Regulation of Phagocytosis in Macrophages by Membrane Ethanolamine Plasmalogens

Macrophages, as professional phagocytes of the immune system, possess the ability to detect and clear invading pathogens and apoptotic cells through phagocytosis. Phagocytosis involves membrane reorganization and remodeling events on the cell surface, which play an essential role in innate immunity and tissue homeostasis and the control of inflammation. In this work, we report that cells deficient in membrane ethanolamine plasmalogen demonstrate a reduced capacity to phagocytize opsonized zymosan particles. Amelioration of plasmalogen deficiency in these cells by incubation with lysoplasmalogen results in a significant augmentation of the phagocytic capacity of the cells. In parallel with these increases, restoration of plasmalogen levels in the cells also increases the number and size of lipid rafts in the membrane, reduces membrane fluidity down to levels found in cells containing normal plasmalogen levels, and improves receptor-mediated signaling. Collectively, these results suggest that membrane plasmalogen level determines characteristics of the plasma membrane such as fluidity and the formation of microdomains that are necessary for efficient signal transduction leading to optimal phagocytosis by macrophages

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

Sequestration of 9-hydroxystearic acid in FAHFA (Fatty Acid Esters of Hydroxy Fatty Acids) as a protective mechanism for colon carcinoma cells to avoid apoptotic cell death

Hydroxy fatty acids are known to cause cell cycle arrest and apoptosis. The best studied of them, 9-hydroxystearic acid (9-HSA), induces apoptosis in cell lines by acting through mechanisms involving different targets. Using mass spectrometry-based lipidomic approaches, we show in this study that 9-HSA levels in human colorectal tumors are diminished when compared with normal adjacent tissue. Since this decrease could be compatible with an escape mechanism of tumors from 9-HSA-induced apoptosis, we investigated different features of the utilization of this hydroxyfatty acid in colon. We show that in colorectal tumors and related cell lines such as HT-29 and HCT-116, 9-HSA is the only hydroxyfatty acid constituent of branched fatty acid esters of hydroxyfatty acids (FAHFA), a novel family of lipids with anti-inflammatory properties. Importantly, FAHFA levels in tumors are elevated compared with normal tissue and, unlike 9-HSA, they do not induce apoptosis of colorectal cell lines over a wide range of concentrations. Further, the addition of 9-HSA to colon cancer cell lines augments the synthesis of different FAHFA before the cells commit to apoptosis, suggesting that FAHFA formation may function as a buffer system that sequesters the hydroxyacid into an inactive form, thereby restricting apoptosis.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers SAF2016-80883-R and SAF2015-73000-EXP. C.G. was funded by a predoctoral fellowship from the University of Valladolid (Plan de Formación de Personal Investigador). CIBERDEM is an initiative of Instituto de Salud Carlos IIIWe acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Activation of lysophosphatidic acid receptor type 1 (LPA1) contributes to pathophysiology of spinal cord injury

Altres ajuts: NIH/NS084398Lysophosphatidic acid (LPA) is an extracellular lipid mediator involved in many physiological functions that signals through six known G-protein-coupled receptors (LPA1-LPA6). A wide range of LPA effects have been identified in the CNS, including neural progenitor cell physiology, astrocyte and microglia activation, neuronal cell death, axonal retraction, and development of neuropathic pain. However, little is known about the involvement of LPA in CNS pathologies. Herein, we demonstrate for the first time that LPA signaling via LPA1 contributes to secondary damage after spinal cord injury. LPA levels increase in the contused spinal cord parenchyma during the first 14 d. To model this potential contribution of LPA in the spinal cord, we injected LPA into the normal spinal cord, revealing that LPA induces microglia/macrophage activation and demyelination. Use of a selective LPA1 antagonist or mice lacking LPA1 linked receptor-mediated signaling to demyelination, which was in part mediated by microglia. Finally, we demonstrate that selective blockade of LPA1 after spinal cord injury results in reduced demyelination and improvement in locomotor recovery. Overall, these results support LPA-LPA1 signaling as a novel pathway that contributes to secondary damage after spinal cord contusion in mice and suggest that LPA1 antagonism might be useful for the treatment of acute spinal cord injur

ECM deposition is driven by caveolin-1-dependent regulation of exosomal biogenesis and cargo sorting.

