@article{ZeitlerYeAndreyevaetal.2019, author = {Zeitler, Stefanie and Ye, Lian and Andreyeva, Aksana and Schumacher, Fabian and Monti, Juliana and N{\"u}rnberg, Bernd and Nowak, Gabriel and Kleuser, Burkhard and Reichel, Martin and Fejtova, Anna and Kornhuber, Johannes and Rhein, Cosima and Friedland, Kristina}, title = {Acid sphingomyelinase - a regulator of canonical transient receptor potential channel 6 (TRPC6) activity}, series = {Journal of neurochemistry}, volume = {150}, journal = {Journal of neurochemistry}, number = {6}, publisher = {Wiley}, address = {Hoboken}, issn = {0022-3042}, doi = {10.1111/jnc.14823}, pages = {678 -- 690}, year = {2019}, abstract = {Recent investigations propose the acid sphingomyelinase (ASM)/ceramide system as a novel target for antidepressant action. ASM catalyzes the breakdown of the abundant membrane lipid sphingomyelin to the lipid messenger ceramide. This ASM-induced lipid modification induces a local shift in membrane properties, which influences receptor clustering and downstream signaling. Canonical transient receptor potential channels 6 (TRPC6) are non-selective cation channels located in the cell membrane that play an important role in dendritic growth, synaptic plasticity and cognition in the brain. They can be activated by hyperforin, an ingredient of the herbal remedy St. John's wort for treatment of depression disorders. Because of their role in the context of major depression, we investigated the crosstalk between the ASM/ceramide system and TRPC6 ion channels in a pheochromocytoma cell line 12 neuronal cell model (PC12 rat pheochromocytoma cell line). Ca2+ imaging experiments indicated that hyperforin-induced Ca2+ influx through TRPC6 channels is modulated by ASM activity. While antidepressants, known as functional inhibitors of ASM activity, reduced TRPC6-mediated Ca2+ influx, extracellular application of bacterial sphingomyelinase rebalanced TRPC6 activity in a concentration-related way. This effect was confirmed in whole-cell patch clamp electrophysiology recordings. Lipidomic analyses revealed a decrease in very long chain ceramide/sphingomyelin molar ratio after ASM inhibition, which was connected with changes in the abundance of TRPC6 channels in flotillin-1-positive lipid rafts as visualized by western blotting. Our data provide evidence that the ASM/ceramide system regulates TRPC6 channels likely by controlling their recruitment to specific lipid subdomains and thereby fine-tuning their physical properties.}, language = {en} } @article{BeckmannBeckerKadowetal.2019, author = {Beckmann, Nadine and Becker, Katrin Anne and Kadow, Stephanie and Schumacher, Fabian and Kramer, Melanie and Kuehn, Claudine and Schulz-Schaeffer, Walter J. and Edwards, Michael J. and Kleuser, Burkhard and Gulbins, Erich and Carpinteiro, Alexander}, title = {Acid Sphingomyelinase Deficiency Ameliorates Farber Disease}, series = {International journal of molecular sciences}, volume = {20}, journal = {International journal of molecular sciences}, number = {24}, publisher = {MDPI}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms20246253}, pages = {18}, year = {2019}, abstract = {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.}, language = {en} } @article{MuellerFinkeEbertetal.2018, author = {M{\"u}ller, S. M. and Finke, Hannah and Ebert, Franziska and Kopp, Johannes Florian and Schumacher, Fabian and Kleuser, Burkhard and Francesconi, Kevin A. and Raber, G. and Schwerdtle, Tanja}, title = {Arsenic-containing hydrocarbons}, series = {Archives of toxicology : official journal of EUROTOX}, volume = {92}, journal = {Archives of toxicology : official journal of EUROTOX}, number = {5}, publisher = {Springer}, address = {Heidelberg}, issn = {0340-5761}, doi = {10.1007/s00204-018-2194-z}, pages = {1751 -- 1765}, year = {2018}, abstract = {Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids found in fish and algae, elicit substantial toxic effects