Rapid Movement of Palmitoleic Acid from Phosphatidylcholine to Phosphatidylinositol in Activated Human Monocytes.

This work describes a novel route for phospholipid fatty acid remodeling involving the monounsaturated fatty acid palmitoleic acid. When administered to human monocytes, palmitoleic acid rapidly incorporates into membrane phospholipids, notably into phosphatidylcholine (PC). In resting cells, palmitoleic acid remains within the phospholipid pools where it was initially incorporated, showing no further movement. However, stimulation of the human monocytes with either receptor-directed (opsonized zymosan) or soluble (calcium ionophore A23187) agonists results in the rapid transfer of palmitoleic acid moieties from PC to phosphatidylinositol (PI). This is due to the activation of a coenzyme A-dependent remodeling route involving two different phospholipase A2 enzymes that act on different substrates to generate free palmitoleic acid and lysoPI acceptors. The stimulated enrichment of specific PI molecular species with palmitoleic acid unveils a hitherto-unrecognized pathway for lipid turnover in human monocytes which may play a role in regulating lipid signaling during innate immune activation.

Lipin-2 regulates the antiviral and anti-inflammatory responses to interferon.

Interferons (IFN) are crucial antiviral and immunomodulatory cytokines that exert their function through the regulation of a myriad of genes, many of which are not yet characterized. Here, we reveal that lipin-2, a phosphatidic acid phosphatase whose mutations produce an autoinflammatory syndrome known as Majeed syndrome in humans, is regulated by IFN in a STAT-1-dependent manner. Lipin-2 inhibits viral replication both in vitro and in vivo. Moreover, lipin-2 also acts as a regulator of inflammation in a viral context by reducing the signaling through TLR3 and the generation of ROS and release of mtDNA that ultimately activate the NLRP3 inflammasome. Inhibitors of mtDNA release from mitochondria restrict IL-1ß production in lipin-2-deficient animals in a model of viral infection. Finally, analyses of databases from COVID-19 patients show that LPIN2 expression levels negatively correlate with the severity of the disease. Overall, these results uncover novel regulatory mechanisms of the IFN response driven by lipin-2 and open new perspectives for the future management of patients with LPIN2 mutations.

CHOP upregulation and dysregulation of the mature form of the SNAT2 amino acid transporter in the placentas from small for gestational age newborns.

BACKGROUND: The placentas from newborns that are small for gestational age (SGA; birth weight < -2 SD for gestational age) may display multiple pathological characteristics. A key determinant of fetal growth and, therefore, birth weight is placental amino acid transport, which is under the control of the serine/threonine kinase mechanistic target of rapamycin (mTOR). The effects of endoplasmic reticulum (ER) stress on the mTOR pathway and the levels of amino acid transporters are not well established. METHODS: Placentas from SGA and appropriate for gestational age (AGA) newborns and the human placental BeWo cell line exposed to the ER stressor tunicamycin were used. RESULTS: We detected a significant increase in the levels of C/EBP homologous protein (CHOP) in the placentas from SGA newborns compared with those from AGA newborns, while the levels of other ER stress markers were barely affected. In addition, placental mTOR Complex 1 (mTORC1) activity and the levels of the mature form of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) were also reduced in the SGA group. Interestingly, CHOP has been reported to upregulate growth arrest and DNA damage-inducible protein 34 (GADD34), which in turn suppresses mTORC1 activity. The GADD34 inhibitor guanabenz attenuated the increase in CHOP protein levels and the reduction in mTORC1 activity caused by the ER stressor tunicamycin in the human placental cell line BeWo, but it did not recover mature SNAT2 protein levels, which might be reduced as a result of defective glycosylation. CONCLUSIONS: Collectively, these data reveal that GADD34A activity and glycosylation are key factors controlling mTORC1 signaling and mature SNAT2 levels in trophoblasts, respectively, and might contribute to the SGA condition. Video Abstract.

Dynamics of Docosahexaenoic Acid Utilization by Mouse Peritoneal Macrophages.

