Publications

Protty MB, Jenkins PV, Collins PW, O'Donnell VB Signalling

Phospholipids (PLs) are found in all cell types and are required for structural support and cell activation signalling pathways. In resting cells, PLs are asymmetrically distributed throughout the plasma membrane with native procoagulant aminophospholipids (aPLs) being actively maintained in the inner leaflet of the membrane. Upon platelet activation, aPLs rapidly externalize to the outer leaflet and are essential for supporting the coagulation cascade by providing binding sites for factors in the cell-based model. More recent work has uncovered a role for enzymatically oxidized PLs (eoxPLs) in facilitating coagulation, working in concert with native aPLs. Despite this, the role of aPLs and eoxPLs in thrombo-inflammatory conditions, such as arterial and venous thrombosis, has not been fully elucidated. In this review, we describe the biochemical structures, distribution and regulation of aPL externalization and summarize the literature on eoxPL generation in circulating blood cells. We focus on the currently understood role of these lipids in mediating coagulation reactions , and in human thrombotic disease. Finally, we highlight gaps in our understanding in how these lipids vary in health and disease, which may place them as future therapeutic targets for the management of thrombo-inflammatory conditions.

+view abstract Open biology, PMID: 35440201 Apr 2022

Saud Z, Tyrrell VJ, Zaragkoulias A, Protty MB, Statkute E, Rubina A, Bentley K, White DA, Rodrigues PDS, Murphy RC, Köfeler H, Griffiths WJ, Alvarez-Jarreta J, Brown RW, Newcombe RG, Heyman J, Pritchard M, Mcleod RW, Arya A, Lynch CA, Owens D, Jenkins PV, Buurma NJ, O'Donnell VB, Thomas DW, Stanton RJ Signalling

The lipid envelope of SARS-CoV-2 is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of anti-viral agents, as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Using lipidomics analyses, we revealed that the virus envelope comprised mainly phospholipids (PL), with little cholesterol or sphingolipids, indicating significant differences from the composition of host membranes. Unlike cellular membranes, procoagulant aminophospholipids were present on the external side of the viral envelope at levels exceeding those on activated platelets. As a result, virions directly promoted blood coagulation. To investigate whether these differences could enable selective targeting of the viral envelope in vivo, we tested whether oral rinses containing lipid-disrupting chemicals could reduce viral infectivity. Products containing PL-disrupting surfactants (such as cetylpyridinium chloride (CPC)) met European virucidal standards in vitro; however, components that altered the critical micelle concentration reduced efficacy, and products containing essential oils, PVP-I, or Chlorhexidine were ineffective. This result was recapitulated in vivo, where a 30-second oral rinse with CPC mouthwash eliminated live virus in the oral cavity of COVID-19 patients for at least one hour, while PVP-Iodine and saline mouthwashes were found ineffective. We conclude the SARS-CoV-2 lipid envelope (i) is distinct from the host plasma membrane, which may enable design of selective anti-viral approaches; (ii) contains exposed PE and PS, which may influence thrombosis, pathogenicity, and inflammation; and (iii) can be selectively targeted in vivo by specific oral rinses.

+view abstract Journal of lipid research, PMID: 35436499 15 Apr 2022

Schebb NH, Kühn H, Kahnt AS, Rund KM, O'Donnell VB, Flamand N, Peters-Golden M, Jakobsson PJ, Weylandt KH, Rohwer N, Murphy RC, Geisslinger G, FitzGerald GA, Hanson J, Dahlgren C, Alnouri MW, Offermanns S, Steinhilber D Signalling

Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.