in various human cell lines and have a considerable impact on cellular energy levels. The underlying mode of action, however, is still unknown. The present study analyzes the effects of two AsHCs (AsHC 332 and AsHC 360) on the expression of 44 genes covering DNA repair, stress response, cell death, autophagy, and epigenetics via RT-qPCR in human liver (HepG2) cells. Both AsHCs affected the gene expression, but to different extents. After treatment with AsHC 360, flap structure-specific endonuclease 1 (FEN1) as well as xeroderma pigmentosum group A complementing protein (XPA) and (cytosine-5)-methyltransferase 3A (DNMT3A) showed time- and concentration-dependent alterations in gene expression, thereby indicating an impact on genomic stability. In the subsequent analysis of epigenetic markers, within 72 h, neither AsHC 332 nor AsHC 360 showed an impact on the global DNA methylation level, whereas incubation with AsHC 360 increased the global DNA hydroxymethylation level. Analysis of cell extracts and cell media by HPLC-mass spectrometry revealed that both AsHCs were considerably biotransformed. The identified metabolites include not only the respective thioxo-analogs of the two AsHCs, but also several arsenic-containing fatty acids and fatty alcohols, contributing to our knowledge of biotransformation mechanisms of arsenolipids.}, language = {en} } @article{SchoenauerLarpinBabiychuketal.2019, author = {Schoenauer, Roman and Larpin, Yu and Babiychuk, Eduard B. and Drucker, Patrick and Babiychuk, Viktoriia S. and Avota, Elita and Schneider-Schaulies, Sibylle and Schumacher, Fabian and Kleuser, Burkhard and Koffel, Rene and Draeger, Annette}, title = {Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins}, series = {The FASEB journal : the official journal of the Federation of American Societies for Experimental Biology}, volume = {33}, journal = {The FASEB journal : the official journal of the Federation of American Societies for Experimental Biology}, number = {1}, publisher = {Federation of American Societies for Experimental Biology}, address = {Bethesda}, issn = {0892-6638}, doi = {10.1096/fj.201800033R}, pages = {275 -- 285}, year = {2019}, abstract = {Bacterial pore-forming toxins compromise plasmalemmal integrity, leading to Ca2+ influx, leakage of the cytoplasm, and cell death. Such lesions can be repaired by microvesicular shedding or by the endocytic uptake of the injured membrane sites. Cells have at their disposal an entire toolbox of repair proteins for the identification and elimination of membrane lesions. Sphingomyelinases catalyze the breakdown of sphingomyelin into ceramide and phosphocholine. Sphingomyelin is predominantly localized in the outer leaflet, where it is hydrolyzed by acid sphingomyelinase (ASM) after lysosomal fusion with the plasma membrane. The magnesium-dependent neutral sphingomyelinase (NSM)-2 is found at the inner leaflet of the plasmalemma. Because either sphingomyelinase has been ascribed a role in the cellular stress response, we investigated their role in plasma membrane repair and cellular survival after treatment with the pore-forming toxins listeriolysin O (LLO) or pneumolysin (PLY). Jurkat T cells, in which ASM or NSM-2 was down-regulated [ASM knockdown (KD) or NSM-2 KD cells], showed inverse reactions to toxin-induced membrane damage: ASM KD cells displayed reduced toxin resistance, decreased viability, and defects in membrane repair. In contrast, the down-regulation of NSM-2 led to an increase in viability and enhanced plasmalemmal repair. Yet, in addition to the increased plasmalemmal repair, the enhanced toxin resistance of NSM-2 KD cells also appeared to be dependent on the activation of p38/MAPK, which was constitutively activated, whereas in ASM KD cells, the p38/MAPK activation was constitutively blunted.Schoenauer, R., Larpin, Y., Babiychuk, E. B., Drucker, P., Babiychuk, V. S., Avota, E., Schneider-Schaulies, S., Schumacher, F., Kleuser, B., Koffel, R., Draeger, A. Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.}, language = {en} } @article{HausmannZoschkeWolffetal.2019, author = {Hausmann, Christian and Zoschke, Christian and Wolff, Christopher and Darvin, Maxim E. and Sochorova, Michaela and Kovacik, Andrej and Wanjiku, Barbara and Schumacher, Fabian and Tigges, Julia and Kleuser, Burkhard and Lademann, Juergen and Fritsche, Ellen and Vavrova, Katerina and Ma, Nan and Schaefer-Korting, Monika}, title = {Fibroblast origin shapes tissue homeostasis, epidermal differentiation, and drug uptake}, series = {Scientific reports}, volume = {9}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-019-39770-6}, pages = {10}, year = {2019}, abstract = {Preclinical studies frequently lack predictive value for human conditions. Human cell-based disease models that reflect patient heterogeneity may reduce the high failure rates of preclinical research. Herein, we investigated the impact of primary cell age and body region on skin homeostasis, epidermal differentiation, and drug uptake. Fibroblasts derived from the breast skin of female 20- to 30-yearolds or 60- to 70-year-olds and fibroblasts from juvenile foreskin (<10 years old) were compared in cell monolayers and in reconstructed human skin (RHS). RHS containing aged fibroblasts differed from its juvenile and adult counterparts, especially in terms of the dermal extracellular matrix composition and interleukin-6 levels. The site from which the fibroblasts were derived appeared to alter fibroblast-keratinocyte crosstalk by affecting, among other things, the levels of granulocyte-macrophage colony-stimulating factor. Consequently, the epidermal expression of filaggrin and e-cadherin was increased in RHS containing breast skin fibroblasts, as were lipid levels in the stratum corneum. In conclusion, the region of the body from which fibroblasts are derived appears to affect the epidermal differentiation of RHS, while the age of the fibroblast donors determines the expression of proteins involved in wound healing. Emulating patient heterogeneity in preclinical studies might improve the treatment of age-related skin conditions.}, language = {en} } @article{MeinersPalmieriKlopfleischetal.2019, author = {Meiners, Jana and Palmieri, Vittoria and Klopfleisch, Robert and Ebel, Jana-Fabienne and Japtok, Lukasz and Schumacher, Fabian and Yusuf, Ayan Mohamud and Becker, Katrin Anne and Z{\"o}ller, Julia and Hose, Matthias and Kleuser, Burkhard and Hermann, Dirk Matthias and Kolesnick, Richard N. and Buer, Jan and Hansen, Wiebke and Westendorf, Astrid M.}, title = {Intestinal acid sphingomyelinase protects from severe Pathogen-Driven Colitis}, series = {Frontiers in immunology}, volume = {10}, journal = {Frontiers in immunology}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {1664-3224}, doi = {10.3389/fimmu.2019.01386}, pages = {14}, year = {2019}, abstract = {Inflammatory diseases of the gastrointestinal tract are emerging as a global problem with increased evidence and prevalence in numerous countries. A dysregulated sphingolipid metabolism occurs in patients with ulcerative colitis and is discussed to contribute to its pathogenesis. In the present study, we determined the impact of acid sphingomyelinase (Asm), which catalyzes the hydrolysis of sphingomyelin to ceramide, on the course of Citrobacter (C.) rodentium-driven colitis. C. rodentium is an enteric pathogen and induces colonic inflammation very similar to the pathology in patients with ulcerative colitis. We found that mice with Asm deficiency or Asm inhibition were strongly susceptible to C. rodentium infection. These mice showed increased levels of C. rodentium in the feces and were prone to bacterial spreading to the systemic organs. In addition, mice lacking Asm activity showed an uncontrolled inflammatory T(h)1 and T(h)17 response, which was accompanied by a stronger colonic pathology compared to infected wild type mice. These findings identified Asm as an essential regulator of mucosal immunity to the enteric pathogen C. rodentium.