Myeloid-Specific Deficiency of Long-Chain Acyl CoA Synthetase 4 Reduces Inflammation by Remodeling Phospholipids and Reducing Production of Arachidonic Acid-Derived Proinflammatory Lipid Mediators.

In response to infection or tissue damage, resident peritoneal macrophages (rpMACs) produce inflammatory lipid mediators from the polyunsaturated fatty acid (PUFA), arachidonic acid (AA). Long-chain acyl-CoA synthetase 4 (ACSL4) catalyzes the covalent addition of a CoA moiety to fatty acids, with a strong preference for AA and other PUFAs containing three or more double bonds. PUFA-CoA can be incorporated into phospholipids, which is the source of PUFA for lipid mediator synthesis. In this study, we demonstrated that deficiency of Acsl4 in mouse rpMACs resulted in a significant reduction of AA incorporated into all phospholipid classes and a reciprocal increase in incorporation of oleic acid and linoleic acid. After stimulation with opsonized zymosan (opZym), a diverse array of AA-derived lipid mediators, including leukotrienes, PGs, hydroxyeicosatetraenoic acids, and lipoxins, were produced and were significantly reduced in Acsl4-deficient rpMACs. The Acsl4-deficient rpMACs stimulated with opZym also demonstrated an acute reduction in mRNA expression of the inflammatory cytokines, Il6, Ccl2, Nos2, and Ccl5 When Acsl4-deficient rpMACs were incubated in vitro with the TLR4 agonist, LPS, the levels of leukotriene B4 and PGE2 were also significantly decreased. In LPS-induced peritonitis, mice with myeloid-specific Acsl4 deficiency had a significant reduction in leukotriene B4 and PGE2 levels in peritoneal exudates, which was coupled with reduced infiltration of neutrophils in the peritoneal cavity as compared with wild-type mice. Our data demonstrate that chronic deficiency of Acsl4 in rpMACs reduces the incorporation of AA into phospholipids, which reduces lipid mediator synthesis and inflammation.

Neutrophil-induced ferroptosis promotes tumor necrosis in glioblastoma progression.

Tumor necrosis commonly exists and predicts poor prognoses in many cancers. Although it is thought to result from chronic ischemia, the underlying nature and mechanisms driving the involved cell death remain obscure. Here, we show that necrosis in glioblastoma (GBM) involves neutrophil-triggered ferroptosis. In a hyperactivated transcriptional coactivator with PDZ-binding motif-driven GBM mouse model, neutrophils coincide with necrosis temporally and spatially. Neutrophil depletion dampens necrosis. Neutrophils isolated from mouse brain tumors kill cocultured tumor cells. Mechanistically, neutrophils induce iron-dependent accumulation of lipid peroxides within tumor cells by transferring myeloperoxidase-containing granules into tumor cells. Inhibition or depletion of myeloperoxidase suppresses neutrophil-induced tumor cell cytotoxicity. Intratumoral glutathione peroxidase 4 overexpression or acyl-CoA synthetase long chain family member 4 depletion diminishes necrosis and aggressiveness of tumors. Furthermore, analyses of human GBMs support that neutrophils and ferroptosis are associated with necrosis and predict poor survival. Thus, our study identifies ferroptosis as the underlying nature of necrosis in GBMs and reveals a pro-tumorigenic role of ferroptosis. Together, we propose that certain tumor damage(s) occurring during early tumor progression (i.e. ischemia) recruits neutrophils to the site of tissue damage and thereby results in a positive feedback loop, amplifying GBM necrosis development to its fullest extent.

NHR-49 Transcription Factor Regulates Immunometabolic Response and Survival of Caenorhabditis elegans during Enterococcus faecalis Infection.

