Publications by authors named "Pauline Le Faouder"

26 Publications

  • Page 1 of 1

Untargeted Lipidomic Profiling of Dry Blood Spots Using SFC-HRMS.

Metabolites 2021 May 11;11(5). Epub 2021 May 11.

MetaboHUB-MetaToul-Lipidomique, MetaboHUB-ANR-11-INBS-0010, Inserm U1297/Université Paul Sabatier Toulouse III, 31432 Toulouse, France.

Lipids are essential cellular constituents that have many critical roles in physiological functions. They are notably involved in energy storage and cell signaling as second messengers, and they are major constituents of cell membranes, including lipid rafts. As a consequence, they are implicated in a large number of heterogeneous diseases, such as cancer, diabetes, neurological disorders, and inherited metabolic diseases. Due to the high structural diversity and complexity of lipid species, the presence of isomeric and isobaric lipid species, and their occurrence at a large concentration scale, a complete lipidomic profiling of biological matrices remains challenging, especially in clinical contexts. Using supercritical fluid chromatography coupled with high-resolution mass spectrometry, we have developed and validated an untargeted lipidomic approach to the profiling of plasma and blood. Moreover, we have tested the technique using the Dry Blood Spot (DBS) method and found that it allows for the easy collection of blood for analysis. To develop the method, we performed the optimization of the separation and detection of lipid species on pure standards, reference human plasma (SRM1950), whole blood, and DBS. These analyses allowed an in-house lipid data bank to be built. Using the MS-Dial software, we developed an automatic process for the relative quantification of around 500 lipids species belonging to the 6 main classes of lipids (including phospholipids, sphingolipids, free fatty acids, sterols, and fatty acyl-carnitines). Then, we compared the method using the published data for SRM 1950 and a mouse blood sample, along with another sample of the same blood collected using the DBS method. In this study, we provided a method for blood lipidomic profiling that can be used for the easy sampling of dry blood spots.
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http://dx.doi.org/10.3390/metabo11050305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151068PMC
May 2021

Lipid exchanges drove the evolution of mutualism during plant terrestrialization.

Science 2021 05;372(6544):864-868

Laboratoire de Recherche en Sciences Végétales (LRSV), Université de Toulouse, CNRS, UPS, 31326 Castanet-Tolosan, France.

Symbiosis with arbuscular mycorrhizal fungi (AMF) improves plant nutrition in most land plants, and its contribution to the colonization of land by plants has been hypothesized. Here, we identify a conserved transcriptomic response to AMF among land plants, including the activation of lipid metabolism. Using gain of function, we show the transfer of lipids from the liverwort to AMF and its direct regulation by the transcription factor WRINKLED (WRI). Arbuscules, the nutrient-exchange structures, were not formed in loss-of-function mutants in , leading to aborted mutualism. Our results show the orthology of the symbiotic transfer of lipids across land plants and demonstrate that mutualism with arbuscular mycorrhizal fungi was present in the most recent ancestor of land plants 450 million years ago.
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http://dx.doi.org/10.1126/science.abg0929DOI Listing
May 2021

Uropathogenic E. coli induces DNA damage in the bladder.

PLoS Pathog 2021 02 25;17(2):e1009310. Epub 2021 Feb 25.

IRSD, INSERM, Université de Toulouse, INRA, ENVT, UPS, Toulouse, France.

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells.
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http://dx.doi.org/10.1371/journal.ppat.1009310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906301PMC
February 2021

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

Gut 2021 Jun 25;70(6):1088-1097. Epub 2020 Sep 25.

IRSD, INSERM, INRA, INP-ENVT, Toulouse University 3 Paul Sabatier, Toulouse, France

Objective: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action.

Design: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis.

Results: Among all the LCFAs quantified by mass spectrometry in Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in , and . In culture, produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma.

Conclusion: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.
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http://dx.doi.org/10.1136/gutjnl-2020-321173DOI Listing
June 2021

Improving lipid mapping in Genome Scale Metabolic Networks using ontologies.

Metabolomics 2020 03 25;16(4):44. Epub 2020 Mar 25.

UMR1331, Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, 31300, Toulouse, France.

Introduction: To interpret metabolomic and lipidomic profiles, it is necessary to identify the metabolic reactions that connect the measured molecules. This can be achieved by putting them in the context of genome-scale metabolic network reconstructions. However, mapping experimentally measured molecules onto metabolic networks is challenging due to differences in identifiers and level of annotation between data and metabolic networks, especially for lipids.

Objectives: To help linking lipids from lipidomics datasets with lipids in metabolic networks, we developed a new matching method based on the ChEBI ontology. The implementation is freely available as a python library and in MetExplore webserver.

Methods: Our matching method is more flexible than an exact identifier-based correspondence since it allows establishing a link between molecules even if a different level of precision is provided in the dataset and in the metabolic network. For instance, it can associate a generic class of lipids present in the network with the molecular species detailed in the lipidomics dataset. This mapping is based on the computation of a distance between molecules in ChEBI ontology.