}, language = {en} } @article{CastanoMartinezSchumacherSchumacheretal.2019, author = {Casta{\~n}o Mart{\´i}nez, Mar{\´i}a Teresa and Schumacher, Fabian and Schumacher, Silke and Kochlik, Bastian Max and Weber, Daniela and Grune, Tilman and Biemann, Ronald and McCann, Adrian and Abraham, Klaus and Weikert, Cornelia and Kleuse, Burkhard and Sch{\"u}rmann, Annette and Laeger, Thomas}, title = {Methionine restriction prevents onset of type 2 diabetes in NZO mice}, series = {The FASEB journal : the official journal of the Federation of American Societies for Experimental Biology}, volume = {33}, journal = {The FASEB journal : the official journal of the Federation of American Societies for Experimental Biology}, number = {6}, publisher = {Federation of American Societies for Experimental Biology}, address = {Bethesda}, issn = {0892-6638}, doi = {10.1096/fj.201900150R}, pages = {7092 -- 7102}, year = {2019}, abstract = {Dietary methionine restriction (MR) is well known to reduce body weight by increasing energy expenditure (EE) and insulin sensitivity. An elevated concentration of circulating fibroblast growth factor 21 (FGF21) has been implicated as a potential underlying mechanism. The aims of our study were to test whether dietary MR in the context of a high-fat regimen protects against type 2 diabetes in mice and to investigate whether vegan and vegetarian diets, which have naturally low methionine levels, modulate circulating FGF21 in humans. New Zealand obese (NZO) mice, a model for polygenic obesity and type 2 diabetes, were placed on isocaloric high-fat diets (protein, 16 kcal\%; carbohydrate, 52 kcal\%; fat, 32 kcal\%) that provided methionine at control (Con; 0.86\% methionine) or low levels (0.17\%) for 9 wk. Markers of glucose homeostasis and insulin sensitivity were analyzed. Among humans, low methionine intake and circulating FGF21 levels were investigated by comparing a vegan and a vegetarian diet to an omnivore diet and evaluating the effect of a short-term vegetarian diet on FGF21 induction. In comparison with the Con group, MR led to elevated plasma FGF21 levels and prevented the onset of hyperglycemia in NZO mice. MR-fed mice exhibited increased insulin sensitivity, higher plasma adiponectin levels, increased EE, and up-regulated expression of thermogenic genes in subcutaneous white adipose tissue. Food intake and fat mass did not change. Plasma FGF21 levels were markedly higher in vegan humans compared with omnivores, and circulating FGF21 levels increased significantly in omnivores after 4 d on a vegetarian diet. These data suggest that MR induces FGF21 and protects NZO mice from high-fat diet-induced glucose intolerance and type 2 diabetes. The normoglycemic phenotype in vegans and vegetarians may be caused by induced FGF21. MR akin to vegan and vegetarian diets in humans may offer metabolic benefits via increased circulating levels of FGF21 and merits further investigation.-Castano-Martinez, T., Schumacher, F., Schumacher, S., Kochlik, B., Weber, D., Grune, T., Biemann, R., McCann, A., Abraham, K., Weikert, C., Kleuser, B., Schurmann, A., Laeger, T. Methionine restriction prevents onset of type 2 diabetes in NZO mice.}, language = {en} } @article{BeckmannKadowSchumacheretal.2018, author = {Beckmann, Nadine and Kadow, Stephanie and Schumacher, Fabian and Goethert, Joachim R. and Kesper, Stefanie and Draeger, Annette and Schulz-Schaeffer, Walter J. and Wang, Jiang and Becker, Jan U. and Kramer, Melanie and Kuehn, Claudine and Kleuser, Burkhard and Becker, Katrin Anne and Gulbins, Erich and Carpinteiro, Alexander}, title = {Pathological manifestations of Farber disease in a new mouse model}, series = {Biological chemistry}, volume = {399}, journal = {Biological chemistry}, number = {10}, publisher = {De Gruyter}, address = {Berlin}, issn = {1431-6730}, doi = {10.1515/hsz-2018-0170}, pages = {1183 -- 1202}, year = {2018}, abstract = {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. Asah1(tmEx1) 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.