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Background: Maladapted endothelial cells (ECs) secrete ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin)—a lysophospholipase D that generates lysophosphatidic acids (LPAs). ENPP2 derived from the arterial wall promotes atherogenic monocyte adhesion induced by generating LPAs, such as arachidonoyl-LPA (LPA20:4), from oxidized lipoproteins. Here, we aimed to determine the role of endothelial ENPP2 in the production of LPAs and atherosclerosis. Methods: We quantified atherosclerosis in mice harboring loxP-flanked Enpp2 alleles crossed with Apoe –/– mice expressing tamoxifen-inducible Cre recombinase under the control of the EC-specific bone marrow X kinase promoter after 12 weeks of high-fat diet feeding. Results: A tamoxifen-induced EC-specific Enpp2 knockout decreased atherosclerosis, accumulation of lesional macrophages, monocyte adhesion, and expression of endothelial CXCL (C-X-C motif chemokine ligand) 1 in male and female Apoe –/– mice. In vitro, ENPP2 mediated the mildly oxidized LDL (low-density lipoprotein)-induced expression of CXCL1 in aortic ECs by generating LPA20:4, palmitoyl-LPA (LPA16:0), and oleoyl-LPA (LPA18:1). ENPP2 and its activity were detected on the endothelial surface by confocal imaging. The expression of endothelial Enpp2 established a strong correlation between the plasma levels of LPA16:0, stearoyl-LPA (LPA18:0), and LPA18:1 and plaque size and a strong negative correlation between the LPA levels and ENPP2 activity in the plasma. Moreover, endothelial Enpp2 knockout increased the weight of high-fat diet–fed male Apoe –/– mice. Conclusions: We demonstrated that the expression of ENPP2 in ECs promotes atherosclerosis and endothelial inflammation in a sex-independent manner. This might be due to the generation of LPA20:4, LPA16:0, and LPA18:1 from mildly oxidized lipoproteins on the endothelial surface.

Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post–myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A–mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia–sera/immunoglobulin G–triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.

An altered lipidome in tumors may affect not only tumor cells themselves but also their microenvironment. In this study, a lipidomics screen reveals increased amounts of phosphatidylserine (PS), particularly ether-PS (ePS), in murine mammary tumors compared with normal tissue. PS was produced by phosphatidylserine synthase 1 (PTDSS1), and depletion of Ptdss1 from tumor cells in mice reduced ePS levels accompanied by stunted tumor growth and decreased tumor-associated macrophage (TAM) abundance. Ptdss1-deficient tumor cells exposed less PS during apoptosis, which was recognized by the PS receptor MERTK. Mammary tumors in macrophage-specific Mertk−/− mice showed similarly suppressed growth and reduced TAM infiltration. Transcriptomic profiles of TAMs from Ptdss1-knockdown tumors and Mertk−/− TAMs revealed that macrophage proliferation was reduced when the Ptdss1/Mertk pathway was targeted. Moreover, PTDSS1 expression correlated positively with TAM abundance but negatively with breast carcinoma patient survival. PTDSS1 thus may be a target to modify tumor-promoting inflammation.Significance:This study shows that inhibiting the production of ether-phosphatidylserine by targeting phosphatidylserine synthase PTDSS1 limits tumor-associated macrophage expansion and breast tumor growth.

Supplementary Table from Phosphatidylserine Synthase PTDSS1 Shapes the Tumor Lipidome to Maintain Tumor-Promoting Inflammation

Supplementary Figure from Phosphatidylserine Synthase PTDSS1 Shapes the Tumor Lipidome to Maintain Tumor-Promoting Inflammation

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.

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.

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. SAMHD1 inhibits murine cytomegalovirus replication in vivo and its activity is counteracted by the viral kinase M97. Phosphorylation of SAMHD1 by M97 correlates with reduced dNTP hydrolase activity and a loss of viral restriction in infected cells.

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., In acute myeloid leukemia, hypomethylating agents decitabine and azacytidine are used interchangeably. Here, the authors show that the major metabolite of decitabine, but not azacytidine, is subject to SAMHD1 inactivation, highlighting SAMHD1 as a potential biomarker and therapeutic target

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.

SummaryBackgroundSAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 substrate, in the context of SAMHD1.MethodsCNDAC was tested in 13 acute myeloid leukaemia (AML cell lines), in 26 acute lymphoblastic leukaemia cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation.ResultsAlthough the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance.ConclusionIntrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

Summary 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., Graphical Abstract, Highlights • K6PC-5, a sphingosine kinase 1 activator, inhibits Ebola virus infection • Sphingosine 1-phosphate, the product of SphK1, attenuates the viral entry • Inhibiton/activation of S1P receptors has no influence on Ebola virus entry • These data support the endogen effect of S1P in Ebola virus infection, Virology ; Molecular Microbiology ; Cell Biology

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.

Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.

An altered lipidome in tumors may affect not only tumor cells themselves but also their microenvironment. In this study, a lipidomics screen reveals increased amounts of phosphatidylserine (PS), particularly ether-PS (ePS), in murine mammary tumors compared with normal tissue. PS was produced by phosphatidylserine synthase 1 (PTDSS1), and depletion of Ptdss1 from tumor cells in mice reduced ePS levels accompanied by stunted tumor growth and decreased tumor-associated macrophage (TAM) abundance. Ptdss1-deficient tumor cells exposed less PS during apoptosis, which was recognized by the PS receptor MERTK. Mammary tumors in macrophage-specific Mertk−/− mice showed similarly suppressed growth and reduced TAM infiltration. Transcriptomic profiles of TAMs from Ptdss1-knockdown tumors and Mertk−/− TAMs revealed that macrophage proliferation was reduced when the Ptdss1/Mertk pathway was targeted. Moreover, PTDSS1 expression correlated positively with TAM abundance but negatively with breast carcinoma patient survival. PTDSS1 thus may be a target to modify tumor-promoting inflammation. Significance: This study shows that inhibiting the production of ether-phosphatidylserine by targeting phosphatidylserine synthase PTDSS1 limits tumor-associated macrophage expansion and breast tumor growth.

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 Gq-coupled receptors and constitutively active G&alpha, q/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 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 PLC&beta, 3, but accelerated by wild-type PLC&beta, 3 and the PLC&delta, PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at G&alpha, q. Embryonic fibroblasts from G&alpha, q/11 double-deficient mice were used to show that amino acids W263 and T257 of G&alpha, q, which interact directly with PLC&beta, 3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100&ndash, 105 in human SphK1a), which resembles the G&alpha, q binding motif, ALXXPI, in PLC&beta, and p63RhoGEF. After M3 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 PLC&beta, /PLC&delta, (PH) expression are important for regulation of SphK1 by Gq/11.

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.

The putative cannabinoid receptor GPR55 has been shown to play a tumor-promoting role in various cancers, and is involved in many physiological and pathological processes of the gastrointestinal (GI) tract. While the cannabinoid receptor 1 (CB1 ) has been reported to suppress intestinal tumor growth, the role of GPR55 in the development of GI cancers is unclear. We, therefore, aimed at elucidating the role of GPR55 in colorectal cancer (CRC), the third most common cancer worldwide. Using azoxymethane (AOM)- and dextran sulfate sodium (DSS)-driven CRC mouse models, we found that GPR55 plays a tumor-promoting role that involves alterations of leukocyte populations, i.e. myeloid-derived suppressor cells and T lymphocytes, within the tumor tissues. Concomitantly, expression levels of COX-2 and STAT3 were reduced in tumor tissue of GPR55 knockout mice, indicating reduced presence of tumor-promoting factors. By employing the experimental CRC models to CB1 knockout and CB1 /GPR55 double knockout mice, we can further show that GPR55 plays an opposing role to CB1 . We report that GPR55 and CB1 mRNA expression are differentially regulated in the experimental models and in a cohort of 86 CRC patients. Epigenetic methylation of CNR1 and GPR55 was also differentially regulated in human CRC tissue compared to control samples. Collectively, our data suggest that GPR55 and CB1 play differential roles in colon carcinogenesis where the former seems to act as oncogene and the latter as tumor suppressor.