Results: We applied our method to a chemical library (968 lipids) and an experimental dataset (32 modulated lipids) and showed that using ontology-based mapping improves and facilitates the link with genome scale metabolic networks. Beyond network mapping, the results provide ways for improvements in terms of network curation and lipidomics data annotation.

Conclusion: This new method being generic, it can be applied to any metabolomics data and therefore improve our comprehension of metabolic modulations.
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http://dx.doi.org/10.1007/s11306-020-01663-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096385PMC
March 2020

Heteroleptic Ruthenium(II) Complexes with Bathophenanthroline and Bathophenanthroline Disulfonate Disodium Salt as Fluorescent Dyes for In-Gel Protein Staining.

Inorg Chem 2020 Apr 17;59(7):4527-4535. Epub 2020 Mar 17.

Univ. Lille, CNRS, USR 3290, MSAP, Miniaturisation pour la Synthèse l'Analyse et la Protèomique, F-59 000 Lille, France.

The in-gel detection of proteins for various proteomic experiments is commonly done with the fluorescent Ru tris(bathophenanthroline disulfonate) complex (Ru(BPS)), which is more cost-effective compared to commercial Ru-based formulations but requires tedious procedures for its preparation and strongly acidic staining conditions. Herein, we report the synthesis and characterization of heteroleptic Ru complexes Ru(BPS)(BP) and Ru(BPS)(BP) containing bathophenanthroline (BP) and bathophenanthroline disulfonate disodium salt (BPS) in comparison with Ru(BPS). It was shown by fluorescent and UV-vis measurements that novel Ru complexes were excitable in both UV and visible light, close to emission bands of classical lasers, which is important for successful in-gel protein detection. Novel fluorescent dyes demonstrated improved protein detection in comparison with commercially available SYPRO Ruby staining solution. In addition, unlike commonly used staining protocols, staining with Ru(BPS)(BP) can be performed at nearly neutral pH, thereby reducing artificial post-translational modifications (PTMs).
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http://dx.doi.org/10.1021/acs.inorgchem.9b03679DOI Listing
April 2020

Platelet Activation Is Limited during Transcatheter Aortic Valve Implantation in Patients on Aspirin Monotherapy and without per Procedural Clinical Complications.

TH Open 2019 Apr 30;3(2):e146-e152. Epub 2019 May 30.

INSERM, U1048 and Université Toulouse 3, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France.

Transcatheter aortic valve implantation (TAVI) is an established treatment option for symptomatic patients with severe aortic valve stenosis (AS). During and early after the procedure, both ischemic events (predominantly stroke) and bleedings remain prevalent. The optimal antithrombotic regimen is still debated. Single- versus dual-antiplatelet therapy is associated with a lower rate of severe bleeding, without difference in thrombotic complications. Although platelets have been empirically targeted, little is known on their contribution to these events primarily related to embolization of thrombotic material and tissue-derived debris from the wounded aortic valve and large vessels. The objective of this study was to assess local platelet activation in blood sampled in the ascending aorta immediately before and within minutes postimplantation. A series of 18 patients with AS on monotherapy with aspirin successfully underwent TAVI with the self-expandable Medtronic CoreValve by transfemoral route. No clinical thrombotic complication occurred at 30-day follow-up. Compared with patients with stable coronary artery disease unscathed of AS and similarly treated by low-dose aspirin, AS patients displayed a chronic state of platelet activation before TAVI, assessed in venous blood using various biomarkers. However, per procedure, in aortic blood, no change occurred between the two time points in the plasma levels of serotonin or 12-lipoxgenase products, or membrane exposure of granule markers CD62-P and CD63. Our results suggest that local acute platelet activation is limited during TAVI on monotherapy with aspirin.
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http://dx.doi.org/10.1055/s-0039-1692142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598084PMC
April 2019

Polyunsaturated fatty acid metabolites: biosynthesis in and role in parasite/host interaction.

J Lipid Res 2019 03 9;60(3):636-647. Epub 2019 Jan 9.

IRSD, Université de Toulouse, INSERM, INRA, INP-ENVT, 31024 Toulouse, France

Inside the human host, infection starts with phagocytosis of infective promastigotes by macrophages. In order to survive, has developed several strategies to manipulate macrophage functions. Among these strategies, as a source of bioactive lipids has been poorly explored. Herein, we assessed the biosynthesis of polyunsaturated fatty acid metabolites by infective and noninfective stages of and further explored the role of these metabolites in macrophage polarization. The concentration of docosahexaenoic acid metabolites, precursors of proresolving lipid mediators, was increased in the infective stage of the parasite compared with the noninfective stage, and cytochrome P450-like proteins were shown to be implicated in the biosynthesis of these metabolites. The treatment of macrophages with lipids extracted from the infective forms of the parasite led to M2 macrophage polarization and blocked the differentiation into the M1 phenotype induced by IFN-γ. In conclusion, polyunsaturated fatty acid metabolites, produced by cytochrome P450-like protein activity, are implicated in parasite/host interactions by promoting the polarization of macrophages into a proresolving M2 phenotype.
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http://dx.doi.org/10.1194/jlr.M091736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399491PMC
March 2019

5-oxoETE triggers nociception in constipation-predominant irritable bowel syndrome through MAS-related G protein-coupled receptor D.

