Life Sciences Research for Lifelong Health


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Title / Authors / Details Open Access Download

Frontline Science: TNF-α and GM-CSF1 priming augments the role of SOS1/2 in driving activation of Ras, PI3K-γ, and neutrophil proinflammatory responses.
Suire S, Baltanas FC, Segonds-Pichon A, Davidson K, Santos E, Hawkins PT, Stephens LR

Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen-killing responses. In neutrophils G protein-coupled receptor (GPCR)-driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP ), are highly dependent on PI3K-γ, a Ras-GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR-triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR-PIP signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM-CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation.

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Journal of leukocyte biology, , 1938-3673, , 2019

PMID: 30720883

The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication.
Zhuang X, Magri A, Hill M, Lai AG, Kumar A, Rambhatla SB, Donald CL, Lopez-Clavijo AF, Rudge S, Pinnick K, Chang WH, Wing PAC, Brown R, Qin X, Simmonds P, Baumert TF, Ray D, Loudon A, Balfe P, Wakelam M, Butterworth S, Kohl A, Jopling CL, Zitzmann N, McKeating JA

The circadian clock regulates immune responses to microbes and affects pathogen replication, but the underlying molecular mechanisms are not well understood. Here we demonstrate that the circadian components BMAL1 and REV-ERBα influence several steps in the hepatitis C virus (HCV) life cycle, including particle entry into hepatocytes and RNA genome replication. Genetic knock out of Bmal1 and over-expression or activation of REV-ERB with synthetic agonists inhibits the replication of HCV and the related flaviruses dengue and Zika via perturbation of lipid signaling pathways. This study highlights a role for the circadian clock component REV-ERBα in regulating flavivirus replication.

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Nature communications, 10, 2041-1723, 377, 2019

PMID: 30670689

Open Access

LIPID MAPS: Serving the next generation of lipid researchers with tools, resources, data, and training.
O'Donnell VB, Dennis EA, Wakelam MJO, Subramaniam S

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.

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Science signaling, 12, 1937-9145, , 2019

PMID: 30622195

Imaging Noncanonical Autophagy and LC3-Associated Phagocytosis in Cultured Cells.
Jacquin E, Fletcher K, Florey O

Monitoring of ATG8 proteins by western blotting and immunofluorescence microscopy are the most common methods to monitor the autophagy pathway. However, it has recently been shown that ATG8 proteins can be lipidated to non-autophagosome, single-membrane compartments through a noncanonical autophagy pathway. This is commonly found to occur during macro-endocytic processes such as phagocytosis, where it has been termed LC3-associated phagocytosis, and upon lysosomotropic drug treatment. Therefore, care is required when interpreting data based on ATG8 in order to conclude whether a signal relates to the canonical or noncanonical pathway. Here we provide methods to monitor noncanonical autophagy through fluorescence microscopy.

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Methods in molecular biology (Clifton, N.J.), 1880, 1940-6029, 295-303, 2019

PMID: 30610705

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK.
Hey F, Andreadi C, Noble C, Patel B, Jin H, Kamata T, Straatman K, Luo J, Balmanno K, Jones DTW, Collins VP, Cook SJ, Caunt CJ, Pritchard C

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of mutations in human cancer, the most common being , although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (BRAF) were detected at lower levels in the nucleus while full-length BRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-BRAF and GFP-BRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of .

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Heliyon, 4, 2405-8440, e01065, 2018

PMID: 30603699

The double life of autophagy proteins.
Florey O

Nature microbiology, 3, 2058-5276, 1334-1335, 2018

PMID: 30478385

3D growth of cancer cells elicits sensitivity to kinase inhibitors but not lipid metabolism modifiers.
Jones DT, Valli A, Haider S, Zang Q, Smethurst E, Schug ZT, Peck B, Aboagye EO, Critchlow SE, Schulze A, Gottlieb E, Wakelam MJO, Adrian HL