In this work, the incorporation of docosahexaenoic acid (DHA) in mouse resident peritoneal macrophages and its redistribution within the various phospholipid classes were investigated. Choline glycerophospholipids (PC) behaved as the major initial acceptors of DHA. Prolonged incubation with the fatty acid resulted in the transfer of DHA from PC to ethanolamine glycerophospholipids (PE), reflecting phospholipid remodeling. This process resulted in the cells containing similar amounts of DHA in PC and PE in the resting state. Mass spectrometry-based lipidomic analyses of phospholipid molecular species indicated a marked abundance of DHA in ether phospholipids. Stimulation of the macrophages with yeast-derived zymosan resulted in significant decreases in the levels of all DHA-containing PC and PI species; however, no PE or PS molecular species were found to decrease. In contrast, the levels of an unusual DHA-containing species, namely PI(20:4/22:6), which was barely present in resting cells, were found to markedly increase under zymosan stimulation. The levels of this phospholipid also significantly increased when the calcium-ionophore A23187 or platelet-activating factor were used instead of zymosan to stimulate the macrophages. The study of the route involved in the synthesis of PI(20:4/22:6) suggested that this species is produced through deacylation/reacylation reactions. These results define the increases in PI(20:4/22:6) as a novel lipid metabolic marker of mouse macrophage activation, and provide novel information to understand the regulation of phospholipid fatty acid turnover in activated macrophages.

Elafibranor upregulates the EMT-inducer S100A4 via PPARß/δ.

Elafibranor is a dual peroxisome proliferator-activated receptor (PPAR)α and ß/δ agonist that has reached a phase III clinical trial for the treatment of metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we examined the effects of elafibranor in mice fed a choline-deficient high-fat diet (CD-HFD), a model of metabolic dysfunction-associated steatohepatitis (MASH) that presents obesity and insulin resistance. Our findings revealed that elafibranor treatment ameliorated steatosis, inflammation, and fibrogenesis in the livers of CD-HFD-fed mice. Unexpectedly, elafibranor also increased the levels of the epithelial-mesenchymal transition (EMT)-promoting protein S100A4 via PPARß/δ activation. The increase in S100A4 protein levels caused by elafibranor was accompanied by changes in the levels of markers associated with the EMT program. The S100A4 induction caused by elafibranor was confirmed in the BRL-3A rat liver cells and a mouse primary hepatocyte culture. Furthermore, elafibranor reduced the levels of ASB2, a protein that promotes S100A4 degradation, while ASB2 overexpression prevented the stimulating effect of elafibranor on S100A4. Collectively, these findings reveal an unexpected hepatic effect of elafibranor on increasing S100A4 and promoting the EMT program.

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. Impact and Implications: Small extracellular vesicles (sEV) released by the hepatocytes under non-alcoholic fatty liver disease (NAFLD) conditions cause liver inflammation and insulin resistance in hepatocytes via paracrine hepatocyte-macrophage-hepatocyte crosstalk. We identified sEV as transporters of saturated fatty acids (SFAs) and potent lipotoxic inducers of liver inflammation. TLR4 deficiency or its pharmacological inhibition ameliorated liver inflammation induced by hepatocyte-derived lipotoxic sEV. Evidence of this macrophage-hepatocyte interactome was also found in patients with NAFLD, pointing to the relevance of sEV in SFA-mediated lipotoxicity in NAFLD.

Compartmentalized regulation of lipid signaling in oxidative stress and inflammation: Plasmalogens, oxidized lipids and ferroptosis as new paradigms of bioactive lipid research.