+view abstract Frontiers in pharmacology, PMID: 35308198 2022

O'Donnell VB Signalling

The Lands Pathway is a fundamental biochemical process named for its discovery by William EM Lands and revealed in a series of seminal papers published in the Journal of Biological Chemistry between 1958-65. It describes the selective placement in phospholipids of acyl chains, by phospholipid acyltransferases. This pathway has formed a core component of our knowledge of phospholipid and also diglyceride metabolism in mammalian tissues for over 60 years now. Our understanding of how the Lands pathways are enzymatically mediated via large families of related gene products that display both substrate and tissue specificity has grown exponentially since. Recent studies building on this are starting to reveal key roles for the Lands pathway in specific scenarios, in particular inflammation, immunity and inflammation. This review will cover the Lands cycle from historical perspectives first, then present new information on how this important cycle forms a central regulatory node connecting fatty acyl and phospholipid metabolism and how its altered regulation may present new opportunities for therapeutic intervention in human disease.

+view abstract Biochemical Society transactions, PMID: 35225335 28 Feb 2022

Misheva M, Kotzamanis K, Davies LC, Tyrrell VJ, Rodrigues PRS, Benavides GA, Hinz C, Murphy RC, Kennedy P, Taylor PR, Rosas M, Jones SA, McLaren JE, Deshpande S, Andrews R, Schebb NH, Czubala MA, Gurney M, Aldrovandi M, Meckelmann SW, Ghazal P, Darley-Usmar V, White DA, O'Donnell VB Signalling

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial β-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin β-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by β-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial β-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

+view abstract Nature communications, PMID: 35013270 10 Jan 2022

Diskin C, Corcoran SE, Tyrrell VJ, McGettrick AF, Zaslona Z, O'Donnell VB, Nolan DP, O'Neill LAJ Signalling

The protozoan parasite is the causative agent of the neglected tropical disease human African trypanosomiasis, otherwise known as sleeping sickness. Trypanosomes have evolved many immune-evasion mechanisms to facilitate their own survival, as well as prolonging host survival to ensure completion of the parasitic life cycle. A key feature of the bloodstream form of is the secretion of aromatic keto acids, which are metabolized from tryptophan. In this study, we describe an immunomodulatory role for one of these keto acids, indole-3-pyruvate (I3P). We demonstrate that I3P inhibits the production of PGs in activated macrophages. We also show that, despite the reduction in downstream PGs, I3P augments the expression of cyclooxygenase (COX2). This increase in COX2 expression is mediated in part via inhibition of PGs relieving a negative-feedback loop on COX2. Activation of the aryl hydrocarbon receptor also participates in this effect. However, the increase in COX2 expression is of little functionality, as we also provide evidence to suggest that I3P targets COX activity. This study therefore details an evasion strategy by which a trypanosome-secreted metabolite potently inhibits macrophage-derived PGs, which might promote host and trypanosome survival.

+view abstract Journal of immunology, PMID: 34635586 11 Oct 2021

Diskin C, Zotta A, Tyrrell VJ, Zaslona Z, O'Donnell VB, O'Neill LAJ Signalling

PGs are important proinflammatory lipid mediators, the significance of which is highlighted by the widespread and efficacious use of nonsteroidal anti-inflammatory drugs in the treatment of inflammation. 4-Octyl itaconate (4-OI), a derivative of the Krebs cycle-derived metabolite itaconate, has recently garnered much interest as an anti-inflammatory agent. In this article, we show that 4-OI limits PG production in murine macrophages stimulated with the TLR1/2 ligand Pam3CSK4. This decrease in PG secretion is due to a robust suppression of cyclooxygenase 2 (COX2) expression by 4-OI, with both mRNA and protein levels decreased. Dimethyl fumarate, a fumarate derivative used in the treatment of multiple sclerosis, with properties similar to itaconate, replicated the phenotype observed with 4-OI. We also demonstrate that the decrease in COX2 expression and inhibition of downstream PG production occurs in an NRF2-independent manner. Our findings provide a new insight into the potential of 4-OI as an anti-inflammatory agent and also identifies a novel anti-inflammatory function of dimethyl fumarate.