Tumour cells exhibit altered lipid metabolism compared to normal cells. Cell signalling kinases are important for regulating lipid synthesis and energy storage. How upstream kinases regulate lipid content, versus direct targeting of lipid metabolising enzymes, is currently unexplored. We evaluated intracellular lipid concentrations in prostate and breast tumour spheroids, treated with drugs directly inhibiting metabolic enzymes FASN, ACC, DGAT and PDHK, or cell signalling kinase enzymes PI3K, AKT and mTOR with lipidomic analysis. We assessed whether baseline lipid profiles corresponded to inhibitors' effectiveness in modulating lipid profiles in 3D-growth, and their relationship to therapeutic activity. Inhibitors against PI3K, AKT and mTOR significantly inhibited MDA-MB-468 and PC3 cell growth in 2D and 3D spheroid growth, while moderately altering lipid content. Conversely, metabolism inhibitors against FASN and DGAT altered lipid content most effectively, while only moderately inhibiting growth compared to kinase inhibitors. The FASN and ACC inhibitors' effectiveness in MDA-MB-468, versus PC3, suggested the former depended more on synthesis whereas the latter may salvage lipids. Although baseline lipid profiles didn't predict growth effects, lipid changes on therapy matched the growth effects of FASN and DGAT inhibitors. Several phospholipids, including phosphatidylcholine, were also upregulated following treatment, possibly via the Kennedy pathway. As this promotes tumour growth, combination studies should include drugs targeting it. Two-dimensional drug screening may miss important metabolism inhibitors or underestimate their potency. Clinical studies should consider serial measurements of tumour lipids to prove target modulation. Pre-therapy tumour classification by de novo lipid synthesis versus uptake may help demonstrate efficacy.

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Molecular cancer therapeutics, , 1538-8514, , 2018

PMID: 30478149

Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.
Tsolakos N, Durrant TN, Chessa T, Suire SM, Oxley D, Kulkarni S, Downward J, Perisic O, Williams RL, Stephens L, Hawkins PT

Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing "excess p85," p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.

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Proceedings of the National Academy of Sciences of the United States of America, 115, 1091-6490, 12176-12181, 2018

PMID: 30442661

Open Access

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.
Bussi C, Peralta Ramos JM, Arroyo DS, Gallea JI, Ronchi P, Kolovou A, Wang JM, Florey O, Celej MS, Schwab Y, Ktistakis NT, Iribarren P

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most work was done in neurons and not in microglial cells. Here we report that exogenous fibrillar but not monomeric alpha-synuclein (AS) induces autophagy in microglial cells. We extensively studied the dynamics of this response by both live-cell imaging and correlative light-electron microscopy (CLEM) and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and Optineurin (OPTN) to ubiquitinated lysosomes. In addition, we observed that LC3 recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.

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Journal of cell science, , 1477-9137, , 2018

PMID: 30404831

The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis.
Rai S, Arasteh M, Jefferson M, Pearson T, Wang Y, Zhang W, Bicsak B, Divekar D, Powell PP, Nauman R, Beraza N, Carding SR, Florey O, Mayer U, Wileman T

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.

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Autophagy, , 1554-8635, 1-14, 2018

PMID: 30403914

CD151 regulates expression of FGFR2 in breast cancer cells via PKC-dependent pathways.
Sadej R, Lu X, Turczyk L, Novitskaya V, Lopez-Clavijo AF, Kordek R, Potemski P, Wakelam MJO, Romanska H, Berditchevski F

Expression of the tetraspanin CD151 is frequently upregulated in epithelial malignancies and correlates with poor prognosis. Here we report that CD151 is involved in regulation of the expression of fibroblast growth factor receptor 2 (FGFR2). Depletion of CD151 in breast cancer cells resulted in an increased level of FGFR2. Accordingly, an inverse correlation between CD151 and FGFR2 was observed in breast cancer tissues. CD151-dependent regulation of the FGFR2 expression relies on post-transcriptional mechanisms involving HuR/ELAVL1, a multifunctional RNA binding protein, and the assembly of processing bodies (P-bodies). Depletion of CD151 correlated with inhibition of PKC, a well-established downstream target of CD151. Accordingly, the levels of dialcylglycerol species were decreased in CD151-negative cells, and inhibition of PKC resulted in the increased expression of FGFR2. Whilst expression of FGFR2 itself did not correlate with any of the clinicopathological data, the FGFR2-/CD151+ patients are more likely to develop lymph node metastasis. Conversely, FGFR2-/CD151- patients demonstrated better overall survival. These results illustrate functional interdependency between CD151 complexes and FGFR2 and suggest a previously unsuspected role of CD151 in breast tumourigenesis.

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Journal of cell science, , 1477-9137, , 2018

PMID: 30257985

Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells.
Koohy H, Bolland DJ, Matheson LS, Schoenfelder S, Stellato C, Dimond A, Várnai C, Chovanec P, Chessa T, Denizot J, Manzano Garcia R, Wingett SW, Freire-Pritchett P, Nagano T, Hawkins P, Stephens L, Elderkin S, Spivakov M, Fraser P, Corcoran AE, Varga-Weisz PD

Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice.

