ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2.

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Association between arachidonate lipoxygenase 15,c.-292 C > T gene polymorphism and non-cystic fibrosis bronchiectasis in children: a pilot study on the effects on airway lipoxin A4 and disease phenotype.

BACKGROUND: Persistent airway inflammation is a central feature of bronchiectasis. Arachidonate 15-lipoxygenase (ALOX-15) controls production of endogenous lipid mediators, including lipoxins that regulate airway inflammation. Mutations at various positions in ALOX-15 gene can influence airway disease development. We investigated association between ALOX-15,c.-292 C > T gene polymorphism and bronchiectasis unrelated to cystic fibrosis in Egyptian children. Also, lipoxin A4 (LXA4) level in bronchoalveolar lavage (BAL) was studied in relation to polymorphism genotypes and disease phenotypes determined by clinical, pulmonary functions, and radiological severity parameters. METHODS: This was an exploratory study that included 60 participants. Thirty children with non-cystic fibrosis bronchiectasis (NCFB) were compared with 30 age and sex-matched controls. ALOX-15,c.-292 C > T polymorphism was genotyped using TaqMan-based Real-time PCR. LXA4 was measured in BAL using ELISA method. RESULTS: There was no significant difference between patients and controls regarding ALOX-15,c.-292 C > T polymorphism genotypes and alleles (OR = 1.75; 95% CI (0.53-5.7), P = 0.35) (OR = 1; 95% CI (0.48-2), p = 1). BAL LXA4 level was significantly lower in patients, median (IQR) of 576.9 (147.6-1510) ng/ml compared to controls, median (IQR) of 1675 (536.8-2542) (p = 0.002). Patients with severe bronchiectasis had a significantly lower LXA4 level (p < 0.001). There were significant correlations with exacerbations frequency (r=-0.54, p = 0.002) and FEV1% predicted (r = 0.64, p = 0.001). Heterozygous CT genotype carriers showed higher LXA4 levels compared to other genotypes(p = 0.005). CONCLUSIONS: Low airway LXA4 in children with NCFB is associated with severe disease phenotype and lung function deterioration. CT genotype of ALOX-15,c.-292 C > T polymorphism might be a protective genetic factor against bronchiectasis development and/or progression due to enhanced LXA4 production.

Identifying ALOX15-initiated lipid peroxidation increases susceptibility to ferroptosis in asthma epithelial cells.

Ferroptosis is a programmed form of cell death regulated by iron and has been linked to the development of asthma. However, the precise mechanisms driving ferroptosis in asthma remain elusive. To gain deeper insights, we conducted an analysis of nasal epithelial and sputum samples from the GEO database using three machine learning methods. Our investigation identified a pivotal gene, Arachidonate 15-lipoxygenase (ALOX15), associated with ferroptosis in asthma. Through both in vitro and in vivo experiments, we further confirmed the significant role of ALOX15 in ferroptosis in asthma. Our results demonstrate that ferroptosis manifests in an HDM/LPS-induced allergic airway inflammation (AAI) mouse model, mimicking human asthma, and in HDM/LPS-stimulated 16HBE cells. Moreover, we observed an up-regulation of ALOX15 expression in HDM/LPS-induced mice and cells. Notably, silencing ALOX15 markedly decreased HDM/LPS-induced ferroptosis in 16HBE cells. These findings indicate that ferroptosis may be implicated in the onset and progression of asthma, with ALOX15-induced lipid peroxidation raising the susceptibility to ferroptosis in asthmatic epithelial cells.

Rhythm gene PER1 mediates ferroptosis and lipid metabolism through SREBF2/ALOX15 axis in polycystic ovary syndrome.

