cancer cells in vitro and in vivo. The combination of both drugs results in a marked inhibition of the acid sphingomyelinase and accumulation of cellular sphingomyelin in vitro and in vivo in orthotopic and flank pancreas cancers. Mechanistically, PAPTP and FTY-720 cause a disruption of both mitochondria and lysosomes, an alteration of mitochondrial bioenergetics and accumulation of cytoplasmic Ca 2+ , events that collectively mediate cell death. Our findings point to an unexpected cross-talk between lysosomes and mitochondria mediated by sphingolipid metabolism. We show that the combination of PAPTP and FTY-720 induces massive death of pancreas cancer cells, thereby leading to a substantially delayed and reduced PDAC growth in vivo. Key messages FTY-720 inhibits acid sphingomyelinase in pancreas cancer cells (PDAC). FTY-720 induces sphingomyelin accumulation and lysosomal dysfunction. The mitochondrial Kv1.3 inhibitor PAPTP disrupts mitochondrial functions. PAPTP and FTY-720 synergistically kill PDAC in vitro. The combination of FTY-720 and PAPTP greatly delays PDAC growth in vivo.

Prior evidence indicates the potential central role of the acid sphingomyelinase (ASM)/ceramide system in the infection of cells with SARS-CoV-2. We conducted a multicenter retrospective observational study including 72,105 adult patients with laboratory-confirmed SARS-CoV-2 infection who were admitted to 36 AP-HP (Assistance Publique–Hôpitaux de Paris) hospitals from 2 May 2020 to 31 August 2022. We examined the association between the ongoing use of medications functionally inhibiting acid sphingomyelinase (FIASMA), which reduces the infection of cells with SARS-CoV-2 in vitro, upon hospital admission with 28-day all-cause mortality in a 1:1 ratio matched analytic sample based on clinical characteristics, disease severity and other medications (N = 9714). The univariate Cox regression model of the matched analytic sample showed that FIASMA medication use at admission was associated with significantly lower risks of 28-day mortality (HR = 0.80; 95% CI = 0.72–0.88; p < 0.001). In this multicenter observational study, the use of FIASMA medications was significantly and substantially associated with reduced 28-day mortality among adult patients hospitalized with COVID-19. These findings support the continuation of these medications during the treatment of SARS-CoV-2 infections. Randomized clinical trials (RCTs) are needed to confirm these results, starting with the molecules with the greatest effect size in the study, e.g., fluoxetine, escitalopram, and amlodipine.

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.

Pancreatic adenocarcinoma (PDAC) is one of the most common cancers worldwide. Unfortunately, the prognosis of PDAC is rather poor, and for instance, in the USA, over 47,000 people die because of pancreatic cancer annually. Here, we demonstrate that high expression of acid sphingomyelinase in PDAC strongly correlates with long-term survival of patients, as revealed by the analysis of two independent data sources. The positive effects of acid sphingomyelinase expression on long-term survival of PDAC patients were independent of patient demographics as well as tumor grade, lymph node involvement, perineural invasion, tumor stage, lymphovascular invasion, and adjuvant therapy. We also demonstrate that genetic deficiency or pharmacological inhibition of the acid sphingomyelinase promotes tumor growth in an orthotopic mouse model of PDAC. This is mirrored by a poorer pathologic response, as defined by the College of American Pathologists (CAP) score for pancreatic cancer, to neoadjuvant therapy of patients co-treated with functional inhibitors of the acid sphingomyelinase, in particular tricyclic antidepressants and selective serotonin reuptake inhibitors, in a retrospective analysis. Our data indicate expression of the acid sphingomyelinase in PDAC as a prognostic marker for tumor progression. They further suggest that the use of functional inhibitors of the acid sphingomyelinase, at least of tricyclic antidepressants and selective serotonin reuptake inhibitors in patients with PDAC, is contra-indicated. Finally, our data also suggest a potential novel treatment of PDAC patients with recombinant acid sphingomyelinase.Key messagesPancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis.Expression of acid sphingomyelinase (ASM) determines outcome of PDAC.Genetic deficiency or pharmacologic inhibition of ASM promotes tumor growth in a mouse model.Inhibition of ASM during neoadjuvant treatment for PDAC correlates with worse pathology.ASM expression is a prognostic marker and potential target in PDAC.

BACKGROUND:Ex-vivo lung perfusion (EVLP) is a save way to verify performance of donor lungs prior to implantation. A major problem of lung transplantation is a donor-to-recipient-transmission of bacterial cultures. Thus, a broadspectrum anti-infective treatment with sphingosine in EVLP might be a novel way to prevent such infections. Sphingosine inhalation might provide a reliable anti-infective treatment option in EVLP. Here, antimicrobial potency of inhalative sphingosine in an infection EVLP model was tested.METHODS:A 3-hour EVLP run using pig lungs was performed. Bacterial infection was initiated 1-hour before sphingosine inhalation. Biopsies were obtained 60 and 120 min after infection with Pseudomonas aeruginosa. Aliquots of broncho-alveolar lavage (BAL) before and after inhalation of sphingosine were plated and counted, tissue samples were fixed in paraformaldehyde, embedded in paraffin and sectioned. Immunostainings were performed.RESULTS:Sphingosine inhalation in the setting of EVLP rapidly resulted in a 6-fold decrease of P. aeruginosa CFU in the lung (p = 0.016). We did not observe any negative side effects of sphingosine.CONCLUSION:Inhalation of sphingosine induced a significant decrease of Pseudomonas aeruginosa at the epithelial layer of tracheal and bronchial cells. The inhalation has no local side effects in ex-vivo perfused and ventilated pig lungs.