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Genome biology, 19, 1474-760X, 126, 2018

PMID: 30180872

Open Access

Targeting IKKβ in Cancer: Challenges and Opportunities for the Therapeutic Utilisation of IKKβ Inhibitors.
Prescott JA, Cook SJ

Deregulated NF-κB signalling is implicated in the pathogenesis of numerous human inflammatory disorders and malignancies. Consequently, the NF-κB pathway has attracted attention as an attractive therapeutic target for drug discovery. As the primary, druggable mediator of canonical NF-κB signalling the IKKβ protein kinase has been the historical focus of drug development pipelines. Thousands of compounds with activity against IKKβ have been characterised, with many demonstrating promising efficacy in pre-clinical models of cancer and inflammatory disease. However, severe on-target toxicities and other safety concerns associated with systemic IKKβ inhibition have thus far prevented the clinical approval of any IKKβ inhibitors. This review will discuss the potential reasons for the lack of clinical success of IKKβ inhibitors to date, the challenges associated with their therapeutic use, realistic opportunities for their future utilisation, and the alternative strategies to inhibit NF-κB signalling that may overcome some of the limitations associated with IKKβ inhibition.

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Cells, 7, 2073-4409, , 2018

PMID: 30142927

Open Access

MS-based lipidomics of human blood plasma - a community-initiated position paper to develop accepted guidelines.
Burla B, Arita M, Arita M, Bendt AK, Cazenave-Gassiot A, Dennis EA, Ekroos K, Han X, Ikeda K, Liebisch G, Lin MK, Loh TP, Meikle PJ, Orešič M, Quehenberger O, Shevchenko A, Torta F, Wakelam MJO, Wheelock CE, Wenk MR

Human blood is a self-regenerating, lipid-rich biologic fluid that is routinely collected in hospital settings. The inventory of lipid molecules found in blood plasma (plasma lipidome) offers insights into individual metabolism and physiology in health and disease. Disturbances in lipid metabolism also occur in conditions that are not directly linked to lipid metabolism; therefore, plasma lipidomics based on mass spectrometry (MS) is an emerging tool in an array of clinical diagnostics and disease management. However, challenges exist in the translation of such lipidomic data to clinical applications. These relate to the reproducibility, accuracy, and precision of lipid quantitation, study design, sample handling, and data sharing. This position paper emerged from a workshop that initiated a community-led process to elaborate and define a set of generally accepted guidelines for quantitative MS-based lipidomics of blood plasma or serum, with harmonization of data acquired on different instrumentation platforms in independent laboratories across laboratories as an ultimate goal. We hope that other fields may benefit from and follow such a precedent.

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Journal of lipid research, , 1539-7262, , 2018

PMID: 30115755

Open Access

C. elegans Eats Its Own Intestine to Make Yolk Leading to Multiple Senescent Pathologies.
Ezcurra M, Benedetto A, Sornda T, Gilliat AF, Au C, Zhang Q, van Schelt S, Petrache AL, Wang H, de la Guardia Y, Bar-Nun S, Tyler E, Wakelam MJ, Gems D

Aging (senescence) is characterized by the development of numerous pathologies, some of which limit lifespan. Key to understanding aging is discovery of the mechanisms (etiologies) that cause senescent pathology. In C. elegans, a major senescent pathology of unknown etiology is atrophy of its principal metabolic organ, the intestine. Here we identify a cause of not only this pathology but also of yolky lipid accumulation and redistribution (a form of senescent obesity): autophagy-mediated conversion of intestinal biomass into yolk. Inhibiting intestinal autophagy or vitellogenesis rescues both visceral pathologies and can also extend lifespan. This defines a disease syndrome leading to multimorbidity and contributing to late-life mortality. Activation of gut-to-yolk biomass conversion by insulin/IGF-1 signaling (IIS) promotes reproduction and senescence. This illustrates how major, IIS-promoted senescent pathologies in C. elegans can originate not from damage accumulation but from direct effects of futile, continued action of a wild-type biological program (vitellogenesis).

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Current biology : CB, , 1879-0445, , 2018

PMID: 30100339

Open Access

Removing physiological motion from intravital and clinical functional imaging data.
Warren SC, Nobis M, Magenau A, Mohammed YH, Herrmann D, Moran I, Vennin C, Conway JR, Mélénec P, Cox TR, Wang Y, Morton JP, Welch HC, Strathdee D, Anderson KI, Phan TG, Roberts MS, Timpson P

Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark , a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.