Immune response to pathogens is energetically expensive to the host; however, the cellular source of energy to fuel immune response remains unknown. In this study, we show that Caenorhabditis elegans exposed to pathogenic Gram-positive and Gram-negative bacteria or yeast rapidly utilizes lipid droplets, the major energy reserve. The nematode's response to the pathogenic bacterium Enterococcus faecalis entails metabolic rewiring for the upregulation of several genes involved in lipid utilization and downregulation of lipid synthesis genes. Genes encoding acyl-CoA synthetase ACS-2, involved in lipid metabolism, and flavin monooxygenase FMO-2, involved in detoxification, are two highly upregulated genes during E. faecalis infection. We find that both ACS-2 and FMO-2 are necessary for survival and rely on NHR-49, a peroxisome proliferator-activated receptor alpha (PPARα) ortholog, for upregulation during E. faecalis infection. Thus, NHR-49 regulates an immunometabolic axis of survival in C. elegans by modulating breakdown of lipids as well as immune effector production upon E. faecalis exposure.

Long-Chain Acyl-CoA Synthetase 1 Role in Sepsis and Immunity: Perspectives From a Parallel Review of Public Transcriptome Datasets and of the Literature.

A potential role for the long-chain acyl-CoA synthetase family member 1 (ACSL1) in the immunobiology of sepsis was explored during a hands-on training workshop. Participants first assessed the robustness of the potential gap in biomedical knowledge identified via an initial screen of public transcriptome data and of the literature associated with ACSL1. Increase in ACSL1 transcript abundance during sepsis was confirmed in several independent datasets. Querying the ACSL1 literature also confirmed the absence of reports associating ACSL1 with sepsis. Inferences drawn from both the literature (via indirect associations) and public transcriptome data (via correlation) point to the likely participation of ACSL1 and ACSL4, another family member, in inflammasome activation in neutrophils during sepsis. Furthermore, available clinical data indicate that levels of ACSL1 and ACSL4 induction was significantly higher in fatal cases of sepsis. This denotes potential translational relevance and is consistent with involvement in pathways driving potentially deleterious systemic inflammation. Finally, while ACSL1 expression was induced in blood in vitro by a wide range of pathogen-derived factors as well as TNF, induction of ACSL4 appeared restricted to flagellated bacteria and pathogen-derived TLR5 agonists and IFNG. Taken together, this joint review of public literature and omics data records points to two members of the acyl-CoA synthetase family potentially playing a role in inflammasome activation in neutrophils. Translational relevance of these observations in the context of sepsis and other inflammatory conditions remain to be investigated.

Neurotoxic potential of reactive astrocytes in canine distemper demyelinating leukoencephalitis.

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP)+ astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5+/GFAP+, and serglycin+/GFAP+ cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Giardia lamblia: Identification of peroxisomal-like proteins.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.

Neonicotinoid insecticides alter induced defenses and increase susceptibility to spider mites in distantly related crop plants.

BACKGROUND: Chemical suppression of arthropod herbivores is the most common approach to plant protection. Insecticides, however, can cause unintended, adverse consequences for non-target organisms. Previous studies focused on the effects of pesticides on target and non-target pests, predatory arthropods, and concomitant ecological disruptions. Little research, however, has focused on the direct effects of insecticides on plants. Here we demonstrate that applications of neonicotinoid insecticides, one of the most important insecticide classes worldwide, suppress expression of important plant defense genes, alter levels of phytohormones involved in plant defense, and decrease plant resistance to unsusceptible herbivores, spider mites Tetranychus urticae (Acari: Tetranychidae), in multiple, distantly related crop plants. METHODOLOGY/PRINCIPAL FINDINGS: Using cotton (Gossypium hirsutum), corn (Zea mays) and tomato (Solanum lycopersicum) plants, we show that transcription of phenylalanine ammonia lyase, coenzyme A ligase, trypsin protease inhibitor and chitinase are suppressed and concentrations of the phytohormone OPDA and salicylic acid were altered by neonicotinoid insecticides. Consequently, the population growth of spider mites increased from 30% to over 100% on neonicotinoid-treated plants in the greenhouse and by nearly 200% in the field experiment. CONCLUSIONS/SIGNIFICANCE: Our findings are important because applications of neonicotinoid insecticides have been associated with outbreaks of spider mites in several unrelated plant species. More importantly, this is the first study to document insecticide-mediated disruption of plant defenses and link it to increased population growth of a non-target herbivore. This study adds to growing evidence that bioactive agrochemicals can have unanticipated ecological effects and suggests that the direct effects of insecticides on plant defenses should be considered when the ecological costs of insecticides are evaluated.