Perturbations in lipid homeostasis combined with conditions favoring oxidative stress constitute a hallmark of the inflammatory response. In this review we focus on the most recent results concerning lipid signaling in various oxidative stress-mediated responses and inflammation. These include phagocytosis and ferroptosis. The best characterized event, common to these responses, is the synthesis of oxygenated metabolites of arachidonic acid and other polyunsaturated fatty acids. Major developments in this area have highlighted the importance of compartmentalization of the enzymes and lipid substrates in shaping the appropriate response. In parallel, other relevant lipid metabolic pathways are also activated and, until recently, there has been a general lack of knowledge on the enzyme regulation and molecular mechanisms operating in these pathways. Specifically, data accumulated in recent years on the regulation and biological significance of plasmalogens and oxidized phospholipids have expanded our knowledge on the involvement of lipid metabolism in the progression of disease and the return to homeostasis. These recent major developments have helped to establish the concept of membrane phospholipids as cellular repositories for the compartmentalized production of bioactive lipids involved in cellular regulation. Importantly, an enzyme classically described as being involved in regulating the homeostatic turnover of phospholipids, namely the group VIA Ca2+-independent phospholipase A2 (iPLA2ß), has taken center stage in oxidative stress and inflammation research owing to its key involvement in regulating metabolic and ferroptotic signals arising from membrane phospholipids. Understanding the role of iPLA2ß in ferroptosis and metabolism not only broadens our knowledge of disease but also opens possible new horizons for this enzyme as a target for therapeutic intervention.

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.

Differential Mobilization of the Phospholipid and Triacylglycerol Pools of Arachidonic Acid in Murine Macrophages.

Innate immune cells such as monocytes and macrophages contain high levels of arachidonic acid (AA), part of which can be mobilized during cellular activation for the formation of a vast array of bioactive oxygenated metabolites. Monocytes and macrophages present in inflammatory foci typically incorporate large amounts of AA, not only in membrane phospholipids, but also in neutral lipids such as triacylglycerol. Thus, it was of interest to investigate the metabolic fate of these two AA pools in macrophages. Utilizing a variety of radiolabeling techniques to distinguish the phospholipid and triacylglycerol pools, we show in this paper that during an acute stimulation of the macrophages with yeast-derived zymosan, the membrane phospholipid AA pool acts as the major, if not the only, source of releasable AA. On the contrary, the AA pool in triacylglycerol appears to be used at a later stage, when the zymosan-stimulated response has declined, as a source to replenish the phospholipid pools that were consumed during the activation process. Thus, phospholipids and triacylglycerol play different in roles AA metabolism and dynamics during macrophage activation.

Roles of Palmitoleic Acid and Its Positional Isomers, Hypogeic and Sapienic Acids, in Inflammation, Metabolic Diseases and Cancer.

In the last few years, the monounsaturated hexadecenoic fatty acids are being increasingly considered as biomarkers of health with key functions in physiology and pathophysiology. Palmitoleic acid (16:1n-7) and sapienic acid (16:1n-10) are synthesized from palmitic acid by the action of stearoyl-CoA desaturase-1 and fatty acid desaturase 2, respectively. A third positional isomer, hypogeic acid (16:1n-9) is produced from the partial ß-oxidation of oleic acid. In this review, we discuss the current knowledge of the effects of palmitoleic acid and, where available, sapienic acid and hypogeic acid, on metabolic diseases such as diabetes, cardiovascular disease, and nonalcoholic fatty liver disease, and cancer. The results have shown diverse effects among studies in cell lines, animal models and humans. Palmitoleic acid was described as a lipokine able to regulate different metabolic processes such as an increase in insulin sensitivity in muscle, ß cell proliferation, prevention of endoplasmic reticulum stress and lipogenic activity in white adipocytes. Numerous beneficial effects have been attributed to palmitoleic acid, both in mouse models and in cell lines. However, its role in humans is not fully understood, and is sometimes controversial. Regarding sapienic acid and hypogeic acid, studies on their biological effects are still scarce, but accumulating evidence suggests that they also play important roles in metabolic regulation. The multiplicity of effects reported for palmitoleic acid and the compartmentalized manner in which they often occur, may suggest the overlapping actions of multiple isomers being present at the same or neighboring locations.

Ptpn1 deletion protects oval cells against lipoapoptosis by favoring lipid droplet formation and dynamics.