+view abstract Journal of immunology, PMID: 34635585 11 Oct 2021

Contursi A, Schiavone S, Dovizio M, Hinz C, Fullone R, Tacconelli S, Tyrrell VJ, Grande R, Lanuti P, Marchisio M, Zucchelli M, Ballerini P, Lanas A, O'Donnell VB, Patrignani P Signalling

Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.

+view abstract Journal of lipid research, PMID: 34428433 21 Aug 2021

Köfeler HC, Eichmann TO, Ahrends R, Bowden JA, Danne-Rasche N, Dennis EA, Fedorova M, Griffiths WJ, Han X, Hartler J, Holčapek M, Jirásko R, Koelmel JP, Ejsing CS, Liebisch G, Ni Z, O'Donnell VB, Quehenberger O, Schwudke D, Shevchenko A, Wakelam MJO, Wenk MR, Wolrab D, Ekroos K Signalling, Lipidomics

n/a

+view abstract Nature communications, PMID: 34362906 06 Aug 2021

Gaud C, C Sousa B, Nguyen A, Fedorova M, Ni Z, O'Donnell VB, Wakelam MJO, Andrews S, Lopez-Clavijo AF Signalling, Bioinformatics

Lipidomics increasingly describes the quantification using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues.

+view abstract F1000Research, PMID: 33564392 2021

O'Donnell VB, Thomas D, Stanton R, Maillard JY, Murphy RC, Jones SA, Humphreys I, Wakelam MJO, Fegan C, Wise MP, Bosch A, Sattar SA Signalling

Emerging studies increasingly demonstrate the importance of the throat and salivary glands as sites of virus replication and transmission in early COVID-19 disease. SARS-CoV-2 is an enveloped virus, characterized by an outer lipid membrane derived from the host cell from which it buds. While it is highly sensitive to agents that disrupt lipid biomembranes, there has been no discussion about the potential role of oral rinsing in preventing transmission. Here, we review known mechanisms of viral lipid membrane disruption by widely available dental mouthwash components that include ethanol, chlorhexidine, cetylpyridinium chloride, hydrogen peroxide, and povidone-iodine. We also assess existing formulations for their potential ability to disrupt the SARS-CoV-2 lipid envelope, based on their concentrations of these agents, and conclude that several deserve clinical evaluation. We highlight that already published research on other enveloped viruses, including coronaviruses, directly supports the idea that oral rinsing should be considered as a potential way to reduce transmission of SARS-CoV-2. Research to test this could include evaluating existing or specifically tailored new formulations in well-designed viral inactivation assays, then in clinical trials. Population-based interventions could be undertaken with available mouthwashes, with active monitoring of outcome to determine efficacy. This is an under-researched area of major clinical need.

+view abstract Function, PMID: 33215159 2020

O'Donnell VB, FitzGerald GA, Murphy RC, Liebisch G, Dennis EA, Quehenberger O, Subramaniam S, Wakelam MJO Signalling, Lipidomics

None listed

+view abstract Circulation. Genomic and precision medicine, PMID: 33196315 16 Nov 2020

Liebisch G, Fahy E, Aoki J, Dennis EA, Durand T, Ejsing C, Fedorova M, Feussner I, Griffiths WJ, Koefeler H, Merrill AH, Murphy RC, O'Donnell VB, Oskolkova OV, Subramaniam S, Wakelam M, Spener F Signalling

A comprehensive and standardized system to report lipid structures analyzed by mass spectrometry is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e. annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labelled compounds in metabolic labelling experiments or as internal standards.This update on lipid classification, nomenclature and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.