}, language = {en} } @article{BoertleinSchumacherKleuseretal.2019, author = {B{\"o}rtlein, Charlene and Schumacher, Fabian and Kleuser, Burkhard and D{\"o}lken, Lars and Avota, Elita}, title = {Role of Neutral Sphingomyelinase-2 (NSM 2) in the Control of T Cell Plasma Membrane Lipid Composition and Cholesterol Homeostasis}, series = {Frontiers in cell and developmental biology}, volume = {7}, journal = {Frontiers in cell and developmental biology}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {2296-634X}, doi = {10.3389/fcell.2019.00226}, pages = {16}, year = {2019}, abstract = {The activity of neutral sphingomyelinase-2 (NSM2) to catalyze the conversion of sphingomyelin (SM) to ceramide and phosphocholine at the cytosolic leaflet of plasma membrane (PM) is important in T cell receptor (TCR) signaling. We recently identified PKC zeta as a major NSM2 downstream effector which regulates microtubular polarization. It remained, however, unclear to what extent NSM2 activity affected overall composition of PM lipids and downstream effector lipids in antigen stimulated T cells. Here, we provide a detailed lipidomics analyses on PM fractions isolated from TCR stimulated wild type and NSM2 deficient (Delta NSM) Jurkat T cells. This revealed that in addition to that of sphingolipids, NSM2 depletion also affected concentrations of many other lipids. In particular, NSM2 ablation resulted in increase of lyso-phosphatidylcholine (LPC) and lyso-phosphatidylethanolamine (LPE) which both govern PM biophysical properties. Crucially, TCR dependent upregulation of the important T cell signaling lipid diacylglycerol (DAG), which is fundamental for activation of conventional and novel PKCs, was abolished in Delta NSM cells. Moreover, NSM2 activity was found to play an important role in PM cholesterol transport to the endoplasmic reticulum (ER) and production of cholesteryl esters (CE) there. Most importantly, CE accumulation was essential to sustain human T cell proliferation. Accordingly, inhibition of CE generating enzymes, the cholesterol acetyltransferases ACAT1/SOAT1 and ACAT2/SOAT2, impaired TCR driven expansion of both CD4(+) and CD8(+) T cells. In summary, our study reveals an important role of NSM2 in regulating T cell functions by its multiple effects on PM lipids and cholesterol homeostasis.}, language = {en} } @article{SpeckmannSchulzHilleretal.2017, author = {Speckmann, Bodo and Schulz, Sarah and Hiller, Franziska and Hesse, Deike and Schumacher, Fabian and Kleuser, Burkhard and Geisel, Juergen and Obeid, Rima and Grune, Tilman and Kipp, Anna Patricia}, title = {Selenium increases hepatic DNA methylation and modulates one-carbon metabolism in the liver of mice}, series = {The journal of nutritional biochemistry}, volume = {48}, journal = {The journal of nutritional biochemistry}, publisher = {Elsevier}, address = {New York}, issn = {0955-2863}, doi = {10.1016/j.jnutbio.2017.07.002}, pages = {112 -- 119}, year = {2017}, abstract = {The average intake of the essential trace element selenium (Se) is below the recommendation in most European countries, possibly causing sub-optimal expression of selenoproteins. It is still unclear how a suboptimal Se status may affect health. To mimic this situation, mice were fed one of three physiologically relevant amounts of Se. We focused on the liver, the organ most sensitive to changes in the Se supply indicated by hepatic glutathione peroxidase activity. In addition, liver is the main organ for synthesis of methyl groups and glutathione via one-carbon metabolism. Accordingly, the impact of Se on global DNA methylation, methylation capacity, and gene expression was assessed. We observed higher global DNA methylation indicated by LINE1 