Free Arachidonic Acid Research Articles

IPLA2β regulates the dual effects of arachidonic acid in thyroid cancer.

Abnormal arachidonic acid metabolism in the tumor microenvironment is closely related to cancer progression; however, thyroid cancer was rarely researched. Through lipidomic analysis, we disclosed that dysregulated arachidonic acid metabolism plays dual effects on thyroid cancer. The promoting role of arachidonic acid in the progression of thyroid cancer cells was evaluated utilizing cell viability (CCK-8 assay) and transwell invasion assays, confirmed by corresponding inhibitors. Lipid peroxidation and the use of various cell death inhibitors confirmed that arachidonic acid confers vulnerability to ferroptosis in thyroid cancer. The roles of arachidonic acid and ferroptosis inducer in thyroid cancer were assessed in a xenograft mouse model. On one hand, arachidonic acid promotes the progression of thyroid cancer through the cyclooxygenase/prostaglandin pathway; on another hand, arachidonic acid confers vulnerability to ferroptosis through lipoxygenases. Moreover, iPLA2β drives converse roles of arachidonic acid between cancer-progression and ferroptosis vulnerability through releasing free arachidonic acid from the cell membrane. Finally, we confirmed high arachidonic acid diet promotes the development of thyroid cancer in vivo, whereas ferroptosis inducer sulfasalazine dramatically reduced tumor growth of mice with feeding arachidonic acid. Our research demonstrated the roles of iPLA2β in conversing dual effects of arachidonic acid in thyroid cancer and provides ferroptosis inducer as a potential therapeutic strategy.

A mouse protozoan boosts antigen-specific mucosal IgA responses in a specific lipid metabolism- and signaling-dependent manner

IgA antibodies play an important role in mucosal immunity. However, there is still no effective way to consistently boost mucosal IgA responses, and the factors influencing these responses are not fully understood. We observed that colonization with the murine intestinal symbiotic protozoan Tritrichomonas musculis (T.mu) boosted antigen-specific mucosal IgA responses in wild-type C57BL/6 mice. This enhancement was attributed to the accumulation of free arachidonic acid (ARA) in the intestinal lumen, which served as a signal to stimulate the production of antigen-specific mucosal IgA. When ARA was prevented from undergoing its downstream metabolic transformation using the 5-lipoxygenase inhibitor zileuton or by blocking its downstream biological signaling through genetic deletion of the Leukotriene B4 receptor 1 (Blt1), the T.mu-mediated enhancement of antigen-specific mucosal IgA production was suppressed. Moreover, both T.mu transfer and dietary supplementation of ARA augmented the efficacy of an oral vaccine against Salmonella infection, with this effect being dependent on Blt1. Our findings elucidate a tripartite circuit linking nutrients from the diet or intestinal microbiota, host lipid metabolism, and the mucosal humoral immune response.

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Arachidonic Acid Mobilization and Peroxidation Promote Microglial Dysfunction in Aβ Pathology.

Aberrant increase of arachidonic acid (ARA) has long been implicated in the pathology of Alzheimer's disease (AD), while the underlying causal mechanism remains unclear. In this study, we revealed a link between ARA mobilization and microglial dysfunction in Aβ pathology. Lipidomic analysis of primary microglia from AppNL-GF mice showed a marked increase in free ARA and lysophospholipids (LPLs) along with a decrease in ARA-containing phospholipids, suggesting increased ARA release from phospholipids (PLs). To manipulate ARA-containing PLs in microglia, we genetically deleted lysophosphatidylcholine acyltransferase 3 (Lpcat3), the main enzyme catalyzing the incorporation of ARA into PLs. Loss of microglial Lpcat3 reduced the levels of ARA-containing PLs, free ARA and LPLs, leading to a compensatory increase in monounsaturated fatty acid (MUFA)-containing PLs in both male and female App NL-GF mice. Notably, the reduction of ARA in microglia significantly ameliorated oxidative stress and inflammatory responses while enhancing the phagocytosis of Aβ plaques and promoting the compaction of Aβ deposits. Mechanistically, scRNA seq suggested that LPCAT3 deficiency facilitates phagocytosis by facilitating de novo lipid synthesis while protecting microglia from oxidative damage. Collectively, our study reveals a novel mechanistic link between ARA mobilization and microglial dysfunction in AD. Lowering brain ARA levels through pharmacological or dietary interventions may be a potential therapeutic strategy to slow down AD progression.

