Publications by authors named "Nerea Ferreirós"

119 Publications

The sphingosine kinase 1 activator, K6PC-5, attenuates Ebola virus infection.

iScience 2021 Apr 5;24(4):102266. Epub 2021 Mar 5.

Institute of General Pharmacology and Toxicology, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt am Main 60590, Germany.

Ebola virus (EBOV) is responsible for outbreaks with case fatality rates of up to 90% and for an epidemic in West Africa with more than ten thousand deaths. EBOV glycoprotein (EBOV-GP) is the only viral surface protein and is responsible for viral entry into cells. Here, by employing pseudotyped EBOV-GP viral particles, we uncover a critical role for sphingolipids in inhibiting viral entry. Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). The administration of the SphK1 activator, K6PC-5, or S1P, or the overexpression of SphK1 consistently exhibited striking inhibitory effects in EBOV-GP-driven entry in diverse cell lines. Finally, K6PC-5 markedly reduced the EBOV titer in infected cells and the de novo production of viral proteins. These data present K6PC-5 as an efficient tool to inhibit EBOV infection in endothelial cells and suggest further studies to evaluate its systemic effects.
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http://dx.doi.org/10.1016/j.isci.2021.102266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005759PMC
April 2021

Bacterial and Fungal Toll-Like Receptor Activation Elicits Type I IFN Responses in Mast Cells.

Front Immunol 2020 12;11:607048. Epub 2021 Feb 12.

Institute of Clinical Pharmacology, University Hospital Goethe-University Frankfurt, Frankfurt, Germany.

Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-β synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-β was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-β, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.
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http://dx.doi.org/10.3389/fimmu.2020.607048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907501PMC
February 2021

Lipidomic analysis of local inflammation models shows a specific systemic acute phase response to lipopolysaccharides.

Biochim Biophys Acta Mol Cell Biol Lipids 2021 01 1;1866(1):158822. Epub 2020 Oct 1.

Institute of Clinical Pharmacology, University Hospital Goethe University Frankfurt, Germany; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Frankfurt, Germany; Fraunhofer Cluster of Excellence for Immune-Mediated Diseases (CIMD), Frankfurt, Germany. Electronic address:

Toll-like receptors (TLR) are crucial for recognizing bacterial, viral or fungal pathogens and to orchestrate the appropriate immune response. The widely expressed TLR2 and TLR4 differentially recognize various pathogens to initiate partly overlapping immune cascades. To better understand the physiological consequences of both immune responses, we performed comparative lipidomic analyses of local paw inflammation in mice induced by the TLR2 and TLR4 agonists, zymosan and lipopolysaccharide (LPS), respectively, which are commonly used in models for inflammation and inflammatory pain. Doses for both agonists were chosen to cause mechanical hypersensitivity with identical strength and duration. Lipidomic analysis showed 5 h after LPS or zymosan injection in both models an increase of ether-phosphatidylcholines (PC O) and their corresponding lyso species with additional lipids being increased only in response to LPS. However, zymosan induced stronger immune cell recruitment and edema formation as compared to LPS. Importantly, only in LPS-induced inflammation the lipid profile in the contralateral paw was altered. Fittingly, the plasma level of various cytokines and chemokines, including IL-1β and IL-6, were significantly increased only in LPS-treated mice. Accordingly LPS induced distinct changes in the lipid profiles of ipsilateral and contralateral paws. Here, oxydized fatty acids, phosphatidylcholines and phosphatidylethanolamines were uniquely upregulated on the contralateral side. Thus, both models cause increased levels of PC O and lyso-PC O lipids at the site of inflammation pointing at a common role in inflammation. Also, LPS initiates systemic changes, which can be detected by changes in the lipid profiles.
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http://dx.doi.org/10.1016/j.bbalip.2020.158822DOI Listing
January 2021

Dissecting G-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1.

Cells 2020 09 29;9(10). Epub 2020 Sep 29.

Institut für Allgemeine Pharmakologie und Toxikologie, Universitätsklinikum, Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that G-coupled receptors and constitutively active Gα specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical G/phospholipase C (PLC) signaling. Here, we further characterized G regulation of SphK1. SphK1 translocation by the M receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCβ, but accelerated by wild-type PLCβ and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M receptor-stimulated inositol phosphate production, suggesting competition at Gα. Embryonic fibroblasts from Gα double-deficient mice were used to show that amino acids W263 and T257 of Gα, which interact directly with PLCβ and p63RhoGEF, were important for bradykinin B receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gα binding motif, ALXXPI, in PLCβ and p63RhoGEF. After M receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by G.
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http://dx.doi.org/10.3390/cells9102201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599897PMC
September 2020

Exercise-Induced Changes in Bioactive Lipids Might Serve as Potential Predictors of Post-Exercise Hypotension. A Pilot Study in Healthy Volunteers.

Cells 2020 09 16;9(9). Epub 2020 Sep 16.

Pharmazentrum Frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany.

