ALOX5 contributes to glioma progression by promoting 5-HETE-mediated immunosuppressive M2 polarization and PD-L1 expression of glioma-associated microglia/macrophages.

BACKGROUND: Oxylipin metabolism plays an essential role in glioma progression and immune modulation in the tumor microenvironment. Lipid metabolic reprogramming has been linked to macrophage remodeling, while the understanding of oxylipins and their catalyzed enzymes lipoxygenases in the regulation of glioma-associated microglia/macrophages (GAMs) remains largely unexplored. METHODS: To explore the pathophysiological relevance of oxylipin in human glioma, we performed Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) analysis in human glioma and non-tumor brain tissues. To comprehensively investigate the role of arachidonate lipoxygenase 5 (ALOX5) in glioma, we performed in vivo bioluminescent imaging, immunofluorescence staining and flow cytometry analysis on tumors from orthotopic glioma-bearing mice. We developed an ALOX5-targeted nanobody, and tested its anti-glioma efficacy of combination therapy with α-programmed cell death protein-1 (PD-1). RESULTS: In this study, we found that ALOX5 and its oxylipin 5-hydroxyeicosatetraenoic acid (5-HETE) are upregulated in glioma, accumulating programmed death-ligand 1 (PD-L1)+ M2-GAMs and orchestrating an immunosuppressive tumor microenvironment. Mechanistically, 5-HETE derived from ALOX5-overexpressing glioma cells, promotes GAMs migration, PD-L1 expression, and M2 polarization by facilitating nuclear translocation of nuclear factor erythroid 2-related factor 2. Additionally, a nanobody targeting ALOX5 is developed that markedly suppresses 5-HETE efflux from glioma cells, attenuates M2 polarization of GAMs, and consequently ameliorates glioma progression. Furthermore, the combination therapy of the ALOX5-targeted nanobody plus α-PD-1 exhibits superior anti-glioma efficacy. CONCLUSIONS: Our findings reveal a pivotal role of the ALOX5/5-HETE axis in regulating GAMs and highlight the ALOX5-targeted nanobody as a potential therapeutic agent, which could potentiate immune checkpoint therapy for glioma.

Stereoselective Syntheses of Polyunsaturated Fatty Acids, 13-(S)-HODE and 15-(S)-HETE.

Polyunsaturated fatty acids and their metabolites have been reported in which their pathway has potential for the modulation of cancer cell growth. 13-(S)-HODE and 15-(S)-HETE, both of which are main metabolites of 15-LOXs, play an important role as endogenous ligands in biological systems. However, the modification of 13-(S)-HODE and 15-(S)-HETE in pharmaceutical applications has not been explored widely. Herein, we report the stereoselective syntheses of 13-(S)-HODE, 15-(S)-HETE, and its derivatives to enable the synthesis of bioactive fatty acid analogues.

Modulation of Angiotensin II-Induced Cellular Hypertrophy by Cannflavin-C: Unveiling the Impact on Cytochrome P450 1B1 and Arachidonic Acid Metabolites.

This research aimed to clarify the impacts of cannflavin-C on angiotensin II (Ang II)-induced cardiac hypertrophy and their potential role in modulating cytochrome P450 1B1 (CYP1B1) and arachidonic acid (AA) metabolites. Currently there is no evidence to suggest that cannflavin-C, a prenylated flavonoid, has any significant effects on the heart or cardiac hypertrophy. The metabolism of arachidonic acid (AA) into midchain hydroxyeicosatetraenoic acids (HETEs), facilitated by CYP1B1 enzyme, plays a role in the development of cardiac hypertrophy, which is marked by enlarged cardiac cells. Adult human ventricular cardiomyocyte (AC16) cell line was cultured and exposed to cannflavin-C in the presence and absence of Ang II. The assessment of mRNA expression pertaining to cardiac hypertrophic markers and cytochromes P450 (P450s) was conducted via real-time polymerase chain reaction (PCR), whereas the quantification of P450 protein levels was carried out through western blot analysis. Ang II induced hypertrophic markers myosin heavy chain (ß/α-MHC), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) and increased cell surface area, whereas cannflavin-C mitigated these effects. Gene and protein expression analysis revealed that cannflavin-C downregulated CYP1B1 gene expression, protein level, and enzyme activity assessed by 7-methoxyresorufin O-deethylase (MROD). Arachidonic acid metabolites analysis, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), demonstrated that Ang II increased midchain (R/S)-HETE concentrations, which were attenuated by cannflavin-C. This study provides novel insights into the potential of cannflavin-C in modulating arachidonic acid metabolites and attenuating Ang II-induced cardiac hypertrophy, highlighting the importance of this compound as potential therapeutic agents for cardiac hypertrophy. SIGNIFICANCE STATEMENT: This study demonstrates that cannflavin-C offers protection against cellular hypertrophy induced by angiotensin II. The significance of this research lies in its novel discovery, which elucidates a mechanistic pathway involving the inhibition of CYP1B1 by cannflavin-C. This discovery opens up new avenues for leveraging this compound in the treatment of heart failure.