Sci Signal 2018 12 18;11(561). Epub 2018 Dec 18.

INSERM, UMR1220, IRSD, Université de Toulouse, INRA, ENVT, UPS, Toulouse, France.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is characterized by chronic abdominal pain concurrent with altered bowel habit. Polyunsaturated fatty acid (PUFA) metabolites are increased in abundance in IBS and are implicated in the alteration of sensation to mechanical stimuli, which is defined as visceral hypersensitivity. We sought to quantify PUFA metabolites in patients with IBS and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxoeicosatetraenoic acid (5-oxoETE) only in biopsies taken from patients with IBS with predominant constipation (IBS-C). Local administration of 5-oxoETE to mice induced somatic and visceral hypersensitivity to mechanical stimuli without causing tissue inflammation. We found that 5-oxoETE directly acted on both human and mouse sensory neurons as shown by lumbar splanchnic nerve recordings and Ca imaging of dorsal root ganglion (DRG) neurons. We showed that 5-oxoETE selectively stimulated nonpeptidergic, isolectin B4 (IB4)-positive DRG neurons through a phospholipase C (PLC)- and pertussis toxin-dependent mechanism, suggesting that the effect was mediated by a G protein-coupled receptor (GPCR). The MAS-related GPCR D (Mrgprd) was found in mouse colonic DRG afferents and was identified as being implicated in the noxious effects of 5-oxoETE. Together, these data suggest that 5-oxoETE, a potential biomarker of IBS-C, induces somatic and visceral hyperalgesia without inflammation in an Mrgprd-dependent manner. Thus, 5-oxoETE may play a pivotal role in the abdominal pain associated with IBS-C.
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http://dx.doi.org/10.1126/scisignal.aal2171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411128PMC
December 2018

Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.

Gut 2019 03 9;68(3):522-532. Epub 2018 Oct 9.

INSERM-UMR1149, Centre de Recherche sur l'Inflammation, Paris, France.

Objective: Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury.

Design: C57BL/6J mice and mice with global invalidation of MAGL (MAGL ), or myeloid-specific deletion of either MAGL (MAGL), ATG5 (ATG) or CB2 (CB2), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells.

Results: MAGL or MAGL mice exposed to CCl or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6C macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGL BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGL mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2 mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5 BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited.

Conclusion: MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.
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http://dx.doi.org/10.1136/gutjnl-2018-316137DOI Listing
March 2019

Cyclooxygenases and lipoxygenases are used by the fungus Podospora anserina to repel nematodes.

Biochim Biophys Acta Gen Subj 2018 10 17;1862(10):2174-2182. Epub 2018 Jul 17.

Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de Demain (LIED), Univ. Paris Diderot, Paris F-75205, France. Electronic address:

Oxylipins are secondary messengers used universally in the living world for communication and defense. The paradigm is that they are produced enzymatically for the eicosanoids and non-enzymatically for the isoprostanoids. They are supposed to be degraded into volatile organic compounds (VOCs) and to participate in aroma production. Some such chemicals composed of eight carbons are also envisoned as alternatives to fossil fuels. In fungi, oxylipins have been mostly studied in Aspergilli and shown to be involved in signalling asexual versus sexual development, mycotoxin production and interaction with the host for pathogenic species. Through targeted gene deletions of genes encoding oxylipin-producing enzymes and chemical analysis of oxylipins and volatile organic compounds, we show that in the distantly-related ascomycete Podospora anserina, isoprostanoids are likely produced enzymatically. We show the disappearance in the mutants lacking lipoxygenases and cyclooxygenases of the production of 10-hydroxy-octadecadienoic acid and that of 1-octen-3-ol, a common volatile compound. Importantly, this was correlated with the inability of the mutants to repel nematodes as efficiently as the wild type. Overall, our data show that in this fungus, oxylipins are not involved in signalling development but may rather be used directly or as precursors in the production of odors against potential agressors.

Significance: We analyzse the role in inter-kingdom communication of lipoxygenase (lox) and cyclooxygenase (cox) genes in the model fungus Podospora anserina. Through chemical analysis we define the oxylipins and volatile organic compounds (VOCs)produce by wild type and mutants for cox and lox genes, We show that the COX and LOX genes are required for the production of some eight carbon VOCs. We show that COX and LOX genes are involved in the production of chemicals repelling nematodes. This role is very different from the ones previously evidenced in other fungi.
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http://dx.doi.org/10.1016/j.bbagen.2018.07.012DOI Listing
October 2018

Identification of an analgesic lipopeptide produced by the probiotic Escherichia coli strain Nissle 1917.

Nat Commun 2017 11 3;8(1):1314. Epub 2017 Nov 3.

IRSD, Université de Toulouse, INSERM, INRA, INP-ENVT, Université de Toulouse 3 Paul Sabatier, 31024, Toulouse, France.

