@article{FinkSchumacherSchlegeletal.2021, author = {Fink, Julian and Schumacher, Fabian and Schlegel, Jan and Stenzel, Philipp and Wigger, Dominik and Sauer, Markus and Kleuser, Burkhard and Seibel, J{\"u}rgen}, title = {Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis}, series = {Organic \& biomolecular chemistry : an international journal of synthetic, physical and biomolecular organic chemistry}, volume = {19}, journal = {Organic \& biomolecular chemistry : an international journal of synthetic, physical and biomolecular organic chemistry}, number = {10}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1477-0520}, doi = {10.1039/d0ob02592e}, pages = {2203 -- 2212}, year = {2021}, abstract = {Here were report the combination of biocompatible click chemistry of omega-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20\% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that omega-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, omega-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.}, language = {en} } @article{NaserKadowSchumacheretal.2021, author = {Naser, Eyad and Kadow, Stephanie and Schumacher, Fabian and Mohamed, Zainelabdeen H. and Kappe, Christian and Hessler, Gabriele and Pollmeier, Barbara and Kleuser, Burkhard and Arenz, Christoph and Becker, Katrin Anne and Gulbins, Erich and Carpinteiro, Alexander}, title = {Characterization of the small molecule ARC39}, series = {Journal of Lipid Research}, volume = {61}, journal = {Journal of Lipid Research}, number = {6}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {1539-7262}, doi = {10.1194/jlr.RA120000682}, pages = {896 -- 910}, year = {2021}, abstract = {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.}, language = {en} } @article{WetzelScholtkaSchumacheretal.2021, author = {Wetzel, Alexandra Nicole and Scholtka, Bettina and Schumacher, Fabian and Rawel, Harshadrai Manilal and Geisend{\"o}rfer, Birte and Kleuser, Burkhard}, title = {Epigenetic DNA methylation of EBI3 modulates human interleukin-35 formation via NFkB signaling}, series = {International journal of molecular sciences}, volume = {22}, journal = {International journal of molecular sciences}, number = {10}, publisher = {MDPI}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms22105329}, pages = {21}, year = {2021}, abstract = {Ulcerative colitis (UC), a severe chronic disease with unclear etiology that is associated with increased risk for colorectal cancer, is accompanied by dysregulation of cytokines. Epstein-Barr virus-induced gene 3 (EBI3) encodes a subunit in the unique heterodimeric IL-12 cytokine family of either pro- or anti-inflammatory function. After having recently demonstrated that upregulation of EBI3 by histone acetylation alleviates disease symptoms in a dextran sulfate sodium (DSS)-treated mouse model of chronic colitis, we now aimed to examine a possible further epigenetic regulation of EBI3 by DNA methylation under inflammatory conditions. Treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC) and TNF alpha led to synergistic upregulation of EBI3 in human colon epithelial cells (HCEC). Use of different signaling pathway inhibitors indicated NF kappa B signaling was necessary and proportional to the synergistic EBI3 induction. MALDI-TOF/MS and HPLC-ESIMS/MS analysis of DAC/TNF alpha-treated HCEC identified IL-12p35 as the most probable binding partner to form a functional protein. EBI3/IL-12p35 heterodimers (IL-35) induce their own gene upregulation, something that was indeed observed in HCEC cultured with media from previously DAC/TNF alpha-treated HCEC. These results suggest that under inflammatory and demethylating conditions the upregulation of EBI3 results in the formation of anti-inflammatory IL-35, which might be considered as a therapeutic target in colitis.}, language = {en} } @article{WardelmannRathCastroetal.2021, author = {Wardelmann, Kristina and Rath, Michaela and Castro, Jos{\´e} Pedro and Bl{\"u}mel, Sabine and Schell, Mareike and Hauffe, Robert and Schumacher, Fabian and Flore, Tanina and Ritter, Katrin and Wernitz, Andreas and Hosoi, Toru and Ozawa, Koichiro and Kleuser, Burkhard and Weiß, J{\"u}rgen and Sch{\"u}rmann, Annette and Kleinridders, Andr{\´e}}, title = {Central acting Hsp10 regulates mitochondrial function, fatty acid metabolism and insulin sensitivity in the hypothalamus}, series = {Antioxidants}, volume = {10}, journal = {Antioxidants}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2076-3921}, doi = {10.3390/antiox10050711}, pages = {22}, year = {2021}, abstract = {Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.