The enzyme encoded by Myrmecia incisa, a green microalga, phospholipase A2 gene preferentially hydrolyzes arachidonic acid at the sn-2 position of phosphatidylcholine

The enzyme phospholipase A2 (PLA2) plays a crucial role in acyl remodeling of phospholipids via the Lands’ cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A2 (MiPLA2) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA2 contained 3 introns. Mature MiPLA2 (mMiPLA2) had a conserved Ca2+-binding loop and a catalytic site motif that has been recognized in plant secretory PLA2 (sPLA2) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA2 was clustered within GroupXIA of plant sPLA2 proteins. To ascertain the function of MiPLA2, the cDNA coding for mMiPLA2 was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA2 in Escherichia coli. Recombinant mMiPLA2 was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA2 indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA2. Although recombinant mMiPLA2 displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA2 illustrated that MiPLA2 was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA2 transcription and free ArA levels were established. Consequently, the role of mMiPLA2 in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.

MBOAT7 expression is associated with disease progression in COVID-19 patients

Background and aimThe emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 caused a pandemic of acute respiratory disease, named coronavirus disease 2019 (COVID-19). COVID-19 became one of the most challenging health emergencies, hence the necessity to find different prognostic factors for disease progression, and severity. Membrane bound O-acyltransferase domain containing 7 (MBOAT7) demonstrates anti-inflammatory effects through acting as a fine-tune regulator of the amount of cellular free arachidonic acid. We aimed in this study to evaluate MBOAT7 expression in COVID-19 patients and to correlate it with disease severity and outcomes.MethodsThis case-control study included 56 patients with confirmed SARS-CoV-2 diagnosis and 28 control subjects. Patients were further classified into moderate (n = 28) and severe (n = 28) cases. MBOAT7, tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) mRNA levels were evaluated in peripheral blood mononuclear cells (PBMC) samples isolated from patients and control subjects by real time quantitative polymerase chain reaction (RT-qPCR). In addition, circulating MBOAT7 protein levels were assayed by enzyme-linked immunosorbent assay (ELISA).ResultsSignificant lower levels of circulating MBOAT7 mRNA and protein were observed in COVID-19 patients compared to control subjects with severe COVID-19 cases showing significant lower levels compared to moderate cases. Moreover, severe cases showed a significant upregulation of TNF-α and IL-1ß mRNA. MBOAT7 mRNA and protein levels were significantly correlated with inflammatory markers (TNF-α, IL-1ß, C-reactive protein (CRP), and ferritin), liver enzymes, severity, and oxygen saturation levels.ConclusionCOVID-19 is associated with downregulation of MBAOT7, which correlates with disease severity.

Screening of insect immune suppressors using a recombinant phospholipase A2 of a lepidopteran insect.

Phospholipase A2 (PLA2 ) catalyzes phospholipids at the sn-2 position to release free fatty acids, including arachidonic acid (AA) or its precursor. The free AA is then oxygenated into different eicosanoids, which mediate the diverse physiological processes in insects. Any inhibition of the PLA2 catalysis would give rise to serious malfunctioning in insect growth and development. An onion moth, Acrolepiopsis sapporensis, encodes four different PLA2 genes (As-PLA2 A-As-PLA2 D), in which As-PLA2 A is dominantly expressed at all developmental stages and in different larval tissues. RNA interference of the As-PLA2 A expression significantly reduced the PLA2 activity of A. sapporensis, which suffered from immunosuppression. A recombinant As-PLA2 A protein was purified from a bacterial expression system, which exhibited a typical Michaelis-Menten kinetics and hence susceptible to a specific inhibitor to sPLA2 and dithiothreitol. A total of 19 bacterial metabolites derived from Xenorhabdus and Photorhabdus were screened against the recombinant As-PLA2 A. Five potent metabolites were highly inhibitory and followed a competitive enzyme inhibition. These five inhibitors suppressed the immune responses of A. sapporensis by inhibiting hemocyte-spreading behavior and phenoloxidase activity. However, an addition of AA could significantly rescue the immunosuppression induced by the selected inhibitors. These studies suggest that the recombinant As-PLA2 A protein can be applied for high-throughput screening of insect immunosuppressive compounds.