Administration of the probiotic Escherichia coli strain Nissle 1917 (EcN) decreases visceral pain associated with irritable bowel syndrome. Mutation of clbA, a gene involved in the biosynthesis of secondary metabolites, including colibactin, was previously shown to abrogate EcN probiotic activity. Here, we show that EcN, but not an isogenic clbA mutant, produces an analgesic lipopeptide. We characterize lipoamino acids and lipopeptides produced by EcN but not by the mutant by online liquid chromatography mass spectrometry. One of these lipopeptides, C12AsnGABAOH, is able to cross the epithelial barrier and to inhibit calcium flux induced by nociceptor activation in sensory neurons via the GABA receptor. C12AsnGABAOH inhibits visceral hypersensitivity induced by nociceptor activation in mice. Thus, EcN produces a visceral analgesic, which could be the basis for the development of new visceral pain therapies.
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http://dx.doi.org/10.1038/s41467-017-01403-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5670229PMC
November 2017

The importance of blood platelet lipid signaling in thrombosis and in sepsis.

Adv Biol Regul 2018 01 29;67:66-73. Epub 2017 Sep 29.

Inserm U1048, I2MC, Université Paul Sabatier, 31024 Toulouse Cedex 03, France; CHU de Toulouse, Laboratoire d'Hématologie, 31059 Toulouse Cedex 03, France. Electronic address:

Blood platelets are the first line of defense against hemorrhages and are also strongly involved in the processes of arterial thrombosis, a leading cause of death worldwide. Besides their well-established roles in hemostasis, vascular wall repair and thrombosis, platelets are now recognized as important players in other processes such as inflammation, healing, lymphangiogenesis, neoangiogenesis or cancer. Evidence is accumulating they are key effector cells in immune and inflammatory responses to host infection. To perform their different functions platelets express a wide variety of membrane receptors triggering specific intracellular signaling pathways and largely use lipid signaling systems. Lipid metabolism is highly active in stimulated platelets including the phosphoinositide metabolism with the phospholipase C (PLC) and the phosphoinositide 3-kinase (PI3K) pathways but also other enzymatic systems producing phosphatidic acid, lysophosphatidic acid, platelet activating factor, sphingosine 1-phosphate and a number of eicosanoids. While several of these bioactive lipids regulate intracellular platelet signaling mechanisms others are released by activated platelets acting as autocrine and/or paracrine factors modulating neighboring cells such as endothelial and immune cells. These bioactive lipids have been shown to play important roles in hemostasis and thrombosis but also in vessel integrity and dynamics, inflammation, tissue remodeling and wound healing. In this review, we will discuss some important aspects of platelet lipid signaling in thrombosis and during sepsis that is an important cause of death in intensive care unit. We will particularly focus on the implication of the different isoforms of PI3Ks and on the generation of eicosanoids released by activated platelets.
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http://dx.doi.org/10.1016/j.jbior.2017.09.011DOI Listing
January 2018

Acid Ceramidase Deficiency in Mice Results in a Broad Range of Central Nervous System Abnormalities.

Am J Pathol 2017 Apr;187(4):864-883

Dominick P. Purpura Department of Neuroscience, Rose F. Kennedy Intellectual and Developmental Disabilities Research Center, Albert Einstein College of Medicine, Bronx, New York. Electronic address:

Farber disease is a rare autosomal recessive disorder caused by acid ceramidase deficiency that usually presents as early-onset progressive visceral and neurologic disease. To understand the neurologic abnormality, we investigated behavioral, biochemical, and cellular abnormalities in the central nervous system of Asah1 mice, which serve as a model of Farber disease. Behaviorally, the mutant mice had reduced voluntary locomotion and exploration, increased thigmotaxis, abnormal spectra of basic behavioral activities, impaired muscle grip strength, and defects in motor coordination. A few mutant mice developed hydrocephalus. Mass spectrometry revealed elevations of ceramides, hydroxy-ceramides, dihydroceramides, sphingosine, dihexosylceramides, and monosialodihexosylganglioside in the brain. The highest accumulation was in hydroxy-ceramides. Storage compound distribution was analyzed by mass spectrometry imaging and morphologic analyses and revealed involvement of a wide range of central nervous system cell types (eg, neurons, endothelial cells, and choroid plexus cells), most notably microglia and/or macrophages. Coalescing and mostly perivascular granuloma-like accumulations of storage-laden CD68 microglia and/or macrophages were seen as early as 3 weeks of age and located preferentially in white matter, periventricular zones, and meninges. Neurodegeneration was also evident in specific cerebral areas in late disease. Overall, our central nervous system studies in Asah1 mice substantially extend the understanding of human Farber disease and suggest that this model can be used to advance therapeutic approaches for this currently untreatable disorder.
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http://dx.doi.org/10.1016/j.ajpath.2016.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397689PMC
April 2017

Liquid Chromatography-High Resolution Mass Spectrometry Method to Study Sphingolipid Metabolism Changes in Response to CD95L.

Methods Mol Biol 2017 ;1557:213-217

INSERM UMR 1037, CRCT, 31037, Toulouse, France.

Sphingolipids are sphingoid base-containing lipids, among which some metabolites behave as bioactive molecules in various biological processes, including cell death. Whereas ceramide is now viewed as an anti-oncometabolite, leading to cancer cell death, CD95L-induced apoptosis is associated with sphingolipid changes, which likely contribute to caspase-dependent signaling pathway activation. Here, we describe Liquid Chromatography-high resolution mass spectrometry method (LC-HRMS) to analyze sphingolipid metabolism changes triggered by CD95L.
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http://dx.doi.org/10.1007/978-1-4939-6780-3_20DOI Listing
February 2018

Atypical cleavage of protonated N-fatty acyl amino acids derived from aspartic acid evidenced by sequential MS experiments.