Physical exercise has been repeatedly associated with decreased nociceptive responses but the underlying mechanisms have still not been fully clarified. In this study, we investigated exercise-induced effects after a single bout of treadmill running on the mouse model of formalin-induced inflammatory nociception. As potential molecular mediators, we focused on endogenous endocannabinoids as well as AMP-activated protein kinase (AMPK). Our results showed that wild type mice display a reduced nociceptive response in the formalin test after treadmill running, while exercise had no effect on inflammatory nociception in AMPKα2 knockout mice. Levels of the endocannabinoid anandamide (AEA) were increased after physical activity in both wild type and AMPKα2 knockout mice, in association with decreased expression of the AEA-hydrolyzing enzyme FAAH and an increased level of the cannabinoid receptor 1 (CB1). Accordingly, treatment of wild type mice with the CB1 inverse agonist AM251 prior to the treadmill running reversed exercise-induced antinociception. However, if mice received AM251 in combination with the AMPK activator 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR), the positive effect of treadmill running on inflammatory nociception was restored, indicating that AMPK affects exercise-induced antinociception downstream of endocannabinoids. This assumption was further supported by cell culture experiments showing AMPK activation after stimulation of neuronal cells with AEA. In conclusion, our data suggest that AMPK is an intermediate effector in endocannabinoid-mediated exercise-induced antinociception. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".

Platelets are well known for their role in hemostasis but are also increasingly recognized for their supporting role in innate immune responses. Here, we studied the role of platelets in the development of peripheral inflammation and found that platelets colocalize with macrophages in the inflamed tissue outside of blood vessels in different animal models for cutaneous inflammation. Collagen-treatment of macrophages isolated from paws during zymosan-induced inflammation induced thromboxane synthesis through the platelet-expressed collagen receptor glycoprotein VI. Deletion of glycoprotein VI or its downstream effector thromboxane A2 receptor (TP) reduced zymosan-induced mechanical allodynia without altering macrophage recruitment or formation of macrophage/platelet complexes. Instead, macrophages in inflamed paws of glycoprotein VI- and TP-deficient mice exhibited an increased expression of anti-inflammatory markers and synthesized less proinflammatory mediators (prostaglandin E2 and IL6). TP expression on platelets was necessary to mediate increased prostaglandin E2 and IL6 synthesis, whereas TP expression on macrophages was sufficient to decrease the expression of the anti-inflammatory macrophage marker CD206, showing that TP activation on platelets and macrophages regulates different aspects of macrophage activation.

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 (Tregs) 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 Tregs, which in turn inhibit neutrophils.

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 Ca2+ 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.

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

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.

The role of homocysteic acid (HCA) in severe diseases like Alzheimer's disease is under discussion and some recent studies correlate elevated HCA concentrations with the diagnosis of Alzheimer's. However, non-selective and insufficiently sensitive methods have been used to quantitate HCA and results of different studies show large differences in the determined HCA concentration in samples from patients and controls, and therefore non-comparable results. An accurate and precise quantitation method for the determination of HCA in human serum, urine and CSF has been developed by using a combination of protein precipitation and solid phase extraction for sample preparation followed by an LC-MS/MS analysis using a combination of a HILIC separation and tandem mass spectrometry. The developed method has been fully validated in accordance with the guidelines provided by the US Food and Drug administration FDA and the European Medicines Agency EMA. Furthermore, the method has demonstrated its ability to determine the endogenous HCA concentration in serum and urine samples from healthy volunteers.

Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.

Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 μg/100 μl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1α LysM-/- macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1α LysM-/- macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 μg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.

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.

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.

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 Gq-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 Gq regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gαq to phospholipase C (PLC)-β and the thromboxane A2 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 M3 and bradykinin B2 receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca2+]i increases. Surprisingly, WDR36 also augmented M3 receptor-induced [Ca2+]i increases, which was due to increased Ca2+ mobilization while the Ca2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by Gq-coupled M3, B2 and H1 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 B2 receptors. It is concluded that WDR36 reduces signalling of Gq-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gαq and PLCβ. Our results support a role of WDR36 in orchestration of Gq signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.