OBJECTIVE: This work aimed to investigate the role of rhythm gene PER1 in mediating granulosa cell ferroptosis and lipid metabolism of polycystic ovary syndrome (PCOS). METHODS: We injected dehydroepiandrosterone and Ferrostatin-1 (Fer-1) into mice to explore the mechanism of ferroptosis in PCOS. The effect of PER1 on ferroptosis-like changes in granulosa cells was explored by overexpression of PER1 plasmid transfection and Fer-1 treatment. RESULTS: We found that Fer-1 ameliorated the characteristic polycystic ovary morphology, suppressed ferroptosis in the PCOS mice. PER1 and ALOX15 were highly expressed in PCOS, whereas SREBF2 was lowly expressed. Overexpression of PER1 decreased granulosa cell viability and inhibited proliferation. Meanwhile, overexpression of PER1 increased lipid reactive oxygen species, 4-Hydroxynonenal (4-HNE), Malondialdehyde (MDA), total Fe, and Fe2+ levels in granulosa cells and decreased Glutathione (GSH) content. Fer-1, SREBF2 overexpression, or ALOX15 silencing treatment reversed the effects of PER1 overexpression on granulosa cells. PER1 binds to the SREBF2 promoter and represses SREBF2 transcription. SREBF2 binds to the ALOX15 promoter and represses ALOX15 transcription. Correlation analysis of clinical trials showed that PER1 was positively correlated with total cholesterol, low-density lipoprotein cholesterol, luteinizing hormone, testosterone, 4-HNE, MDA, total Fe, Fe2+, and ALOX15. In contrast, PER1 was negatively correlated with SREBF2, high-density lipoprotein cholesterol, follicle-stimulating hormone, progesterone, and GSH. CONCLUSION: This study demonstrates that the rhythm gene PER1 promotes ferroptosis and dysfunctional lipid metabolism in granulosa cells in PCOS by inhibiting SREBF2/ALOX15 signaling.

Loss of 15-Lipoxygenase in Retinodegenerative RCS Rats.

Retinitis pigmentosa (RP) is a retinal degenerative disease associated with a diversity of genetic mutations. In a natural progression study (NPS) evaluating the molecular changes in Royal College of Surgeons (RCS) rats using lipidomic profiling, RNA sequencing, and gene expression analyses, changes associated with retinal degeneration from p21 to p60 were evaluated, where reductions in retinal ALOX15 expression corresponded with disease progression. This important enzyme catalyzes the formation of specialized pro-resolving mediators (SPMs) such as lipoxins (LXs), resolvins (RvDs), and docosapentaenoic acid resolvins (DPA RvDs), where reduced ALOX15 corresponded with reduced SPMs. Retinal DPA RvD2 levels were found to correlate with retinal structural and functional decline. Retinal RNA sequencing comparing p21 with p60 showed an upregulation of microglial inflammatory pathways accompanied by impaired damage-associated molecular pattern (DAMP) clearance pathways. This analysis suggests that ALXR/FPR2 activation can ameliorate disease progression, which was supported by treatment with an LXA4 analog, NAP1051, which was able to promote the upregulation of ALOX12 and ALOX15. This study showed that retinal inflammation from activated microglia and dysregulation of lipid metabolism were central to the pathogenesis of retinal degeneration in RP, where ALXR/FPR2 activation was able to preserve retinal structure and function.

A Multicenter Long-Term Cohort Study of Eosinophilic Esophagitis Variants and Their Progression to Eosinophilic Esophagitis Over Time.

INTRODUCTION: Eosinophilic esophagitis (EoE) variants have been recently characterized as conditions with symptoms of esophageal dysfunction resembling EoE, but absence of significant esophageal eosinophilia. Their disease course and severity have yet to be determined. METHODS: Patients from 6 EoE centers with symptoms of esophageal dysfunction, but peak eosinophil counts of <15/hpf in esophageal biopsies and absence of gastroesophageal reflux disease with at least one follow-up visit were included. Clinical, (immuno)histological, and molecular features were determined and compared with EoE and healthy controls. RESULTS: We included 54 patients with EoE variants (EoE-like esophagitis 53.7%; lymphocytic esophagitis 13.0%; and nonspecific esophagitis 33.3%). In 8 EoE-like esophagitis patients, EoE developed after a median of 14 months (interquartile range 3.6-37.6). Such progression increased over time (17.6% year 1, 32.0% year 3, and 62.2% year 6). Sequential RNA sequencing analyses revealed only 7 genes associated with this progression (with TSG6 and ALOX15 among the top 3 upregulated genes) with upregulation of a previously attenuated Th2 pathway. Immunostaining confirmed the involvement of eosinophil-associated proteins (TSG6 and ALOX15) and revealed a significantly increased number of GATA3-positive cells during progression, indicating a Th1/Th2 switch. Transition from one EoE variant (baseline) to another variant (during follow-up) was seen in 35.2% (median observation time of 17.3 months). DISCUSSION: Transition of EoE variants to EoE suggests the presence of a disease spectrum. Few genes seem to be associated with the progression to EoE with upregulation of a previously attenuated Th2 signal. These genes, including GATA3 as a Th1/Th2 switch regulator, may represent potential therapeutic targets in early disease pathogenesis.