The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor-stroma interaction critically depends on cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). We show that caveolin-1 (Cav1) centrally regulates exosome biogenesis and exosomal protein cargo sorting through the control of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets, including Tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently deposit ECM and promote tumor invasion. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling but also the generation of distant ECM-enriched stromal niches in vivo. Cav1 acts as a cholesterol rheostat in MVBs, determining sorting of ECM components into specific exosome pools and thus ECM deposition. This supports a model by which Cav1 is a central regulatory hub for tumor-stroma interactions through a novel exosome-dependent ECM deposition mechanism.This study was supported by the Ministerio de Ciencia, Innovación y Universidades (CSD2009-0016, SAF2014-51876-R, SAF2017-83130-R, BFU2016-81912-REDC, and IGP-SO-MINSEV1512-07-2016); the Fundació La Marató de TV3 (385/C/2019); the Worldwide Cancer Research Foundation (AICR 15-0404); and Fondo Europeo de Desarrollo Regional “Una manera de hacer Europa” (to M.Á. del Pozo). M.Á. del Pozo’s group received funding from the European Union Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant agreement no. 641639. M.Á. del Pozo is a member of the Tec4Bio consortium (ref. S2018/NMT4443; Actividades de I+D entre Grupos de Investigación en Tecnologías, Comunidad Autónoma de Madrid/FEDER, Spain). J. Balsinde was supported by Ministerio de Ciencia, Innovación y Universidades grants SAF2013-48201-R and SAF2016-80883-R, and G. Orend was supported by Institut National de la Santé et de la Recherche Médicale, the University of Strasbourg, the Ligue Contre le Cancer, and the Institut National du Cancer (ref. TENPLAMET). L. Albacete-Albacete was supported by a Ministerio de Ciencia, Innovación y Universidades predoctoral fellowship associated with the Severo Ochoa Excellence program (ref. SVP-2013-06789). I. Navarro-Lérida was supported by a postdoctoral fellowship from the Asociación Española Contra el Cáncer (ref. INVES191NAVA). The Centro Nacional de Investigaciones Cardiovasculares Carlos III is supported by the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Saturated fatty acid-enriched small extracellular vesicles mediate a crosstalk inducing liver inflammation and hepatocyte insulin resistance