Cytosolic phospholipase A2 regulates lipid homeostasis under osmotic stress through PPARγ.

Physiologically, renal medullary cells are surrounded by a hyperosmolar interstitium. However, different pathological situations can induce abrupt changes in environmental osmolality, causing cell stress. Therefore, renal cells must adapt to survive in this new condition. We previously demonstrated that, among the mechanisms involved in osmoprotection, renal cells upregulate triglyceride biosynthesis (which helps preserve glycerophospholipid synthesis and membrane homeostasis) and cyclooxygenase-2 (which generates prostaglandins from arachidonic acid) to maintain lipid metabolism in renal tissue. Herein, we evaluated whether hyperosmolality modulates phospholipase A2 (PLA2 ) activity, leading to arachidonic acid release from membrane glycerophospholipid, and investigated its possible role in hyperosmolality-induced triglyceride synthesis and accumulation. We found that hyperosmolality induced PLA2 expression and activity in Madin-Darby canine kidney cells. Cytosolic PLA2 (cPLA2) inhibition, but not secreted or calcium-independent PLA2 (sPLA2 or iPLA2 , respectively), prevented triglyceride synthesis and reduced cell survival. Inhibition of prostaglandin synthesis with indomethacin not only failed to prevent hyperosmolality-induced triglyceride synthesis but also exacerbated it. Similar results were observed with the peroxisomal proliferator activated receptor gamma (PPARγ) agonist rosiglitazone. Furthermore, hyperosmolality increased free intracellular arachidonic acid levels, which were even higher when prostaglandin synthesis was inhibited by indomethacin. Blocking PPARγ with GW-9662 prevented the effects of both indomethacin and rosiglitazone on triglyceride synthesis and even reduced hyperosmolality-induced triglyceride synthesis, suggesting that arachidonic acid may stimulate triglyceride synthesis through PPARγ activation. These results highlight the role of cPLA2 in osmoprotection, since it is essential to provide arachidonic acid, which is involved in PPARγ-regulated triglyceride synthesis, thus guaranteeing cell survival.

Multi-enzyme Co-expressed Dual-Atom Nanozymes Induce Cascade Immunogenic Ferroptosis via Activating Interferon-γ and Targeting Arachidonic Acid Metabolism

Immunotherapy is currently the most promising treatment strategy for long-term tumor regression. However, current cancer immunotherapy shows low response rates due to insufficient immunogenicity of tumor cells. Herein, we report a strategy to keep tumor cells highly immunogenic by triggering cascade immunogenic tumor ferroptosis. We developed a six-enzyme co-expressed nanoplatform: lipoxygenase (LOX) and phospholipase A2 (PLA2)-co-loaded FeCo/Fe-Co dual-metal atom nanozyme (FeCo/Fe-Co DAzyme/PL), which can not only induce initial immunogenic tumor ferroptosis through its own multi-enzyme mimetic activities but also up-regulate arachidonic acid (AA) expression to synergize with CD8+ T cell-derived IFN-γ to induce ACSL4-mediated immunogenic tumor ferroptosis. During this process, FeCo/Fe-Co DAzyme/PL can induce lipid peroxidation (LPO) by efficiently generating reactive oxygen species (ROS) and depleting GSH and GPX4 at tumor sites. Additionally, free AA released from PLA2 catalysis is converted into arachidonyl-CoA under the activation of ACSL4 stimulated by IFN-γ, which is further incorporated into phospholipids on membranes and peroxidized with the participation of LOX. Consequently, FeCo/Fe-Co DAzyme/PL can promote irreversible cascade immunogenic ferroptosis through multiple ROS storms, GSH/GPX4 depletion, LOX catalysis, and IFN-γ-mediated ACSL4 activation, constructing an effective pathway to overcome the drawbacks of current immunotherapy.