15-Lipoxygenase promotes resolution of inflammation in lymphedema by controlling Treg cell function through IFN-ß.

Lymphedema (LD) is characterized by the accumulation of interstitial fluid, lipids and inflammatory cell infiltrate in the limb. Here, we find that LD tissues from women who developed LD after breast cancer exhibit an inflamed gene expression profile. Lipidomic analysis reveals decrease in specialized pro-resolving mediators (SPM) generated by the 15-lipoxygenase (15-LO) in LD. In mice, the loss of SPM is associated with an increase in apoptotic regulatory T (Treg) cell number. In addition, the selective depletion of 15-LO in the lymphatic endothelium induces an aggravation of LD that can be rescued by Treg cell adoptive transfer or ALOX15-expressing lentivector injections. Mechanistically, exogenous injections of the pro-resolving cytokine IFN-ß restores both 15-LO expression and Treg cell number in a mouse model of LD. These results provide evidence that lymphatic 15-LO may represent a therapeutic target for LD by serving as a mediator of Treg cell populations to resolve inflammation.

Knock-in mice expressing a humanized arachidonic acid 15-lipoxygenase (Alox15) carry a partly dysfunctional erythropoietic system.

Arachidonic acid 15-lipoxygenases (ALOX15) play a role in mammalian erythropoiesis but they have also been implicated in inflammatory processes. Seven intact Alox genes have been detected in the mouse reference genome and the mouse Alox15 gene is structurally similar to the orthologous genes of other mammals. However, mouse and human ALOX15 orthologs have different functional characteristics. Human ALOX15 converts C20 polyenoic fatty acids like arachidonic acid mainly to the n-6 hydroperoxide. In contrast, the n-9 hydroperoxide is the major oxygenation product formed by mouse Alox15. Previous experiments indicated that Leu353Phe exchange in recombinant mouse Alox15 humanized the catalytic properties of the enzyme. To investigate whether this functional humanization might also work in vivo and to characterize the functional consequences of mouse Alox15 humanization we generated Alox15 knock-in mice (Alox15-KI), in which the Alox15 gene was modified in such a way that the animals express the arachidonic acid 15-lipoxygenating Leu353Phe mutant instead of the arachidonic acid 12-lipoxygenating wildtype enzyme. These mice develop normally, they are fully fertile but display modified plasma oxylipidomes. In young individuals, the basic hematological parameters were not different when Alox15-KI mice and outbred wildtype controls were compared. However, when growing older male Alox15-KI mice develop signs of dysfunctional erythropoiesis such as reduced hematocrit, lower erythrocyte counts and attenuated hemoglobin concentration. These differences were paralleled by an improved ex vivo osmotic resistance of the peripheral red blood cells. Interestingly, such differences were not observed in female individuals suggesting gender specific effects. In summary, these data indicated that functional humanization of mouse Alox15 induces defective erythropoiesis in aged male individuals.

Epigenetic regulation of 15-lipoxygenase-1 expression in human chondrocytes by promoter methylation.

OBJECTIVE AND DESIGN: 15-Lipoxygenase-1 (15-LOX-1) catalyzes the biosynthesis of many anti-inflammatory and immunomodulatory lipid mediators and was reported to have protective properties in several inflammatory conditions, including osteoarthritis (OA). This study was designed to evaluate the expression of 15-LOX-1 in cartilage from normal donors and patients with OA, and to determine whether it is regulated by DNA methylation. METHODS: Cartilage samples were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee joint replacement surgery. The expression of 15-LOX-1 was evaluated using real-time polymerase chain reaction (PCR). The role of DNA methylation in 15-LOX-1 expression was assessed using the DNA methyltransferase inhibitor 5-Aza-2'-desoxycytidine (5-Aza-dC). The effect of CpG methylation on 15-LOX-1 promoter activity was evaluated using a CpG-free luciferase vector. The DNA methylation status of the 15-LOX-1 promoter was determined by pyrosequencing. RESULTS: Expression of 15-LOX-1 was upregulated in OA compared to normal cartilage. Treatment with 5-Aza-dC increased 15-LOX-1 mRNA levels in chondrocytes, and in vitro methylation decreased 15-LOX-1 promoter activity. There was no difference in the methylation status of the 15-LOX-1 gene promoter between normal and OA cartilage. CONCLUSION: The expression level of 15-LOX-1 was elevated in OA cartilage, which may be part of a repair process. The upregulation of 15-LOX-1 in OA cartilage was not associated with the methylation status of its promoter, suggesting that other mechanisms are involved in its upregulation.