Amino Acids 2016 12 26;48(12):2717-2729. Epub 2016 Aug 26.

IPCM UMR-CNRS 8232, University Paris VI (UPMC), Paris, France.

Lipidomics calls for information on detected lipids and conjugates whose structural elucidation by mass spectrometry requires to rationalization of their gas phase dissociations toward collision-induced dissociation (CID) processes. This study focused on activated dissociations of two lipoamino acid (LAA) systems composed of N-palmitoyl acyl coupled with aspartic and glutamic acid mono ethyl esters (as LAA and LAA). Although in MS/MS, their CID spectra show similar trends, e.g., release of water and ethanol, the [(LAA+H)-CHOH] product ions dissociate via distinct pathways in sequential MS experiments. The formation of all the product ions is rationalized by charge-promoted cleavages often involving stepwise processes with ion isomerization into ion-dipole prior to dissociation. The latter explains the maleic anhydride or ketene neutral losses from N-palmitoyl acyl aspartate and glutamate anhydride fragment ions, respectively. Consequently, protonated palmitoyl acid amide is generated from LAA whereas LAA leads to the [*E+H-HO] anhydride. The former releases ammonia to provide acylium, which gives the C H and C H carbenium series. This should offer structural information, e.g., to locate either unsaturation(s) or alkyl group branching present on the various fatty acyl moieties of lipo-aspartic acid in further studies based on MS experiments.
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http://dx.doi.org/10.1007/s00726-016-2286-0DOI Listing
December 2016

Simultaneous quantitative profiling of 20 isoprostanoids from omega-3 and omega-6 polyunsaturated fatty acids by LC-MS/MS in various biological samples.

Anal Chim Acta 2016 05 19;921:46-58. Epub 2016 Mar 19.

MetaToul-Lipidomic Core Facility, MetaboHUB, Inserm U1048, Toulouse, France. Electronic address:

Isoprostanoids are a group of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids. It belongs to oxylipins group, which are important lipid mediators in biological processes, such as tissue repair, blood clotting, blood vessel permeability, inflammation and immunity regulation. Recently, isoprostanoids from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively have attracted attention because of their putative contribution to health. Since isoprostanoids are derived from different substrate of PUFAs and can have similar or opposing biological consequences, a total isoprostanoids profile is essential to understand the overall effect in the testing model. However, the concentration of most isoprostanoids range from picogram to nanogram, therefore a sensitive method to quantify 20 isoprostanoids simultaneously was formulated and measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The lipid portion from various biological samples was extracted prior to LC-MS/MS evaluation. For all the isoprostanoids LOD and LOQ, and the method was validated on plasma samples for matrix effect, yield of extraction and reproducibility were determined. The methodology was further tested for the isoprostanoids profiles in brain and liver of LDLR(-/-) mice with and without docosahexaenoic acid (DHA) supplementation. Our analysis showed similar levels of total F2-isoprostanes and F4-neuroprostanes in the liver and brain of non-supplemented LDLR(-/-) mice. The distribution of different F2-isoprostane isomers varied between tissues but not for F4-neuroprostanes which were predominated by the 4(RS)-4-F4t-neuroprostane isomer. DHA supplementation to LDLR(-/-) mice concomitantly increased total F4-neuroprostanes levels compared to F2-isoprostanes but this effect was more pronounced in the liver than brain.
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http://dx.doi.org/10.1016/j.aca.2016.03.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517622PMC
May 2016

A novel jaspine B-ceramide hybrid modulates sphingolipid metabolism.

Chem Biodivers 2015 Jul;12(7):1115-25

LSPCMIB, UMR-5068, CNRS, Université de Toulouse, UPS, FR-31062, Toulouse, (phone: +33-561-556299; fax: +33-561-556011).

A new sphingolipid hybrid molecule was designed to assemble, within a tail-to-tail double-chain structure, the ceramide hydrophilic moiety and the tetrahydrofuran pharmacophore of jaspine B, a natural product known to interfere with sphingolipid metabolism. This compound was prepared through acylation of sphingosine with a jaspine B derivative bearing a COOH group in the terminal position of the aliphatic backbone. This new hybrid molecule was evaluated for its capacities to affect melanoma cell viability and sphingolipid metabolism. While retaining the cytotoxicity of ceramide itself, this compound was shown to lower the sphingomyelin cellular levels and significantly enhance the production of sphingosine-1-phosphate, thus representing a novel sphingolipid metabolism modulator.
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http://dx.doi.org/10.1002/cbdv.201400357DOI Listing
July 2015

Cytomegalovirus Infection Triggers the Secretion of the PPARγ Agonists 15-Hydroxyeicosatetraenoic Acid (15-HETE) and 13-Hydroxyoctadecadienoic Acid (13-HODE) in Human Cytotrophoblasts and Placental Cultures.