Activation of oval cells (OCs) has been related to hepatocyte injury during chronic liver diseases including non-alcoholic fatty liver disease (NAFLD). However, OCs plasticity can be affected under pathological environments. We previously found protection against hepatocyte cell death by inhibiting protein tyrosine phosphatase 1B (PTP1B). Herein, we investigated the molecular and cellular processes involved in the lipotoxic susceptibility in OCs expressing or not PTP1B. Palmitic acid (PA) induced apoptotic cell death in wild-type (Ptpn1+/+) OCs in parallel to oxidative stress and impaired autophagy. This lipotoxic effect was attenuated in OCs lacking Ptpn1 that showed upregulated antioxidant defences, increased unfolded protein response (UPR) signaling, higher endoplasmic reticulum (ER) content and elevated stearoyl CoA desaturase (Scd1) expression and activity. These effects in Ptpn1-/- OCs concurred with an active autophagy, higher mitochondrial efficiency and a molecular signature of starvation, favoring lipid droplet (LD) formation and dynamics. Autophagy blockade in Ptpn1-/- OCs reduced Scd1 expression, mitochondrial fitness, LD formation and restored lipoapoptosis, an effect also recapitulated by Scd1 silencing. PTP1B immunostaining was detected in OCs from mouse liver and, importantly, LDs were found in OCs from Ptpn1-/- mice with NAFLD. In conclusion, we demonstrated that Ptpn1 deficiency restrains lipoapoptosis in OCs through a metabolic rewiring towards a "starvation-like" fate, favoring autophagy, mitochondrial fitness and LD formation. Dynamic LD-lysosomal interations likely ensure lipid recycling and, overall, these adaptations protect against lipotoxicity. The identification of LDs in OCs from Ptpn1-/- mice with NAFLD opens therapeutic perspectives to ensure OC viability and plasticity under lipotoxic liver damage.

Phosphorylation of cPLA2α at Ser505 Is Necessary for Its Translocation to PtdInsP2-Enriched Membranes.

Group IVA cytosolic phospholipase A2α (cPLA2α) is a key enzyme in physiology and pathophysiology because it constitutes a rate-limiting step in the pathway for the generation of pro- and anti-inflammatory eicosanoid lipid mediators. cPLA2α activity is tightly regulated by multiple factors, including the intracellular Ca2+ concentration, phosphorylation reactions, and cellular phosphatidylinositol (4,5) bisphosphate levels (PtdInsP2). In the present work, we demonstrate that phosphorylation of the enzyme at Ser505 is an important step for the translocation of the enzyme to PtdInsP2-enriched membranes in human cells. Constructs of eGFP-cPLA2 mutated in Ser505 to Ala (S505A) exhibit a delayed translocation in response to elevated intracellular Ca2+, and also in response to increases in intracellular PtdInsP2 levels. Conversely, translocation of a phosphorylation mimic mutant (S505E) is fully observed in response to cellular increases in PtdInsP2 levels. Collectively, these results suggest that phosphorylation of cPLA2α at Ser505 is necessary for the enzyme to translocate to internal membranes and mobilize arachidonic acid for eicosanoid synthesis.

Lipid Droplets, Phospholipase A2, Arachidonic Acid, and Atherosclerosis.

Lipid droplets, classically regarded as static storage organelles, are currently considered as dynamic structures involved in key processes of lipid metabolism, cellular homeostasis and signaling. Studies on the inflammatory state of atherosclerotic plaques suggest that circulating monocytes interact with products released by endothelial cells and may acquire a foamy phenotype before crossing the endothelial barrier and differentiating into macrophages. One such compound released in significant amounts into the bloodstream is arachidonic acid, the common precursor of eicosanoids, and a potent inducer of neutral lipid synthesis and lipid droplet formation in circulating monocytes. Members of the family of phospholipase A2, which hydrolyze the fatty acid present at the sn-2 position of phospholipids, have recently emerged as key controllers of lipid droplet homeostasis, regulating their formation and the availability of fatty acids for lipid mediator production. In this paper we discuss recent findings related to lipid droplet dynamics in immune cells and the ways these organelles are involved in regulating arachidonic acid availability and metabolism in the context of atherosclerosis.