}, language = {en} } @article{WiggerSchumacherSchneiderSchauliesetal.2021, author = {Wigger, Dominik and Schumacher, Fabian and Schneider-Schaulies, Sibylle and Kleuser, Burkhard}, title = {Sphingosine 1-phosphate metabolism and insulin signaling}, series = {Cellular signalling}, volume = {82}, journal = {Cellular signalling}, publisher = {Elsevier Science}, address = {Amsterdam [u.a.]}, issn = {0898-6568}, doi = {10.1016/j.cellsig.2021.109959}, pages = {16}, year = {2021}, abstract = {Insulin is the main anabolic hormone secreted by 13-cells of the pancreas stimulating the assimilation and storage of glucose in muscle and fat cells. It modulates the postprandial balance of carbohydrates, lipids and proteins via enhancing lipogenesis, glycogen and protein synthesis and suppressing glucose generation and its release from the liver. Resistance to insulin is a severe metabolic disorder related to a diminished response of peripheral tissues to the insulin action and signaling. This leads to a disturbed glucose homeostasis that precedes the onset of type 2 diabetes (T2D), a disease reaching epidemic proportions. A large number of studies reported an association between elevated circulating fatty acids and the development of insulin resistance. The increased fatty acid lipid flux results in the accumulation of lipid droplets in a variety of tissues. However, lipid intermediates such as diacylglycerols and ceramides are also formed in response to elevated fatty acid levels. These bioactive lipids have been associated with the pathogenesis of insulin resistance. More recently, sphingosine 1-phosphate (S1P), another bioactive sphingolipid derivative, has also been shown to increase in T2D and obesity. Although many studies propose a protective role of S1P metabolism on insulin signaling in peripheral tissues, other studies suggest a causal role of S1P on insulin resistance. In this review, we critically summarize the current state of knowledge of S1P metabolism and its modulating role on insulin resistance. A particular emphasis is placed on S1P and insulin signaling in hepatocytes, skeletal muscle cells, adipocytes and pancreatic 13-cells. In particular, modulation of receptors and enzymes that regulate S1P metabolism can be considered as a new therapeutic option for the treatment of insulin resistance and T2D.}, language = {en} } @article{BeckmannSchumacherKleuseretal.2021, author = {Beckmann, Nadine and Schumacher, Fabian and Kleuser, Burkhard and Gulbins, Erich and Nomellini, Vanessa and Caldwell, Charles C.}, title = {Burn injury impairs neutrophil chemotaxis through increased ceramide}, series = {Shock : injury, inflammation, and sepsis, laboratory and clinical approaches}, volume = {56}, journal = {Shock : injury, inflammation, and sepsis, laboratory and clinical approaches}, number = {1}, publisher = {Lippincott Williams \& Wilkins}, address = {Hagerstown, Md.}, issn = {1073-2322}, doi = {10.1097/SHK.0000000000001693}, pages = {125 -- 132}, year = {2021}, abstract = {Infection is a common and often deadly complication after burn injury. A major underlying factor is burn-induced immune dysfunction, particularly with respect to neutrophils as the primary responders to infection. Temporally after murine scald injury, we demonstrate impaired bone marrow neutrophil chemotaxis toward CXCL1 ex vivo. Additionally, we observed a reduced recruitment of neutrophils to the peritoneal after elicitation 7 days after injury. We demonstrate that neutrophil ceramide levels increase after burn injury, and this is associated with decreased expression of CXCR2 and blunted chemotaxis. A major signaling event upon CXCR2 activation is Akt phosphorylation and this was reduced when ceramide was elevated. In contrast, PTEN levels were elevated and PTEN-inhibition elevated phospho-Akt levels and mitigated the burn-induced neutrophil chemotaxis defect. Altogether, this study identifies a newly described pathway of ceramide-mediated suppression of neutrophil chemotaxis after burn injury and introduces potential targets to mitigate this defect and reduce infection-related morbidity and mortality after burn.