Novel lipid mediators of innate immunity and inflammation

Trabajo presentado a la L Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology) celebrada en Rosario (Argentina) del 11 al 14 de noviembre de 2014.Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. LD are formed by two different routes, namely (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling leading to increased neutral lipid formation utilizing fatty acids coming from the de novo biosynthetic route. The latter predominates, accounting for 60-70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2, pointing out this enzyme as a key regulator of AA-induced signaling. In other work, we applied mass spectrometrybased lipid profiling to study the levels of AA-containing phospholipids in macrophages. We identified an unusual inositol phospholipid molecule, PI(20:4/20:4), the levels of which do not decrease but actually increase by 300% after activation of the cells. Elevating the intracellular concentration of PI(20:4/20:4) by introducing the lipid into the cells results in enhancement of the microbicidal capacity of macrophages but does not change gene expression in response to inflammatory stimuli, highlighting the selectivity of action of PI(20:4/20:4). These findings suggest that PI(20:4/20:4) is a novel bioactive inositol phospholipid molecule that regulates innate immune responses in macrophages.Peer Reviewe

Phospholipase A2-regulated lipid droplet formation in leukocytes

Trabajo presentado al 5th International Conference on Phospholipase A2 Mediated Signaling in Translational Medicine celebrado en New Orleans (US) del 20 al 21 de mayo de 2013.[Background]: Lipid droplets (LD) are cytosolic inclusions present in most eukaryotic cells that contain a core rich in neutral lipids such as triacylglycerol (TAG) and cholesteryl esters (CE) and are surrounded by a phospholipid monolayer decorated with a variety of proteins. [Objective]: We have examined the pathways for LD biosynthesis in human monocytes exposed to free arachidonic acid (AA), and studied the signaling cascade and intracellular events leading to LD formation in human monocytes. [Methods]: Mass spectrometry analyses of neutral lipids were conducted to delineate the composition of LD in monocytes exposed to AA. [Results]: Exposure of human peripheral blood monocytes to AA results in the rapid induction of LD formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LD are formed by two different routes, namely (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. LD formation can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2α (cPLA2α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA2α activation by phosphorylation. [Conclusions]: These results suggest that concomitant activation of both p38 and JNK by AA cooperate to activate cPLA2α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis.This work was supported by the Spanish Ministry of Science and Education and the Spanish National Network on Diabetes and Associated Metabolic Disorders (CIBERDEM).Peer Reviewe

Overexpression of cytosolic group IVA phospholipase A2 protects cells from Ca2+ -dependent death

El pdf del artículo es la versión post-print.-- et al.The calcium ionophore ionomycin induces apoptosis-like events in the human embryonic kidney cell line at early times. Plasma membrane blebbing, mitochondrial depolarization, externalization of phosphatidylserine, and nuclear permeability changes can all be observed within 15 min of treatment. However, there is no activation of caspases or chromatin condensation. Expression of a fusion protein containing the enhanced green fluorescent protein (EGFP) and human cytosolic Group IVA phospholipase A2α (EGFP-cPLA 2α) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA 2α mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA2α-expressing human embryonic kidney cells can be spared from ionomycin-induced cell death by pretreating them with exogenous arachidonic acid. Moreover, during calcium overload, mitochondrial depolarization is significantly lower in the EGFP-cPLA2α- expressing cells than in cells expressing normal amounts of cPLA 2α. These results suggest that early cell death events promoted by an overload of calcium can be prevented by the presence of high levels of arachidonic acid. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Grant BMC2001- 2244 from the Spanish Ministry of Science and Technology, Grants BFU2004-01886/BMC, SAF2004-04676, and SAF2004-01232 from the Spanish Ministry of Education and Science, and Red Brucella, Red Respira, and Red Temática de Investigación Cardiovascular, from the Instituto de Salud Carlos III.Peer Reviewe

Chitosan enhances transcellular permeability in human and rat intestine epithelium

The intestinal epithelium regulates the transit of molecules from and into the organism. Several agentsact as absorption enhancers inducing changes in both transcellular and paracellular routes. Chitosan isa non-toxic biocompatible polysaccharide widely used as dietary supplement and mucosal delivery.Chitosan triggers both the activation of intestinal epithelial cells and the release of regulatory factors relevantfor its immunomodulatory activity. Yet, the interaction of chitosan with intestinal epithelial cells ispoorly characterized. We studied the uptake of this polysaccharide, and we evaluated its effects in boththe net water and ion movements across human and rat colon samples and the epithelial permeability.Herein, we demonstrate that chitosan increases the transcellular permeability to ions, water and proteinmarkers in human and rat intestinal mucosa and decreases the water permeability across the paracellularpathway. These findings are relevant to understand the activity of the polysaccharide in the mucosalenvironment.Fil: Canali, María Magdalena. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pedrotti, Luciano Pablo. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Balsinde, Jesús. Universidad de Valladolid; EspañaFil: Ibarra, Cristina Adriana. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Correa, Silvia Graciela. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Lipin-1 integrates lipid synthesis with proinflammatory responses during TLR activation in macrophages

Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase involved in the de novo synthesis of phospholipids and triglycerides. Using macrophages from lipin-1-deficient animals and human macrophages deficient in the enzyme, we show in this work that this phosphatase acts as a proinflammatory mediator during TLR signaling and during the development of in vivo inflammatory processes. After TLR4 stimulation lipin-1-deficient macrophages showed a decreased production of diacylglycerol and activation of MAPKs and AP-1. Consequently, the generation of proinflammatory cytokines like IL-6, IL-12, IL-23, or enzymes like inducible NO synthase and cyclooxygenase 2, was reduced. In addition, animals lacking lipin-1 had a faster recovery from endotoxin administration concomitant with a reduced production of harmful molecules in spleen and liver. These findings demonstrate an unanticipated role for lipin-1 as a mediator of macrophage proinflammatory activation and support a critical link between lipid biosynthesis and systemic inflammatory responses.This work was supported by the Spanish Ministry of Science and Innovation (Grants SAF2007-60055, SAF2010-18831, and BFU2010-18826) and the Regional Government of Castile and Leon (Grants BIO39/VA04/10 and CSI168A12-1). L.P. and G.L. were supported by predoctoral fellowships from the Spanish Ministry of Science and Innovation (Plan de Formación de Personal Investigador and Plan de Formación de Profesorado Universitario programs). M.V. was supported by a predoctoral fellowship from the Regional Government of Castile and Leon. E.E. was supported by a predoctoral fellowship from the Spanish National Research Council (Junta de Ampliación de Estudios Program). C.G. was supported by a predoctoral fellowship from the University of Valladolid.Peer Reviewe

Essential Role for Ethanolamine Plasmalogen Hydrolysis in Bacterial Lipopolysaccharide Priming of Macrophages for Enhanced Arachidonic Acid Release

Due to their high content in esterified arachidonic acid (AA), macrophages provide large amounts of eicosanoids during innate immune reactions. Bacterial lipopolysaccharide (LPS) is a poor trigger of AA mobilization in macrophages but does have the capacity to prime these cells for greatly increased AA release upon subsequent stimulation. In this work, we have studied molecular mechanisms underlying this phenomenon. By using mass spectrometry-based lipidomic analyses, we show in this work that LPS-primed zymosan-stimulated macrophages exhibit an elevated consumption of a particular phospholipid species, i.e., the ethanolamine plasmalogens, which results from reduced remodeling of phospholipids via coenzyme A-independent transacylation reactions. Importantly however, LPS-primed macrophages show no changes in their capacity to directly incorporate AA into phospholipids via CoA-dependent acylation reactions. The essential role for ethanolamine plasmalogen hydrolysis in LPS priming is further demonstrated by the use of plasmalogen-deficient cells. These cells, while responding normally to zymosan by releasing quantities of AA similar to those released by cells expressing normal plasmalogen levels under the same conditions, fail to show an LPS-primed response to the same stimulus, thus unambiguously demonstrating a cause–effect relationship between LPS priming and plasmalogen hydrolysis. Collectively, these results suggest a hitherto unrecognized role for ethanolamine plasmalogen hydrolysis and CoA-independent transacylation reactions in modulating the eicosanoid biosynthetic response

ISG15 Is a Novel Regulator of Lipid Metabolism during Vaccinia Virus Infection.

Interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like modifier that binds to target proteins in a process termed ISGylation. ISG15, first described as an antiviral molecule against many viruses, participates in numerous cellular processes, from immune modulation to the regulation of genome stability. Interestingly, the role of ISG15 as a regulator of cell metabolism has recently gained strength. We previously described ISG15 as a regulator of mitochondrial functions in bone marrow-derived macrophages (BMDMs) in the context of Vaccinia virus (VACV) infection. Here, we demonstrate that ISG15 regulates lipid metabolism in BMDMs and that ISG15 is necessary to modulate the impact of VACV infection on lipid metabolism. We show that Isg15-/- BMDMs demonstrate alterations in the levels of several key proteins of lipid metabolism that result in differences in the lipid profile compared with Isg15+/+ (wild-type [WT]) BMDMs. Specifically, Isg15-/- BMDMs present reduced levels of neutral lipids, reflected by decreased lipid droplet number. These alterations are linked to increased levels of lipases and are independent of enhanced fatty acid oxidation (FAO). Moreover, we demonstrate that VACV causes a dysregulation in the proteomes of BMDMs and alterations in the lipid content of these cells, which appear exacerbated in Isg15-/- BMDMs. Such metabolic changes are likely caused by increased expression of the metabolic regulators peroxisome proliferator-activated receptor-γ (PPARγ) and PPARγ coactivator-1α (PGC-1α). In summary, our results highlight that ISG15 controls BMDM lipid metabolism during viral infections, suggesting that ISG15 is an important host factor to restrain VACV impact on cell metabolism. IMPORTANCE The functions of ISG15 are continuously expanding, and growing evidence supports its role as a relevant modulator of cell metabolism. In this work, we highlight how the absence of ISG15 impacts macrophage lipid metabolism in the context of viral infections and how poxviruses modulate metabolism to ensure successful replication. Our results open the door to new advances in the comprehension of macrophage immunometabolism and the interaction between VACV and the host.We thank the expert technical assistance of Sara Sandoval. We are grateful to Miguel Sánchez-Álvarez who has kindly provided several commercial reagents. We would like to thank the Spanish National Plan for Scientific and Technical Research and Innovation (Plan Estatal de Investigación Científica y Técnica y de Innovación), (Ministry of Health of Spain, State Secretary of R1D and FEDER/FSE).S