15-lipoxygenase and cyclooxygenase expression profile and their related modulators in COVID-19 infection.

BACKGROUND: The role of the lipoxygenase (LOX) and cyclooxygenase (COX) enzymes in maintaining cellular homeostasis and regulating immune responses promoted us in this study to analyze the pattern of changes in 15-lipoxygenase and cyclooxygenase isoforms and their related cytokines in SARS-CoV-2 infection. METHODS: 15-LOX-1, 15-LOX-2, COX-1 and COX-2 gene expression levels were determined using qRT-PCR in nasopharynx specimens from patients with severe [N = 40] and non-severe [N = 40] confirmed SARS-CoV-2 infections and healthy controls. Circulating levels of lL-6, lL-10, PGE2, and IFN-γ were measured in patients and healthy controls using ELISA assay. The associations between the measured variables and the patient's clinic-pathological characteristics were assessed for all groups. RESULTS: The expression level of 15-LOX-1 was elevated significantly in male patients with severe infection; although female patients showed a different expression profile. 15-LOX-2 expression level was considerably increased in male patients with severe infection; while changes in its expression remained inconclusive in female patients. The relationship between 15-LOX expression and the male gender was prominent. Both COX isoforms expression showed elevation in male and female patients that were correlated with disease severity. The simultaneous increase in lL-6, PGE2 and IFN-γ levels also decrease in lL-10 in patients with severe infection indicating the possible regulatory network related to the COX and 15-LOX enzymes in the output of the SARS-CoV-2 infection. CONCLUSION: The results of this study determined the pattern of possible changes in key enzymes of prostaglandin and eicosanoids synthesis pathway and their mediators, which can be helpful in mapping the SARS-CoV-2 pathogenicity and pharmaceutical approaches.

Baicalein, a component of banxia xiexin decoction, alleviates CPT-11-induced gastrointestinal dysfunction by inhibiting ALOX15-mediated ferroptosis.

Baicalein, one of the active ingredients of banxia xiexin decoction, has good therapeutic efficacy in treating diarrhea and improving gastrointestinal dysfunction. The role and mechanism of Baicalein on irinotecan (CPT-11)-induced gastrointestinal dysfunction are the focus of this study. Concretely, CPT-11 induced delayed diarrhea rat model and intestinal epithelial cell (IEC)-6 cell injury model with Baicalein treatment as needed. Colonic pathological changes were analyzed by hematoxylin-eosin staining, and inflammatory factor expressions in serum were determined by enzyme-linked immunosorbent assay. Immunohistochemistry and western blot were performed to quantify ferroptosis-related protein expressions. Thiobarbituric acid reactive substances (TBARS) kits and colorimetric assay kit were applied to detect lipid peroxidation levels and Fe2+ content, respectively. In vitro experiments also included quantitative real-time polymerase chain reaction, cell counting kit-8, and C11 BODIPY staining. CPT-11 induced aggravation of intestinal tissue damage, inflammatory factor release, Fe2+ accumulation, upregulation of lipid peroxidation and 15-Lipoxygenase (ALOX15) expression, and downregulation of glutathione peroxidase 4 (Gpx4) and SLC7A11 in vivo in rats; however, Baicalein dose-dependently reversed the effects of CPT-11. Baicalein elevated cell viability, reduced lipid peroxidation and Fe2+ accumulation, and elevated Gpx4 and SLC7A11 levels, whereas ALOX15 overexpression reversed the effects of Baicalein on a CPT-11-induced IEC-6 cell injury model. In conclusion, Baicalein plays a mitigating role in CPT-11-induced delayed diarrhea via ALOX15-mediated ferroptosis.