PLoS One 2015 14;10(7):e0132627. Epub 2015 Jul 14.

Centre de Physiopathologie de Toulouse Purpan, INSERM U1043, Toulouse, France; CNRS U5282, Toulouse, France; Université Toulouse III Paul-Sabatier, Toulouse, France.

Introduction: Congenital infection by human cytomegalovirus (HCMV) is a leading cause of congenital abnormalities of the central nervous system. Placenta infection by HCMV allows for viral spread to fetus and may result in intrauterine growth restriction, preeclampsia-like symptoms, or miscarriages. We previously reported that HCMV activates peroxisome proliferator-activated receptor gamma (PPARγ) for its own replication in cytotrophoblasts. Here, we investigated the molecular bases of PPARγ activation in infected cytotrophoblasts.

Results: We show that onboarded cPLA2 carried by HCMV particles is required for effective PPARγ activation in infected HIPEC cytotrophoblasts, and for the resulting inhibition of cell migration. Natural PPARγ agonists are generated by PLA2 driven oxidization of linoleic and arachidonic acids. Therefore, using HPLC coupled with mass spectrometry, we disclosed that cellular and secreted levels of 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE) were significantly increased in and from HIPEC cytotrophoblasts at soon as 6 hours post infection. 13-HODE treatment of uninfected HIPEC recapitulated the effect of infection (PPARγ activation, migration impairment). We found that infection of histocultures of normal, first-term, human placental explants resulted in significantly increased levels of secreted 15-HETE and 13-HODE.

Conclusion: Our findings reveal that 15-HETE and 13-HODE could be new pathogenic effectors of HCMV congenital infection They provide a new insight about the pathogenesis of congenital infection by HCMV.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132627PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501751PMC
April 2016

Quantification and Potential Functions of Endogenous Agonists of Transient Receptor Potential Channels in Patients With Irritable Bowel Syndrome.

Gastroenterology 2015 Aug 22;149(2):433-44.e7. Epub 2015 Apr 22.

Inserm, U1043, Toulouse, France; CNRS, U5282, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France; University of Calgary, Department of Pharmacology and Physiology, Calgary, Alberta, Canada. Electronic address:

Background & Aims: In mice, activation of the transient receptor potential cation channels (TRP) TRPV1, TRPV4, and TRPA1 causes visceral hypersensitivity. These receptors and their agonists might be involved in development of irritable bowel syndrome (IBS). We investigated whether polyunsaturated fatty acid (PUFA) metabolites, which activate TRPs, are present in colon tissues from patients with IBS and act as endogenous agonists to induce hypersensitivity.

Methods: We analyzed colon biopsy samples from 40 patients with IBS (IBS biopsies) and 11 healthy individuals undergoing colorectal cancer screening (controls), collected during colonoscopy at the University of Bologna, Italy. Levels of the PUFA metabolites that activate TRPV1 (12-hydroperoxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 5-hydroxyeicosatetraenoic acid, and leukotriene B4), TRPV4 (5,6-epoxyeicosatrienoic acid [EET] and 8,9-EET), and TRPA1 (PGA1, 8-iso-prostaglandin A2, and 15-deoxy-Δ-prostaglandin J2) were measured in biopsies and their supernatants using liquid chromatography and tandem mass spectrometry; we also measured levels of the PUFA metabolites prostaglandin E2 (PGE2) and resolvins. C57Bl6 mice were given intrathecal injections of small interfering RNAs to reduce levels of TRPV4, or control small interfering RNAs, along with colonic injections of biopsy supernatants; visceral hypersensitivity was measured based on response to colorectal distension. Mouse sensory neurons were cultured and incubated with biopsy supernatants and lipids extracted from biopsies or colons of mice. Immunohistochemistry was used to detect TRPV4 in human dorsal root ganglia samples (from the National Disease Research Interchange).

Results: Levels of the TRPV4 agonist 5,6-EET, but not levels of TRPV1 or TRPA1 agonists, were increased in IBS biopsies compared with controls; increases correlated with pain and bloating scores. Supernatants from IBS biopsies, but not from controls, induced visceral hypersensitivity in mice. Small interfering RNA knockdown of TRPV4 in mouse primary afferent neurons inhibited the hypersensitivity caused by supernatants from IBS biopsies. Levels of 5,6-EET and 15-HETE were increased in colons of mice with, but not without, visceral hypersensitivity. PUFA metabolites extracted from IBS biopsies or colons of mice with visceral hypersensitivity activated mouse sensory neurons in vitro, by activating TRPV4. Mouse sensory neurons exposed to supernatants from IBS biopsies produced 5,6-EET via a mechanism that involved the proteinase-activated receptor-2 and cytochrome epoxygenase. In human dorsal root ganglia, TPV4 was expressed by 35% of neurons.

Conclusions: Colon tissues from patients with IBS have increased levels of specific PUFA metabolites. These stimulate sensory neurons from mice and generate visceral hypersensitivity via activation of TRPV4.
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http://dx.doi.org/10.1053/j.gastro.2015.04.011DOI Listing
August 2015

Nonredundant protective properties of FPR2/ALX in polymicrobial murine sepsis.