Arachidonic acid is associated with dyslipidemia and cholesterol-related lipoprotein metabolism signatures.

Abnormal lipoprotein metabolism is associated with a variety of diseases, cardiovascular disease in particular. Free fatty acids (FAs) and triglycerides (TGs) are the principal lipid species in adipocytes and are the major components of lipoproteins. However, in routine clinical laboratory testing, only the total plasma concentrations of FAs and TGs are typically measured. We collected 965 individuals with hyperlipidemia plasma and clinical characteristics; high-throughput metabolomics permits the accurate qualitative and quantitative assessment of a variety of specific FAs and TGs and their association with lipoproteins; through regression analysis, the correlation between multiple metabolites and routine measured lipid parameters was found. Mice were fed a diet containing AA, and the concentrations of TC and TG in the plasma of mice were detected by enzyme method, western blot and qRT-PCR detected the protein and mRNA levels of cholesterol synthesis and metabolism in mice. Using LC-MS/MS identified eight free FA and 27 TG species in plasma samples, the plasma concentrations of free arachidonic acid (AA) and AA-enriched TG species were significantly associated with the plasma low-density lipoprotein-cholesterol, apolipoprotein B (ApoB), and total cholesterol (TC) concentrations after adjustment for age, sex, the use of lipid-lowering therapy, and body mass index. AA-rich diet significantly increased the plasma concentrations of TC and ApoB and the liver expression of ApoB protein and reduced the protein expression of ATP binding cassette subfamily G members 5 and 8 in mice. In this study, it was clarified that the plasma concentrations of free AA- and AA-enriched TG species were significantly associated with the plasma low-density lipoprotein-cholesterol, ApoB, and TC concentrations in individuals with hyperlipidemia, and it was verified that AA could increase the plasma TC level in mice. Taken together, these findings suggest a potential role of AA in the regulation of plasma cholesterol and lipoprotein concentrations.

Modeling the Interaction of Human Lipoxygenase LOX 15 with Free Arachidonic Acid

Modeling the Interaction of Human Lipoxygenase LOX 15 with Free Arachidonic Acid

Oncometabolite-Independent Anti-Apoptotic Feature Via Phospholipid Metabolic Adaptation in IDH2 Mutant AML Cells