}, language = {en} } @article{HalilbasicFuerstHeidenetal.2020, author = {Halilbasic, Emina and Fuerst, Elisabeth and Heiden, Denise and Japtok, Lukasz and Diesner, Susanne C. and Trauner, Michael and Kulu, Askin and Jaksch, Peter and Hoetzenecker, Konrad and Kleuser, Burkhard and Kazemi-Shirazi, Lili and Untersmayr, Eva}, title = {Plasma levels of the bioactive sphingolipid metabolite S1P in adult cystic fibrosis patients}, series = {Nutrients}, volume = {12}, journal = {Nutrients}, number = {3}, publisher = {MDPI}, address = {Basel}, issn = {2072-6643}, doi = {10.3390/nu12030765}, pages = {11}, year = {2020}, abstract = {Recent research has linked sphingolipid (SL) metabolism with cystic fibrosis transmembrane conductance regulator (CFTR) activity, affecting bioactive lipid mediator sphingosine-1-phosphate (S1P). We hypothesize that loss of CFTR function in cystic fibrosis (CF) patients influenced plasma S1P levels. Total and unbound plasma S1P levels were measured in 20 lung-transplanted adult CF patients and 20 healthy controls by mass spectrometry and enzyme-linked immunosorbent assay (ELISA). S1P levels were correlated with CFTR genotype, routine laboratory parameters, lung function and pathogen colonization, and clinical symptoms. Compared to controls, CF patients showed lower unbound plasma S1P, whereas total S1P levels did not differ. A positive correlation of total and unbound S1P levels was found in healthy controls, but not in CF patients. Higher unbound S1P levels were measured in Delta F508-homozygous compared to Delta F508-heterozygous CF patients (p = 0.038), accompanied by higher levels of HDL in Delta F508-heterozygous patients. Gastrointestinal symptoms were more common in Delta F508 heterozygotes compared to Delta F508 homozygotes. This is the first clinical study linking plasma S1P levels with CFTR function and clinical presentation in adult CF patients. Given the emerging role of immunonutrition in CF, our study might pave the way for using S1P as a novel biomarker and nutritional target in CF.}, language = {en} } @article{WetzelScholtkaGereckeetal.2020, author = {Wetzel, Alexandra Nicole and Scholtka, Bettina and Gerecke, Christian and Kleuser, Burkhard}, title = {Epigenetic histone modulation contributes to improvements in inflammatory bowel disease via EBI3}, series = {Cellular and molecular life sciences}, volume = {77}, journal = {Cellular and molecular life sciences}, number = {23}, publisher = {Springer International Publishing AG}, address = {Cham (ZG)}, issn = {1420-682X}, doi = {10.1007/s00018-020-03451-9}, pages = {5017 -- 5030}, year = {2020}, abstract = {Ulcerative colitis (UC) is characterized by relapsing-remitting inflammatory episodes paralleled by varying cytokine levels, suggesting that switching epigenetic processes might be involved. However, the epigenetic impact on cytokine levels in colitis is mostly unexplored. The heterodimeric interleukin (IL)-12 cytokine family have various functions in both pro- and anti-inflammatory processes. The family member IL-35 (EBI3/IL-12p35) was recently reported to play an anti-inflammatory role in UC. Therefore, we aimed to investigate a possible epigenetic regulation of the IL-35 subunits in vitro and in vivo, and to examine the epigenetic targeting of EBI3 expression as a therapeutic option for UC. Exposure to either the pro-inflammatory TNF alpha or to histone deacetylase inhibitors (HDACi) significantly increased EBI3 expression in Human Colon Epithelial Cells (HCEC) generated from healthy tissue. When applied in combination, a drastic upregulation of EBI3 expression occurred, suggesting a synergistic mechanism. Consequently, IL-35 was increased as well. In vivo, the intestines of HDACi-treated wild-type mice exhibited reduced pathological signs of colitis compared to non-treated colitic mice. However, the improvement by HDACi treatment was completely lost in Ebi3-deficient mice (Ebi3(-/-)). In fact, HDACi appeared to exacerbate the disease phenotype in Ebi3(-/-). In conclusion, our results reveal that under inflammatory conditions, EBI3 is upregulated by the epigenetic mechanism of histone acetylation. The in vivo data show that the deficiency of EBI3 plays a key role in colitis manifestation. Concordantly, our data suggest that conditions promoting histone acetylation, such as upon HDACi application, improve colitis by a mechanism involving the local formation of the anti-inflammatory cytokine IL-35.