TY - JOUR A1 - Hasan, Ahmed Abdallah Abdalrahman Mohamed A1 - von Websky, Karoline A1 - Reichetzeder, Christoph A1 - Tsuprykov, Oleg A1 - Gaballa, Mohamed Mahmoud Salem Ahmed A1 - Guo, Jingli A1 - Zeng, Shufei A1 - Delic, Denis A1 - Tammen, Harald A1 - Klein, Thomas A1 - Kleuser, Burkhard A1 - Hocher, Berthold T1 - Mechanisms of GLP-1 receptor-independent renoprotective effects of the dipeptidyl peptidase type 4 inhibitor linagliptin in GLP-1 receptor knockout mice with 5/6 nephrectomy JF - Kidney international : official journal of the International Society of Nephrology N2 - Dipeptidyl peptidase type 4 (DPP-4) inhibitors were reported to have beneficial effects in experimental models of chronic kidney disease. The underlying mechanisms are not completely understood. However, these effects could be mediated via the glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP1R) pathway. Here we investigated the renal effects of the DPP-4 inhibitor linagliptin in Glp1r-/- knock out and wild-type mice with 5/6 nephrectomy (5/6Nx). Mice were allocated to groups: sham + wild type + placebo; 5/6Nx+ wild type + placebo; 5/6Nx+ wild type + linagliptin; sham + knock out+ placebo; 5/6Nx + knock out+ placebo; 5/6Nx + knock out+ linagliptin. 5/6Nx caused the development of renal interstitial fibrosis, significantly increased plasma cystatin C and creatinine levels and suppressed renal gelatinase/collagenase, matrix metalloproteinase-1 and -13 activities; effects counteracted by linagliptin treatment in wildtype and Glp1r-/- mice. Two hundred ninety-eight proteomics signals were differentially regulated in kidneys among the groups, with 150 signals specific to linagliptin treatment as shown by mass spectrometry. Treatment significantly upregulated three peptides derived from collagen alpha-1(I), thymosin beta 4 and heterogeneous nuclear ribonucleoprotein Al (HNRNPA1) and significantly downregulated one peptide derived from Y box binding protein-1 (YB-1). The proteomics results were further confirmed using western blot and immunofluorescence microscopy. Also, 5/6Nx led to significant up-regulation of renal transforming growth factor-beta 1 and pSMAD3 expression in wild type mice and linagliptin significantly counteracted this up-regulation in wild type and GIplr-/- mice. Thus, the renoprotective effects of linagliptin cannot solely be attributed to the GLP-1/GLP1R pathway, highlighting the importance of other signaling pathways (collagen I homeostasis, HNRNPA1,YB-1,thymosin beta 4 and TGF-beta 1) influenced by DPP-4 inhibition. KW - chronic kidney disease KW - collagen I KW - fibrosis KW - Glp1r(-/-) mice KW - HNRNPA1 KW - linagliptin KW - proteomic analysis KW - TGF-beta 1 KW - thymosin beta 4 KW - YB-1 Y1 - 2019 U6 - https://doi.org/10.1016/j.kint.2019.01.010 SN - 0085-2538 SN - 1523-1755 VL - 95 IS - 6 SP - 1373 EP - 1388 PB - Elsevier CY - New York ER - TY - JOUR A1 - Schoenauer, Roman A1 - Larpin, Yu A1 - Babiychuk, Eduard B. A1 - Drucker, Patrick A1 - Babiychuk, Viktoriia S. A1 - Avota, Elita A1 - Schneider-Schaulies, Sibylle A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Koffel, Rene A1 - Draeger, Annette T1 - Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins JF - The FASEB journal : the official journal of the Federation of American Societies for Experimental Biology N2 - 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. KW - membrane repair KW - blebbing KW - calcium KW - bacterial toxins KW - annexins Y1 - 2018 U6 - https://doi.org/10.1096/fj.201800033R SN - 0892-6638 SN - 1530-6860 VL - 33 IS - 1 SP - 275 EP - 285 PB - Federation of American Societies for Experimental Biology CY - Bethesda ER - TY - JOUR A1 - Solger, Franziska A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Paprotka, Kerstin A1 - Pfister, Pauline A1 - Hagen, Franziska A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Seibel, Jürgen A1 - Rudel, Thomas T1 - A role of sphingosine in the intracellular survival of Neisseria gonorrhoeae JF - Frontiers in Cellular and Infection Microbiology N2 - Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells. KW - Neisseria gonorrhoeae KW - sphingosine KW - sphingolipids KW - sphingosine kinases KW - invasion KW - survival KW - click chemistry Y1 - 2020 U6 - https://doi.org/10.3389/fcimb.2020.00215 SN - 2235-2988 VL - 10 PB - Frontiers Media CY - Lausanne ER - TY - JOUR A1 - Derakhshani, Shaghayegh A1 - Kurz, Andreas A1 - Japtok, Lukasz A1 - Schumacher, Fabian A1 - Pilgram, Lisa A1 - Steinke, Maria A1 - Kleuser, Burkhard A1 - Sauer, Markus A1 - Schneider-Schaulies, Sibylle