The determination of endocannabinoids and endocannabinoid-like substances in biological human samples is a vibrant field of research with great significance due to postulated relevance of these substances in diseases such as Alzheimer's disease, multiple sclerosis, cancer and cardiovascular diseases. For a possible use as biomarker in early prediction or diagnosis of a disease as well as examination of a successful treatment, the valid determination of the analytes in common accessible human samples, such as plasma or serum, is of great importance. A method for the determination of arachidonoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, 1-arachidonoyl glycerol and 2-arachidonoyl glycerol in human K3EDTA plasma using liquid-liquid-extraction in combination with liquid chromatography-tandem-mass spectrometry has been developed and validated for the quantification of the aforementioned analytes. Particular emphasis was placed on the chromatographic separation of the isomers 1-arachidonoyl glycerol and 2-arachidonoyl glycerol, arachidonoyl ethanolamide and O-arachidonoyl ethanolamine (virodhamine) as well as oleoyl ethanolamide and vaccenic acid ethanolamide. During the validation process, increasing concentrations of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol while storing plasma samples were observed. In-depth investigation of pre-analytical sample handling revealed rising concentrations for both analytes in plasma and for arachidonoyl ethanolamide, oleoyl ethanolamide and palmitoyl ethanolamide in whole blood, dependent on the period and temperature of storage. Prevention of the increase in concentration was not possible, raising the question whether human K3EDTA plasma is suitable for the determination of endocannabinoids and endocannabinoid-like substances. Especially the common practice to calculate the concentration of 2-arachidonoyl glycerol as sum of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol is highly questionable because the concentrations of both analytes increase unequally while storing the plasma samples in the fridge.

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 (

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.

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.

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.

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.

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 samhd1 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. Electronic supplementary material The online version of this article (10.1186/s13100-018-0116-5) contains supplementary material, which is available to authorized users.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in non-cycling 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 analogue 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 find 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 analogues 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 regards to nucleotide analogues, which can be used to improve current cancer and antiviral therapies.SignificanceNucleoside analogue drugs are widely used to treat a variety of cancers and viral infections. With an essential role in regulating the nucleotide pool in the cell by degrading cellular nucleotides, SAMHD1 has the potential to decrease the cellular concentration of frequently prescribed nucleotide analogues and thereby decrease their clinical efficacy in cancer therapy. To improve future nucleotide analogue treatments, it is important to understand SAMHD1 interactions with these drugs. Our work thoroughly examines the extent to which nucleotide analogues interact with the catalytic and allosteric sites of SAMHD1. This work contributes to the assessment of SAMHD1 as a potential therapeutic target for cancer therapy and the future design of SAMHD1 modulators that might improve the efficacy of existing therapies.

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 samhd1 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.

We have recently shown that major alterations of serum sphingolipid metabolites in chronic liver disease associate significantly with the stage of liver fibrosis in corresponding patients. In the current study we assessed via mass spectrometry serum concentrations of sphingolipid metabolites in a series of 122 patients with hepatocellular carcinoma (HCC) compared to an age- and sex-matched series of 127 patients with cirrhosis. We observed a highly significant upregulation of long and very long chain ceramides (C16-C24) in the serum of patients with HCC as compared to patients with cirrhosis (P < 0.001). Accordingly, dihydro-ceramides, synthetic precursors of ceramides and notably sphingosine, sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (SA1P) were upregulated in patients with HCC (P < 0.001). Especially the diagnostic accuracy of C16-ceramide and S1P, assessed by receiver operating curve (ROC) analysis, showed a higher area under the curve (AUC) value as compared to alpha fetoprotein (AFP) (0.999 and 0.985 versus 0.823, P < 0.001 respectively). In conclusion, serum levels of sphingolipid metabolites show a significant upregulation in patients with HCC as compared to patients with cirrhosis. Particularly C16-ceramide and S1P may serve as novel diagnostic markers for the identification of HCC in patients with liver diseases. Our data justify further investigations on the role of sphingolipids in HCC.