}, language = {en} } @article{StepanovskaZivkovicEnzmannetal.2020, author = {Stepanovska, Bisera and Zivkovic, Aleksandra and Enzmann, Gaby and Tietz, Silvia and Homann, Thomas and Kleuser, Burkhard and Engelhardt, Britta and Stark, Holger and Huwiler, Andrea}, title = {Morpholino analogues of fingolimod as novel and selective S1P1 ligands with in vivo efficacy in a mouse model of experimental antigen-induced encephalomyelitis}, series = {International journal of molecular sciences}, volume = {21}, journal = {International journal of molecular sciences}, number = {18}, publisher = {MDPI}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms21186463}, pages = {17}, year = {2020}, abstract = {Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya(R)), which acts as a functional S1P(1) antagonist on T lymphocytes to deplete these cells from the blood. In this study, we synthesized two novel structures, ST-1893 and ST-1894, which are derived from fingolimod and chemically feature a morpholine ring in the polar head group. These compounds showed a selective S1P(1) activation profile and a sustained S1P(1) internalization in cultures of S1P(1)-overexpressing Chinese hamster ovary (CHO)-K1 cells, consistent with a functional antagonism. In vivo, both compounds induced a profound lymphopenia in mice. Finally, these substances showed efficacy in the EAE model, where they reduced clinical symptoms of the disease, and, on the molecular level, they reduced the T-cell infiltration and several inflammatory mediators in the brain and spinal cord. In summary, these data suggest that S1P(1)-selective compounds may have an advantage over fingolimod and siponimod, not only in MS but also in other autoimmune diseases.}, language = {en} } @article{SamahaHamdoCongetal.2020, author = {Samaha, Doaa and Hamdo, Housam H. and Cong, Xiaojing and Schumacher, Fabian and Banhart, Sebastian and Aglar, {\"O}znur and M{\"o}ller, Heiko Michael and Heuer, Dagmar and Kleuser, Burkhard and Saied, Essa M. and Arenz, Christoph}, title = {Liposomal FRET assay identifies potent drug-like inhibitors of the Ceramide Transport Protein (CERT)}, series = {Chemistry - a European journal}, volume = {26}, journal = {Chemistry - a European journal}, number = {70}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {0947-6539}, doi = {10.1002/chem.202003283}, pages = {16616 -- 16621}, year = {2020}, abstract = {Ceramide transfer protein (CERT) mediates non-vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate-limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non-homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Forster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT-mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96-well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.}, language = {en} } @article{LangBohnBhatetal.2020, author = {Lang, Judith and Bohn, Patrick and Bhat, Hilal and Jastrow, Holger and Walkenfort, Bernd and Cansiz, Feyza and Fink, Julian and Bauer, Michael and Schumacher, Fabian and Kleuser, Burkhard and Lang, Karl S.}, title = {Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease}, series = {Nature Communications}, volume = {11}, journal = {Nature Communications}, number = {1}, publisher = {Nature Publishing Group UK}, address = {London}, issn = {2041-1723}, doi = {10.1038/s41467-020-15072-8}, pages = {1 -- 15}, year = {2020}, abstract = {Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1(-/-) mice results in replication of HSV-1 and Asah1(-/-) mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.