Post-exercise hypotension (PEH) is the phenomenon of lowered blood pressure after a single bout of exercise. Only a fraction of people develops PEH but its occurrence correlates well with long-term effects of sports on blood pressure. Therefore, PEH has been suggested as a suitable predictor for the effectivity of exercise as therapy in hypertension. Local vascular bioactive lipids might play a potential role in this context. We performed a cross-over clinical pilot study with 18 healthy volunteers to investigate the occurrence of PEH after a single short-term endurance exercise. Furthermore, we investigated the plasma lipid profile with focus on arachidonic acid (AA)-derived metabolites as potential biomarkers of PEH. A single bout of ergometer cycling induced a significant PEH in healthy volunteers with the expected high inter-individual variability. Targeted lipid spectrum analysis revealed significant upregulation of several lipids in the direct post-exercise phase. Among these changes, only 15- hydroxyeicosatetranoic acid (HETE) correlated significantly with the extent of PEH but in an AA-independent manner, suggesting that 15-HETE might act as specific PEH-marker. Our data indicate that specific lipid modulation might facilitate the identification of patients who will benefit from exercise activity in hypertension therapy. However, larger trials including hypertonic patients are necessary to verify the clinical value of this hypothesis.
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http://dx.doi.org/10.3390/cells9092111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563406PMC
September 2020

A Sphingosine 1-Phosphate Gradient Is Linked to the Cerebral Recruitment of T Helper and Regulatory T Helper Cells during Acute Ischemic Stroke.

Int J Mol Sci 2020 Aug 28;21(17). Epub 2020 Aug 28.

Institute of General Pharmacology and Toxicology, Pharmazentrum Frankfurt, Goethe University Frankfurt, 60528 Frankfurt am Main, Germany.

Emerging evidence suggests a complex relationship between sphingosine 1-phosphate (S1P) signaling and stroke. Here, we show the kinetics of S1P in the acute phase of ischemic stroke and highlight accompanying changes in immune cells and S1P receptors (S1P). Using a C57BL/6 mouse model of middle cerebral artery occlusion (MCAO), we assessed S1P concentrations in the brain, plasma, and spleen. We found a steep S1P gradient from the spleen towards the brain. Results obtained by qPCR suggested that cells expressing the S1P type 1 (S1P) were the predominant population deserting the spleen. Here, we report the cerebral recruitment of T helper (T) and regulatory T (T) cells to the ipsilateral hemisphere, which was associated with differential regulation of cerebral S1P expression patterns in the brain after MCAO. This study provides insight that the S1P-S1P axis facilitates splenic T cell egress and is linked to the cerebral recruitment of S1P T and T cells. Further insights by which means the S1P-S1P-axis orchestrates neuronal positioning may offer new therapeutic perspectives after ischemic stroke.
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http://dx.doi.org/10.3390/ijms21176242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503682PMC
August 2020

Ether lipid and sphingolipid expression patterns are estrogen receptor-dependently altered in breast cancer cells.

Int J Biochem Cell Biol 2020 10 19;127:105834. Epub 2020 Aug 19.

Pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, Theodor Stern-Kai 7, 60590 Frankfurt am Main, Germany; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia. Electronic address:

Identifying co-expression of lipid species is challenging, but indispensable to identify novel therapeutic targets for breast cancer treatment. Lipid metabolism is often dysregulated in cancer cells, and changes in lipid metabolism affect cellular processes such as proliferation, autophagy, and tumor development. In addition to mRNA analysis of sphingolipid metabolizing enzymes, we performed liquid chromatography time-of-flight mass spectrometry analysis in three breast cancer cell lines. These breast cancer cell lines differ in estrogen receptor and G-protein coupled estrogen receptor 1 status. Our data show that sphingolipids and non-sphingolipids are strongly increased in SKBr3 cells. SKBr3 cells are estrogen receptor negative and G-protein coupled estrogen receptor 1 positive. Treatment with G15, a G-protein coupled estrogen receptor 1 antagonist, abolishes the effect of increased sphingolipid and non-sphingolipid levels in SKBr3 cells. In particular, ether lipids are expressed at much higher levels in cancer compared to normal cells and are strongly increased in SKBr3 cells. Our analysis reveals that this is accompanied by increased sphingolipid levels such as ceramide, sphingadiene-ceramide and sphingomyelin. This shows the importance of focusing on more than one lipid class when investigating molecular mechanisms in breast cancer cells. Our analysis allows unbiased screening for different lipid classes leading to identification of co-expression patterns of lipids in the context of breast cancer. Co-expression of different lipid classes could influence tumorigenic potential of breast cancer cells. Identification of co-regulated lipid species is important to achieve improved breast cancer treatment outcome.
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http://dx.doi.org/10.1016/j.biocel.2020.105834DOI Listing
October 2020

12/15-Lipoxygenase choreographs the resolution of IgG-mediated skin inflammation.

J Autoimmun 2020 12 4;115:102528. Epub 2020 Aug 4.