Serum levels of hydroxylated metabolites of arachidonic acid cross-sectionally and longitudinally predict knee pain progression: an observational cohort study.

OBJECTIVE: To examine associations between serum oxylipins, which regulate tissue repair and pain signalling, and knee pain/radiographic osteoarthritis (OA) at baseline and knee pain at 3 year follow-up. METHOD: Baseline, and 3 year follow-up, knee pain phenotypes were assessed from 154 participants in the Knee Pain in the Community (KPIC) cohort study. Serum and radiographic Kellgren and Lawrence (KL) and Nottingham line drawing atlas OA scores were collected at baseline. Oxylipin levels were quantified using liquid chromatography coupled with mass spectrometry. Associations were measured by linear regression and receiver operating characteristics (ROC). RESULTS: Serum levels of 8,9-epoxyeicosatrienoic acid (EET) (ß(95% confidence intervals (CI)) = 1.809 (-0.71 to 2.91)), 14,15-dihydroxyeicosatrienoic acid (DHET) (ß(95%CI) = 0.827 (0.34-1.31)), and 12-hydroxyeicosatetraenoic acid (HETE) (ß(95%CI) = 4.090 (1.92-6.26)) and anandamide (ß(95%CI) = 3.060 (1.35-4.77)) were cross-sectionally associated with current self-reported knee pain scores (numerical rating scale (NRS) item 3, average pain). Serum levels of 9- (ß(95%CI) = 0.467 (0.18-0.75)) and 15-HETE (ß(95%CI) = 0.759 (0.29-1.22)), 14-hydroxydocosahexaenoic acid (ß(95%CI) = 0.483(0.24-0.73)), and the ratio of 8,9-EET:DHET (ß(95%CI) = 0.510(0.19-0.82)) were cross-sectionally associated with KL scores. Baseline serum concentrations of 8,9-EET (ß(95%CI) = 2.166 (0.89-3.44)), 5,6-DHET (ß(95%CI) = 152.179 (69.39-234.97)), and 5-HETE (ß(95%CI) = 1.724 (0.677-2.77) showed positive longitudinal associations with follow-up knee pain scores (NRS item 3, average pain). Combined serum 8,9-EET and 5-HETE concentration showed the strongest longitudinal association (ß(95%CI) = 1.156 (0.54-1.77) with pain scores at 3 years, and ROC curves distinguished between participants with no pain and high pain scores at follow-up (area under curve (95%CI) = 0.71 (0.61-0.82)). CONCLUSIONS: Serum levels of a combination of hydroxylated metabolites of arachidonic acid may have prognostic utility for knee pain, providing a potential novel approach to identify people who are more likely to have debilitating pain in the future.

Enantioselectivity in some physiological and pathophysiological roles of hydroxyeicosatetraenoic acids.

The phenomenon of chirality has been shown to greatly impact drug activities and effects. Different enantiomers may exhibit different effects in a certain biological condition or disease state. Cytochrome P450 (CYP) enzymes metabolize arachidonic acid (AA) into a large variety of metabolites with a wide range of activities. Hydroxylation of AA by CYP hydroxylases produces hydroxyeicosatetraenoic acids (HETEs), which are classified into mid-chain (5, 8, 9, 11, 12, and 15-HETE), subterminal (16-, 17-, 18- and 19-HETE) and terminal (20-HETE) HETEs. Except for 20-HETE, these metabolites exist as a racemic mixture of R and S enantiomers in the physiological system. The two enantiomers could have different degrees of activity or sometimes opposing effects. In this review article, we aimed to discuss the role of mid-chain and subterminal HETEs in different organs, importantly the heart and the kidneys. Moreover, we summarized their effects in some conditions such as neutrophil migration, inflammation, angiogenesis, and tumorigenesis, with a focus on the reported enantiospecific effects. We also reported some studies using genetically modified models to investigate the roles of HETEs in different conditions.

20-HETE mediated TRPV1 activation drives allokinesis via MrgprA3+ neurons in chronic dermatitis.