Glucocorticoids regulate lipid mediator networks by reciprocal modulation of 15-lipoxygenase isoforms affecting inflammation resolution.

Glucocorticoids (GC) are potent anti-inflammatory agents, broadly used to treat acute and chronic inflammatory diseases, e.g., critically ill COVID-19 patients or patients with chronic inflammatory bowel diseases. GC not only limit inflammation but also promote its resolution although the underlying mechanisms are obscure. Here, we reveal reciprocal regulation of 15-lipoxygenase (LOX) isoform expression in human monocyte/macrophage lineages by GC with respective consequences for the biosynthesis of specialized proresolving mediators (SPM) and their 15-LOX-derived monohydroxylated precursors (mono-15-OH). Dexamethasone robustly up-regulated pre-mRNA, mRNA, and protein levels of ALOX15B/15-LOX-2 in blood monocyte-derived macrophage (MDM) phenotypes, causing elevated SPM and mono-15-OH production in inflammatory cell types. In sharp contrast, dexamethasone blocked ALOX15/15-LOX-1 expression and impaired SPM formation in proresolving M2-MDM. These dexamethasone actions were mimicked by prednisolone and hydrocortisone but not by progesterone, and they were counteracted by the GC receptor (GR) antagonist RU486. Chromatin immunoprecipitation (ChIP) assays revealed robust GR recruitment to a putative enhancer region within intron 3 of the ALOX15B gene but not to the transcription start site. Knockdown of 15-LOX-2 in M1-MDM abolished GC-induced SPM formation and mono-15-OH production. Finally, ALOX15B/15-LOX-2 upregulation was evident in human monocytes from patients with GC-treated COVID-19 or patients with IBD. Our findings may explain the proresolving GC actions and offer opportunities for optimizing GC pharmacotherapy and proresolving mediator production.

Transgenic mice overexpressing human ALOX15 under the control of the aP2 promoter are partly protected in the complete Freund's adjuvant-induced paw inflammation model.

BACKGROUND, OBJECTIVES AND DESIGN: Arachidonic acid 15-lipoxygenase (ALOX15) has been implicated in the pathogenesis of inflammatory diseases but since pro- and anti-inflammatory roles have been suggested, the precise function of this enzyme is still a matter of discussion. To contribute to this discussion, we created transgenic mice, which express human ALOX15 under the control of the activating protein 2 promoter (aP2-ALOX15 mice) and compared the sensitivity of these gain-of-function animals in two independent mouse inflammation models with Alox15-deficient mice (loss-of-function animals) and wildtype control animals. MATERIALS AND METHODS: Transgenic aP2-ALOX15 mice were tested in comparison with Alox15 knockout mice (Alox15-/-) and corresponding wildtype control animals (C57BL/6J) in the complete Freund's adjuvant induced hind-paw edema model and in the dextran sulfate sodium induced colitis (DSS-colitis) model. In the paw edema model, the degree of paw swelling and the sensitivity of the inflamed hind-paw for mechanic (von Frey test) and thermal (Hargreaves test) stimulation were quantified as clinical readout parameters. In the dextran sodium sulfate induced colitis model the loss of body weight, the colon lengths and the disease activity index were determined. RESULTS: In the hind-paw edema model, systemic inactivation of the endogenous Alox15 gene intensified the inflammatory symptoms, whereas overexpression of human ALOX15 reduced the degree of hind-paw inflammation. These data suggest anti-inflammatory roles for endogenous and transgenic ALOX15 in this particular inflammation model. As mechanistic reason for the protective effect downregulation of the pro-inflammatory ALOX5 pathways was suggested. However, in the dextran sodium sulfate colitis model, in which systemic inactivation of the Alox15 gene protected female mice from DSS-induced colitis, transgenic overexpression of human ALOX15 did hardly impact the intensity of the inflammatory symptoms. CONCLUSION: The biological role of ALOX15 in the pathogenesis of inflammation is variable and depends on the kind of the animal inflammation model.

Overexpression of LOX-1 in hepatocytes protects vascular smooth muscle cells from phenotype transformation and wire injury induced carotid neoatherosclerosis through ALOX15.