Isocitrate dehydrogenase (IDH) gene mutation is found in approximately 20% of adult acute myeloid leukemia (AML) patients. IDH gene mutations give a pathological enzymatic activity producing oncometabolite, which impair hematopoietic differentiation while promoting leukemogenesis via epigenetic changes. Targeting mutant (mut) form of IDH enzymes, small molecule inhibitors have been developed. Among these inhibitors, AG-221 (enasidenib), which is the first-in-class, selective and orally available mut IDH2 inhibitor (Yen et al., Cancer Discov. 2017), has been tested in clinical trial. The clinical trial showed overall response rate was approximately 40% and more than half of the patient didn't respond to the treatment. This clinical study also showed clear correlation between clearance of IDH2 mut clones and complete clinical response rate (Stein et al., Blood 2019). Relapse cases under the AG-221 treatment, in which additional mutation in IDH2 or IDH1 gene occurred in the residual IDH2 mut clone have been reported (Intlekofer et al., Nature 2018, Harding et al., Cancer Discov. 2018). These reports suggested that IDH2 mut cells have an oncometabolite-independent survival advantage which contributes to refractoriness and relapse. However, most of the studies on IDH mut AML have been focused on oncometabolite-related mechanisms and oncometabolite-independent mechanism has not been investigated in detail so far. IDH gene mutations frequently coexist with other oncogenic mutations in AML patient cells, which make it difficult to analyze the precise role of IDH gene mutation in leukemogenesis. To address this issue, we utilized the cytokine-dependent AML cell line, TF-1 cells and its isogenic cell line with IDH2 gene mutation, which showed cytokine independent growth. Though AG-221 suppressed the cytokine independent growth in IDH2 mut cells, the growth of AG-221 treated IDH2 mut cells remained significantly faster than IDH2 wild type (WT) cells. These data indicated that IDH2 mut cells have a growth advantage machinery in an oncometabolite-independent manner. Metabolome analysis clearly separated AG-221 treated IDH2 mut cells from IDH2 WT cells and identified that inositol phosphate metabolism pathway showed significant difference between these two groups. High throughput drug screening using metabolic inhibitors also revealed that inositol phosphate related enzyme, phospholipase C (PLC) specific inhibitor promoted IDH2 mut cell proliferation. These data suggested that suppressed PLC activity induced oncometabolite-independent growth advantage in IDH2 mut AML cells. Since PLC indirectly release free arachidonic acid (AA) from phospholipid bilayer of cellular membrane and intracellular AA level has positive correlation to apoptosis (Cao et al. PNAS 2000), we analyzed intracellular AA and apoptosis in IDH2 mut cells. As expected, these analyses showed significantly lower intracellular AA and reduced Annexin V+ apoptotic cells in IDH2 mut cells irrespective of AG-221 treatment. Supporting these data, PLC activity related gene sets were negatively enriched and anti-apoptosis related genes were positively enriched in IDH2 mut cells in RNA seq analysis. Based on these novel mechanisms, we tested FDA-approved anti-inflammatory drugs, COX-2 and 5-LOX specific inhibitors (celecoxib and zileuton) to upregulate intracellular AA and induce apoptosis of TF-1 cells in vitro and in vivo xenograft. These drugs in combination with AG-221 successfully suppressed IDH2 mut cell proliferation and induced apoptosis both in vitro and in vivo. The mice treated with these triple drugs showed no sign of cytopenia nor therapy-associated complication, securing safety of this combination therapy. In this study, we identified unappreciated oncometabolite-independent anti-apoptotic feature acquisition via phospholipid metabolic adaptation in IDH2 mut AML cells. Moreover, targeting this metabolic adaptation, we identified that FDA-approved anti-inflammatory drugs in combination with mut IDH2 inhibitor could eradicate IDH2 mut AML cells by efficiently inducing apoptosis. Since these anti-inflammatory drugs have a track record of safety in clinical practice, the triple drug combination therapy could be an alternative therapeutic option for the IDH2 mut AML patients who couldn't tolerate existing therapy.

Altered Cord Blood Lipid Concentrations Correlate with Birth Weight and Doppler Velocimetry of Fetal Vessels in Human Fetal Growth Restriction Pregnancies.