[Background & Aims]: Lipotoxicity triggers non-alcoholic fatty liver disease (NAFLD) progression owing to the accumulation of toxic lipids in hepatocytes including saturated fatty acids (SFAs), which activate pro-inflammatory pathways. We investigated the impact of hepatocyte- or circulating-derived small extracellular vesicles (sEV) secreted under NAFLD conditions on liver inflammation and hepatocyte insulin signalling. [Methods]: sEV released by primary mouse hepatocytes, characterised and analysed by lipidomics, were added to mouse macrophages/Kupffer cells (KC) to monitor internalisation and inflammatory responses. Insulin signalling was analysed in hepatocytes exposed to conditioned media from sEV-loaded macrophages/KC. Mice were i.v. injected sEV to study liver inflammation and insulin signalling. Circulating sEV from mice and humans with NAFLD were used to evaluate macrophage–hepatocyte crosstalk. [Results]: Numbers of sEV released by hepatocytes increased under NAFLD conditions. Lipotoxic sEV were internalised by macrophages through the endosomal pathway and induced pro-inflammatory responses that were ameliorated by pharmacological inhibition or deletion of Toll-like receptor-4 (TLR4). Hepatocyte insulin signalling was impaired upon treatment with conditioned media from macrophages/KC loaded with lipotoxic sEV. Both hepatocyte-released lipotoxic sEV and the recipient macrophages/KC were enriched in palmitic (C16:0) and stearic (C18:0) SFAs, well-known TLR4 activators. Upon injection, lipotoxic sEV rapidly reached KC, triggering a pro-inflammatory response in the liver monitored by Jun N-terminal kinase (JNK) phosphorylation, NF-κB nuclear translocation, pro-inflammatory cytokine expression, and infiltration of immune cells into the liver parenchyma. sEV-mediated liver inflammation was attenuated by pharmacological inhibition or deletion of TLR4 in myeloid cells. Macrophage inflammation and subsequent hepatocyte insulin resistance were also induced by circulating sEV from mice and humans with NAFLD. [Conclusions]: We identified hepatocyte-derived sEV as SFA transporters targeting macrophages/KC and activating a TLR4-mediated pro-inflammatory response enough to induce hepatocyte insulin resistance.This work was supported by grants PID2021-122766OB-I00 (AMV), PID2019-105989RB-I00 (JB), PID2020-113238RB-I00 (LB), PID2019-106581RB-I00 (MAM), PID2020-114148RB-I00 (MI), PID2019-107036RB-I00 (RF), and RD21/0006/0001 (ISCIII) (IL) funded by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación/10.13039/501100011033 and ERDF ‘A way of making Europe’ by the European Union (MICINN/AEI/FEDER, EU), grant EFSD/Boehringer Ingelheim European Research Programme on ‘Multi-System Challenges in Diabetes’ from the European Foundation for the Study of Diabetes (AMV), P2022/BMD-7227 (Comunidad de Madrid, Spain) (AMV), Fundación Ramón Areces (Spain) (AMV), CIBERdem (AMV and JB), CIBERhed (RF), and CIBERcv (LB) (ISCIII, Spain). LB and AMV belong to the Spanish National Research Council’s (CSIC’s) Cancer Hub. We also acknowledge the Spanish Ministry of Economy and Competitiveness (MINECO) postdoctoral contract IJCI-2015-24758 to IGM and the Spanish Ministry of Education, Culture and Sport (MECD) FPU17/02786 grant to RA

Asmase Regulates autophagy and lysosomal membrane permeabilization and its inhibition prevents early stage nonalcoholic steatohepatitis

Background & Aims: Acid sphingomyelinase (ASMase) is activated in nonalcoholic steatohepatitis (NASH). However, ASMase's contribution to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined ASMase's role in high fat diet (HFD)-induced NASH. Methods: Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase-/- mice fed HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed HFD. Results: ASMase deficiency determined resistance to HFD or methionine and choline deficient diet-mediated hepatic steatosis. ASMase-/- mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress and steatosis, indicating selectivity in the resistance of ASMase-/- mice to ER stress. Autophagic flux determined in the presence of rapamycin and/or chloroquine was lower in primary mouse hepatocytes (PMH) from ASMase-/- mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldinA caused ER stress in PMH from ASMase+/+ mice but not ASMase-/- mice. ASMase-/- PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by the oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD- induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. Conclusions: These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH

HypoxamiRs Profiling Identify miR-765 as a Regulator of the Early Stages of Vasculogenic Mimicry in SKOV3 Ovarian Cancer Cells