Lipin-1-derived diacylglycerol activates intracellular TRPC3 which is critical for inflammatory signaling.

Exposure to Gram-negative bacterial LPS exacerbates host immune responses and may lead to sepsis, a life-threatening condition. Despite its high mortality and morbidity, no drugs specifically directed to treating sepsis are currently available. Using human cell genetic depletion, pharmacological inhibition, live-cell microscopy and organelle-targeted molecular sensors we present evidence that the channel TRPC3 is activated intracellularly during macrophage exposure to LPS and is essential for Ca2+ release from internal stores. In this manner, TRPC3 participates in cytosolic Ca2+ elevations, activation of the transcription factor NF-κB and cytokine upregulation. We also report that TRPC3 is activated by diacylglycerol generated by the phosphatidic acid phosphatase lipin-1. In accord with this, lipin-1-deficient cells exhibit reduced Ca2+ responses to LPS challenge. Finally, pharmacological inhibition of TRPC3 reduces systemic inflammation induced by LPS in mice. Collectively, our study unveils a central component of LPS-triggered Ca2+ signaling that involves intracellular sensing of lipin-1-derived DAG by TRPC3, and opens new opportunities for the development of strategies to treat LPS-driven inflammation.

The Hypoxic Microenvironment Induces Stearoyl-CoA Desaturase-1 Overexpression and Lipidomic Profile Changes in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cell carcinoma (RCC). It is characterized by a high cell proliferation and the ability to store lipids. Previous studies have demonstrated the overexpression of enzymes associated with lipid metabolism, including stearoyl-CoA desaturase-1 (SCD-1), which increases the concentration of unsaturated fatty acids in tumor cells. In this work, we studied the expression of SCD-1 in primary ccRCC tumors, as well as in cell lines, to determine its influence on the tumor lipid composition and its role in cell proliferation. The lipidomic analyses of patient tumors showed that oleic acid (18:1n-9) is one of the major fatty acids, and it is particularly abundant in the neutral lipid fraction of the tumor core. Using a ccRCC cell line model and in vitro-generated chemical hypoxia, we show that SCD-1 is highly upregulated (up to 200-fold), and this causes an increase in the cellular level of 18:1n-9, which, in turn, accumulates in the neutral lipid fraction. The pharmacological inhibition of SCD-1 blocks 18:1n-9 synthesis and compromises the proliferation. The addition of exogenous 18:1n-9 to the cells reverses the effects of SCD-1 inhibition on cell proliferation. These data reinforce the role of SCD-1 as a possible therapeutic target.

Phospholipases: From Structure to Biological Function.

Phospholipases are enzymes that cleave ester bonds within phospholipids [...].

Choline Glycerophospholipid-Derived Prostaglandins Attenuate TNFα Gene Expression in Macrophages via a cPLA2α/COX-1 Pathway.

Macrophages are professional antigen presenting cells with intense phagocytic activity, strategically distributed in tissues and cavities. These cells are capable of responding to a wide variety of innate inflammatory stimuli, many of which are signaled by lipid mediators. The distribution of arachidonic acid (AA) among glycerophospholipids and its subsequent release and conversion into eicosanoids in response to inflammatory stimuli such as zymosan, constitutes one of the most studied models. In this work, we used liquid and/or gas chromatography coupled to mass spectrometry to study the changes in the levels of membrane glycerophospholipids of mouse peritoneal macrophages and the implication of group IVA cytosolic phospholipase A2 (cPLA2α) in the process. In the experimental model used, we observed that the acute response of macrophages to zymosan stimulation involves solely the cyclooxygenase-1 (COX-1), which mediates the rapid synthesis of prostaglandins E2 and I2. Using pharmacological inhibition and antisense inhibition approaches, we established that cPLA2α is the enzyme responsible for AA mobilization. Zymosan stimulation strongly induced the hydrolysis of AA-containing choline glycerophospholipids (PC) and a unique phosphatidylinositol (PI) species, while the ethanolamine-containing glycerophospholipids remained constant or slightly increased. Double-labeling experiments with 3H- and 14C-labeled arachidonate unambiguously demonstrated that PC is the major, if not the exclusive source, of AA for prostaglandin E2 production, while both PC and PI appeared to contribute to prostaglandin I2 synthesis. Importantly, in this work we also show that the COX-1-derived prostaglandins produced during the early steps of macrophage activation restrict tumor necrosis factor-α production. Collectively, these findings suggest new approaches and targets to the selective inhibition of lipid mediator production in response to fungal infection.