Department of Dermatology, Allergy, and Venereology, University of Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany; Center for Research on Inflammation of the Skin (CRIS), University of Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany. Electronic address:

Autoimmune diseases are defined by an immune response against a specific autoantigen, driven by antigen-specific T cells or antibodies. While the mechanisms resolving brief episodes of acute inflammation elicited by microbial components or tissue injury are well understood, the mechanisms resolving tissue inflammation in autoimmune diseases are still largely elusive. We have, therefore, addressed the mechanisms of resolution in IgG-mediated autoimmune diseases using a mouse model of the pemphigoid disease "bullous pemphigoid-like epidermolysis bullosa acquisita" (BP-like EBA) as prototypical example. We found that 12/15-LO is induced in skin lesions of BP-like EBA and is predominantly expressed in eosinophils. Dependent on the expression of 12/15-LO, large amounts of proresolving lipid mediators, are biosynthesized in the skin by the point disease peaks. Their production is timely correlated to the gradual reversal of tissue inflammation. Genetic deficiency in Alox15, the gene encoding 12/15-LO, disrupts this process significantly protracting and aggravating disease. This protraction is associated reduced recruitment of regulatory T cells (T) into lesional skin. Intriguingly, Alox15 mice also exhibit reduced recruitment of eosinophils into the skin, and the chemotaxis of cultured Alox15 eosinophils towards CCL11/eotaxin-1 is compromised. Finally, we demonstrate that 15-lipoxygenase-1, the human homologue of 12/15-LO is induced in granulocytes in lesional skin of patients suffering from a pemphigoid disease. Collectively, our result uncover key mechanisms resolving IgG-mediated skin inflammation. These mechanisms are orchestrated by 12/15-LO expressed in eosinophils promoting the recruitment of eosinophils and T, which in turn inhibit neutrophils.
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http://dx.doi.org/10.1016/j.jaut.2020.102528DOI Listing
December 2020

UGCG overexpression leads to increased glycolysis and increased oxidative phosphorylation of breast cancer cells.

Sci Rep 2020 05 18;10(1):8182. Epub 2020 May 18.

Pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, Theodor Stern-Kai 7, 60590, Frankfurt am Main, Germany.

The only enzyme in the glycosphingolipid (GSL) metabolic pathway, which produces glucosylceramide (GlcCer) de novo is UDP-glucose ceramide glucosyltransferase (UGCG). UGCG is linked to pro-cancerous processes such as multidrug resistance development and increased proliferation in several cancer types. Previously, we showed an UGCG-dependent glutamine metabolism adaption to nutrient-poor environment of breast cancer cells. This adaption includes reinforced oxidative stress response and fueling the tricarboxylic acid (TCA) cycle by increased glutamine oxidation. In the current study, we investigated glycolytic and oxidative metabolic phenotypes following UGCG overexpression (OE). UGCG overexpressing MCF-7 cells underwent a metabolic shift from quiescent/aerobic to energetic metabolism by increasing both glycolysis and oxidative glucose metabolism. The energetic metabolic phenotype was not associated with increased mitochondrial mass, however, markers of mitochondrial turnover were increased. UGCG OE altered sphingolipid composition of the endoplasmic reticulum (ER)/mitochondria fractions that may contribute to increased mitochondrial turnover and increased cell metabolism. Our data indicate that GSL are closely connected to cell energy metabolism and this finding might contribute to development of novel therapeutic strategies for cancer treatment.
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http://dx.doi.org/10.1038/s41598-020-65182-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7234995PMC
May 2020

The WD40 repeat protein, WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-β activation by G-coupled receptors.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 07 31;1865(7):158704. Epub 2020 Mar 31.

Institut für Allgemeine Pharmakologie und Toxikologie, Universitätsklinikum, Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address:

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed G regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gα to phospholipase C (PLC)-β and the thromboxane A receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M and bradykinin B receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca] increases. Surprisingly, WDR36 also augmented M receptor-induced [Ca] increases, which was due to increased Ca mobilization while the Ca content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by G-coupled M, B and H receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B receptors. It is concluded that WDR36 reduces signalling of G-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gα and PLCβ. Our results support a role of WDR36 in orchestration of G signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.
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http://dx.doi.org/10.1016/j.bbalip.2020.158704DOI Listing
July 2020

Long-term stimulation of toll-like receptor-2 and -4 upregulates 5-LO and 15-LO-2 expression thereby inducing a lipid mediator shift in human monocyte-derived macrophages.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 09 25;1865(9):158702. Epub 2020 Mar 25.

Institute of Pharmaceutical Chemistry/ZAFES, Max-von-Laue-Straße 9, Goethe University, 60438 Frankfurt/Main, Germany. Electronic address:

Macrophage polarization switches during the course of inflammation along with the lipid mediators released. We investigated the lipid mediator formation in human monocyte-derived macrophages during in vitro differentiation and pathogen stimulation. For this, peripheral blood monocytes were differentiated into M1 (CSF-2/IFNγ) or M2 (CSF-1/IL-4) macrophages followed by stimulation with the toll-like receptor (TLR) ligands zymosan (TLR-2), Poly(I:C) (TLR-3) or bacterial lipopolysaccharides (TLR-4) mimicking fungal, viral and bacterial infection, respectively. Expression of enzymes involved in lipid mediator formation such as 5- and 15-lipoxygenases (LO), the 5-LO activating protein and cyclooxygenase-2 (COX-2) was monitored on mRNA and protein level and lipid mediator formation was assessed. In addition, cytokine release was measured. In vitro differentiation of human peripheral blood monocytes to M1 and M2 macrophages considerably attenuated 5-LO activity. Furthermore, while TLR-2 and -4 stimulation of M1 macrophages primarily triggered pro-inflammatory cytokines and lipid mediators, persistent stimulation (16 h) of human M2 macrophages induced a coordinated upregulation of 5- and 15-LO-2 expression. This was accompanied by a marked increase in IL-10 and monohydroxylated 15-LO products in the conditioned media of the cells. After additional stimulation with Ca ionophore combined with supplementation of arachidonic, eicosapentaenoic and docosahexaenoic acid these cells also released small amounts of SPM such as lipoxins and resolvins. From this we conclude that activation of TLR-2 or -4 triggers the biosynthesis of pro-inflammatory 5-LO and COX-2 derived lipid mediators in human monocyte-derived M1 macrophages while persistent stimulation of M2 macrophages induces a shift towards pro-resolving 15-LO derived oxylipins.
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http://dx.doi.org/10.1016/j.bbalip.2020.158702DOI Listing
September 2020

Tumors Provoke Inflammation and Perineural Microlesions at Adjacent Peripheral Nerves.