+view abstract Journal of lipid research, PMID: 33037133 09 Oct 2020

Alvarez-Jarreta J, Rodrigues PRS, Fahy E, O'Connor A, Price A, Gaud C, Andrews S, Benton P, Siuzdak G, Hawksworth JI, Valdivia-Garcia M, Allen SM, O'Donnell VB Bioinformatics

We present LipidFinder 2.0, incorporating four new modules that apply artefact filters, remove lipid and contaminant stacks, in-source fragments and salt clusters, and a new isotope deletion method which is significantly more sensitive than available open-access alternatives. We also incorporate a novel false discovery rate (FDR) method, utilizing a target-decoy strategy, which allows users to assess data quality. A renewed lipid profiling method is introduced which searches three different databases from LIPID MAPS and returns bulk lipid structures only, and a lipid category scatter plot with color blind friendly pallet. An API interface with XCMS Online is made available on LipidFinder's online version. We show using real data that LipidFinder 2.0 provides a significant improvement over non-lipid metabolite filtering and lipid profiling, compared to available tools.

+view abstract Bioinformatics, PMID: 33027502 07 Oct 2020

Ipseiz N, Pickering RJ, Rosas M, Tyrrell VJ, Davies LC, Orr SJ, Czubala MA, Fathalla D, Robertson AA, Bryant CE, O'Donnell V, Taylor PR Signalling

The alarm cytokine interleukin-1β (IL-1β) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1β production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1β; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1β from its pro-form. However, despite the important role of IL-1β in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1β processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1β processing, providing a previously unrecognized control of IL-1β in tissue-resident macrophages.

+view abstract The EMBO journal, PMID: 32484988 15 Jul 2020

Meckelmann SW, Hawksworth JI, White D, Andrews R, Rodrigues P, O'Connor A, Alvarez-Jarreta J, Tyrrell VJ, Hinz C, Zhou Y, Williams J, Aldrovandi M, Watkins WJ, Engler AJ, Lo Sardo V, Slatter DA, Allen SM, Acharya J, Mitchell J, Cooper J, Aoki J, Kano K, Humphries SE, O'Donnell VB Signalling

Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA.

+view abstract Circulation. Genomic and precision medicine, PMID: 32396387 06 2020

O'Donnell VB, Ekroos K, Liebisch G, Wakelam M Signalling,

Lipids are essential for all facets of life. They play three major roles: energy metabolism, structural, and signaling. They are dynamic molecules strongly influenced by endogenous and exogenous factors including genetics, diet, age, lifestyle, drugs, disease and inflammation. As precision medicine starts to become mainstream, there is a huge burgeoning interest in lipids and their potential to act as unique biomarkers or prognostic indicators. Lipids comprise a large component of all metabolites (around one-third), and our expanding knowledge about their dynamic behavior is fueling the hope that mapping their regulatory biochemical pathways on a systems level will revolutionize our ability to prevent, diagnose, and stratify major human diseases. Up to now, clinical lipid measurements have consisted primarily of total cholesterol or triglycerides, as a measure for cardiovascular risk and response to lipid lowering drugs. Nowadays, we are able to measure thousands of individual lipids that make up the lipidome. nuclear magnetic resonance spectrometry (NMR) metabolomics is also being increasingly used in large cohort studies where it can report on total levels of selected lipid classes, and relative levels of fatty acid saturation. To support the application of lipidomics research, LIPID MAPS was established in 2003, and since then has gone on to become the go-to resource for several lipid databases, lipid drawing tools, data deposition, and more recently lipidomics informatics tools, and a lipid biochemistry encyclopedia, LipidWeb. Alongside this, the recently established Lipidomics Standards Initiative plays a key role in standardization of lipidomics methodologies. This article is categorized under: Laboratory Methods and Technologies > Metabolomics Analytical and Computational Methods > Analytical Methods.