A1 - Avota, Elita T1 - Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium JF - Frontiers in immunology N2 - Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit. KW - dendritic cell KW - cell migration KW - measles virus KW - 3D tissue model KW - sphingosine-1-phosphate Y1 - 2019 U6 - https://doi.org/10.3389/fimmu.2019.01294 SN - 1664-3224 VL - 10 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Meiners, Jana A1 - Palmieri, Vittoria A1 - Klopfleisch, Robert A1 - Ebel, Jana-Fabienne A1 - Japtok, Lukasz A1 - Schumacher, Fabian A1 - Yusuf, Ayan Mohamud A1 - Becker, Katrin Anne A1 - Zöller, Julia A1 - Hose, Matthias A1 - Kleuser, Burkhard A1 - Hermann, Dirk Matthias A1 - Kolesnick, Richard N. A1 - Buer, Jan A1 - Hansen, Wiebke A1 - Westendorf, Astrid M. T1 - Intestinal acid sphingomyelinase protects from severe Pathogen-Driven Colitis JF - Frontiers in immunology N2 - 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. KW - Citrobacter rodentium KW - colitis KW - acid sphingomyelinase KW - amitriptyline KW - T(h)1 KW - T(h)17 Y1 - 2019 U6 - https://doi.org/10.3389/fimmu.2019.01386 SN - 1664-3224 VL - 10 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Hausmann, Christian A1 - Zoschke, Christian A1 - Wolff, Christopher A1 - Darvin, Maxim E. A1 - Sochorova, Michaela A1 - Kovacik, Andrej A1 - Wanjiku, Barbara A1 - Schumacher, Fabian A1 - Tigges, Julia A1 - Kleuser, Burkhard A1 - Lademann, Juergen A1 - Fritsche, Ellen A1 - Vavrova, Katerina A1 - Ma, Nan A1 - Schaefer-Korting, Monika T1 - Fibroblast origin shapes tissue homeostasis, epidermal differentiation, and drug uptake JF - Scientific reports N2 - 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. Y1 - 2019 U6 - https://doi.org/10.1038/s41598-019-39770-6 SN - 2045-2322 VL - 9 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Beckmann, Nadine A1 - Becker, Katrin Anne A1 - Kadow, Stephanie A1 - Schumacher, Fabian A1 - Kramer, Melanie A1 - Kuehn, Claudine A1 - Schulz-Schaeffer, Walter J. A1 - Edwards, Michael J. A1 - Kleuser, Burkhard A1 - Gulbins, Erich A1 - Carpinteiro, Alexander T1 - Acid Sphingomyelinase Deficiency Ameliorates Farber Disease JF - International journal of molecular sciences N2 - 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. KW - Farber disease KW - lysosomal storage disorders KW - acid ceramidase KW - acid sphingomyelinase KW - amitriptyline Y1 - 2019 U6 - https://doi.org/10.3390/ijms20246253 SN - 1422-0067 VL - 20 IS - 24 PB - MDPI CY - Basel ER - TY - JOUR A1 - Zeitler, Stefanie A1 - Ye, Lian A1 - Andreyeva, Aksana A1 - Schumacher, Fabian A1 - Monti, Juliana A1 - Nürnberg, Bernd A1 - Nowak, Gabriel A1 - Kleuser, Burkhard A1 - Reichel, Martin A1 - Fejtova, Anna A1 - Kornhuber, Johannes A1 - Rhein, Cosima A1 - Friedland, Kristina T1 - Acid sphingomyelinase - a regulator of canonical transient receptor potential channel 6 (TRPC6) activity JF - Journal of neurochemistry N2 - 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. KW - acid sphingomyelinase KW - antidepressants KW - ceramide KW - hyperforin KW - lipid rafts KW - TRPC6 Y1 - 2019 U6 - https://doi.org/10.1111/jnc.14823 SN - 0022-3042 SN - 1471-4159 VL - 150 IS - 6 SP - 678 EP - 690 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Grafen, Anika A1 - Schumacher, Fabian A1 - Chithelen, Janice A1 - Kleuser, Burkhard A1 - Beyersdorf, Niklas A1 - Schneider-Schaulies, Jürgen T1 - Use of Acid Ceramidase and Sphingosine Kinase Inhibitors as Antiviral Compounds Against Measles Virus Infection of Lymphocytes in vitro JF - Frontiers in Cell and Developmental Biology N2 - 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. KW - measles virus KW - sphingolipids KW - acid ceramidase KW - acid ceramidase inhibitor ceranib-2 KW - sphingosine kinase KW - sphingosine kinase inhibitor SKI-II Y1 - 2019 U6 - https://doi.org/10.3389/fcell.2019.00218 SN - 2296-634X VL - 7 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Börtlein, Charlene A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Dölken, Lars A1 - Avota, Elita T1 - Role of Neutral Sphingomyelinase-2 (NSM 2) in the Control of T Cell Plasma Membrane Lipid Composition and Cholesterol Homeostasis JF - Frontiers in cell and developmental biology N2 - 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. KW - neutral sphingomyelinase-2 KW - T cell receptor KW - plasma membrane KW - lyso-phospholipids KW - diacylglycerol KW - cholesteryl ester Y1 - 2019 U6 - https://doi.org/10.3389/fcell.2019.00226 SN - 2296-634X VL - 7 PB - Frontiers Research Foundation CY - Lausanne ER -