Neoatherosclerosis (NA), the main pathological basis of late stent failure, is the main limitation of interventional therapy. However, the specific pathogenesis and treatment remain unclear. In vivo, NA model was established by carotid wire injury and high-fat feeding in ApoE-/- mice. Oxidized low-density lipoprotein receptor-1/lectin-like oxidized low-density lipoprotein receptor-1 (OLR1/LOX-1), a specific receptor for oxidized low-density lipoprotein (ox-LDL), was specifically ectopically overexpressed in hepatocytes by portal vein injection of adeno-associated serotype 8 (AAV8)-thyroid binding globulin (TBG)-Olr1 and the protective effect against NA was examined. In vitro, LOX-1 was overexpressed on HHL5 using lentivirus (LV)-OLR1 and the vascular smooth muscle cells (VSMCs)-HHL5 indirect co-culture system was established to examine its protective effect on VSMCs and the molecular mechanism. Functionally, we found that specific ectopic overexpression of LOX-1 by hepatocytes competitively engulfed and metabolized ox-LDL, alleviating its resulting phenotypic transformation of VSMCs including migration, downregulation of contractile shape markers (smooth muscle α-actin (SMαA) and smooth muscle-22α (SM22α)), and upregulation of proliferative/migratory shape markers (osteopontin (OPN) and Vimentin) as well as foaminess and apoptosis, thereby alleviating NA, which independent of low-density lipoprotein (LDL) lowering treatment (evolocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9)). Mechanistically, we found that overexpression of LOX-1 in hepatocytes competitively engulfed and metabolized ox-LDL through upregulation of arachidonate-15-lipoxygenase (ALOX15), which further upregulated scavenger receptor class B type I (SRBI) and ATP-binding cassette transporter A1 (ABCA1). In conclusion, the overexpression of LOX-1 in liver protects VSMCs from phenotypic transformation and wire injury induced carotid neoatherosclerosis through ALOX15.

Hematopoietic 12/15-lipoxygenase activity negatively contributes to fungal-associated allergic asthma.

Asthma is one of the most common noncommunicable diseases in the world. Approximately 30% of severe cases are associated with fungal sensitization, often associated with allergy to the opportunistic mold Aspergillus fumigatus. Leukotrienes, immunopathogenic mediators derived from the metabolism of arachidonic acid (AA) by 5-lipoxygenase (5-LOX), are often elevated in severe asthma. As such, these mediators are Food and Drug Administration-approved therapeutic targets of the antiasthmatic drugs Zileuton/Zyflo and Singulair/Montelukast. A second enzyme involved in AA metabolism is 12/15-lipoxygenase (12/15-LOX; Alox15). Here, C57BL/6 wild-type (WT) mice subjected to experimental fungal asthma had increased expression of Alox15 mRNA and increased levels of 12-HETE, a product of 12/15-LOX activity, in the lung when compared with naïve and vehicle-treated mice. Mice deficient in 12/15-LOX (Alox15-/-) demonstrated better lung function, as measured by airway hyperresponsiveness (AHR), during fungal asthma. Histological assessment revealed reduced inflammation in the lungs of Alox15-/- mice compared with WT mice, which was corroborated by flow cytometric analysis of multiple myeloid (eosinophils and neutrophils) and lymphoid (CD4+ T and γδ T) cell populations. This was further supported by decreased levels of specific chemokines that promote the recruitment of these cells. Likewise, type 1 and 2, but not type 17 cytokines, were significantly lower in the lungs of Alox15-/- mice. Bone marrow chimera studies revealed that the presence of 12/15-LOX in hematopoietic cells contributed to AHR during fungal asthma. Taken together, our data support the hypothesis that hematopoietic-associated 12/15-LOX contributes to type 1 and 2 responses and exacerbation of allergic fungal asthma.NEW & NOTEWORTHY Humans with asthma sensitized to fungi often have more severe asthma than those who are not sensitized to fungi. Products of arachidonic acid generated via 5-lipoxygenase are often elevated in severe asthma and are successful FDA-approved drug targets. Less understood is the role of products generated via 12/15-lipoxygenase. We demonstrate that 12/15-lipoxygenase expression in hematopoietic cells contributes to type 1 and 2 responses and impaired lung function during allergic fungal asthma.

12/15-lipoxygenase activity promotes efficient inflammation resolution in a murine model of Lyme arthritis.