A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunity.

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H(2)O(2) and O(2) (-), are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H(2)O(2) was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses.

A novel 78-kDa fatty acyl-CoA synthetase (ACS1) of Babesia bovis stimulates memory CD4+ T lymphocyte responses in B. bovis-immune cattle.

Antigen-specific CD4+ T lymphocyte responses contribute to protective immunity against Babesia bovis, however the antigens that induce these responses remain largely unknown. A proteomic approach was used to identify novel B. bovis antigens recognized by memory CD4+ T cells from immune cattle. Fractions obtained from merozoites separated by continuous-flow electrophoresis (CFE) that contained proteins ranging from 20 to 83 kDa were previously shown to stimulate memory CD4+ lymphocyte responses in B. bovis-immune cattle. Expression library screening with rabbit antiserum raised against an immunostimulatory CFE fraction identified a clone encoding a predicted 78 kDa protein. BLAST analysis revealed sequence identity of this B. bovis protein with Plasmodium falciparum fatty acyl coenzyme A synthetase (ACS) family members (PfACS1-PfACS11), and the protein was designated B. bovis acyl-CoA synthetase 1 (ACS1). Southern blot analysis indicated that B. bovis ACS1 is encoded by a single gene, although BLAST analysis of the preliminary B. bovis genome sequence identified two additional family members, ACS2 and ACS3. Peripheral blood lymphocytes and CD4+ T cell lines from B. bovis-immune cattle proliferated significantly against recombinant ACS1 protein, consistent with its predicted involvement in protective immunity. However, immune sera from cattle recovered from B. bovis infection did not react with ACS1, indicating that epitopes may be conformationally dependent.

Tyrosine phosphates act on steroidogenesis through the activation of arachidonic acid release.

The ACTH signaling pathway includes both PKA activation as well as PKA-dependent tyrosine phosphatase activation. In addition, the action of this hormone also includes the regulation of the intracellular levels of arachidonic acid (AA) by the concerted action of two enzymes: an acylCoA-thioesterase and an acyl-CoA-synthetase (ACS4). This work describes the production and characterization of a specific ACS4 antibody, which was used to analyze the effect of ACTH on ACS4 protein level in Y1 adrenocortical cells and the putative relationship between tyrosine phosphatases and ACS4. The antiserum was obtained from rabbits immunized with the recombinant ACS4. This immunogen was produced in bacteria and eluted from an acrylamide gel after SDS-PAGE separation of a partially purified bacteria lysate. When used in Western blot analysis, the antibody obtained specifically recognized only one protein of the molecular mass corresponding to ACS4, in Y1 cells and in several rat tissues. Using the antibody described here, we analyzed the effect of ACTH stimulation on ACS4 protein level. The hormone produced an increase of this acyl-CoA synthetase in Y1 adrenocortical cells. Moreover, this effect was mimicked by cAMP and partially reduced by a tyrosine phosphatase inhibitor. We propose that ACTH regulates ACS4 protein levels through a PKA-dependent mechanism that could involve also PTP activity.

Impaired expression of acyl-CoA-synthetase 5 in epithelial tumors of the small intestine.

Fatty acids are implicated in tumorigenesis, but data are limited concerning endogenous fatty acid metabolism of tumor cells in adenomas and adenocarcinomas of the small intestine. The recently cloned human acyl-CoA-synthetase 5 (ACS5) is predominantly found in the small intestine and represents a key enzyme in providing cytosolic acyl-CoA thioesters. Protein synthesis and mRNA expression of ACS5 were studied in human intestinal tissues using different methods, including a newly established monoclonal antibody. In the healthy small intestine, expression of ACS5 was restricted to the villus surface epithelium but was not detectable in enterocytes lining crypts. ACS5 protein and mRNA were progressively diminished in epithelial cells of adenomas and adenocarcinomas of the small intestine. In conclusion, altered expression of ACS5 is probably related to the adenoma-carcinoma sequence of small intestinal epithelial tumors due to an impaired acyl-CoA thioester synthesis.

Fatty acid-CoA ligase 4 is overexpressed in human hepatocellular carcinoma.