Rationale: Noxious stimuli are often perceived as itchy in patients with chronic dermatitis (CD); however, itch and pain mechanisms of CD are not known. Methods: TRPV1 involvement in CD was analyzed using a SADBE induced CD-like mouse model, and several loss- and gain-of-function mouse models. Trigeminal TRPV1 channel and MrgprA3+ neuron functions were analyzed by calcium imaging and whole-cell patch-clamp recordings. Lesional CD-like skin from mice were analyzed by unbiased metabolomic analysis. 20-HETE availability in human and mouse skin were determined by LC/MS and ELISA. And finally, HET0016, a selective 20-HETE synthase inhibitor, was used to evaluate if blocking skin TRPV1 activation alleviates CD-associated chronic itch or pain. Results: While normally a pain inducing chemical, capsaicin induced both itch and pain in mice with CD condition. DREADD silencing of MrgprA3+ primary sensory neurons in these mice selectively decreased capsaicin induced scratching, but not pain-related wiping behavior. In the mice with CD condition, MrgprA3+ neurons showed elevated ERK phosphorylation. Further experiments showed that MrgprA3+ neurons from MrgprA3;Braf mice, which have constitutively active BRAF in MrgprA3+ neurons, were significantly more excitable and responded more strongly to capsaicin. Importantly, capsaicin induced both itch and pain in MrgprA3;Braf mice in an MrgprA3+ neuron dependent manner. Finally, the arachidonic acid metabolite 20-HETE, which can activate TRPV1, was significantly elevated in the lesional skin of mice and patients with CD. Treatment with the selective 20-HETE synthase inhibitor HET0016 alleviated itch in mice with CD condition. Conclusion: Our results demonstrate that 20-HETE activates TRPV1 channels on sensitized MrgprA3+ neurons, and induces allokinesis in lesional CD skin. Blockade of 20-HETE synthesis or silencing of TRPV1-MrgprA3+ neuron signaling offers promising therapeutic strategies for alleviating CD-associated chronic itch.

Eicosanoids and Oxylipin Signature in Hereditary Hemochromatosis Patients Are Similar to Dysmetabolic Iron Overload Syndrome Patients but Are Impacted by Dietary Iron Absorption.

INTRODUCTION: Oxylipins are mediators of oxidative stress. To characterize the underlying inflammatory processes and phenotype effect of iron metabolism disorders, we investigated the oxylipin profile in hereditary hemochromatosis (HH) and dysmetabolic iron overload syndrome (DIOS) patients. METHODS: An LC-MS/MS-based method was performed to quantify plasma oxylipins in 20 HH and 20 DIOS patients in fasting conditions and 3 h after an iron-rich meal in HH patients. RESULTS: Principal component analysis showed no separation between HH and DIOS, suggesting that the clinical phenotype has no direct impact on oxylipin metabolism. 20-HETE was higher in DIOS and correlated with hypertension (p = 0.03). Different oxylipin signatures were observed in HH before and after the iron-rich meal. Discriminant oxylipins include epoxy fatty acids derived from docosahexaenoic acid and arachidonic acid as well as 13-HODE and 9-HODE. Mediation analysis found no major contribution of dietary iron absorption for 16/22 oxylipins significantly affected by the meal. DISCUSSION: The oxylipin profiles of HH and DIOS seemed similar except for 20-HETE, possibly reflecting different hypertension prevalence between the two groups. Oxylipins were significantly affected by the iron-rich meal, but the specific contribution of iron was not clear. Although iron may contribute to oxidative stress and inflammation in HH and DIOS, this does not seem to directly affect oxylipin metabolism.

Lipids and lipid signaling molecules in human milk and infant formula, a chemical characterization of relevant biochemical components.

Breastfeeding is the gold standard in infant nutrition and continuous researches aim to optimize infant formula composition as the best alternative available. Human milk lipid content provides more than 50% of energy requirements for infants together with essential vitamins, polyunsaturated fatty acids, and other bioactive components. While fatty acids and vitamins human milk content has been extensively studied and, when needed those have been added to infant formulas, less is known about polyunsaturated fatty acids functional derivatives and other bioactive components. Here we describe the comparison of lipid compositions in breast milk from 22 healthy volunteers breastfeeding mothers and the six most common infant formula devoting particular attention to two families of signaling lipids, endocannabinoids, and eicosanoids. The main differences between breast milk and formulas lie in a variety of saturated fatty and unsaturated fatty acids, in the total amount (45-95% less in infant formula) and a variety of endocannabinoids and eicosanoids (2-AG, 5(s)HETE, 15(S)-HETE and 14,15-EET).

12-(S)-Hydroxyeicosatetraenoic Acid and GPR31 Signaling in Spinal Cord in Neuropathic Pain.