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eLife, 7, 2050-084X, , 2018

PMID: 29985127

Open Access

Host lipidome analysis during rhinovirus replication in human bronchial epithelial cells identifies potential therapeutic targets.
Nguyen A, Guedan A, Mousnier A, Swieboda D, Zhang Q, Horkai D, Le Novere N, Solari R, Wakelam MJO

In patients with asthma or chronic obstructive pulmonary disease rhinovirus infections can provoke acute worsening of disease and limited treatment options exist. Viral replication in the host cell induces significant remodeling of intracellular membranes, but few studies have explored this mechanistically or as a therapeutic opportunity. We performed unbiased lipidomic analysis on human bronchial epithelial cells infected over a 6 hour period with the RV-A1b strain of rhinovirus to determine changes in 493 distinct lipid species. Through pathway and network analysis we identified temporal changes in the apparent activities of a number of lipid metabolizing and signaling enzymes. In particular, analysis highlighted fatty acid synthesis and ceramide metabolism as potential anti-rhinoviral targets. To validate the importance of these enzymes in viral replication, we explored the effects of commercially-available enzyme inhibitors upon RV-A1b infection and replication. Ceranib-1, D609 and C75 were the most potent inhibitors, which confirmed that fatty acid synthase and ceramidase are potential inhibitory targets in rhinoviral infections. More broadly, this study demonstrates the potential of lipidomics and pathway analysis to identify novel targets to treat human disorders.

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Journal of lipid research, , 1539-7262, , 2018

PMID: 29946055

Open Access

Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence: supporting the next generation of autophagy researchers and fostering international collaborations.
Deretic V, Prossnitz E, Burge M, Campen MJ, Cannon J, Liu KJ, Sklar LA, Allers L, Garcia SA, Baehrecke EH, Behrends C, Cecconi F, Codogno P, Chen GC, Elazar Z, Eskelinen EL, Fourie B, Gozuacik D, Hong W, Hotamisligi G, Jäättelä M, Jo EK, Johansen T, Juhász G, Kimchi A, Ktistakis N, Kroemer G, MIzushima N, Münz C, Reggiori F, Rubinsztein D, Ryan K, Schroder K, Simonsen A, Tooze S, Vaccaro M, Yoshimori T, Yu L, Zhang H, Klionsky DJ

Recently, NIH has funded a center for autophagy research named the Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence, located at the University of New Mexico Health Science Center (UNM HSC), with aspirations to promote autophagy research locally, nationally, and internationally. The center has 3 major missions: (i) to support junior faculty in their endeavors to develop investigations in this area and obtain independent funding; (ii) to develop and provide technological platforms to advance autophagy research with emphasis on cellular approaches for high quality reproducible research; and (iii) to foster international collaborations through the formation of an International Council of Affiliate Members and through hosting national and international workshops and symposia. Scientifically, the AIM center is focused on autophagy and its intersections with other processes, with emphasis on both fundamental discoveries and applied translational research.

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Autophagy, , 1554-8635, , 2018

PMID: 29938597

Profiling of phosphoinositide molecular species in human and mouse platelets identifies new species increasing following stimulation.
Mujalli A, Chicanne G, Bertrand-Michel J, Viars F, Stephens L, Hawkins P, Viaud J, Gaits-Iacovoni F, Severin S, Gratacap MP, Terrisse AD, Payrastre B

Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP and PIP molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110β deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110β and the more subtle role of p110α in the production of PIP molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP during stimulation, opening new perspectives in phosphoinositide signaling in platelets.

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Biochimica et biophysica acta, , 0006-3002, , 2018

PMID: 29902570

Compensation between CSF1R+ macrophages and Foxp3+ Treg cells drives resistance to tumor immunotherapy.
Gyori D, Lim EL, Grant FM, Spensberger D, Roychoudhuri R, Shuttleworth SJ, Okkenhaug K, Stephens LR, Hawkins PT

Redundancy and compensation provide robustness to biological systems but may contribute to therapy resistance. Both tumor-associated macrophages (TAMs) and Foxp3+ regulatory T (Treg) cells promote tumor progression by limiting antitumor immunity. Here we show that genetic ablation of CSF1 in colorectal cancer cells reduces the influx of immunosuppressive CSF1R+ TAMs within tumors. This reduction in CSF1-dependent TAMs resulted in increased CD8+ T cell attack on tumors, but its effect on tumor growth was limited by a compensatory increase in Foxp3+ Treg cells. Similarly, disruption of Treg cell activity through their experimental ablation produced moderate effects on tumor growth and was associated with elevated numbers of CSF1R+ TAMs. Importantly, codepletion of CSF1R+ TAMs and Foxp3+ Treg cells resulted in an increased influx of CD8+ T cells, augmentation of their function, and a synergistic reduction in tumor growth. Further, inhibition of Treg cell activity either through systemic pharmacological blockade of PI3Kδ, or its genetic inactivation within Foxp3+ Treg cells, sensitized previously unresponsive solid tumors to CSF1R+ TAM depletion and enhanced the effect of CSF1R blockade. These findings identify CSF1R+ TAMs and PI3Kδ-driven Foxp3+ Treg cells as the dominant compensatory cellular components of the immunosuppressive tumor microenvironment, with implications for the design of combinatorial immunotherapies.