Fatty acid-CoA ligase 4 (FACL4) is a central enzyme controlling the unesterified arachidonic acid (AA) level in cells. It has been shown that FACL4 blocks apoptosis and promotes colon carcinogenesis by lowering the cellular level of unesterified AA. Consistent with this, FACL4 is upregulated in colon adenocarcinoma. The status of FACL4 in other tumors including hepatocellular carcinoma (HCC) is not known. Here, we report that FACL4 is overexpressed in human HCC compared with adjacent normal liver tissues. FACL4 mRNA and protein were overexpressed in 5 out of 12 (41.7%) and 3 out of 8 (37.5%) cases of HCC, respectively. Immunohistochemical staining showed strong fine granular intracytoplasmic staining in tumor cells, whereas we observed occasional weak staining in normal liver tissues surrounding the tumors. We found that 14 out of 37 (37.8%) HCC expressed moderate to strong FACL4 immunostaining. Both normal adult and fetal liver tissues showed very weak to no detectable staining, whereas 3 out of 10 (30%) cirrhotic livers expressed weak staining. In addition, we found that 4 out of 8 (50%) human hepatoma cell lines expressed high levels of FACL4 by northern blot analysis. Our results show that FACL4 is a new molecular marker for HCC and suggest that the FACL4 pathway may be involved in liver carcinogenesis.

Low activity of mitochondrial HMG-CoA synthase in liver of starved piglets is due to low levels of protein despite high mRNA levels.

The unusually low hepatic ketogenic capacity of piglets has been correlated with lack of expression of the mitochondrial HMG-CoA synthase gene. However, we have shown that starvation of 2-week-old piglets increased the mRNA levels of mitochondrial HMG-CoA synthase to a level similar to that observed in starved rats (S. H. Adams, C. S. Alho, G. Asins, F. G. Hegardt, and P. F. Marrero, 1997, Biochem. J. 324, 65-73). We now report that antibodies against pig mitochondrial HMG-CoA synthase detected the pig enzyme in mitochondria of 2-week-old starved piglets and that the pig mitochondrial HMG-CoA synthase cDNA encodes an active enzyme in the eukaryotic cell line Mev-1, with catalytic behavior similar to that of the rat enzyme when expressed in the same system. We also show that low activity of pig mitochondrial HMG-CoA synthase correlates with low expression of the pig enzyme. The discrepancy in mitochondrial HMG-CoA synthase gene expression between the high levels of mRNA and low levels of enzyme was not associated with differences in transcript maturation, which suggests that an attenuated translation of the pig mRNA is responsible for the diminished ketogenic capacity of pig mitochondria.

Two distinct long-chain-acyl-CoA synthetases in guinea pig Harderian gland.

Two distinct long-chain-acyl-CoA synthetases which have different kinetic properties were identified in the guinea pig Harderian gland. One was localized in the microsomes and the other in the mitochondria. The relative V(max) values of the microsomal enzyme were 8.1, 1.7 and 1 and the apparent Km values were 66.7, 12.0 and 30.0 microM for palmitic, linoleic and arachidonic acids, respectively. The relative V(max) values of the mitochondrial enzyme were 2.7, 3.5 and 1 and the apparent Km values were 33.3, 29.9 and 30.0 microM for palmitic, linoleic and arachidonic acids, respectively. The relative V(max) values for the liver microsomal enzyme were 2.0, 2.5 and 1, while those of the liver mitochondrial enzyme were 4.1, 3.9 and 1 with palmitic, linoleic and arachidonic acids, respectively. There were no difference between the microsomal and the mitochondrial enzymes in the liver, regarding apparent Km values; these were 38.4, 29.9 and 22.0 microM for palmitic, linoleic and arachidonic acids, respectively. Thus, the substrate specificity and catalytic rate of the mitochondrial enzyme in Harderian gland for palmitic, linoleic and arachidonic acids were similar to the liver enzyme, but not to the microsomal enzyme in Harderian gland. On the other hand, the antiserum raised against the rat liver enzyme immune-titrated and immuno-blotted the enzymes from Harderian gland microsomes and liver, but not so the enzyme from Harderian gland mitochondria. Thus, the microsomal enzyme in Harderian gland had a common immunogenic epitope(s) with the liver enzyme, but the mitochondrial enzyme did not. The Harderian gland mitochondrial enzyme was a distinct protein from liver enzymes. The catalytic and immunogenic characteristics suggest that the enzyme proteins in the Harderian gland are unique, that is, different from that in the liver. The large V(max) value of the Harderian gland microsomal enzyme for palmitic acid suggests that it contributes to the synthesis of a large amount of the secretory lipid and the high Km value to maintenance of cellular lipid in this organ. The evidence that long-chain-acyl-CoA synthetase in the mitochondria is distinct from that in the microsomes was first found in guinea pig Harderian gland.