methylation, and an increase of the methylation potential as indicated by higher S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio and by elevated mRNA expression of serine hydroxymethyltransferase in both or either of the Se groups. Furthermore, increasing the Se supply resulted in higher plasma concentrations of triglycerides. Hepatic expression of glycolytic and lipogenic genes revealed consistent Se dependent up-regulation of glucokinase. The sterol regulatory element-binding transcription factor 1 (Srebf1) was also up-regulated by Se. Both effects were confirmed in primary hepatocytes. In contrast to the overall Se-dependent increase of methylation capacity, the up-regulation of Srebf1 expression was paralleled by reduced local methylation of a specific CpG site within the Srebf1 gene. Thus, we provided evidence that Se-dependent effects on lipogenesis involve epigenetic mechanisms. (C) 2017 The Authors. Published by Elsevier Inc.}, language = {en} } @article{GutbierSchoenrockEhrleretal.2018, author = {Gutbier, Birgitt and Sch{\"o}nrock, Stefanie M. and Ehrler, Carolin and Haberberger, Rainer and Dietert, Kristina and Gruber, Achim D. and Kummer, Wolfgang and Michalick, Laura and Kuebler, Wolfgang M. and Hocke, Andreas C. and Szymanski, Kolja and Letsiou, Eleftheria and L{\"u}th, Anja and Schumacher, Fabian and Kleuser, Burkhard and Mitchell, Timothy J. and Bertrams, Wilhelm and Schmeck, Bernd and Treue, Denise and Klauschen, Frederick and Bauer, Torsten T. and T{\"o}nnies, Mario and Weissmann, Norbert and Hippenstiel, Stefan and Suttorp, Norbert and Witzenrath, Martin}, title = {Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2}, series = {Critical care medicine}, volume = {46}, journal = {Critical care medicine}, number = {3}, publisher = {Lippincott Williams \& Wilkins}, address = {Philadelphia}, organization = {CAPNETZ Study Grp}, issn = {0090-3493}, doi = {10.1097/CCM.0000000000002916}, pages = {e258 -- e267}, year = {2018}, abstract = {Objectives: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. Design: Controlled, in vitro, ex vivo, and in vivo laboratory study. Subjects: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. Interventions: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. Measurements and Main Results: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1(-/-) mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. Conclusions: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.}, language = {en} } @article{SeitzSchumacherBakeretal.2019, author = {Seitz, Aaron P. and Schumacher, Fabian and Baker, Jennifer and Soddemann, Matthias and Wilker, Barbara and Caldwell, Charles C. and Gobble, Ryan M. and Kamler, Markus and Becker, Katrin Anne and Beck, Sascha and Kleuser, Burkhard and Edwards, Michael J. and Gulbins, Erich}, title = {Sphingosine-coating of plastic surfaces prevents ventilator-associated pneumonia}, series = {Journal of molecular medicine}, volume = {97}, journal = {Journal of molecular medicine}, number = {8}, publisher = {Springer}, address = {Heidelberg}, issn = {0946-2716}, doi = {10.1007/s00109-019-01800-1}, pages = {1195 -- 1211}, year = {2019}, abstract = {Ventilator-associated pneumonia (VAP) is a major cause of morbidity and mortality in critically ill patients. Here, we employed the broad antibacterial effects of sphingosine to prevent VAP by developing a novel method of coating surfaces of endotracheal tubes with sphingosine and sphingosine analogs. Sphingosine and phytosphingosine coatings of endotracheal tubes prevent adherence and mediate killing of Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus, even in biofilms. Most importantly, sphingosine-coating of endotracheal tubes also prevented P. aeruginosa and S. aureus pneumonia in vivo. Coating of the tubes with sphingosine was stable, without obvious side effects on tracheal epithelial cells and did not induce inflammation. In summary, we describe a novel