Infection of C3H/HeJ (C3H) mice with Borrelia burgdorferi results in the development of a robust inflammatory arthritis that peaks around 3-4 weeks post-infection and then spontaneously resolves over the next few weeks. Mice lacking cyclooxygenase (COX)-2 or 5-lipoxygenase (5-LO) activity develop arthritis similar to wild-type mice but display delayed or prolonged joint resolution. Since 12/15-lipoxygenase (12/15-LO) activity is generally down-stream of both COX-2 and 5-LO activity and results in the production of pro-resolution lipids such as lipoxins and resolvins among others, we investigated the impact of 12/15-LO deficiency on the resolution of Lyme arthritis in mice on a C3H background. We found the expression of Alox15 (12/15-LO gene) peaked around 4-weeks post-infection in C3H mice suggesting a role for 12/15-LO in mediating arthritis resolution. A deficiency in 12/15-LO resulted in exacerbated ankle swelling and arthritis severity during the resolution phase without compromising anti-Borrelia antibody production and spirochete clearance. However, clearance of inflammatory cells was impeded. Therapeutic treatment of B. burgdorferi-infected C3H mice with lipoxin A4 (LXA4) near the peak of disease resulted in significantly decreased ankle swelling and a switch of joint macrophages to a resolving phenotype but did not directly impact arthritis severity. These results demonstrate that 12/15-LO lipid metabolites are important components of inflammatory arthritis resolution in murine Lyme arthritis and may be a therapeutic target for treatment of joint edema and pain for Lyme arthritis patients without compromising spirochete clearance.

Ovariectomy Via 12/15-lipoxygenase Augments Angiotensin II-Induced Hypertension and Its Pathogenesis in Female Mice.

BACKGROUND: Ang II (angiotensin II) releases arachidonic acid from tissue phospholipids that is metabolized by 12/15-lipoxygenase (ALOX15), generating 12(S)- and 15(S)-hydroxyeicosatetraenoic acid (HETE), which have been implicated in cardiovascular and renal diseases. In this study, we tested the hypothesis that ovariectomy augments Ang II-induced hypertension and renal pathophysiological changes via ALOX15 activation in female mice. METHODS: Ang II (700 ng/kg/min) was infused subcutaneously by osmotic pumps for 2 weeks in intact and ovariectomized wild-type and Alox15 knockout (ALOX15KO) female mice for evaluation of hypertension and associated pathogenesis. RESULTS: Ang II increased blood pressure, impaired autonomic function, and increased renal reactive oxygen species production and plasma 12(S)-HETE level without altering renal function in intact wild-type mice. However, in OVX-wild-type mice with depleted plasma 17ß-estradiol, the effects of Ang II on blood pressure, autonomic impairment, renal reactive oxygen species production, and plasma 12(S)- but not 15(S)-HETE was markedly enhanced. In OVX-wild-type mice, Ang II also increased renal alox15 mRNA, urine 12(S)-HETE, water intake, urine output, decreased osmolality, increased urinary excretion of vasopressin prosegment copeptin, protein/creatinine ratio, and caused renal hypertrophy, fibrosis, and inflammation. These effects of Ang II were attenuated in ALOX15KO mice. CONCLUSIONS: These data suggest that 17ß-estradiol protects against Ang II-induced hypertension and associated pathogenesis in female mice, most likely via inhibition of ALOX15-arachidonic acid derived production of 12(S)-HETE. Therefore, the selective inhibitors of ALOX15 or 12(S)-HETE receptor antagonists could be useful for treating hypertension and its pathogenesis in postmenopausal, hypoestrogenic women, or females with ovarian failure.

Functional Characterization of Transgenic Mice Overexpressing Human 15-Lipoxygenase-1 (ALOX15) under the Control of the aP2 Promoter.

Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory, hyperproliferative, neurodegenerative, and metabolic diseases, but the physiological function of ALOX15 still remains a matter of discussion. To contribute to this discussion, we created transgenic mice (aP2-ALOX15 mice) expressing human ALOX15 under the control of the aP2 (adipocyte fatty acid binding protein 2) promoter, which directs expression of the transgene to mesenchymal cells. Fluorescence in situ hybridization and whole-genome sequencing indicated transgene insertion into the E1-2 region of chromosome 2. The transgene was highly expressed in adipocytes, bone marrow cells, and peritoneal macrophages, and ex vivo activity assays proved the catalytic activity of the transgenic enzyme. LC-MS/MS-based plasma oxylipidome analyses of the aP2-ALOX15 mice suggested in vivo activity of the transgenic enzyme. The aP2-ALOX15 mice were viable, could reproduce normally, and did not show major phenotypic alterations when compared with wildtype control animals. However, they exhibited gender-specific differences with wildtype controls when their body-weight kinetics were evaluated during adolescence and early adulthood. The aP2-ALOX15 mice characterized here can now be used for gain-of-function studies evaluating the biological role of ALOX15 in adipose tissue and hematopoietic cells.