+view abstract Wiley interdisciplinary reviews. Systems biology and medicine, PMID: 31646749 2019

Doll S, Freitas FP, Shah R, Aldrovandi M, da Silva MC, Ingold I, Goya Grocin A, Xavier da Silva TN, Panzilius E, Scheel CH, Mourão A, Buday K, Sato M, Wanninger J, Vignane T, Mohana V, Rehberg M, Flatley A, Schepers A, Kurz A, White D, Sauer M, Sattler M, Tate EW, Schmitz W, Schulze A, O'Donnell V, Proneth B, Popowicz GM, Pratt DA, Angeli JPF, Conrad M Signalling

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4) and radical-trapping antioxidants. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints and phospholipid composition contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q, CoQ): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

+view abstract Nature, PMID: 31634899 11 2019

Kornilov A, Kennedy PD, Aldrovandi M, Watson AJA, Hinz C, Harless B, Colombo J, Maxey KM, Tyrrell VJ, Simon M, Aggarwal VK, Boeglin WE, Brash AR, Murphy RC, O'Donnell VB Signalling

Eicosanoids are critical mediators of fever, pain, and inflammation generated by immune and tissue cells. We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS), stable isotope labeling, and GC-MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA). Here, we achieved enzymatic synthesis and H NMR characterization of this compound with results in conflict with the previously proposed structural assignment. Accordingly, by using LC-MS, we screened autoxidation reactions of 11-hydroperoxy-eicosatetraenoic acid (11-HpETE) and thereby identified a candidate sharing the precise reverse-phase chromatographic and MS characteristics of the platelet product. We optimized these methods to increase yield, allowing full structural analysis by H NMR. The revised assignment is presented here as 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE, substituted for the previous name DXA We found that in platelets, the lipid likely forms via dioxolane ring opening with rearrangement to the diepoxy moieties followed by oxygen insertion at C13. We present its enzymatic biosynthetic pathway and MS/MS fragmentation pattern and, using the synthetic compound, demonstrate that it has bioactivity. For the platelet lipid, we estimate 16 isomers based on our current knowledge (and four isomers for the synthetic lipid). Determining the exact isomeric structure of the platelet lipid remains to be undertaken.

+view abstract The Journal of biological chemistry, PMID: 31061099 07 06 2019

Allen-Redpath K, Aldrovandi M, Lauder SN, Gketsopoulou A, Tyrrell VJ, Slatter DA, Andrews R, Watkins WJ, Atkinson G, McNeill E, Gilfedder A, Protty M, Burston J, Johnson SRC, Rodrigues PRS, Jones DO, Lee R, Handa A, Channon K, Obaji S, Alvarez-Jarreta J, Krönke G, Ackermann J, Jenkins PV, Collins PW, O'Donnell VB Signalling

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes ( or ) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. -deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with deletion, and many were absent in mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.

+view abstract Proceedings of the National Academy of Sciences of the United States of America, PMID: 30944221 16 04 2019

Evans RJ, Pline K, Loynes CA, Needs S, Aldrovandi M, Tiefenbach J, Bielska E, Rubino RE, Nicol CJ, May RC, Krause HM, O'Donnell VB, Renshaw SA, Johnston SA Signalling

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.

+view abstract PLoS pathogens, PMID: 30921435 03 2019

O'Donnell VB, Dennis EA, Wakelam MJO, Subramaniam S Signalling, Lipidomics

Lipids are increasingly recognized as dynamic, critical metabolites affecting human physiology and pathophysiology. LIPID MAPS is a free resource dedicated to serving the lipid research community.

+view abstract Science signaling, PMID: 30622195 2019

Fahy E, Alvarez-Jarreta J, Brasher CJ, Nguyen A, Hawksworth JI, Rodrigues P, Meckelmann S, Allen SM, O'Donnell VB

We present LipidFinder online, hosted on the LIPID MAPS website, as a liquid chromatography/mass spectrometry (LC/MS) workflow comprising peak filtering, MS searching and statistical analysis components, highly customized for interrogating lipidomic data. The online interface of LipidFinder includes several innovations such as comprehensive parameter tuning, a MS search engine employing in-house customized, curated and computationally generated databases and multiple reporting/display options. A set of integrated statistical analysis tools which enable users to identify those features which are significantly-altered under the selected experimental conditions, thereby greatly reducing the complexity of the peaklist prior to MS searching is included. LipidFinder is presented as a highly flexible, extensible user-friendly online workflow which leverages the lipidomics knowledge base and resources of the LIPID MAPS website, long recognized as a leading global lipidomics portal.