Neuropathic pain is a pressing unmet medical need requiring novel nonopioid-based therapeutic approaches. Using unbiased transcriptomic analysis, we found that the expression of Gpr31, a G protein-coupled receptor, increased in the dorsal horn of the spinal cord in rats with traumatic nerve injury-induced neuropathic pain. Daily intrathecal injections of siGpr31 reversed behavioral hypersensitivities in a time-dependent manner. GPR31, a Gα i protein-coupled receptor, has recently been cloned and is a receptor for 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. The lack of commercially available GPR31 antagonists has hampered the understanding of this receptor in pathophysiological states, including pain. To investigate this, our first approach was to identify novel GPR31 antagonists. Using a multidisciplinary approach, including in silico modeling, we identified the first highly potent and selective small-molecule GPR31 antagonist, SAH2. Here, we characterize the pharmacological activity in well-described models of neuropathic pain in rodents and provide evidence that 12-(S)-HETE/GPR31-dependent behavioral hypersensitivities are mediated through mitogen-activated protein kinase (MAPK) activation in the spinal cord. Our studies provide the pharmacological rationale for investigating contributions of GPR31 along the pain neuroaxis and the development of nonopioid GPR31-targeted strategies. SIGNIFICANCE STATEMENT: We have identified the first highly selective GPR31 antagonist. Using this antagonist, we have demonstrated that GPR31 signaling in the spinal cord is pronociceptive and MAPK pathways provided signaling mechanisms downstream of GPR31 activation in these processes.

15-Hydroxyeicosatrienoic acid induces nasal congestion by changing vascular functions in mice.

BACKGROUND: Nasal congestion in allergic rhinitis (AR) is caused by vascular hyperpermeability and vascular relaxation of the nasal mucosa. We previously detected high levels of a lipoxygenation metabolite of dihomogammalinolenic acid, 15-hydroxy-8Z,11Z,13E-eicosatrienoic acid (15-HETrE) in the nasal lavage fluid of AR model mice. Here, we investigated the effects of 15-HETrE on vascular functions associated with nasal congestion. METHODS: We measured 15-HETrE levels in the nasal lavage fluid of ovalbumin-induced AR model mice and nasal discharge of patients with AR. We also assessed nasal congestion and vascular relaxation in mice. Vascular contractility was investigated using isolated mouse aortas. RESULTS: Five ovalbumin challenges increased 15-HETrE levels in AR model mice. 15-HETrE was also detected in patients who exhibiting AR-related symptoms. Intranasal administration of 15-HETrE elicited dyspnea-related behavior and decreased the nasal cavity volume in mice. Miles assay and whole-mount immunostaining revealed that 15-HETrE administration caused vascular hyperpermeability and relaxation of the nasal mucosa. Intravital imaging demonstrated that 15-HETrE relaxed the ear vessels that were precontracted via thromboxane receptor stimulation. Moreover, 15-HETrE dilated the isolated mouse aortas, and this effect was attenuated by K+ channel inhibitors and prostaglandin D2 (DP) and prostacyclin (IP) receptor antagonists. Additionally, vasodilatory effects of 15-HETrE were accompanied by an increase in intracellular cAMP levels. CONCLUSIONS: Our results indicate that 15-HETrE, whose levels are elevated in the nasal cavity upon AR, can be a novel lipid mediator that exacerbates nasal congestion. Moreover, it can stimulate DP and IP receptors and downstream K+ channels to dilate the nasal mucosal vasculature.

Brown Adipose Tissue Activation in Humans Increases Plasma Levels of Lipid Mediators.

CONTEXT: Activation of brown adipose tissue (BAT) thermogenesis improves insulin sensitivity and is beneficial in obesity. Emerging evidence indicates that BAT activation increases lipid mediators that play autocrine and endocrine roles to regulate metabolism and inflammation. OBJECTIVE: The goal of the study was to determine the relationship between 2 distinct approaches of BAT activation (cold exposure and mirabegron treatment) with lipid mediators in humans. METHODS: Healthy female subjects (n = 14) were treated with the ß3-adrenergic receptor agonist mirabegron (100 mg) daily for 28 days. A subset of female subjects (n = 8) was additionally exposed to cold temperatures (14-16 °C) for 2 hours using a cooling vest prior to initiating mirabegron treatment. A panel of lipid mediators was assessed in plasma using targeted liquid chromatography-tandem mass spectrometry, and their relationship to anthropometric and metabolic parameters was determined. RESULTS: Activation of BAT with cold exposure acutely increased levels of lipoxygenase and cyclooxygenase products, including 12-hydroxyeicosapentaenoic acid, 12-hydroxyeicosatetraenoic acid (HETE), 5-HETE, 14-hydroxydocosahexaenoic acid (HDHA), an isomer of maresin 2 (MaR2), 17-HDHA, protectin D1 (PD1), and prostaglandin E2. Mirabegron treatment similarly increased these products acutely, although levels of some mediators were blunted after chronic mirabegron treatment. Selected lipid mediators, including an MaR2 isomer, 17-HDHA, 5-HETE, and 15-HETE, positively correlated with nonesterified fatty acids and negatively correlated with the respiratory quotient, while PD1, 15-HETE, and 5-HETE positively correlated with adiponectin. CONCLUSION: These results indicate that selected lipid mediators may serve as biomarkers of BAT activation.