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JCI insight, 3, 2379-3708, , 2018

PMID: 29875321

Open Access

Systems medicine disease maps: community-driven comprehensive representation of disease mechanisms.
Mazein A, Ostaszewski M, Kuperstein I, Watterson S, Le Novère N, Lefaudeux D, De Meulder B, Pellet J, Balaur I, Saqi M, Nogueira MM, He F, Parton A, Lemonnier N, Gawron P, Gebel S, Hainaut P, Ollert M, Dogrusoz U, Barillot E, Zinovyev A, Schneider R, Balling R, Auffray C

The development of computational approaches in systems biology has reached a state of maturity that allows their transition to systems medicine. Despite this progress, intuitive visualisation and context-dependent knowledge representation still present a major bottleneck. In this paper, we describe the Disease Maps Project, an effort towards a community-driven computationally readable comprehensive representation of disease mechanisms. We outline the key principles and the framework required for the success of this initiative, including use of best practices, standards and protocols. We apply a modular approach to ensure efficient sharing and reuse of resources for projects dedicated to specific diseases. Community-wide use of disease maps will accelerate the conduct of biomedical research and lead to new disease ontologies defined from mechanism-based disease endotypes rather than phenotypes.

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NPJ systems biology and applications, 4, 2056-7189, 21, 2018

PMID: 29872544

Open Access

Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels.
Riento K, Zhang Q, Clark J, Begum F, Stephens E, Wakelam MJ, Nichols BJ

Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling.

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PloS one, 13, 1932-6203, e0197401, 2018

PMID: 29787576

Open Access

Extracellular vesicles : lipids as key components of their biogenesis and functions.
Record M, Silvente-Poirot S, Poirot M, Wakelam MJO

Intercellular communication has been known for decades to involve either direct contact between cells or to operate by spreading molecules such as cytokines, growth factors, or lipid mediators. Through the last decade we have begun to appreciate the increasing importance of intercellular communication mediated by extracellular vesicles released by viable cells either from plasma membrane shedding microvesicles, also named microparticles), or from an intracellular compartment (exosomes). Exosomes and microvesicles circulate in all biological fluids and can trigger biological responses at distance. Their effects include a large variety of biological processes such as immune surveillance, modification of tumor microenvironment, or regulation of inflammation. They carry a large set of active molecules, including lipid mediators such as eicosanoids, proteins and nucleic acids, able to modify the phenotype of receiving cells. This review will highlight the role of the various lipidic pathways involved in the biogenesis and functions of microvesicles and exosomes.

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Journal of lipid research, , 1539-7262, , 2018

PMID: 29764923

Open Access

Community-driven roadmap for integrated disease maps.
Ostaszewski M, Gebel S, Kuperstein I, Mazein A, Zinovyev A, Dogrusoz U, Hasenauer J, Fleming RMT, Le Novère N, Gawron P, Ligon T, Niarakis A, Nickerson D, Weindl D, Balling R, Barillot E, Auffray C, Schneider R

The Disease Maps Project builds on a network of scientific and clinical groups that exchange best practices, share information and develop systems biomedicine tools. The project aims for an integrated, highly curated and user-friendly platform for disease-related knowledge. The primary focus of disease maps is on interconnected signaling, metabolic and gene regulatory network pathways represented in standard formats. The involvement of domain experts ensures that the key disease hallmarks are covered and relevant, up-to-date knowledge is adequately represented. Expert-curated and computer readable, disease maps may serve as a compendium of knowledge, allow for data-supported hypothesis generation or serve as a scaffold for the generation of predictive mathematical models. This article summarizes the 2nd Disease Maps Community meeting, highlighting its important topics and outcomes. We outline milestones on the roadmap for the future development of disease maps, including creating and maintaining standardized disease maps; sharing parts of maps that encode common human disease mechanisms; providing technical solutions for complexity management of maps; and Web tools for in-depth exploration of such maps. A dedicated discussion was focused on mathematical modeling approaches, as one of the main goals of disease map development is the generation of mathematically interpretable representations to predict disease comorbidity or drug response and to suggest drug repositioning, altogether supporting clinical decisions.

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Briefings in bioinformatics, , 1477-4054, , 2018

PMID: 29688273

Phospholipid signaling in innate immune cells.
O'Donnell VB, Rossjohn J, Wakelam MJ

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.

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The Journal of clinical investigation, , 1558-8238, , 2018

PMID: 29683435