Cells 2020 01 29;9(2). Epub 2020 Jan 29.

Institute of Clinical Pharmacology, University Hospital Goethe University Frankfurt, 60590 Frankfurt, Germany.

Cancer-induced pain occurs frequently in patients when tumors or their metastases grow in the proximity of nerves. Although this cancer-induced pain states poses an important therapeutical problem, the underlying pathomechanisms are not understood. Here, we implanted adenocarcinoma, fibrosarcoma and melanoma tumor cells in proximity of the sciatic nerve. All three tumor types caused mechanical hypersensitivity, thermal hyposensitivity and neuronal damage. Surprisingly the onset of the hypersensitivity was independent of physical contact of the nerve with the tumors and did not depend on infiltration of cancer cells in the sciatic nerve. However, macrophages and dendritic cells appeared on the outside of the sciatic nerves with the onset of the hypersensitivity. At the same time point downregulation of perineural tight junction proteins was observed, which was later followed by the appearance of microlesions. Fitting to the changes in the epi-/perineurium, a dramatic decrease of triglycerides and acylcarnitines in the sciatic nerves as well as an altered localization and appearance of epineural adipocytes was seen. In summary, the data show an inflammation at the sciatic nerves as well as an increased perineural and epineural permeability. Thus, interventions aiming to suppress inflammatory processes at the sciatic nerve or preserving peri- and epineural integrity may present new approaches for the treatment of tumor-induced pain.
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http://dx.doi.org/10.3390/cells9020320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072456PMC
January 2020

Implementation of lipidomics in clinical routine: Can fluoride/citrate blood sampling tubes improve preanalytical stability?

Talanta 2020 Mar 26;209:120593. Epub 2019 Nov 26.

Pharmazentrum Frankfurt/ ZAFES, Institute of Clinical Pharmacology, Goethe University, Frankfurt, Germany. Electronic address:

The impact of preanalytical sample handling on lipid stability has been assessed in human plasma using targeted LC-MS/MS quantification of endocannabinoids, sphingolipids and LPA, complemented by non-targeted lipidomics screening with LC-QTOFMS. The study involved incubation of whole blood and plasma from healthy volunteers at room temperature or in ice water for time periods ranging from 20 min to 24 h. The impact of two different anticoagulants, K3EDTA and sodium fluoride/citrate, on lipid stability was evaluated. It was found that the concentrations determined for several endogenous lipids vary when whole blood and plasma samples are processed at room temperature, whereas the concentrations of most lipids were stable for 4 h in ice water. Surprisingly, the detected amounts of endocannabinoids 1- and 2-arachidonoyl glycerol and arachidonoyl ethanolamide increased markedly by 60, 95, and 30% in K3EDTA whole blood after storage in ice water for only 20 min. When using sodium fluoride/citrate blood collection tubes, the stability of several lipids, including that of the endocannabinoids, was improved. Accordingly, it is absolutely necessary to keep the blood sampling and plasma processing time below 1 h to avoid ex-vivo formation of endocannabinoids. It is worth mentioning that baseline lipid levels differ when using K3EDTA or sodium fluoride/citrate blood sampling tubes, which emphasizes the importance of traceability of reported plasma concentrations to the used anticoagulant.
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http://dx.doi.org/10.1016/j.talanta.2019.120593DOI Listing
March 2020

A data science approach to the selection of most informative readouts of the human intradermal capsaicin pain model to assess pregabalin effects.

Basic Clin Pharmacol Toxicol 2020 Apr 4;126(4):318-331. Epub 2019 Nov 4.

Institute of Clinical Pharmacology, Goethe University, Frankfurt am Main, Germany.

Persistent and, in particular, neuropathic pain is a major healthcare problem with still insufficient pharmacological treatment options. This triggered research activities aimed at finding analgesics with a novel mechanism of action. Results of these efforts will need to pass through the phases of drug development, in which experimental human pain models are established components e.g. implemented as chemical hyperalgesia induced by capsaicin. We aimed at ranking the various readouts of a human capsaicin-based pain model with respect to the most relevant information about the effects of a potential reference analgesic. In a placebo-controlled, randomized cross-over study, seven different pain-related readouts were acquired in 16 healthy individuals before and after oral administration of 300 mg pregabalin. The sizes of the effect on pain induced by intradermal injection of capsaicin were quantified by calculating Cohen's d. While in four of the seven pain-related parameters, pregabalin provided a small effect judged by values of Cohen's d exceeding 0.2, an item categorization technique implemented as computed ABC analysis identified the pain intensities in the area of secondary hyperalgesia and of allodynia as the most suitable parameters to quantify the analgesic effects of pregabalin. Results of this study provide further support for the ability of the intradermal capsaicin pain model to show analgesic effects of pregabalin. Results can serve as a basis for the designs of studies where the inclusion of this particular pain model and pregabalin is planned.
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http://dx.doi.org/10.1111/bcpt.13337DOI Listing
April 2020

A viral kinase counteracts in vivo restriction of murine cytomegalovirus by SAMHD1.