Biological Roles of 5-Oxo-6,8,11,14-Eicosatetraenoic Acid and the OXE Receptor in Allergic Diseases: Collegium Internationale Allergologicum Update 2024.

BACKGROUND: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-Oxo-ETE) is a metabolite of arachidonic acid shown to promote biological activities in different cell types. SUMMARY: 5-Oxo-ETE is synthesized from the 5-lipoxygenase product 5S-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) in the presence of the nicotinamide adenine dinucleotide phosphate (NADP)+-dependent enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Under some conditions that promote oxidation of NADPH to NADP+, such as the respiratory burst in phagocytic cells, eosinophils, and neutrophils, oxidative stress in monocytes and dendritic cells, and cell death, 5-Oxo-ETE synthesis can be dramatically increased. In addition, 5-Oxo-ETE can also be formed in the absence of 5-lipoxygenase in cells through transcellular biosynthesis by inflammatory cell-derived 5S-HETE. This compound performs its biological activities by the highly selective Gi/o-coupled OXE receptor, which is highly expressed on eosinophils, neutrophils, basophils, and monocytes. As such, 5-Oxo-ETE is a potent chemoattractant for these inflammatory cells, especially for eosinophils. KEY MESSAGES: Although the pathophysiological role of 5-Oxo-ETE is not clearly understood, 5-Oxo-ETE may be a significant mediator in allergic diseases, such as allergic asthma, allergic rhinitis, and atopic dermatitis. And targeting the OXE receptor may be a novel therapy for this kind of inflammatory condition. Nowadays, selective OXE receptor antagonists are currently under investigation and could become potential therapeutic agents in allergy.

Deciphering the Role of Fatty Acid-Metabolizing CYP4F11 in Lung Cancer and Its Potential As a Drug Target.

Lung cancer is the leading cause of cancer deaths worldwide. We found that the cytochrome P450 isoform CYP4F11 is significantly overexpressed in patients with lung squamous cell carcinoma. CYP4F11 is a fatty acid ω-hydroxylase and catalyzes the production of the lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. 20-HETE promotes cell proliferation and migration in cancer. Inhibition of 20-HETE-generating cytochrome P450 enzymes has been implicated as novel cancer therapy for more than a decade. However, the exact role of CYP4F11 and its potential as drug target for lung cancer therapy has not been established yet. Thus, we performed a transient knockdown of CYP4F11 in the lung cancer cell line NCI-H460. Knockdown of CYP4F11 significantly inhibits lung cancer cell proliferation and migration while the 20-HETE production is significantly reduced. For biochemical characterization of CYP4F11-inhibitor interactions, we generated recombinant human CYP4F11. Spectroscopic ligand binding assays were conducted to evaluate CYP4F11 binding to the unselective CYP4A/F inhibitor HET0016. HET0016 shows high affinity to recombinant CYP4F11 and inhibits CYP4F11-mediated 20-HETE production in vitro with a nanomolar IC 50 Cross evaluation of HET0016 in NCI-H460 cells shows that lung cancer cell proliferation is significantly reduced together with 20-HETE production. However, HET0016 also displays antiproliferative effects that are not 20-HETE mediated. Future studies aim to establish the role of CYP4F11 in lung cancer and the underlying mechanism and investigate the potential of CYP4F11 as a therapeutic target for lung cancer. SIGNIFICANCE STATEMENT: Lung cancer is a deadly cancer with limited treatment options. Cytochrome P450 4F11 (CYP4F11) is significantly upregulated in lung squamous cell carcinoma. Knockdown of CYP4F11 in a lung cancer cell line significantly attenuates cell proliferation and migration with reduced production of the lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE). Studies with the unselective inhibitor HET0016 show a high inhibitory potency of CYP4F11-mediated 20-HETE production using recombinant enzyme. Overall, our studies demonstrate the potential of targeting CYP4F11 for new transformative lung cancer treatment.