}, language = {en} } @article{ZoicasSchumacherKleuseretal.2020, author = {Zoicas, Iulia and Schumacher, Fabian and Kleuser, Burkhard and Reichel, Martin and Gulbins, Erich and Fejtova, Anna and Kornhuber, Johannes and Rhein, Cosima}, title = {The forebrain-specific overexpression of acid sphingomyelinase induces depressive-like symptoms in mice}, series = {Cells}, volume = {9}, journal = {Cells}, number = {5}, publisher = {MDPI}, address = {Basel}, pages = {12}, year = {2020}, abstract = {Human and murine studies identified the lysosomal enzyme acid sphingomyelinase (ASM) as a target for antidepressant therapy and revealed its role in the pathophysiology of major depression. In this study, we generated a mouse model with overexpression of Asm (Asm-tg(fb)) that is restricted to the forebrain to rule out any systemic effects of Asm overexpression on depressive-like symptoms. The increase in Asm activity was higher in male Asm-tg(fb) mice than in female Asm-tg(fb) mice due to the breeding strategy, which allows for the generation of wild-type littermates as appropriate controls. Asm overexpression in the forebrain of male mice resulted in a depressive-like phenotype, whereas in female mice, Asm overexpression resulted in a social anxiogenic-like phenotype. Ceramides in male Asm-tg(fb) mice were elevated specifically in the dorsal hippocampus. mRNA expression analyses indicated that the increase in Asm activity affected other ceramide-generating pathways, which might help to balance ceramide levels in cortical brain regions. This forebrain-specific mouse model offers a novel tool for dissecting the molecular mechanisms that play a role in the pathophysiology of major depression.}, language = {en} } @article{DwiPutraReichetzederHasanetal.2020, author = {Dwi Putra, Sulistyo Emantoko and Reichetzeder, Christoph and Hasan, Ahmed Abdallah Abdalrahman Mohamed and Slowinski, Torsten and Chu, Chang and Kr{\"a}mer, Bernhard K. and Kleuser, Burkhard and Hocher, Berthold}, title = {Being born large for gestational age is associated with increased global placental DNA methylation}, series = {Scientific Reports}, volume = {10}, journal = {Scientific Reports}, number = {1}, publisher = {Springer Nature}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-020-57725-0}, pages = {1 -- 10}, year = {2020}, abstract = {Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DNA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DNA methylation are lacking. The aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p<0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p<0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p=0.001).}, language = {en} } @article{HasanvonWebskyReichetzederetal.2019, author = {Hasan, Ahmed Abdallah Abdalrahman Mohamed and von Websky, Karoline and Reichetzeder, Christoph and Tsuprykov, Oleg and Gaballa, Mohamed Mahmoud Salem Ahmed and Guo, Jingli and Zeng, Shufei and Delic, Denis and Tammen, Harald and Klein, Thomas and Kleuser, Burkhard and Hocher, Berthold}, title = {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}, series = {Kidney international : official journal of the International Society of Nephrology}, volume = {95}, journal = {Kidney international : official journal of the International Society of Nephrology}, number = {6}, publisher = {Elsevier}, address = {New York}, issn = {0085-2538}, doi = {10.1016/j.kint.2019.01.010}, pages = {1373 -- 1388}, year = {2019}, abstract = {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.}, 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{SolgerKunzFinketal.2019, author = {Solger, Franziska and Kunz, Tobias C. and Fink, Julian and Paprotka, Kerstin and Pfister, Pauline and Hagen, Franziska and Schumacher, Fabian and Kleuser, Burkhard and Seibel, J{\"u}rgen and Rudel, Thomas}, title = {A role of sphingosine in the intracellular survival of Neisseria gonorrhoeae}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {10}, journal = {Frontiers in Cellular and Infection Microbiology}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {2235-2988}, doi = {10.3389/fcimb.2020.00215}, pages = {12}, year = {2019}, abstract = {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.}, language = {en} } @article{DerakhshaniKurzJaptoketal.2019, author = {Derakhshani, Shaghayegh and Kurz, Andreas and Japtok, Lukasz and Schumacher, Fabian and Pilgram, Lisa and Steinke, Maria and Kleuser, Burkhard and Sauer, Markus and Schneider-Schaulies, Sibylle and Avota, Elita}, title = {Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium}, series = {Frontiers in immunology}, volume = {10}, journal = {Frontiers in immunology}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {1664-3224}, doi = {10.3389/fimmu.2019.01294}, pages = {14}, year = {2019}, abstract = {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.}, 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{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{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} }