Nat Microbiol 2019 12 23;4(12):2273-2284. Epub 2019 Sep 23.

Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.
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http://dx.doi.org/10.1038/s41564-019-0529-zDOI Listing
December 2019

Selective inactivation of hypomethylating agents by SAMHD1 provides a rationale for therapeutic stratification in AML.

Nat Commun 2019 08 2;10(1):3475. Epub 2019 Aug 2.

Institute of Medical Virology, University of Frankfurt, Frankfurt, 60590, Germany.

Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms of action remain incompletely understood, and predictive biomarkers for HMA efficacy are lacking. Here, we show that the bioactive metabolite decitabine triphosphate, but not azacytidine triphosphate, functions as activator and substrate of the triphosphohydrolase SAMHD1 and is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis of bone marrow specimens from AML patients at diagnosis revealed that SAMHD1 expression in leukemic cells inversely correlates with clinical response to decitabine, but not to azacytidine. SAMHD1 ablation increases the antileukemic activity of decitabine in AML cell lines, primary leukemic blasts, and xenograft models. AML cells acquire resistance to decitabine partly by SAMHD1 up-regulation. Together, our data suggest that SAMHD1 is a biomarker for the stratified use of hypomethylating agents in AML patients and a potential target for the treatment of decitabine-resistant leukemia.
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http://dx.doi.org/10.1038/s41467-019-11413-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677770PMC
August 2019

Enzymatic Activity of HPGD in Treg Cells Suppresses Tconv Cells to Maintain Adipose Tissue Homeostasis and Prevent Metabolic Dysfunction.

Immunity 2019 05 23;50(5):1232-1248.e14. Epub 2019 Apr 23.

LIMES-Institute, Immunology & Environment, University of Bonn, Carl-Troll-Str. 31, 53115 Bonn, Germany.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E (PGE) into the metabolite 15-keto PGE, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.
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http://dx.doi.org/10.1016/j.immuni.2019.03.014DOI Listing
May 2019

Members of the endocannabinoid system are distinctly regulated in inflammatory bowel disease and colorectal cancer.

Sci Rep 2019 02 20;9(1):2358. Epub 2019 Feb 20.

Otto Loewi Research Center, Division of Pharmacology, Medical University of Graz, Graz, Austria.

Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the 'endocannabinoidome' in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn's disease (CD) patients while 2-arachidonoylglycerol (2-AG) was elevated in patients with CD, but not UC. 2-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI 20:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the 2-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.
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http://dx.doi.org/10.1038/s41598-019-38865-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382821PMC
February 2019

Role of ceramide synthase 2 in G-CSF signaling and G-CSF-R translocation into detergent-resistant membranes.

Sci Rep 2019 01 24;9(1):747. Epub 2019 Jan 24.

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and Pharmacology TMP, Theodor-Stern-Kai 7, 60596, Frankfurt am Main, Germany.

Ceramides are sphingolipids with defined acyl chain lengths, which are produced by corresponding ceramide synthases (CerS1-6). In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), the ablation of CerS2 suppresses EAE-pathology by reducing neutrophil migration into the central nervous system. This migration is induced by granulocyte-colony stimulating factor (G-CSF) signaling. G-CSF signaling leads to a signal cascade including the phosphorylation of Lyn kinase and STAT3. This in turn regulates expression of the neutrophil surface receptor chemokine receptor 2 (CXCR2) and causes translocation of the receptor into detergent-resistant membranes (DRMs). In this study we investigated the role of ceramides in G-CSF signaling. We found, that G-CSF treatment of wild type bone marrow cells (BMCs) leads to translocation of G-CSF-receptor (G-CSF-R) into DRMs. G-CSF also induces downregulation of ceramides in WT and CerS2 null BMCs, as well as upregulation of very long chain lactosylceramides. However, in CerS2 null BMCs, G-CSF failed to induce translocation of G-CSF-R into DRMs, leading to reduced phosphorylation of Lyn and reduced CXCR2 expression. Interestingly, G-CSF signaling in CerS6 null BMCs was not affected. In conclusion, very long chain ceramides are important for G-CSF signaling and translocation of G-CSF-R into DRMs.
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http://dx.doi.org/10.1038/s41598-018-37342-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345911PMC
January 2019

Thromboxane-Induced α-CGRP Release from Peripheral Neurons Is an Essential Positive Feedback Loop in Capsaicin-Induced Neurogenic Inflammation.

J Invest Dermatol 2019 03 26;139(3):656-664. Epub 2018 Oct 26.

Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, University Hospital Frankfurt, Germany; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Frankfurt/Main, Germany. Electronic address:

α-CGRP is synthesized by sensory nerves in the dermis and its release can cause vasodilation and local inflammation. Its vasorelaxant effects are based on the direct activation of smooth muscle and endothelial cells, as well as the activation of mast cells causing the release of vasoactive and proinflammatory mediators. Here, we show that in the capsaicin model for neurogenic inflammation, capsaicin-induced edema formation is mediated by α-CGRP and mast cells, but is absent in thromboxane receptor-deficient mice. Capsaicin treatment of mice induced a thromboxane synthesis, which was mediated by α-CGRP and mast cells. Fittingly, α-CGRP induced thromboxane synthesis in mast cells and the thromboxane receptor agonist I-BOP caused edema formation independently of mast cells, suggesting that mast cells are the source of thromboxane. Most importantly, I-BOP-induced edema formation was mediated by α-CGRP and I-BOP was able to stimulate through calcineurin the α-CGRP release from peripheral neurons. Likewise, the signaling pathway, including α-CGRP, thromboxane receptor, and mast cells, also mediated capsaicin-induced mechanical hypersensitivity, a common symptom of capsaicin treatment. Taken together, the thromboxane-induced α-CGRP release from neurons forms a positive feedback loop causing prolonged α-CGRP release and edema formation during capsaicin-induced neurogenic inflammation.
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http://dx.doi.org/10.1016/j.jid.2018.10.011DOI Listing
March 2019

Cytokine-induced endogenous production of prostaglandin D is essential for human group 2 innate lymphoid cell activation.

J Allergy Clin Immunol 2019 06 19;143(6):2202-2214.e5. Epub 2018 Dec 19.

Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden; Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden. Electronic address:

Background: Group 2 innate lymphoid cells (ILC2s) play a key role in the initiation and maintenance of type 2 immune responses. The prostaglandin (PG) D-chemoattractant receptor-homologous molecule expressed on T2 cells (CRTH2) receptor axis potently induces cytokine production and ILC2 migration.

Objective: We set out to examine PG production in human ILC2s and the implications of such endogenous production on ILC2 function.

Methods: The effects of the COX-1/2 inhibitor flurbiprofen, the hematopoietic prostaglandin D synthase (HPGDS) inhibitor KMN698, and the CRTH2 antagonist CAY10471 on human ILC2s were determined by assessing receptor and transcription factor expression, cytokine production, and gene expression with flow cytometry, ELISA, and quantitative RT-PCR, respectively. Concentrations of lipid mediators were measured by using liquid chromatography-tandem mass spectrometry and ELISA.

Results: We show that ILC2s constitutively express HPGDS and upregulate COX-2 upon IL-2, IL-25, and IL-33 plus thymic stromal lymphopoietin stimulation. Consequently, PGD and its metabolites can be detected in ILC2 supernatants. We reveal that endogenously produced PGD is essential in cytokine-induced ILC2 activation because blocking of the COX-1/2 or HPGDS enzymes or the CRTH2 receptor abolishes ILC2 responses.

Conclusion: PGD produced by ILC2s is, in a paracrine/autocrine manner, essential in cytokine-induced ILC2 activation. Hence we provide the detailed mechanism behind how CRTH2 antagonists represent promising therapeutic tools for allergic diseases by controlling ILC2 function.
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http://dx.doi.org/10.1016/j.jaci.2018.10.069DOI Listing
June 2019

The G2A Receptor Controls Polarization of Macrophage by Determining Their Localization Within the Inflamed Tissue.

Front Immunol 2018 1;9:2261. Epub 2018 Oct 1.

Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, University Hospital Frankfurt, Frankfurt, Germany.

Macrophages are highly versatile cells, which acquire, depending on their microenvironment, pro- (M1-like), or antiinflammatory (M2-like) phenotypes. Here, we studied the role of the G-protein coupled receptor G2A (GPR132), in chemotactic migration and polarization of macrophages, using the zymosan-model of acute inflammation. G2A-deficient mice showed a reduced zymosan-induced thermal hyperalgesia, which was reversed after macrophage depletion. Fittingly, the number of M1-like macrophages was reduced in the inflamed tissue in G2A-deficient mice. However, G2A activation was not sufficient to promote M1-polarization in bone marrow-derived macrophages. While the number of monocyte-derived macrophages in the inflamed paw was not altered, G2A-deficient mice had less macrophages in the direct vicinity of the origin of inflammation, an area marked by the presence of zymosan, neutrophil accumulation and proinflammatory cytokines. Fittingly neutrophil efferocytosis was decreased in G2A-deficient mice and several lipids, which are released by neutrophils and promote G2A-mediated chemotaxis, were increased in the inflamed tissue. Taken together, G2A is necessary to position macrophages in the proinflammatory microenvironment surrounding the center of inflammation. In absence of G2A the macrophages are localized in an antiinflammatory microenvironment and macrophage polarization is shifted toward M2-like macrophages.
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http://dx.doi.org/10.3389/fimmu.2018.02261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174245PMC
October 2019

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1.

Proc Natl Acad Sci U S A 2018 10 10;115(43):E10022-E10031. Epub 2018 Oct 10.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520;

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.
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http://dx.doi.org/10.1073/pnas.1805593115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205433PMC
October 2018

Machine-learning based lipid mediator serum concentration patterns allow identification of multiple sclerosis patients with high accuracy.

Sci Rep 2018 10 5;8(1):14884. Epub 2018 Oct 5.

DataBionics Research Group, University of Marburg, Hans - Meerwein - Straße 22, 35032, Marburg, Germany.