Correlation between immune-related Tryptophan-Kynurenine pathway and severity of severe pneumonia and inflammation-related polyunsaturated fatty acids.

BACKGROUND: Immune dysfunction and oxidative stress caused by severe pneumonia can lead to multiple organ dysfunction and even death, causing a significant impact on health and the economy. Currently, great progress has been made in the diagnosis and treatment of this disease, but the mortality rate remains high (approximately 50%). Therefore, there is still potential for further exploration of the immune response mechanisms against severe pneumonia. OBJECTIVE: This study analyzed the difference in serum metabolic profiles between patients with severe pneumonia and health individuals through metabolomics, aiming to uncover the correlation between the Tryptophan-Kynurenine pathway and the severity of severe pneumonia, as well as N-3/N-6 polyunsaturated fatty acids (PUFAs). METHODS: In this study, 44 patients with severe pneumonia and 37 health controls were selected. According to the changes in the disease symptoms within the 7 days of admission, the patients were divided into aggravation (n = 22) and remission (n = 22) groups. Targeted metabolomics techniques were performed to quantify serum metabolites and analyze changes between groups. RESULTS: Metabolomics analysis showed that serum kynurenine and kynurenine/tryptophan (K/T) were significantly increased and tryptophan was significantly decreased in patients with severe pneumonia; HETE and HEPE in lipids increased significantly, while eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), α-linolenic acid (linolenic acid, α-LNA), arachidonic acid (ARA), Dihomo-γ-linolenic acid (DGLA), and 13(s)-hydroperoxylinoleic acid (HPODE) decreased significantly. Additionally, the longitudinal comparison revealed that Linolenic acid, DPA, and Tryptophan increased significantly in the remission group, while and kynurenine and K/T decreased significantly. In the aggravation group, Kynurenine and K/T increased significantly, while ARA, 8(S)-hydroxyeicosatetraenoic acid (HETE), 11(S)-HETE, and Tryptophan decreased significantly. The correlation analysis matrix demonstrated that Tryptophan was positively correlated with DGLA, 12(S)-hydroxyeicosapentaenoic acid (HEPE), ARA, EPA, α-LNA, DHA, and DPA. Kynurenine was positively correlated with 8(S)-HETE and negatively correlated with DHA. Additionally, K/T was negatively correlated with DGLA, ARA, EPA, α-LNA, DHA, and DPA. CONCLUSION: This study revealed that during severe pneumonia, the Tryptophan-Kynurenine pathway was activated and was positively correlated with the disease progression. On the other hand, the activation of the Tryptophan-Kynurenine pathway was negatively correlated with N-3/N-6 PUFAs.

Exosomes drive ferroptosis by stimulating iron accumulation to inhibit bacterial infection in crustaceans.

Ferroptosis, characterized by iron-dependent cell death, has recently emerged as a critical defense mechanism against microbial infections. The present study aims to investigate the involvement of exosomes in the induction of ferroptosis and the inhibition of bacterial infection in crustaceans. Our findings provide compelling evidence for the pivotal role of exosomes in the immune response of crustaceans, wherein they facilitate intracellular iron accumulation and activate the ferroptotic pathways. Using RNA-seq and bioinformatic analysis, we demonstrate that cytochrome P450 (CYP) can effectively trigger ferroptosis. Moreover, by conducting an analysis of exosome cargo proteins, we have identified the participation of six-transmembrane epithelial antigen of prostate 4 in the regulation of hemocyte ferroptotic sensitivity. Subsequent functional investigations unveil that six-transmembrane epithelial antigen of prostate 4 enhances cellular Fe2+ levels, thereby triggering Fenton reactions and accelerating CYP-mediated lipid peroxidation, ultimately culminating in ferroptotic cell death. Additionally, the Fe2+-dependent CYP catalyzes the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid, which activates the peroxisome proliferator-activated receptor. Consequently, the downstream target of peroxisome proliferator-activated receptor, cluster of differentiation 36, promotes intracellular fatty acid accumulation, lipid peroxidation, and ferroptosis. These significant findings shed light on the immune defense mechanisms employed by crustaceans and provide potential strategies for combating bacterial infections in this species.

Apple consumption affects cecal health by regulating 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HETE) levels through modifying the microbiota in rats.