Loss of plasticity-related gene 1 (PRG-1), which regulates synaptic phospholipid signaling, leads to hyperexcitability via increased glutamate release altering excitation/inhibition (E/I) balance in cortical networks. A recently reported SNP in prg-1 (R345T/ mutPRG-1) affects ~5 million European and US citizens in a monoallelic variant. Our studies show that this mutation leads to a loss-of-PRG-1 function at the synapse due to its inability to control lysophosphatidic acid (LPA) levels via a cellular uptake mechanism which appears to depend on proper glycosylation altered by this SNP. PRG-1 +/ mice, which are animal correlates of human PRG-1 +/mut carriers, showed an altered cortical network function and stress-related behavioral changes indicating altered resilience against psychiatric disorders. These could be reversed by modulation of phospholipid signaling via pharmacological inhibition of the LPA-synthesizing molecule autotaxin. In line, EEG recordings in a human population-based cohort revealed an E/I balance shift in monoallelic mutPRG-1 carriers and an impaired sensory gating, which is regarded as an endophenotype of stress-related mental disorders. Intervention into bioactive lipid signaling is thus a promising strategy to interfere with glutamate-dependent symptoms in psychiatric diseases.

The great impact of cardiovascular diseases in human health has led to the development of a huge number of drugs and therapies to improve the treatment of these diseases. Cardiovascular drug analysis in biological fluids constitutes an important challenge for analytical scientists. There is a clear need for reliable methods to carry out both qualitative and quantitative analysis in a short time of analysis. Different problems such as drug monitoring, analysis of metabolites, study of drugs interactions, drugs residues or degradation products, chiral separation, and screening and confirmation of drugs of abuse in doping control must be solved. New trends in sample preparation, instrumental and column technology advances in LC and innovations in MS are described in this work.

Cannabinoid-1 receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cannabinoid-2 receptor (CB2) possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. Pro- and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system.To specifically address the role of CB2 for age-associated obesity, we analyzed metabolic, cardiovascular, immune and neuronal functions in 1.2-1.8-year-old CB2(-/-) and control mice, fed with a standard diet and assessed effects of the CB2 agonist, HU308, during high-fat diet (HFD) in 12-16-week-old mice.The CB2(-/-) mice were obese with hypertrophy of visceral fat, immune cell polarization toward pro-inflammatory subpopulations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. They also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. The CB2 agonist HU308 prevented HFD-evoked hypertension, reduced HFD-evoked polarization of adipose tissue macrophages toward the M1-like pro-inflammatory type and reduced HFD-evoked nociceptive hypersensitivity, but had no effect on weight gain.CB2 agonists may fortify CB2-mediated anti-obesity signaling without the risk of anti-CB1-mediated depression that caused the failure of rimonabant.

Endocannabinoids modulate neuroendocrine networks by directly targeting cannabinoid receptors. The time-hormone melatonin synchronizes these networks with external light condition and guarantees time-sensitive and ecologically well-adapted behaviors. Here, the endocannabinoid arachidonoyl ethanolamide (AEA) showed rhythmic changes in rat pineal glands with higher levels during the light-period and reduced amounts at the onset of darkness. Norepinephrine, the essential stimulus for nocturnal melatonin biosynthesis, acutely down-regulated AEA and other endocannabinoids in cultured pineal glands. These temporal dynamics suggest that AEA exerts time-dependent autocrine and/or paracrine functions within the pineal. Moreover, endocananbinoids may be released from the pineal into the CSF or blood stream.

Understanding of the physiological role of peroxisome proliferator-activated receptor gamma (PPARγ) offers new opportunities for the treatment of cancers, immune disorders and inflammatory diseases. In contrast to PPARγ agonists, few PPARγ antagonists have been studied, though they do exert immunomodulatory effects. Currently, no therapeutically useful PPARγ antagonist is commercially available. The aim of this study was to identify and kinetically characterise a new competitive PPARγ antagonist for therapeutic use. A PPARγ-dependent transactivation assay was used to kinetically characterise (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB) in kidney, T and monocytic cell lines. Cytotoxic effects were analysed and intracellular accumulation of MTTB was assessed by tandem mass spectrometry (LC-MS/MS). Potential interactions of MTTB with the PPARγ protein were suggested by molecular docking analysis. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), MTTB exhibited competitive antagonism against rosiglitazone in HEK293T and Jurkat T cells, with IC50 values in HEK293T cells of 4.3µM and 1.6µM, using the PPARγ ligand binding domain (PPARγ-LBD) and the full PPARγ protein, respectively. In all cell lines used, however, MTTB showed much higher intracellular accumulation than GW9662. MTTB alone exhibited weak partial agonistic effects and low cytotoxicity. Molecular docking of MTTB with the PPARγ-LBD supported direct interaction with the nuclear receptor. MTTB is a promising prototype for a new class of competitive PPARγ antagonists. It has weak partial agonistic and clear competitive antagonistic characteristics associated with rapid cellular uptake. Compared to commercially available PPARγ modulators, this offers the possibility of dose regulation of PPARγ and immune responses.