Proc Natl Acad Sci U S A 2014 Dec 15;111(52):18685-90. Epub 2014 Dec 15.

The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London EC1M 6BQ, United Kingdom;

Sepsis is characterized by overlapping phases of excessive inflammation temporally aligned with an immunosuppressed state, defining a complex clinical scenario that explains the lack of successful therapeutic options. Here we tested whether the formyl-peptide receptor 2/3 (Fpr2/3)--ortholog to human FPR2/ALX (receptor for lipoxin A4)--exerted regulatory and organ-protective functions in experimental sepsis. Coecal ligature and puncture was performed to obtain nonlethal polymicrobial sepsis, with animals receiving antibiotics and analgesics. Clinical symptoms, temperature, and heart function were monitored up to 24 h. Peritoneal lavage and plasma samples were analyzed for proinflammatory and proresolving markers of inflammation and organ dysfunction. Compared with wild-type mice, Fpr2/3(-/-) animals exhibited exacerbation of disease severity, including hypothermia and cardiac dysfunction. This scenario was paralleled by higher levels of cytokines [CXCL1 (CXC receptor ligand 1), CCL2 (CC receptor ligand 2), and TNFα] as quantified in cell-free biological fluids. Reduced monocyte recruitment in peritoneal lavages of Fpr2/3(-/-) animals was reflected by a higher granulocyte/monocyte ratio. Monitoring Fpr2/3(-/-) gene promoter activity with a GFP proxy marker revealed an over threefold increase in granulocyte and monocyte signals at 24 h post-coecal ligature and puncture, a response mediated by TNFα. Treatment with a receptor peptido-agonist conferred protection against myocardial dysfunction in wild-type, but not Fpr2/3(-/-), animals. Therefore, coordinated physio-pharmacological analyses indicate nonredundant modulatory functions for Fpr2/3 in experimental sepsis, opening new opportunities to manipulate the host response for therapeutic development.
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http://dx.doi.org/10.1073/pnas.1410938111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284560PMC
December 2014

The ω6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway.

Mol Metab 2014 Dec 16;3(9):834-47. Epub 2014 Sep 16.

Univ. Nice Sophia Antipolis, iBV, UMR 7277, 06100 Nice, France ; CNRS, iBV, UMR 7277, 06100 Nice, France ; Inserm, iBV, U1091, 06100 Nice, France.

Objective: Brite adipocytes are inducible energy-dissipating cells expressing UCP1 which appear within white adipose tissue of healthy adult individuals. Recruitment of these cells represents a potential strategy to fight obesity and associated diseases.

Methods/results: Using human Multipotent Adipose-Derived Stem cells, able to convert into brite adipocytes, we show that arachidonic acid strongly inhibits brite adipocyte formation via a cyclooxygenase pathway leading to secretion of PGE2 and PGF2α. Both prostaglandins induce an oscillatory Ca(++) signaling coupled to ERK pathway and trigger a decrease in UCP1 expression and in oxygen consumption without altering mitochondriogenesis. In mice fed a standard diet supplemented with ω6 arachidonic acid, PGF2α and PGE2 amounts are increased in subcutaneous white adipose tissue and associated with a decrease in the recruitment of brite adipocytes.

Conclusion: Our results suggest that dietary excess of ω6 polyunsaturated fatty acids present in Western diets, may also favor obesity by preventing the "browning" process to take place.
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http://dx.doi.org/10.1016/j.molmet.2014.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4264041PMC
December 2014

Non-enzymatic lipid oxidation products in biological systems: assessment of the metabolites from polyunsaturated fatty acids.

J Chromatogr B Analyt Technol Biomed Life Sci 2014 Aug 29;964:65-78. Epub 2014 Apr 29.

Institut des Biomolécules Max Mousseron IBMM, UMR 5247 CNRS/Université Montpellier 1/Université Montpellier 2, France. Electronic address:

Metabolites of non-enzymatic lipid peroxidation of polyunsaturated fatty acids notably omega-3 and omega-6 fatty acids have become important biomarkers of lipid products. Especially the arachidonic acid-derived F2-isoprostanes are the classic in vivo biomarker for oxidative stress in biological systems. In recent years other isoprostanes from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic acids have been evaluated, namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively. These have been gaining interest as complementary specific biomarkers in human diseases. Refined extraction methods, robust analysis and elucidation of chemical structures have improved the sensitivity of detection in biological tissues and fluids. Previously the main reliable instrumentation for measurement was gas chromatography-mass spectrometry (GC-MS), but now the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunological techniques is gaining much attention. In this review, the types of prostanoids generated from non-enzymatic lipid peroxidation of some important omega-3 and omega-6 fatty acids and biological samples that have been determined by GC-MS and LC-MS/MS are discussed.
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http://dx.doi.org/10.1016/j.jchromb.2014.04.042DOI Listing
August 2014

Polyunsaturated fatty acid metabolism signature in ischemia differs from reperfusion in mouse intestine.

PLoS One 2013 20;8(9):e75581. Epub 2013 Sep 20.

Inserm, U1043, Toulouse, France ; CNRS, U5282, Toulouse, France ; Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France ; William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, Charterhouse Square, London, United Kingdom.