Based on increasing evidence suggesting that MS pathology involves alterations in bioactive lipid metabolism, the present analysis was aimed at generating a complex serum lipid-biomarker. Using unsupervised machine-learning, implemented as emergent self-organizing maps of neuronal networks, swarm intelligence and Minimum Curvilinear Embedding, a cluster structure was found in the input data space comprising serum concentrations of d = 43 different lipid-markers of various classes. The structure coincided largely with the clinical diagnosis, indicating that the data provide a basis for the creation of a biomarker (classifier). This was subsequently assessed using supervised machine-learning, implemented as random forests and computed ABC analysis-based feature selection. Bayesian statistics-based biomarker creation was used to map the diagnostic classes of either MS patients (n = 102) or healthy subjects (n = 301). Eight lipid-markers passed the feature selection and comprised GluCerC16, LPA20:4, HETE15S, LacCerC24:1, C16Sphinganine, biopterin and the endocannabinoids PEA and OEA. A complex classifier or biomarker was developed that predicted MS at a sensitivity, specificity and accuracy of approximately 95% in training and test data sets, respectively. The present successful application of serum lipid marker concentrations to MS data is encouraging for further efforts to establish an MS biomarker based on serum lipidomics.
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http://dx.doi.org/10.1038/s41598-018-33077-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173715PMC
October 2018

The relevance of ceramides and their synthesizing enzymes for multiple sclerosis.

Clin Sci (Lond) 2018 09 17;132(17):1963-1976. Epub 2018 Aug 17.

Pharmazentrum Frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University Hospital Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany

Ceramide synthases (CerS) synthesize chain length specific ceramides (Cer), which mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that the genetic deletion of CerS2 suppresses EAE pathology by interaction with granulocyte-colony stimulating factor (G-CSF) signaling and CXC motif chemokine receptor 2 (CXCR2) expression, leading to impaired neutrophil migration. In the present study, we investigated the importance of Cers and their synthesizing/metabolizing enzymes in MS. For this purpose, a longitudinal study with 72 MS patients and 25 healthy volunteers was performed. Blood samples were collected from healthy controls and MS patients over 1- or 3-year periods, respectively. Immune cells were counted using flow cytometry, ceramide levels were determined using liquid chromatography-tandem mass spectrometry, and mRNA expression was analyzed using quantitative PCR. In white blood cells, C16-LacCer and C24-Cer were down-regulated in MS patients in comparison with healthy controls. In plasma, C16-Cer, C24:1-Cer, C16-GluCer, and C24:1-GluCer were up-regulated and C16-LacCer was down-regulated in MS patients in comparison with healthy controls. Blood samples from MS patients were characterized by an increased B-cell number. However, there was no correlation between B-cell number and Cer levels. mRNA expression of Cer metabolizing enzymes and G-CSF signaling enzymes was significantly increased in MS patients. Interestingly, G-CSF receptor (G-CSFR) and CXCR2 mRNA expression correlated with CerS2 and UDP-glucose Cer glucosyltransferase (UGCG) mRNA expression. In conclusion, our results indicate that Cer metabolism is linked to G-CSF signaling in MS.
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http://dx.doi.org/10.1042/CS20180506DOI Listing
September 2018

Simultaneous determination of PUFA-derived pro-resolving metabolites and pathway markers using chiral chromatography and tandem mass spectrometry.

Anal Chim Acta 2018 Nov 8;1031:185-194. Epub 2018 May 8.

pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University Frankfurt, Germany. Electronic address:

Lipid mediators play an important role as biological messengers involved in inflammatory processes. Deriving from different polyunsaturated fatty acids, endogenously built mediators featuring both pro- and anti-inflammatory properties as well as pro-resolving lipid mediators and their biological precursors have been investigated. A newly developed method using chiral chromatography-tandem mass spectrometry on human plasma has demonstrated its suitability for the simultaneous determination of prostaglandins, lipoxins, D-series derived resolvins as well as protectins, maresin 1, leukotriene B4 and several precursors of them in order to yield information about metabolic pathways. Due to the matrix complexity, a solid phase extraction method using an octadecyl-modified silica gel cartridge was carried out. The developed method allows the determination of 34 analytes in 25 min showing enough selectivity as well as precision and accuracy (≤15% relative standard deviation, ≤15% relative error) in the calibration range of 0.1-10 ng mL or 0.2-20 ng mL depending on the analytes. Stability of the analytes in plasma has been demonstrated for at least 3 h at room temperature, 72 h in the autosampler and 60 days in the freezer at -80 °C. This method has been validated and shown its suitability for the determination of all studied analytes in human plasma samples.
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http://dx.doi.org/10.1016/j.aca.2018.05.020DOI Listing
November 2018

The prostaglandin E2 receptor EP3 controls CC-chemokine ligand 2-mediated neuropathic pain induced by mechanical nerve damage.

J Biol Chem 2018 06 11;293(25):9685-9695. Epub 2018 May 11.