Fetal growth restriction (FGR) is associated with short- and long-term morbidity, often with fetal compromise in utero, evidenced by abnormal Doppler velocimetry of fetal vessels. Lipids are vital for growth and development, but metabolism in FGR pregnancy, where fetuses do not grow to full genetic potential, is poorly understood. We hypothesize that triglyceride concentrations are increased in placentas and that important complex lipids are reduced in cord plasma from pregnancies producing the smallest babies (birth weight < 5%) and correlate with ultrasound Dopplers. Dopplers (umbilical artery, UA; middle cerebral artery, MCA) were assessed longitudinally in pregnancies diagnosed with estimated fetal weight (EFW) < 10% at ≥29 weeks gestation. For a subset of enrolled women, placentas and cord blood were collected at delivery, fatty acids were extracted and targeted lipid class analysis (triglyceride, TG; phosphatidylcholine, PC; lysophosphatidylcholine, LPC; eicosanoid) performed by LCMS. For this sub-analysis, participants were categorized as FGR (Fenton birth weight, BW ≤ 5%) or SGA “controls” (Fenton BW > 5%). FGRs (n = 8) delivered 1 week earlier (p = 0.04), were 29% smaller (p = 0.002), and had 133% higher UA pulsatility index (PI, p = 0.02) than SGAs (n = 12). FGR plasma TG, free arachidonic acid (AA), and several eicosanoids were increased (p < 0.05); docosahexaenoic acid (DHA)-LPC was decreased (p < 0.01). Plasma TG correlated inversely with BW (p < 0.05). Plasma EET, non-esterified AA, and DHA correlated inversely with BW and directly with UA PI (p < 0.05). Placental DHA-PC and AA-PC correlated directly with MCA PI (p < 0.05). In fetuses initially referred for inadequate fetal growth (EFW < 10%), those with BW ≤ 5% demonstrated distinctly different cord plasma lipid profiles than those with BW > 5%, which correlated with Doppler PIs. This provides new insights into fetal lipidomic response to the FGR in utero environment. The impact of these changes on specific processes of growth and development (particularly fetal brain) have not been elucidated, but the relationship with Doppler PI may provide additional context for FGR surveillance, and a more targeted approach to nutritional management of these infants.

The association of the lipid profile with knee and hand osteoarthritis severity: the IMI-APPROACH cohort

To investigate the association of the lipidomic profile with osteoarthritis (OA) severity, considering the outcomes radiographic knee and hand OA, pain and function. We used baseline data from the Applied Public-Private Research enabling OsteoArthritis Clinical Headway (APPROACH) cohort, comprising persons with knee OA fulfilling the clinical American College of Rheumatology classification criteria. Radiographic knee and hand OA severity was quantified with Kellgren-Lawrence sum scores. Knee and hand pain and function were assessed with validated questionnaires. We quantified fasted plasma higher order lipids and oxylipins with liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based platforms. Using penalised linear regression, we assessed the variance in OA severity explained by lipidomics, with adjustment for clinical covariates (age, sex, body mass index (BMI) and lipid lowering medication), measurement batch and clinical centre. In 216 participants (mean age 66 years, mean BMI 27.3kg/m2, 75% women) we quantified 603 higher order lipids (triacylglycerols, diacylglycerols, cholesteryl esters, ceramides, free fatty acids, sphingomyelins, phospholipids) and 28 oxylipins. Lipidomics explained 3% and 2% of the variance in radiographic knee and hand OA severity, respectively. Lipids were not associated with knee pain or function. Lipidomics accounted for 12% and 6% of variance in hand pain and function, respectively. The investigated OA severity outcomes were associated with the lipidomic fraction of bound and free arachidonic acid, bound palmitoleic acid, oleic acid, linoleic acid and docosapentaenoic acid. Within the APPROACH cohort lipidomics explained a minor portion of the variation in OA severity, which was most evident for the outcome hand pain. Our results suggest that eicosanoids may be involved in OA severity.

Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures

Survival of ovarian carcinoma is associated with the abundance of immunosuppressed CD163highCD206high tumor‐associated macrophages (TAMs) and high levels of arachidonic acid (AA) in the tumor microenvironment. Here, we show that both associations are functionally linked. Transcriptional profiling revealed that high CD163 and CD206/MRC1 expression in TAMs is strongly associated with an inhibition of cytokine‐triggered signaling, mirrored by an impaired transcriptional response to interferons and IL‐6 in monocyte‐derived macrophages by AA. This inhibition of pro‐inflammatory signaling is caused by dysfunctions of the cognate receptors, indicated by the inhibition of JAK1, JAK2, STAT1, and STAT3 phosphorylation, and by the displacement of the interferon receptor IFNAR1, STAT1 and other immune‐regulatory proteins from lipid rafts. AA exposure led to a dramatic accumulation of free AA in lipid rafts, which appears to be mechanistically crucial, as the inhibition of its incorporation into phospholipids did not affect the AA‐mediated interference with STAT1 phosphorylation. Inhibition of interferon‐triggered STAT1 phosphorylation by AA was reversed by water‐soluble cholesterol, known to prevent the perturbation of lipid raft structure by AA. These findings suggest that the pharmacologic restoration of lipid raft functions in TAMs may contribute to the development new therapeutic approaches.