Apples are rich in many nutrients and functional components. However, the mechanism of the effect of fresh apple consumption on rats remains unclear. In the present study, fresh apples (10 g kg-1) were added to the diet of Wistar rats, and changes in the microbiota and metabolite content of the cecum were analyzed after 28 days of feeding, and changes in the 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HETE) content and indicators related to inflammation, oxidative stress, and apoptosis were detected. Subsequently, a fecal microbiota transplantation (FMT) protocol was designed and carried out to verify the relationship between the microbiota and 12(S)-HETE, the cecal structure, and inflammatory factors. The results show that apple consumption significantly reduced the serum levels of alanine aminotransferase (ALT) and immunoglobulin G (IgG), altered the cecal histomorphology, and significantly upregulated the gene expression of claudin-1 and zonula occludens-1 (ZO-1), which encode tight junction proteins. Apple consumption also changed the structure of the cecal microbiota, increasing the abundance of some species (such as Shuttleworthia) and decreasing the abundance of others (such as Alphaproteobacteria). Metabolomic screening identified 64 significantly different metabolites. The FMT results showed that apple consumption reduced 12(S)-HETE metabolite levels in the cecal contents, improved the intestinal structure, and reduced the levels of proinflammatory factor expression by altering the cecal microbiota. In conclusion, this study provides further insight into the effects of apples on animals using rats as experimental animals. It provides basic data for future exploration of the mechanisms of the effect of apple consumption on humans.

The Effects of 16-HETE Enantiomers on Hypertrophic Markers in Human Fetal Ventricular Cardiomyocytes, RL-14 Cells.

BACKGROUND: Cytochrome P450 (CYP) metabolizes arachidonic acid to produce bioactive metabolites such as EETs and HETEs: mid-chain, subterminal, and terminal HETEs. Recent studies have revealed the role of CYP1B1 and its associated cardiotoxic mid-chain HETE metabolites in developing cardiac hypertrophy and heart failure. Subterminal HETEs have also been involved in various physiological and pathophysiological processes; however, their role in cardiac hypertrophy has not been fully defined. OBJECTIVE: The objective of the current study is to determine the possible effect of subterminal HETEs, R and S enantiomers of 16-HETE, on CYP1B1 expression in vitro using human cardiomyocytes RL-14 cells. METHODS: In the study, RL14 cell line was treated with vehicle and either of the 16-HETE enantiomers for 24 h. Subsequently, the following markers were assessed: cell viability, cellular size, hypertrophic markers, CYP1B1 gene expression (at mRNA, protein, and activity levels), luciferase activity, and CYP1B1 mRNA and protein half-lives. RESULTS: The results of the study showed that 16-HETE enantiomers significantly increased hypertrophic markers and upregulated CYP1B1 mRNA and protein expressions in RL-14 cell line. The upregulation of CYP1B1 by 16-HETE enantiomers occurs via a transcriptional mechanism as evidenced by transcriptional induction and luciferase reporter assay. Furthermore, neither post-transcriptional nor post-translational modification was involved in such modulation since there was no change in CYP1B1 mRNA and protein stabilities upon treatment with 16-HETE enantiomers. CONCLUSION: The current study provides the first evidence that 16R-HETE and 16S-HETE increase CYP1B1 gene expression through a transcriptional mechanism.

P2X7 signaling influences the production of pro-resolving and pro-inflammatory lipid mediators in alveolar macrophages derived from individuals with asthma.

Few new therapeutics exist to target airway inflammation in mild-to-moderate asthma. Alveolar macrophages regulate airway inflammation by producing proresolving eicosanoids. We hypothesized that stimulation of the purinergic receptor P2X7 in macrophages from individuals with asthma produces eicosanoids associated with airway inflammation and resolution, and that these responses are predicted, in part, by P2X7 pore function. Study subjects were recruited in an Institutional Review Board (IRB)-approved study. Alveolar macrophages were recovered from bronchoalveolar lavage fluid following bronchoscopy. Purinergic receptor classification was performed using flow cytometry and fluorescent cell assay. Macrophages were stimulated in vitro and eicosanoids were measured via ELISA or enzyme immunoassay (EIA) in the presence and absence of P2X7-specific agonist [2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate tri(triethylammonium) salt (Bz-ATP)] and antagonist (AZD9056). Functional P2X7 pore status was confirmed in a live cell assay using P2X7-specific agonists and antagonists. Alveolar macrophages produced increased quantities of the oxylipins lipoxin A4 (LXA4), resolvin D1 (RvD1), and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) following stimulation with Bz-ATP compared with vehicle controls, responses that were attenuated in the presence of the P2X7-selective antagonist, AZD9056. LXA4 and RvD1 production was greatest at 1 h, whereas 15(S)-HETE was maximally produced 24 h. Prostaglandin E-2 and resolvin E1 were minimally produced by P2X7 activation, indicating differential signaling pathways involved in eicosanoid production in alveolar macrophages derived from individuals with asthma. The early production of the proresolving eicosanoids, LXA4 and resolvin D1, is regulated by P2X7, whereas generation of the proinflammatory eicosanoid, 15(S)-HETE, is only partially regulated through P2X7 signaling and reaches maximal production after the peak in proresolving eicosanoids.NEW & NOTEWORTHY Alveolar macrophages obtained from individuals with asthma produce soluble lipid mediators in response to P2X7 purinergic receptor signaling. Proinflammatory mediators may contribute to asthma exacerbations but proresolving mediators may help with resolution of asthma loss of control. These specialized proresolving lipid mediators may serve as future potential therapeutics for asthma exacerbation resolution and recovery.

