Microbial lung infections are the major cause of morbidity and mortality in the hereditary metabolic disorder cystic fibrosis, yet the molecular mechanisms leading from the mutation of cystic fibrosis transmembrane conductance regulator (CFTR) to lung infection are still unclear. Here, we show that ceramide age-dependently accumulates in the respiratory tract of uninfected Cftr-deficient mice owing to an alkalinization of intracellular vesicles in Cftr-deficient cells. This change in pH results in an imbalance between acid sphingomyelinase (Asm) cleavage of sphingomyelin to ceramide and acid ceramidase consumption of ceramide, resulting in the higher levels of ceramide. The accumulation of ceramide causes Cftr-deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi and high susceptibility to severe Pseudomonas aeruginosa infections. Partial genetic deficiency of Asm in Cftr(-/-)Smpd1(+/-) mice or pharmacological treatment of Cftr-deficient mice with the Asm blocker amitriptyline normalizes pulmonary ceramide and prevents all pathological findings, including susceptibility to infection. These data suggest inhibition of Asm as a new treatment strategy for cystic fibrosis.-1 - infection. These data suggest inhibition of Asm as a novel treatment strategy in CF. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis-4 -

The acid sphingomyelinase/ceramide system plays an important role in bacterial and viral infections. Here we report that either pharmacological inhibition of acid sphingomyelinase with amitriptyline, imipramine, fluoxetine, sertraline, escitalopram, or maprotiline, or genetic downregulation of the enzyme prevents infection of cultured cells or freshy isolated human nasal epithelial cells with SARS-CoV-2 or pseudoviral pp-VSV-SARS-CoV-2 particles expressing spike, a bona fide system mimicking SARS-CoV-2 infection. Infection activates acid sphingomyelinase and triggers a release of ceramide on the cell surface. Neutralization or consumption of surface ceramide reduces infection with pp-VSV-SARS-CoV-2 spike. Treating volunteers with a low dose of amitriptyline prevents infection of freshly isolated nasal epithelial cells with pp-VSV-SARS-CoV-2 spike. The data justify clinical studies investigating whether amitriptyline, a safe drug used clinically for almost 60 years, or other antidepressants that functionally block acid sphingomyelinase prevent SARS-CoV-2 infection.

The potassium channel Kv1.3 has recently been located to the inner mitochondrial membrane of lymphocytes. Here, we show that mouse and human cells that are genetically deficient in either Kv1.3 or transfected with siRNA to suppress Kv1.3-expression resisted apoptosis induced by several stimuli, including Bax over-expression. Retransfection of either Kv1.3 or a mitochondrial-targeted Kv1.3 restored cell death. Bax interacted with and functionally inhibited mitochondrial Kv1.3. Incubation of isolated Kv1.3-positive mitochondria with recombinant Bax, t-Bid, or toxins that bind to and inhibit Kv1.3 successively triggered hyperpolarization, formation of reactive oxygen species, release of cytochrome c, and marked depolarization. Kv1.3-deficient mitochondria were resistant to Bax, t-Bid, and the toxins. Mutation of Bax at K128, which corresponds to a conserved lysine in Kv1.3-inhibiting toxins, abrogated its effects on both Kv1.3 and mitochondria. These findings suggest that Bax mediates cytochrome c release and mitochondrial depolarization in lymphocytes, at least in part, via its interaction with mitochondrial Kv1.3

The cell membrane contains very small distinct membrane domains enriched of sphingomyelin and cholesterol that are named rafts. We have shown that the formation of ceramide via activation of the acid sphingomyelinase transforms rafts into ceramide-enriched membrane platforms. These platforms are required for infection of mammalian cells with Pseudomonas aeruginosa, Staphylococcus aureus, or Neisseriae gonorrhoeae. In the present study we determined whether the acid sphingomyelinase, ceramide, and ceramide-enriched membrane platforms are also involved in the infection of human cells with pathogenic rhinoviruses. We demonstrate that infection of human epithelial cells with several rhinovirus strains triggers a rapid activation of the acid sphingomyelinase correlating with microtubules-and microfilament-mediated translocation of the enzyme from an intracellular compartment onto the extracellular leaflet of the cell membrane. The classic fluid mosaic model of the cell membrane suggests a random distribution of lipids and proteins in the cell membrane (1). This view was challenged by many recent studies indicating that sphingolipids and cholesterol spontaneously separate from other glycophospholipids in the cell membrane to form distinct microdomains (2, 3). (Glyco)sphingolipids bind to each other via hydrophilic and hydrophobic interactions between their headgroups and the saturated fatty acid side chains, respectively. Void spaces between the bulky glycosphingolipids seem to be filled with cholesterol via tight interactions of the sterol-ring system with the sphingosine moiety of the sphingolipids and hydrogen bonds between the C3-hydroxyl groups with the hydrophilic headgroups of sphingomyelin. These interactions result in a lateral stabilization and facilitate the formation of distinct membrane domains that were named "rafts" (3), because they float in the "ocean" of the other phospholipids. We have identified a novel membrane domain, i.e. ceramide-enriched membrane platforms (4, 5). Ceramideenriched membrane platforms are initiated by activation of the acid sphingomyelinase, which has been shown to be stimulated by several receptors, including CD95 (4 -9), tumor necrosis factor receptor (10), CD40 (11), or Fc␥RII (12), pathogenic bacteria, including Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Staphylococcus aureus (13-16), Sindbis virus (17), and many stress stimuli, e.g. radiation, UV light, or cytostatic drugs (18 -22). These stimuli do not only activate the enzyme but also induce the translocation of the acid sphingomyelinase from an intracellular compartment onto the extracellular leaflet of the cell membrane (4, 23). The hydrolysis of sphingomyelin that is predominantly located in the outer leaflet of the cell membrane by the acid sphingomyelinase results in the generation of ceramide. The biophysical properties of ceramide predict that ceramide molecules self-associate to small ceramide-enriched domains (4), which spontaneously fuse to one or a few large ceramide-enriched membrane platforms (24). Ce...