Polyunsaturated fatty acid (PUFA) metabolites are bioactive autoacoids that play an important role in the pathogenesis of a vast number of pathologies, including gut diseases. The induction and the resolution of inflammation depend on PUFA metabolic pathways that are favored. Therefore, understanding the profile of n-6 (eicosanoids)/n-3 (docosanoids) PUFA-derived metabolites appear to be as important as gene or protein array approaches, to uncover the molecules potentially implicated in inflammatory diseases. Using high sensitivity liquid chromatography tandem mass spectrometry, we characterized the tissue profile of PUFA metabolites in an experimental model of murine intestinal ischemia reperfusion. We identified temporal and quantitative differences in PUFA metabolite production, which correlated with inflammatory damage. Analysis revealed that early ischemia induces both pro-inflammatory and anti-inflammatory eicosanoid production. Primarily, LOX- (5/15/12/8-HETE, LTB4, LxA4) and CYP- (5, 6-EET) metabolites were produced upon ischemia, but also PGE3, and PDx. This suggests that different lipids simultaneously play a role in the induction and counterbalance of ischemic inflammatory response from its onset. COX-derived metabolites were more present from 2 to 5 hours after reperfusion, fitting with the concomitant inflammatory peaks. All metabolites were decreased 48 hours post-reperfusion except for to the pro-resolving RvE precursor 18-HEPE and the PPAR-γαμμα agonist, 15d-PGJ2. Data obtained through the pharmacological blockade of transient receptor potential vanilloid-4, which can be activated by 5, 6-EET, revealed that the endogenous activation of this receptor modulates post-ischemic intestinal inflammation. Altogether, these results demonstrate that different lipid pathways are involved in intestinal ischemia-reperfusion processes. Some metabolites, which expression is severely changed upon intestinal ischemia-reperfusion could provide novel targets and may facilitate the development of new pharmacological treatments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075581PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779198PMC
May 2014

LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites.

J Chromatogr B Analyt Technol Biomed Life Sci 2013 Aug 15;932:123-33. Epub 2013 Jun 15.

Inserm U1048, Toulouse, France.

Lipid autacoids derived from n-3/n-6 polyunsaturated fatty acids (PUFA) are some of the earliest signals triggered by an inflammatory reaction. They are acting also as essential regulators of numerous biological processes in physiological conditions. With regards to their importance, a robust and rapid procedure to quantify a large variety of PUFA metabolites, applicable to diverse biological components needed to be formulated. We have developed a simple methodology using liquid chromatography-tandem mass spectrometry allowing quantification of low-level of PUFA metabolites including bioactive mediators, inactive products and pathway biomarkers. Solid phase extraction was used for samples preparation with an extraction yield of 80% ranging from 65% to 98%. The method was optimized to obtain a rapid (8.5min) and accurate separation of 26 molecules, with a very high sensitivity of detection and analysis (0.6-155pg). When applied to biological samples, the method enabled characterization of eicosanoids and docosanoids production in epithelial cells or foam macrophages stimulated with LPS, in biological fluids and tissues from mouse models of peritonitis or infectious colitis. Our results demonstrate that this new method can be used in cultured cells, in fluids and in colonic tissues to quantify pro-inflammatory and pro-resolving PUFA metabolites mediators.
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http://dx.doi.org/10.1016/j.jchromb.2013.06.014DOI Listing
August 2013

A vasculo-protective circuit centered on lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 operative in murine microcirculation.

Blood 2013 Jul 3;122(4):608-17. Epub 2013 Jun 3.

William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom.

Endogenous protective pathways mitigate the overshooting of inflammation after sterile or infectious injury. Here we report that formyl peptide receptor 2 (Fpr2/3) null mice display a major phenotype with exacerbated vascular inflammation observed postischemia reperfusion (IR) injury of the mesenteric artery, characterized by marked neutrophil adhesion and extravasation as visualized by intravital microscopy. Analysis of endogenous agonists for Fpr2/3 revealed that lipoxin A4 (LXA4) was generated by platelet/neutrophil aggregates during ischemia: this cellular response was attenuated in Fpr2/3(-/-) mice; hence, LXA4 levels were lower after 30 minutes' ischemia, and associated with augmented vascular inflammation in the reperfusion (45-180 minutes) phase. Exogenous delivery of LXA4 attenuated IR-mediated inflammation in Fpr2/3(+/+) but not Fpr2/3(-/-) mice; conversely, an Fpr2/3 antagonist skewed the vascular phenotype of Fpr2/3(+/+) mice to that of Fpr2/3(-/-) animals. Such LXA4-based circuit could be activated by aspirin (30-100 mg/kg), which triggered formation of 15-epi-LXA4 in wild-type mice, yet it was effective in Fpr2/3(-/-) mice. In summary, we propose that during ischemia, neutrophil Fpr2/3 controls platelet/neutrophil aggregates with the rapid generation of circulating LXA4, which in turn modulates downstream vascular inflammatory responses evident during the reperfusion phase.
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http://dx.doi.org/10.1182/blood-2013-04-496661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724196PMC
July 2013
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