Lower serum 15-HETE level predicts nasal ILC2 accumulation during COX-1 inhibition in AERD.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is associated with high levels of cysteinyl leukotrienes, prostaglandin D2, and low levels of prostaglandin E2. Further, 15-hydroxyeicosatetraenoic acid (15-HETE) levels may have predictive value in therapeutic outcomes of aspirin desensitization. Accumulation of nasal group 2 innate lymphoid cells (ILC2s) has been demonstrated during COX-1 inhibition in AERD, although the relationships between tissue ILC2 accumulation, reaction symptom severity, and novel lipid biomarkers are unknown. OBJECTIVE: We sought to determine whether novel lipid mediators are predictive of nasal ILC2 accumulation and symptom scores during COX-1 inhibitor challenge in patients with AERD. METHODS: Blood and nasal scraping samples from patients with AERD were collected at baseline and COX-1 inhibitor reaction and then processed for flow cytometry for nasal ILC2s and serum for lipidomic analysis. RESULTS: Eight patients with AERD who were undergoing aspirin desensitization were recruited. Of the 161 eicosanoids tested, 42 serum mediators were detected. Baseline levels of 15-HETE were negatively correlated with the change in numbers of airway ILC2s (r = -0.6667; P = .0428). Docosahexaenoic acid epoxygenase metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) was positively correlated with both changes in airway ILC2s (r = 0.7143; P = .0305) and clinical symptom scores (r = 0.5000; P = .0081). CONCLUSION: Low levels of baseline 15-HETE predicted a greater accumulation of airway ILC2s in patients with AERD who were receiving COX-1 inhibition. Further, increases in the cytochrome P pathway metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) were associated with increased symptoms and nasal ILC2 accumulation. Future studies to assess how these mediators might control ILC2s may improve the understanding of AERD pathogenesis.

Associations of phthalates, phthalate replacements, and their mixtures with eicosanoid biomarkers during pregnancy.

Humans are exposed to complex mixtures of phthalates. Gestational exposure to phthalates has been linked to preeclampsia and preterm birth through potential pathways such as endocrine disruption, oxidative stress, and inflammation. Eicosanoids are bioactive signaling lipids that are related to a variety of homeostatic and inflammatory processes. We investigated associations between urinary phthalates and their mixtures with plasma eicosanoid levels during pregnancy using the PROTECT cohort in Puerto Rico (N = 655). After adjusting for covariates, we estimated pair-wise associations between the geometric mean of individual phthalate metabolite concentrations across pregnancy and eicosanoid biomarkers using multivariable linear regression. We used bootstrapping of adaptive elastic net regression (adENET) to evaluate phthalate mixtures associated with eicosanoids and subsequently create environmental risk scores (ERS) to represent weighted sums of phthalate exposure for each individual. After adjusting for false-discovery, in single-pollutant analysis, 14 of 20 phthalate metabolites or parent compound indices showed significant and primarily negative associations with multiple eicosanoids. In our mixture analysis, associations with several metabolites of low molecular weight phthalates - DEP, DBP, and DIBP - became prominent. Additionally, MEHHTP and MECPTP, metabolites of a new phthalate replacement, DEHTP, were selected as important predictors for determining the concentrations of multiple eicosanoids from different pathway groups. A unit increase in phthalate ERS derived from bootstrapping of adENET was positively associated with several eicosanoids mainly from Cytochrome P450 pathway. For example, an increase in ERS was associated with 11(S)-HETE (ß = 1.6, 95% CI: 0.020, 3.180), (±)11,12-DHET (ß = 2.045, 95% CI: 0.250, 3.840), 20(S)-HETE (ß = 0.813, 95% CI: 0.147, 1.479), and 9 s-HODE (ß = 2.381, 95% CI: 0.657, 4.104). Gestational exposure to phthalates and phthalate mixtures were associated with eicosanoid levels during pregnancy. Results from the mixture analyses underscore the complexity of physiological impacts of phthalate exposure and call for further in-depth studies to examine these relationships.