Purification of peroxisomes and subcellular distribution of enzyme activities for activation and oxidation of very-long-chain fatty acids in rat brain.

Brain contains high amounts of very-long-chain (VLC) fatty acids (> C22). Since mitochondria from liver and skin fibroblasts lack lignoceroyl-CoA ligase, in liver and skin fibroblasts fatty acids are exclusively oxidized in peroxisomes. Findings by Poulos and associates [9] suggested that contrary to liver and cultured skin fibroblasts brain mitochondria contain lignoceroyl-CoA ligase and can oxidize lignoceric acid. The present study was undertaken to develop a procedure for the isolation of subcellular organelles of higher purity from brain and to get a better understanding of the subcellular localization of the oxidation of VLC fatty acids in brain. The enzyme activities for activation and oxidation of palmitic and lignoceric acids were determined in peroxisomes, mitochondria, microsomes and a myelin fraction from rat brain and peroxisomes, mitochondria and microsomes purified from rat liver. Like in liver, brain lignoceroyl-CoA ligase activity in microsomes and peroxisomes was approx. 9 times higher than in mitochondria. In addition to palmitoyl-CoA ligase the antibodies against palmitoyl-CoA ligase inhibited the residual mitochondrial lignoceroyl-CoA ligase activity, meaning that lignoceroyl-CoA ligase activity in mitochondria was derived from palmitoyl-CoA ligase. Accordingly, in peroxisomes lignoceric acid was oxidized at 7 times higher rate than in mitochondria. Mitochondria were able to oxidize lignoceric acid efficiently when supplemented with lignoceroyl-CoA ligase activity from microsomes or myelin. These results show that in brain lignoceric acid is oxidized in peroxisomes and that lignoceroyl-CoA ligase activity is localized in peroxisomes and microsomes, but not in mitochondria. Peroxisomes and microsomes contain both lignoceroyl-CoA and palmitoyl-CoA ligases. Similar to peroxisomes and microsomes, the antibodies against palmitoyl-CoA ligase inhibited only the palmitoyl-CoA ligase activity in myelin but not the lignoceroyl-CoA ligase activity. These results suggest that in addition to palmitoyl-CoA ligase, myelin also contains lignoceroyl-CoA ligase.

Identity of long-chain acyl-coenzyme A synthetase of microsomes, mitochondria, and peroxisomes in rat liver.

The identity of long-chain acyl-CoA synthetase in microsomes, mitochondria, and peroxisomes of rat liver was examined by using the antibody raised against a purified preparation of the microsomal enzyme. The enzyme activities of these three organelles and the purified microsomal enzyme were titrated by the antibody in a very similar fashion when the activity was measured in terms of palmitoyl-CoA synthetase activity. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates and by Western blot analysis that the enzymes of all three organelles consisted of a polypeptide with the same molecular weight as that of the purified enzyme, and that the specific enzyme activity of the antigenic protein in all three subcellular compartments was nearly the same. The presence of other palmitoyl-CoA synthetase activity in these organelles could not be confirmed. Immunocytochemical study to locate the antigenic site with protein A-gold complex showed that the gold particles were closely associated with the membranes of these organelles. The cell-free translation product in a rabbit reticulocyte lysate protein-synthesizing system and the subunit of the mature enzyme labeled with [35S]methionine in the liver slices exhibited the same mobility as the subunit of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme in microsomes, mitochondria, and peroxisomes was labeled at nearly the same rate when the liver slices were incubated with [35S]methionine.