Alox15/15-HpETE Aggravates Myocardial Ischemia-Reperfusion Injury by Promoting Cardiomyocyte Ferroptosis.

BACKGROUND: Myocardial ischemia-reperfusion (I/R) injury causes cardiac dysfunction to myocardial cell loss and fibrosis. Prevention of cell death is important to protect cardiac function after I/R injury. The process of reperfusion can lead to multiple types of cardiomyocyte death, including necrosis, apoptosis, autophagy, and ferroptosis. However, the time point at which the various modes of cell death occur after reperfusion injury and the mechanisms underlying ferroptosis regulation in cardiomyocytes are still unclear. METHODS: Using a left anterior descending coronary artery ligation mouse model, we sought to investigate the time point at which the various modes of cell death occur after reperfusion injury. To discover the key molecules involved in cardiomyocyte ferroptosis, we performed a metabolomics study. Loss/gain-of-function approaches were used to understand the role of 15-lipoxygenase (Alox15) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α) in myocardial I/R injury. RESULTS: We found that apoptosis and necrosis occurred in the early phase of I/R injury, and that ferroptosis was the predominant form of cell death during the prolonged reperfusion. Metabolomic profiling of eicosanoids revealed that Alox15 metabolites accumulated in ferroptotic cardiomyocytes. We demonstrated that Alox15 expression was specifically increased in the injured area of the left ventricle below the suture and colocalized with cardiomyocytes. Furthermore, myocardial-specific knockout of Alox15 in mice alleviated I/R injury and restored cardiac function. 15-Hydroperoxyeicosatetraenoic acid (15-HpETE), an intermediate metabolite derived from arachidonic acid by Alox15, was identified as a trigger for cardiomyocyte ferroptosis. We explored the mechanism underlying its effects and found that 15-HpETE promoted the binding of Pgc1α to the ubiquitin ligase ring finger protein 34, leading to its ubiquitin-dependent degradation. Consequently, attenuated mitochondrial biogenesis and abnormal mitochondrial morphology were observed. ML351, a specific inhibitor of Alox15, increased the protein level of Pgc1α, inhibited cardiomyocyte ferroptosis, protected the injured myocardium, and caused cardiac function recovery. CONCLUSIONS: Together, our results established that Alox15/15-HpETE-mediated cardiomyocyte ferroptosis plays an important role in prolonged I/R injury.

Knock-out of 5-lipoxygenase in overexpressing tumor cells-consequences on gene expression and cellular function.

5-Lipoxygenase (5-LO), the central enzyme in the biosynthesis of leukotrienes, is frequently expressed in human solid malignancies even though the enzyme is not present in the corresponding healthy tissues. There is little knowledge on the consequences of this expression for the tumor cells regarding gene expression and cellular function. We established a knockout (KO) of 5-LO in different cancer cell lines (HCT-116, HT-29, U-2 OS) and studied the consequences on global gene expression using next generation sequencing. Furthermore, cell viability, proliferation, migration and multicellular tumor spheroid (MCTS) formation were studied in these cells. Our results show that 5-LO influences the gene expression and cancer cell function in a cell type-dependent manner. The enzyme affected genes involved in cell adhesion, extracellular matrix formation, G protein signaling and cytoskeleton organization. Furthermore, absence of 5-LO elevated TGFß2 expression in HCT-116 cells while MCP-1, fractalkine and platelet-derived growth factor expression was attenuated in U-2 OS cells suggesting that tumor cell-derived 5-LO shapes the tumor microenvironment. In line with the gene expression data, KO of 5-LO had an impact on cell proliferation, motility and MCTS formation. Interestingly, pharmacological inhibition of 5-LO only partly mimicked the KO suggesting that also noncanonical functions are involved.