Major depressive disorder (MDD) is a very common, severe disease with a lifetime prevalence of ~ 10%. The pathogenesis of MDD is unknown and, unfortunately, therapy is often insufficient. We have previously reported that ceramide levels are increased in the blood plasma of patients with MDD and in mice with experimental MDD. Here, we demonstrate that ceramide-enriched exosomes in the blood plasma are increased in mice with stress-induced MDD. Genetic studies reveal that neutral sphingomyelinase 2 is required for the formation of ceramide-enriched exosomes in the blood plasma. Accordingly, induced deficiency of neutral sphingomyelinase 2 prevented mice from the development of stress-induced MDD. Intravenous injection of microparticles from mice with MDD or injection of ceramide-loaded exosomes induced MDD-like behavior in untreated mice, which was abrogated by ex vivo pre-incubation of purified exosomes with anti-ceramide antibodies or ceramidase. Mechanistically, injection of exosomes from mice with MDD or injection of ex vivo ceramide-loaded microparticles inhibited phospholipase D (PLD) in endothelial cells in vitro and in the hippocampus in vivo and thereby decreased phosphatidic acid in the hippocampus, which has been previously shown to mediate MDD by plasma ceramide. In summary, our data indicate that ceramide-enriched exosomes are released by neutral sphingomyelinase 2 into the blood plasma upon stress and mediate stress-induced MDD. Key messages Stress induces ceramide-enriched exosomes in the blood plasma. Ceramide-enriched exosomes mediate major depressive disorder (MDD). Deficiency of neutral sphingomyelinase 2 protects from stress-induced MDD. Neutralization or digestion of ceramide in exosomes prevents stress-induced MDD. Ceramide-enriched exosomes inhibit endothelial phospholipase D in the hippocampus.

Acid sphingomyelinase (Asm) and acid ceramidase (Ac) are parts of the sphingolipid metabolism. Asm hydrolyzes sphingomyelin to ceramide, which is further metabolized to sphingosine by Ac. Ceramide generates ceramide-enriched platforms that are involved in receptor clustering within cellular membranes. However, the impact of cell-intrinsic ceramide on T cell function is not well characterized. By using T cell-specific Asm- or Ac-deficient mice, with reduced or elevated ceramide levels in T cells, we identified ceramide to play a crucial role in T cell function in vitro and in vivo. T cell-specific ablation of Asm in Smpd1fl/fl/Cd4cre/+ (Asm/CD4cre) mice resulted in enhanced tumor progression associated with impaired T cell responses, whereas Asah1fl/fl/Cd4cre/+ (Ac/CD4cre) mice showed reduced tumor growth rates and elevated T cell activation compared to the respective controls upon tumor transplantation. Further in vitro analysis revealed that decreased ceramide content supports CD4+ regulatory T cell differentiation and interferes with cytotoxic activity of CD8+ T cells. In contrast, elevated ceramide concentration in CD8+ T cells from Ac/CD4cre mice was associated with enhanced cytotoxic activity. Strikingly, ceramide co-localized with the T cell receptor (TCR) and CD3 in the membrane of stimulated T cells and phosphorylation of TCR signaling molecules was elevated in Ac-deficient T cells. Hence, our results indicate that modulation of ceramide levels, by interfering with the Asm or Ac activity has an effect on T cell differentiation and function and might therefore represent a novel therapeutic strategy for the treatment of T cell-dependent diseases such as tumorigenesis.

Staphylococcus aureus ( S. aureus ) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca 2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.

BACKGROUND:Development of brain metastases in advanced melanoma patients is a frequent event that limits patients' quality of life and survival. Despite recent insights into melanoma genetics, systematic analyses of genetic alterations in melanoma brain metastasis formation are lacking. Moreover, whether brain metastases harbor distinct genetic alterations beyond those observed at different anatomic sites of the same patient remains unknown.EXPERIMENTAL DESIGN AND RESULTS:In our study, 54 intracranial and 18 corresponding extracranial melanoma metastases were analyzed for mutations using targeted next generation sequencing of 29 recurrently mutated driver genes in melanoma. In 11 of 16 paired samples, we detected nucleotide modifications in brain metastases that were absent in matched metastases at extracranial sites. Moreover, we identified novel genetic variants in ARID1A, ARID2, SMARCA4 and BAP1, genes that have not been linked to brain metastases before; albeit most frequent mutations were found in ARID1A, ARID2 and BRAF. Conclusion: Our data provide new insights into the genetic landscape of intracranial melanoma metastases supporting a branched evolution model of metastasis formation.

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.

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.

This protocol enables the testing of drugs against infection of epithelial cells with SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), using pseudo-typed replication deficient vesicular stomatitis virus particles (pp-VSV) presenting the SARS-CoV-2 spike protein. After treating human volunteers with amitriptyline, an approved antidepressant and inhibitor of the acid sphingomyelinase, freshly isolated nasal epithelial cells were infected ex vivo and infection levels were quantified. This protocol offers the possibility to rapidly test the efficacy of potential drugs in the fight against COVID-19. For complete details on the use and execution of this protocol, please refer to Carpinteiro et al. (2020).

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 (ppVSV-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 [C-14]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.

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.

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.