Exposure to the Brominated Flame Retardant Hexabromocyclododecane Results in Systemic Changes in Polyunsaturated Fatty Acid Metabolism Within Phospholipids of Mice

Brominated flame retardants are used in many household products to reduce flammability, but often leach into the surrounding environment over time. Hexabromocyclododecane (HBCD) is one brominated flame retardant detected in human blood across the world. HBCD exposure can result in neurological problems and altered lipid metabolism, but to date the two remain unlinked. As lipids constitute ~50% of brain dry weight, lipid metabolism plays a critical role in neuronal function and homeostasis. To determine the effect of HBCD exposure on brain lipid metabolism, young adult male mice were exposed to 1 mg/kg HBCD every 3 days for 28 days. HBCD exposure altered lipid abundance of 8 major lipid classes in the dorsal striatum, including fatty acids (FA), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), phosphatidylethanolamines (PE) alkylphosphatidylethanolamines (PEO), phosphatidylethanolamine plasmalogens (PEP), and sphingomyelins (SM). The liver, as a major lipid metabolism hub, and blood were also examined to determine whether these alterations correlated to systemic changes in lipid metabolism. Although several lipid classes changed in the liver and blood, only PE was consistently altered between organs. Despite the lack of commonalities between lipid class alterations of differing organs, phospholipid fatty acid tails displayed a consistent enrichment of Polyunsaturated Fatty Acids (PUFAs) such as Arachidonic Acid (AA) and Docosahexaenoic Acid (DHA) across lipid classes and organs. Free AA was enriched in the blood as well as AA contained within phospholipids of the liver (in PE), blood (in LPC, PEO, and PEP), and dorsal striatum (in PE, PEO). Additionally, although free DHA was unchanged in the blood, DHA‐containing phospholipids were enriched in the liver (in PE), blood (in LPC, LPE), and dorsal striatum (in PE, PEO, PEP, PS). These studies indicate that HBCD can cause systemic alterations in PUFA metabolism. Further study is necessary to determine the mechanisms underlying the lipid alterations and their functional consequences.

Investigation of the characteristic binding mode of prostaglandin endoperoxide H2 formed by cyclooxygenase with a group of terminal prostanoid synthases

Free arachidonic acid mobilized from membrane glycerophospholipids by the cognate phospholipases is transformed into prostaglandin endoperoxide H2 (PGH2) by cyclooxygenase (COX), a common substrate for a group of terminal prostanoid synthases. COXs are thought to exhibit distinct functional coupling with these enzymes, which include prostaglandin E2, prostaglandin D2, prostaglandin F2α, prostacyclin, and thromboxane synthases. Three distinctive PGE2 synthases catalyzing nonoxidative rearrangement of PGH2 to PGE2 are identified, which are cytosolic PGES, two membrane-bound PGESs.

Inhibitors of Human 5-Lipoxygenase Potently Interfere With Prostaglandin Transport.