method to coat plastic surfaces and provide evidence for the application of sphingosine and phytosphingosine as novel antimicrobial coatings to prevent bacterial adherence and induce killing of pathogens on the surface of endotracheal tubes with potential to prevent biofilm formation and VAP.Key messagesNovel dip-coating method to coat plastic surfaces with lipids.Sphingosine and phytosphingosine as novel antimicrobial coatings on plastic surface.Sphingosine coatings of endotracheal tubes prevent bacterial adherence and biofilms.Sphingosine coatings of endotracheal tubes induce killing of pathogens.Sphingosine coatings of endotracheal tubes ventilator-associated pneumonia.}, language = {en} } @article{ZabihiGraffSchumacheretal.2018, author = {Zabihi, Fatemeh and Graff, Patrick and Schumacher, Fabian and Kleuser, Burkhard and Hedtrich, Sarah and Haag, Rainer}, title = {Synthesis of poly(lactide-co-glycerol) as a biodegradable and biocompatible polymer with high loading capacity for dermal drug delivery}, series = {Nanoscale}, volume = {10}, journal = {Nanoscale}, number = {35}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {2040-3364}, doi = {10.1039/c8nr05536j}, pages = {16848 -- 16856}, year = {2018}, abstract = {Due to the low cutaneous bioavailability of tacrolimus (TAC), penetration enhancers are used to improve its penetration into the skin. However, poor loading capacity, non-biodegradability, toxicity, and in some cases inefficient skin penetration are challenging issues that hamper their applications for the dermal TAC delivery. Here we present poly(lactide-co-glycerol) (PLG) as a water soluble, biodegradable, and biocompatible TAC-carrier with high loading capacity (14.5\% w/w for TAC) and high drug delivery efficiencies into the skin. PLG was synthesized by cationic ring-opening copolymerization of a mixture of glycidol and lactide and showed 35 nm and 300 nm average sizes in aqueous solutions before and after loading of TAC, respectively. Delivery experiments on human skin, quantified by fluorescence microscopy and LC-MS/MS, showed a high ability for PLG to deposit Nile red and TAC into the stratum corneum and viable epidermis of skin in comparison with Protopic (R) (0.03\% w/w, TAC ointment). The cutaneous distribution profile of delivered TAC proved that 80\%, 16\%, and 4\% of the cutaneous drug level was deposited in the stratum corneum, viable epidermis, and upper dermis, respectively. TAC delivered by PLG was able to efficiently decrease the IL-2 and TSLP expressions in human skin models. Taking advantage of the excellent physicochemical and biological properties of PLG, it can be used for efficient dermal TAC delivery and potential treatment of inflammatory skin diseases.}, language = {en} } @article{GrafenSchumacherChithelenetal.2019, author = {Grafen, Anika and Schumacher, Fabian and Chithelen, Janice and Kleuser, Burkhard and Beyersdorf, Niklas and Schneider-Schaulies, J{\"u}rgen}, title = {Use of Acid Ceramidase and Sphingosine Kinase Inhibitors as Antiviral Compounds Against Measles Virus Infection of Lymphocytes in vitro}, series = {Frontiers in Cell and Developmental Biology}, volume = {7}, journal = {Frontiers in Cell and Developmental Biology}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {2296-634X}, doi = {10.3389/fcell.2019.00218}, pages = {14}, year = {2019}, abstract = {As structural membrane components and signaling effector molecules sphingolipids influence a plethora of host cell functions, and by doing so also the replication of viruses. Investigating the effects of various inhibitors of sphingolipid metabolism in primary human peripheral blood lymphocytes (PBL) and the human B cell line BJAB we found that not only the sphingosine kinase (SphK) inhibitor SKI-II, but also the acid ceramidase inhibitor ceranib-2 efficiently inhibited measles virus (MV) replication. Virus uptake into the target cells was not grossly altered by the two