Release of Anti-Inflammatory Palmitoleic Acid and Its Positional Isomers by Mouse Peritoneal Macrophages.

Positional isomers of hexadecenoic acid are considered as fatty acids with anti-inflammatory properties. The best known of them, palmitoleic acid (cis-9-hexadecenoic acid, 16:1n-7), has been identified as a lipokine with important beneficial actions in metabolic diseases. Hypogeic acid (cis-7-hexadecenoic acid, 16:1n-9) has been regarded as a possible biomarker of foamy cell formation during atherosclerosis. Notwithstanding the importance of these isomers as possible regulators of inflammatory responses, very little is known about the regulation of their levels and distribution and mobilization among the different lipid pools within the cell. In this work, we describe that the bulk of hexadecenoic fatty acids found in mouse peritoneal macrophages is esterified in a unique phosphatidylcholine species, which contains palmitic acid at the sn-1 position, and hexadecenoic acid at the sn-2 position. This species markedly decreases when the macrophages are activated with inflammatory stimuli, in parallel with net mobilization of free hexadecenoic acid. Using pharmacological inhibitors and specific gene-silencing approaches, we demonstrate that hexadecenoic acids are selectively released by calcium-independent group VIA phospholipase A2 under activation conditions. While most of the released hexadecenoic acid accumulates in free fatty acid form, a significant part is also transferred to other phospholipids to form hexadecenoate-containing inositol phospholipids, which are known to possess growth-factor-like-properties, and are also used to form fatty acid esters of hydroxy fatty acids, compounds with known anti-diabetic and anti-inflammatory properties. Collectively, these data unveil new pathways and mechanisms for the utilization of palmitoleic acid and its isomers during inflammatory conditions, and raise the intriguing possibility that part of the anti-inflammatory activity of these fatty acids may be due to conversion to other lipid mediators.

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.

Phospholipid Arachidonic Acid Remodeling During Phagocytosis in Mouse Peritoneal Macrophages.

Macrophages contain large amounts of arachidonic acid (AA), which distributes differentially across membrane phospholipids. This is largely due to the action of coenzyme A-independent transacylase (CoA-IT), which transfers the AA primarily from diacyl choline-containing phospholipids to ethanolamine-containing phospholipids. In this work we have comparatively analyzed glycerophospholipid changes leading to AA mobilization in mouse peritoneal macrophages responding to either zymosan or serum-opsonized zymosan (OpZ). These two phagocytic stimuli promote the cytosolic phospholipase A2-dependent mobilization of AA by activating distinct surface receptors. Application of mass spectrometry-based lipid profiling to identify changes in AA-containing phospholipids during macrophage exposure to both stimuli revealed significant decreases in the levels of all major choline phospholipid molecular species and a major phosphatidylinositol species. Importantly, while no changes in ethanolamine phospholipid species were detected on stimulation with zymosan, significant decreases in these species were observed when OpZ was used. Analyses of CoA-IT-mediated AA remodeling revealed that the process occurred faster in the zymosan-stimulated cells compared with OpZ-stimulated cells. Pharmacological inhibition of CoA-IT strongly blunted AA release in response to zymosan but had only a moderate effect on the OpZ-mediated response. These results suggest a hitherto undescribed receptor-dependent role for CoA-independent AA remodeling reactions in modulating the eicosanoid biosynthetic response of macrophages. Our data help define novel targets within the AA remodeling pathway with potential use to control lipid mediator formation.