+view abstract Bioinformatics, PMID: 30101336 2019

O'Donnell VB, Rossjohn J, Wakelam MJ Signalling

Phospholipids comprise a large body of lipids that define cells and organelles by forming membrane structures. Importantly, their complex metabolism represents a highly controlled cellular signaling network that is essential for mounting an effective innate immune response. Phospholipids in innate cells are subject to dynamic regulation by enzymes, whose activities are highly responsive to activation status. Along with their metabolic products, they regulate multiple aspects of innate immune cell biology, including shape change, aggregation, blood clotting, and degranulation. Phospholipid hydrolysis provides substrates for cell-cell communication, enables regulation of hemostasis, immunity, thrombosis, and vascular inflammation, and is centrally important in cardiovascular disease and associated comorbidities. Phospholipids themselves are also recognized by innate-like T cells, which are considered essential for recognition of infection or cancer, as well as self-antigens. This Review describes the major phospholipid metabolic pathways present in innate immune cells and summarizes the formation and metabolism of phospholipids as well as their emerging roles in cell biology and disease.

+view abstract The Journal of clinical investigation, PMID: 29683435 2018

Slatter DA, Percy CL, Allen-Redpath K, Gajsiewicz JM, Brooks NJ, Clayton A, Tyrrell VJ, Rosas M, Lauder SN, Watson A, Dul M, Garcia-Diaz Y, Aldrovandi M, Heurich M, Hall J, Morrissey JH, Lacroix-Desmazes S, Delignat S, Jenkins PV, Collins PW, O'Donnell VB

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.

+view abstract JCI insight, PMID: 29563336 22 03 2018

Lauder SN, Allen-Redpath K, Slatter DA, Aldrovandi M, O'Connor A, Farewell D, Percy CL, Molhoek JE, Rannikko S, Tyrrell VJ, Ferla S, Milne GL, Poole AW, Thomas CP, Obaji S, Taylor PR, Jones SA, de Groot PG, Urbanus RT, Hörkkö S, Uderhardt S, Ackermann J, Vince Jenkins P, Brancale A, Krönke G, Collins PW, O'Donnell VB Signalling

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein β2GP1 (β2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.

+view abstract Science signaling, PMID: 29184033 28 Nov 2017

Lue HW, Podolak J, Kolahi K, Cheng L, Rao S, Garg D, Xue CH, Rantala JK, Tyner JW, Thornburg KL, Martinez-Acevedo A, Liu JJ, Amling CL, Truillet C, Louie SM, Anderson KE, Evans MJ, O'Donnell VB, Nomura DK, Drake JM, Ritz A, Thomas GV

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.

+view abstract Genes & development, PMID: 29138276 15 10 2017

Uderhardt S, Ackermann JA, Fillep T, Hammond VJ, Willeit J, Santer P, Mayr M, Biburger M, Miller M, Zellner KR, Stark K, Zarbock A, Rossaint J, Schubert I, Mielenz D, Dietel B, Raaz-Schrauder D, Ay C, Gremmel T, Thaler J, Heim C, Herrmann M, Collins PW, Schabbauer G, Mackman N, Voehringer D, Nadler JL, Lee JJ, Massberg S, Rauh M, Kiechl S, Schett G, O'Donnell VB, Krönke G Signalling

Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase-derived hydroxyeicosatetraenoic acid-phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease.

+view abstract The Journal of experimental medicine, PMID: 28566277 03 Jul 2017

Klionsky DJ, Ktistakis NT, O'Donnell VB, et al Signalling

n/a

+view abstract Autophagy, PMID: 26799652 2016