inhibitors, while titers of newly synthesized MV were reduced by approximately 1 log (90\%) in PBL and 70-80\% in BJAB cells. Lipidomic analyses revealed that in PBL SKI-II led to increased ceramide levels, whereas in BJAB cells ceranib-2 increased ceramides. SKI-II treatment decreased sphingosine-1-phosphate (S1P) levels in PBL and BJAB cells. Furthermore, we found that MV infection of lymphocytes induced a transient (0.5-6 h) increase in S1P, which was prevented by SKI-II. Investigating the effect of the inhibitors on the metabolic (mTORC1) activity we found that ceranib-2 reduced the phosphorylation of p70 S6K in PBL, and that both inhibitors, ceranib-2 and SKI-II, reduced the phosphorylation of p70 S6K in BJAB cells. As mTORC1 activity is required for efficient MV replication, this effect of the inhibitors is one possible antiviral mechanism. In addition, reduced intracellular S1P levels affect a number of signaling pathways and functions including Hsp90 activity, which was reported to be required for MV replication. Accordingly, we found that pharmacological inhibition of Hsp90 with the inhibitor 17-AAG strongly impaired MV replication in primary PBL. Thus, our data suggest that treatment of lymphocytes with both, acid ceramidase and SphK inhibitors, impair MV replication by affecting a number of cellular activities including mTORC1 and Hsp90, which alter the metabolic state of the cells causing a hostile environment for the virus.}, language = {en} } @article{WienholdMacriNouaillesetal.2018, author = {Wienhold, Sandra-Maria and Macri, Mario and Nouailles, Geraldine and Dietert, Kristina and Gurtner, Corinne and Gruber, Achim D. and Heimesaat, Markus M. and Lienau, Jasmin and Schumacher, Fabian and Kleuser, Burkhard and Opitz, Bastian and Suttorp, Norbert and Witzenrath, Martin and M{\"u}ller-Redetzky, Holger C.}, title = {Ventilator-induced lung injury is aggravated by antibiotic mediated microbiota depletion in mice}, series = {Critical Care}, volume = {22}, journal = {Critical Care}, number = {282}, publisher = {BMC}, address = {London}, issn = {1466-609X}, doi = {10.1186/s13054-018-2213-8}, pages = {12}, year = {2018}, abstract = {BackgroundAntibiotic exposure alters the microbiota, which can impact the inflammatory immune responses. Critically ill patients frequently receive antibiotic treatment and are often subjected to mechanical ventilation, which may induce local and systemic inflammatory responses and development of ventilator-induced lung injury (VILI). The aim of this study was to investigate whether disruption of the microbiota by antibiotic therapy prior to mechanical ventilation affects pulmonary inflammatory responses and thereby the development of VILI.MethodsMice underwent 6-8weeks of enteral antibiotic combination treatment until absence of cultivable bacteria in fecal samples was confirmed. Control mice were housed equally throughout this period. VILI was induced 3 days after completing the antibiotic treatment protocol, by high tidal volume (HTV) ventilation (34ml/kg; positive end-expiratory pressure=2 cmH(2)O) for 4h. Differences in lung function, oxygenation index, pulmonary vascular leakage, macroscopic assessment of lung injury, and leukocyte and lymphocyte differentiation were assessed. Control groups of mice ventilated with low tidal volume and non-ventilated mice were analyzed accordingly.ResultsAntibiotic-induced microbiota depletion prior to HTV ventilation led to aggravation of VILI, as shown by increased pulmonary permeability, increased oxygenation index, decreased pulmonary compliance, enhanced macroscopic lung injury, and increased cytokine/chemokine levels in lung homogenates.ConclusionsDepletion of the microbiota by broad-spectrum antibiotics prior to HTV ventilation renders mice more susceptible to developing VILI, which could be clinically relevant for critically ill patients frequently receiving broad-spectrum antibiotics.}, language = {en} }