5-Lipoxygenase (5-LO) is the key enzyme in the formation of pro-inflammatory leukotrienes (LT) which play an important role in a number of inflammatory diseases. Accordingly, 5-LO inhibitors are frequently used to study the role of 5-LO and LT in models of inflammation and cancer. Interestingly, the therapeutic efficacy of these inhibitors is highly variable. Here we show that the frequently used 5-LO inhibitors AA-861, BWA4C, C06, CJ-13,610 and the FDA approved compound zileuton as well as the pan-LO inhibitor nordihydroguaiaretic acid interfere with prostaglandin E2 (PGE2) release into the supernatants of cytokine-stimulated (TNFα/IL-1β) HeLa cervix carcinoma, A549 lung cancer as well as HCA-7 colon carcinoma cells with similar potencies compared to their LT inhibitory activities (IC50 values ranging from 0.1–9.1 µM). In addition, AA-861, BWA4C, CJ-13,610 and zileuton concentration-dependently inhibited bacterial lipopolysaccharide triggered prostaglandin (PG) release into human whole blood. Western Blot analysis revealed that inhibition of expression of enzymes involved in PG synthesis was not part of the underlying mechanism. Also, liberation of arachidonic acid which is the substrate for PG synthesis as well as PGH2 and PGE2 formation were not impaired by the compounds. However, accumulation of intracellular PGE2 was found in the inhibitor treated HeLa cells suggesting inhibition of PG export as major mechanism. Further, experiments showed that the PG exporter ATP-binding cassette transporter multidrug resistance protein 4 (MRP-4) is targeted by the inhibitors and may be involved in the 5-LO inhibitor-mediated PGE2 inhibition. In conclusion, the pharmacological effects of a number of 5-LO inhibitors are compound-specific and involve the potent inhibition of PGE2 export. Results from experimental models on the role of 5-LO in inflammation and pain using 5-LO inhibitors may be misleading and their use as pharmacological tools in experimental models has to be revisited. In addition, 5-LO inhibitors may serve as new scaffolds for the development of potent prostaglandin export inhibitors.

The role of 5-lipoxygenase in the pathophysiology of COVID-19 and its therapeutic implications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, known as coronavirus disease 2019 (COVID-19) causes cytokine release syndrome (CRS), leading to acute respiratory distress syndrome (ARDS), acute kidney and cardiac injury, liver dysfunction, and multiorgan failure. Although several studies have discussed the role of 5-lipoxygenase (5-LOX) in viral infections, such as influenzae and SARS, it remains unexplored in the pathophysiology of COVID-19. 5-LOX acts on free arachidonic acid (AA) to form proinflammatory leukotrienes (LTs). Of note, numerous cells involved with COVID-19 (e.g., inflammatory and smooth muscle cells, platelets, and vascular endothelium) widely express leukotriene receptors. Moreover, 5-LOX metabolites induce the release of cytokines (e.g., tumour necrosis factor-α [TNF-α], interleukin-1α [IL-1α], and interleukin-1β [IL-1β]) and express tissue factor on cell membranes and activate plasmin. Since macrophages, monocytes, neutrophils, and eosinophils can express lipoxygenases, activation of 5-LOX and the subsequent release of LTs may contribute to the severity of COVID-19. This review sheds light on the potential implications of 5-LOX in SARS-CoV-2-mediated infection and the anticipated therapeutic role of 5-LOX inhibitors.

Molecular Mechanisms for the Inflammation-Resolving Actions of Lenabasum

A first-in-class cannabinoid analog called lenabasum that is a CB2 agonist is being developed as an inflammation-resolving drug candidate. Thus far, specific therapeutic targets include scleroderma, cystic fibrosis, dermatomyositis, and lupus, all of which represent unmet medical needs. Two somewhat-independent molecular mechanisms for this type of action are here proposed. Both pathways initially involve the release of free arachidonic acid after activation of the CB2 receptor and phospholipase A2 by lenabasum. The pathways then diverge into a cyclooxygenase 2-mediated and a lipoxygenase-mediated route. The former leads to increased levels of the cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin-J2 that can activate the NLPR3 inflammasome, which in turn releases caspase-3, leading to apoptosis and the resolution of chronic inflammation. The lipoxygenase-mediated pathway stimulates the production of lipoxin A4 as well as other signaling molecules called specialized proresolving mediators. These also have inflammation-resolving actions. It is not well understood under which conditions each of these mechanisms operates and whether there is crosstalk between them. Thus, much remains to be learned about the mechanisms describing the actions of lenabasum. SIGNIFICANCE STATEMENT: The resolution of chronic inflammation is a major unmet medical need. The synthetic nonpsychoactive cannabinoid lenabasum could provide a safe and effective drug for this purpose. Two putative molecular mechanisms are suggested to better understand how lenabasum produces this action. In both, different metabolites of arachidonic acid act as mediators.