From the Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, University Hospital Frankfurt, 60590 Frankfurt, Germany,

Prostaglandin (PG) E is an important lipid mediator that is involved in several pathophysiological processes contributing to fever, inflammation, and pain. Previous studies have shown that early and continuous application of nonsteroidal anti-inflammatory drugs significantly reduces pain behavior in the spared nerve injury (SNI) model for trauma-induced neuropathic pain. However, the role of PGE and its receptors in the development and maintenance of neuropathic pain is incompletely understood but may help inform strategies for pain management. Here, we sought to define the nociceptive roles of the individual PGE receptors (EP1-4) in the SNI model using EP knockout mice. We found that PGE levels at the site of injury were increased and that the expression of the terminal synthase for PGE, cytosolic PGE synthase was up-regulated in resident positive macrophages located within the damaged nerve. Only genetic deletion of the EP3 receptor affected nociceptive behavior and reduced the development of late-stage mechanical allodynia as well as recruitment of immune cells to the injured nerve. Importantly, EP3 activation induced the release of CC-chemokine ligand 2 (CCL2), and antagonists against the CCL2 receptor reduced mechanical allodynia in WT but not in EP3 knockout mice. We conclude that selective inhibition of EP3 might present a potential approach for reducing chronic neuropathic pain.
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http://dx.doi.org/10.1074/jbc.RA118.002492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016472PMC
June 2018

The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation.

Mob DNA 2018 28;9:11. Epub 2018 Mar 28.

1Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany.

Background: The restriction factor SAMHD1 regulates intracellular nucleotide level by degrading dNTPs and blocks the replication of retroviruses and DNA viruses in non-cycling cells, like macrophages or dendritic cells. In patients, inactivating mutations in are associated with the autoimmune disease Aicardi-Goutières Syndrome (AGS). The accumulation of intracellular nucleic acids derived from endogenous retroelements thriving in the absence of SAMHD1 has been discussed as potential trigger of the autoimmune reaction. In vitro, SAMHD1 has been found to restrict endogenous retroelements, like LINE-1 elements (L1). The mechanism, however, by which SAMHD1 blocks endogenous retroelements, is still unclear.

Results: Here, we show that SAMHD1 inhibits the replication of L1 and other endogenous retroelements in cycling cells. By applying GFP- and neomycin-based reporter assays we found that the anti-L1 activity of SAMHD1 is regulated by phosphorylation at threonine 592 (T592). Similar to the block of HIV, the cofactor binding site and the enzymatic active HD domain of SAMHD1 proofed to be essential for restriction of L1 elements. However, phosphorylation at T592 did not correlate with the dNTP hydrolase activity of SAMHD1 in cycling 293T cells suggesting an alternative mechanism of regulation. Interestingly, we found that SAMHD1 binds to ORF2 protein of L1 and that this interaction is regulated by T592 phosphorylation. Together with the finding that the block is also active in cycling cells, our results suggest that the SAMHD1-mediated inhibition of L1 is similar but not identical to HIV restriction.

Conclusion: Our findings show conclusively that SAMHD1 restricts the replication of endogenous retroelements in vitro. The results suggest that SAMHD1 is important for maintaining genome integrity and support the idea of an enhanced replication of endogenous retroelements in the absence of SAMHD1 in vivo, potentially triggering autoimmune diseases like AGS. Our analysis also contributes to the better understanding of the activities of SAMHD1 in antiviral defense and nucleotide metabolism. The finding that the phosphorylation of SAMHD1 at T592 regulates its activity against retroelements but not necessarily intracellular dNTP level suggests that the dNTP hydrolase activity might not be the only function of SAMHD1 important for its antiviral activity and for controlling autoimmunity.
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http://dx.doi.org/10.1186/s13100-018-0116-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872582PMC
March 2018

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors.

Cell Physiol Biochem 2018 27;45(6):2516-2528. Epub 2018 Mar 27.

Department of Medicine 1, Goethe-University Hospital Frankfurt, Frankfurt, Germany.

Background/aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.

Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.

Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.

Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.
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http://dx.doi.org/10.1159/000488270DOI Listing
June 2018

Mutual inversion of flurbiprofen enantiomers in various rat and mouse strains.

Chirality 2018 May 6;30(5):632-641. Epub 2018 Feb 6.

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Research Center for Translational Medicine and Pharmacology TMP, Industriepark Höchst, Frankfurt am Main, Germany.

Flurbiprofen (F) is a nonsteroidal anti-inflammatory drug (NSAID) used therapeutically as the racemate of (R)-enantiomer and (S)-enantiomer. The inversion of RF to SF and vice versa was investigated in C57Bl/6 and SJL mice and Dark Agouti and Lewis rats. The enzyme α-methylacyl-CoA racemase (AMACR) is involved in the chiral inversion pathway that converts members of the 2-arylpropionic acid NSAIDs from the R-enantiomer to the S-enantiomer. We studied C57Bl/6 mice deficient in AMACR postulating that they should show reduced inversion of RF to SF. In line with the data of others in mice, (R)-inversion to (S)-inversion was relatively high in both the C57Bl/6 and SJL mice (fraction inverted, F  = 37.7% and 24.7%, respectively). In contrast, in AMACR deficient mice, there was no measurable peak for SF after administration of RF. The results in both rat strains (Dark Agouti and Lewis rats, F  = 1.4% and 4.1%, respectively) confirm the low chiral inversion of the enantiomers of flurbiprofen in the rat, as observed by other authors in the Sprague-Dawley strain (<5%). From the present results, we conclude that for the study of flurbiprofen enantiomers, the rat is more suitable than the mouse as a model for the human in which (R)-inversion to (S)-inversion is negligible.
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http://dx.doi.org/10.1002/chir.22826DOI Listing
May 2018