Vasculogenic mimicry (VM) is a novel cancer hallmark in which malignant cells develop matrix-associated 3D tubular networks with a lumen under hypoxia to supply nutrients needed for tumor growth. Recent studies showed that microRNAs (miRNAs) may have a role in VM regulation. In this study, we examined the relevance of hypoxia-regulated miRNAs (hypoxamiRs) in the early stages of VM formation. Data showed that after 48 h hypoxia and 12 h incubation on matrigel SKOV3 ovarian cancer cells undergo the formation of matrix-associated intercellular connections referred hereafter as 3D channels-like structures, which arose previous to the apparition of canonical tubular structures representative of VM. Comprehensive profiling of 754 mature miRNAs at the onset of hypoxia-induced 3D channels-like structures showed that 11 hypoxamiRs were modulated (FC>1.5; p < 0.05) in SKOV3 cells (9 downregulated and 2 upregulated). Bioinformatic analysis of the set of regulated miRNAs showed that they might impact cellular pathways related with tumorigenesis. Moreover, overall survival analysis in a cohort of ovarian cancer patients (n = 485) indicated that low miR-765, miR-193b, miR-148a and high miR-138 levels were associated with worst patients outcome. In particular, miR-765 was severely downregulated after hypoxia (FC < 32.02; p < 0.05), and predicted to target a number of protein-encoding genes involved in angiogenesis and VM. Functional assays showed that ectopic restoration of miR-765 in SKOV3 cells resulted in a significant inhibition of hypoxia-induced 3D channels-like formation that was associated with a reduced number of branch points and patterned tubular-like structures. Mechanistic studies confirmed that miR-765 decreased the levels of VEGFA, AKT1 and SRC-α transducers and exerted a negative regulation of VEGFA by specific binding to its 3‘UTR. Finally, overall survival analysis of a cohort of ovarian cancer patients (n = 1435) indicates that high levels of VEGFA, AKT1 and SRC-α and low miR-765 expression were associated with worst patients outcome. In conclusion, here we reported a novel hypoxamiRs signature which constitutes a molecular guide for further clinical and functional studies on the early stages of VM. Our data also suggested that miR-765 coordinates the formation of 3D channels-like structures through modulation of VEGFA/AKT1/SRC-α axis in SKOV3 ovarian cancer cells

The PPARβ/δ Activator GW501516 Prevents the Down-Regulation of AMPK Caused by a High-Fat Diet in Liver and Amplifies the PGC-1α-Lipin 1-PPARα Pathway Leading to Increased Fatty Acid Oxidation

12 páginas, 8 figuras.-- et al.Metabolic syndrome-associated dyslipidemia is mainly initiated by hepatic overproduction of the plasma lipoproteins carrying triglycerides. Here we examined the effects of the peroxisome proliferator-activated receptors (PPAR)-β/δ activator GW501516 on high-fat diet (HFD)-induced hypertriglyceridemia and hepatic fatty acid oxidation. Exposure to the HFD caused hypertriglyceridemia that was accompanied by reduced hepatic mRNA levels of PPAR-γ coactivator 1 (PGC-1)-α and lipin 1, and these effects were prevented by GW501516 treatment. GW501516 treatment also increased nuclear lipin 1 protein levels, leading to amplification in the PGC-1α-PPARα signaling system, as demonstrated by the increase in PPARα levels and PPARα-DNA binding activity and the increased expression of PPARα-target genes involved in fatty acid oxidation. These effects of GW501516 were accompanied by an increase in plasma β-hydroxybutyrate levels, demonstrating enhanced hepatic fatty acid oxidation. Moreover, GW501516 increased the levels of the hepatic endogenous ligand for PPARα, 16:0/18:1-phosphatidilcholine and markedly enhanced the expression of the hepatic Vldl receptor. Interestingly, GW501516 prevented the reduction in AMP-activated protein kinase (AMPK) phosphorylation and the increase in phosphorylated levels of ERK1/2 caused by HFD. In addition, our data indicate that the activation of AMPK after GW501516 treatment in mice fed HFD might be the result of an increase in the AMP to ATP ratio in hepatocytes. These findings indicate that the hypotriglyceridemic effect of GW501516 in HFD-fed mice is accompanied by an increase in phospho-AMPK levels and the amplification of the PGC-1α-lipin 1-PPARα pathway.This work was partly supported by funds from the Spanish Ministerio de Ciencia e Innovación (SAF2009-06939 and BFU2010-18826) and the European Union European Regional Development Fund funds. R.R.-C. was supported by a grant from the Fundación Ramón Areces. L.S.-M. was supported by a Formación de Personal Investigador grant from the Spanish Ministerio de Ciencia e Innovación. A.M.A. was supported by a predoctoral fellowship from the Regional Government of Castilla y León (Spain).Peer reviewe

Dynamics of arachidonic acid mobilization by inflammatory cells

The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites. © 2011 Elsevier B.V.Work in our laboratory was supported by the Spanish Ministry of Science and Innovation (Grants BFU2010-18826 and SAF2010-18831).Peer Reviewe