Ceramide synthases (CerS) synthesise ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and its products, C24-ceramides, in CD11b(+) cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 mRNA expression and C24-ceramide levels were significantly increased by 2.2- and 1.5-fold, respectively, in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, since a specific genetic deletion of CerS2 in blood cells or a total genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in their reduced migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired. In conclusion, our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.

Nucleosides and nucleoside triphosphates are the building blocks of nucleic acids and important bioactive metabolites, existing in all living cells. In the present study, two liquid chromatography tandem mass spectrometry methods were developed to quantify both groups of compounds from the same sample with a shared extraction procedure. After a simple protein precipitation with methanol, the nucleosides were separated with reversed phase chromatography on an Atlantis T3 column while for the separation of the nucleoside triphosphates, an anion exchange column (BioBasic AX) was used. No addition of ion pair reagent was required. A 5500 QTrap was used as analyzer, operating as triple quadrupole. The analytical method for the nucleoside triphosphates has been validated according to the guidelines of the US Food and Drug Administration. The lower limit of quantification values were determined as 10 pg on column (0.5 ng/mL in the injection solution) for deoxyadenosine triphosphate and deoxyguanosine triphosphate, 20 pg (1 ng/mL) for deoxycytidine triphosphate and thymidine triphosphate, 100 pg (5 ng/mL) for cytidine triphosphate and guanosine triphosphate, and 500 pg (25 ng/mL) for adenosine triphosphate und uridine triphosphate respectively. This methodology has been applied to the quantitation of nucleosides and nucleoside triphosphates in primary human CD4 T lymphocytes and macrophages. As expected, the concentrations for ribonucleosides and ribonucleoside triphophates were considerably higher than those obtained for the deoxy derivatives. Upon T cell receptor activation, the levels of all analytes, with the notable exceptions of deoxyadenosine triphosphate and deoxyguanosine triphosphate, were found to be elevated in CD4 T cells.

Prostaglandin (PG) E(2) 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(2) 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(2) receptors (EP1–4) in the SNI model using EP knockout mice. We found that PGE(2) levels at the site of injury were increased and that the expression of the terminal synthase for PGE(2), 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.

Purpose Neovascularization is a major cause of blindness in various ocular diseases. Bioactive sphingosine 1-phosphate (S1P), synthesized by two sphingosine kinases (Sphk1, Sphk2), emerged as a key player in a multitude of cellular processes, including cell survival, proliferation, inflammation, migration, and angiogenesis. We investigated the role of Sphk2, S1P, and S1P receptors (S1PR) during retinal neovascularization using the oxygen-induced retinopathy mouse model (OIR). Methods Sphk2 overexpressing (tgSphk2) and Sphk2 knockout (Sphk2-/-) mice were used in the OIR model, exposed to 75% O2 over 5 days from postnatal day (P)7 to 12 to initiate vessel regression. After returning to room air, these mice developed a marked neovascularization. Retinae recovered from untreated and treated eyes at P7, P12, P14, and P17 were used for lectin-stained retinal whole mounts, mass spectrometry, and quantitative real-time PCR. Results tgSphk2 mice showed higher retinal S1P concentrations, accelerated retinal angiogenesis, and increased neovascularization. Expression of S1PR, vascular endothelial growth factor α (VEGFα), and angiopoietin 1 and 2 was differentially regulated during the course of OIR in the different genotypes. Sphk2-/- displayed a markedly reduced retinal angiogenesis and neovascularization as well as decreased VEGFα and angiopoietin expression. Conclusions Using genetic models of Sphk2 overexpression or deletion we demonstrate a strong impact of Sphk2/S1P on retinal vasculopathy and expression of vascular growth factors like VEGF and angiopoietin in the retina. Consequently, Sphk2, S1P, and S1PR may offer attractive novel therapeutic targets for ischemic retinopathies.