Lipin-2 reduces proinflammatory signaling induced by saturated fatty acids in macrophages

Lipin-2 is a member of the lipin family of enzymes, which are key effectors in the biosynthesis of lipids. Mutations in the humanlipin-2 gene are associated with inflammatory-based disorders; however, the role of lipin-2 in cells of the immune system remains obscure. In this study, we have investigated the role of lipin-2 in the proinflammatory action of saturated fatty acids in murine and human macrophages. Depletion of lipin-2 promotes the increased expression of the proinflammatory genes Il6, Ccl2, and Tnfα, which depends on the overstimulation of the JNK1/c-Jun pathway by saturated fatty acids. In contrast, overexpression of lipin-2 reduces the release of proinflammatory factors. Metabolically, the absence of lipin-2 reduces the cellular content of triacylglycerol in saturated fatty acid-overloaded macrophages. Collectively, these studies demonstrate a protective role for lipin-2 in proinflammatory signaling mediated by saturated fatty acids that occurs concomitant with an enhanced cellular capacity for triacylglycerol synthesis. The data provide new insights into the role of lipin-2 in human and murine macrophage biology and may open new avenues for controlling the fatty acid-related low grade inflammation that constitutes the sine qua non of obesity and associated metabolic disorders. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Spanish Ministry of Science and Innovation Grants SAF2010-18831 and BFU2010-18826 and Regional Government of Castile and Leon Grant BIO39/VA04/10. Supported by a predoctoral fellowship from the Regional Government of Castile and Leon, Spain. Supported by a predoctoral fellowship from the Spanish National Research Council (Junta de Ampliación de Estudios Program). Supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation.Peer Reviewe

The Contribution of Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s to Adrenic Acid Mobilization in Murine Macrophages

Producción CientíficaAdrenic acid (AA), the 2-carbon elongation product of arachidonic acid, is present at significant levels in membrane phospholipids of mouse peritoneal macrophages. Despite its abundance and structural similarity to arachidonic acid, very little is known about the molecular mechanisms governing adrenic acid mobilization in cells of the innate immune system. This contrasts with the wide availability of data on arachidonic acid mobilization. In this work, we used mass-spectrometry-based lipidomic procedures to define the profiles of macrophage phospholipids that contain adrenic acid and their behavior during receptor activation. We identified the phospholipid sources from which adrenic acid is mobilized, and compared the data with arachidonic acid mobilization. Taking advantage of the use of selective inhibitors, we also showed that cytosolic group IVA phospholipase A2 is involved in the release of both adrenic and arachidonic acids. Importantly, calcium independent group VIA phospholipase A2 spared arachidonate-containing phospholipids and hydrolyzed only those that contain adrenic acid. These results identify separate mechanisms for regulating the utilization of adrenic and arachidonic acids, and suggest that the two fatty acids may serve non-redundant functions in cells.Ministerio de Economía, Industria y Competitividad (grant SAF2016-80883-R

Differential regulation of phospholipase D and phospholipase A2 by protein kinase C in P388D1 macrophages

Activation of P388D1 macrophages by phorbol myristate acetate (PMA) resulted in the translocation of the protein kinase C (PKC) isoforms alpha, delta, and epsilon from the cytosol to membranes. Furthermore, PMA activated phospholipase D (PLD) in these cells, and potentiated the effect of the inflammatory lipid mediator platelet-activating factor (PAF) on PLD activation. PAF also activated phospholipase A2 (PLA2) and enhanced arachidonic acid (AA) release in P388D1 macrophages, and bacterial lipopolysaccharide (LPS) increased the responsiveness of these cells to PAF. In contrast with PLD, PLA2 activation in P388D1 macrophages was found to take place independently of PKC. This was supported by the following evidence: (i) PMA neither induced AA release nor enhanced the PAF response; (ii) inclusion of PMA along with LPS during priming did not have any effect on PAF-stimulated AA release; (iii) down-regulation of PMA-activatable PKC isoforms by chronic treatment with the phorbol ester had no effect on the PAF response; and (iv) the PKC inhibitor staurosporine did not alter the PAF-induced AA release. The present study provides an example of cells in which the direct activation of PKC by phorbol esters does not lead to a primed and/or enhanced AA release. As a unique example in which PKC activation is neither necessary nor sufficient for AA release to occur, this now allows study of the separate and distinct roles for PLD and PLA2 in signal-transduction processes. This has hitherto been difficult to achieve because of the lack of specific inhibitors of these two phospholipases