Activation of the 15-lipoxygenase pathway in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and an intolerance of medications that inhibit cyclooxygenase-1. Patients with AERD have more severe upper and lower respiratory tract disease than do aspirin-tolerant patients with CRSwNP. A dysregulation in arachidonic acid metabolism is thought to contribute to the enhanced sinonasal inflammation in AERD. OBJECTIVE: Our aim was to utilize an unbiased approach investigating arachidonic acid metabolic pathways in AERD. METHODS: Single-cell RNA sequencing (10× Genomics, Pleasanton, Calif) was utilized to compare the transcriptional profile of nasal polyp (NP) cells from patients with AERD and patients with CRSwNP and map differences in the expression of select genes among identified cell types. Findings were confirmed by traditional real-time PCR. Lipid mediators in sinonasal tissue were measured by mass spectrometry. Localization of various proteins within NPs was assessed by immunofluorescence. RESULTS: The gene encoding for 15-lipooxygenase (15-LO), ALOX15, was significantly elevated in NPs of patients with AERD compared to NPs of patients with CRSwNP (P < .05) or controls (P < .001). ALOX15 was predominantly expressed by epithelial cells. Expression levels significantly correlated with radiographic sinus disease severity (r = 0.56; P < .001) and were associated with asthma. The level of 15-oxo-eicosatetraenoic acid (15-Oxo-ETE), a downstream product of 15-LO, was significantly elevated in NPs from patients with CRSwNP (27.93 pg/mg of tissue) and NPs from patients with AERD (61.03 pg/mg of tissue) compared to inferior turbinate tissue from controls (7.17 pg/mg of tissue [P < .001]). Hydroxyprostaglandin dehydrogenase, an enzyme required for 15-Oxo-ETE synthesis, was predominantly expressed in mast cells and localized near 15-LO+ epithelium in NPs from patients with AERD. CONCLUSIONS: Epithelial and mast cell interactions, leading to the synthesis of 15-Oxo-ETE, may contribute to the dysregulation of arachidonic acid metabolism via the 15-LO pathway and to the enhanced sinonasal disease severity observed in AERD.

12/15-lipoxygenase orchestrates the clearance of apoptotic cells and maintains immunologic tolerance.

Noninflammatory clearance of apoptotic cells (ACs) is crucial to maintain self-tolerance. Here, we have reported a role for the enzyme 12/15-lipoxygenase (12/15-LO) as a central factor governing the sorting of ACs into differentially activated monocyte subpopulations. During inflammation, uptake of ACs was confined to a population of 12/15-LO-expressing, alternatively activated resident macrophages (resMΦ), which blocked uptake of ACs into freshly recruited inflammatory Ly6C(hi) monocytes in a 12/15-LO-dependent manner. ResMΦ exposed 12/15-LO-derived oxidation products of phosphatidylethanolamine (oxPE) on their plasma membranes and thereby generated a sink for distinct soluble receptors for ACs such as milk fat globule-EGF factor 8, which were essential for the uptake of ACs into inflammatory monocytes. Loss of 12/15-LO activity, in turn, resulted in an aberrant phagocytosis of ACs by inflammatory monocytes, subsequent antigen presentation of AC-derived antigens, and a lupus-like autoimmune disease. Our data reveal an unexpected key role for enzymatic lipid oxidation during the maintenance of self-tolerance.

Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.

Although autacoids primarily derived from the cyclooxygenase-2 and 5-lipoxygenase (LOX) pathways are essential mediators of inflammation, endogenous specialized proresolving mediators (SPMs) act as robust agonists of resolution. SPM biosynthesis is initiated by the conversion of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid primarily via the 12/15-LOX pathway. Although 12/15-LOX activity is prominent in the cornea, the role of SPM pathway activation during infection remains largely unknown and is the focus of the current study. Pseudomonas keratitis was induced in resistant BALB/c and susceptible C57BL/6 (B6) mice. Biosynthetic pathways for proinflammatory autacoids and SPMs were assessed. Divergent lipid mediator profiles demonstrate the importance of 15-LOX pathways in the pathogenesis of ocular infectious disease. Results indicate that an imbalance of LOX enzymatic pathways contributes to susceptibility observed in B6 mice where deficient activation of SPM circuits, as indicated by reduced 15-hydroxy-eicosatetraenoic acid and 17-hydroxydocosahexaenoic acid levels, prevented transition toward resolution and led to chronic inflammation. In sharp contrast, BALB/c mice demonstrated a well-balanced axis of 5-LOX/12-LOX/15-LOX pathways, resulting in sufficient proresolving bioactive metabolite formation and immune homeostasis. Furthermore, a novel immunoregulatory role for 15-LOX was revealed in inflammatory cells (polymorphonuclear leukocytes and macrophages), which influenced phagocytic activity. These data provide evidence that SPM circuits are essential for host defense during bacterial keratitis.-Carion, T. W., Greenwood, M., Ebrahim, A. S., Jerome, A., Suvas, S., Gronert, K., Berger, E. A. Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.

Prominent release of lipoxygenase generated mediators in a murine house dust mite-induced asthma model.

The profile of activation of lipid mediator (LM) pathways in asthmatic airway inflammation remains unclear. This experimental study quantified metabolite levels of ω3-, ω6- and ω9-derived polyunsaturated fatty acids in bronchoalveolar lavage fluid (BALF) after 4-weeks of repeated house dust mite (HDM) exposure in a murine (C57BL/6) asthma model. The challenge induced airway hyperresponsiveness, pulmonary eosinophil infiltration, but with low and unchanged mast cell numbers. Of the 112 screened LMs, 26 were increased between 2 to >25-fold in BALF with HDM treatment (p < 0.05, false discovery rate = 5%). While cysteinyl-leukotrienes were the most abundant metabolites at baseline, their levels did not increase after HDM treatment, whereas elevation of PGD2, LTB4 and multiple 12/15-lipoxygenase products, such as 5,15-DiHETE, 15-HEDE and 15-HEPE were observed. We conclude that this model has identified a global lipoxygenase activation signature, not linked to mast cells, but with aspects that mimic chronic allergic airway inflammation in asthma.

Global assessment of oxidized free fatty acids in brain reveals an enzymatic predominance to oxidative signaling after trauma.

Traumatic brain injury (TBI) is a major health problem associated with significant morbidity and mortality. The pathophysiology of TBI is complex involving signaling through multiple cascades, including lipid peroxidation. Oxidized free fatty acids, a prominent product of lipid peroxidation, are potent cellular mediators involved in induction and resolution of inflammation and modulation of vasomotor tone. While previous studies have assessed lipid peroxidation after TBI, to our knowledge no studies have used a systematic approach to quantify the global oxidative changes in free fatty acids. In this study, we identified and quantified 244 free fatty acid oxidation products using a newly developed global liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. This methodology was used to follow the time course of these lipid species in the contusional cortex of our pediatric rat model of TBI. We show that oxidation peaked at 1h after controlled cortical impact and was progressively attenuated at 4 and 24h time points. While enzymatic and non-enzymatic pathways were activated at 1h post-TBI, enzymatic lipid peroxidation was the predominant mechanism with 15-lipoxygenase (LOX) contributing to the majority of total oxidized fatty acid content. Pro-inflammatory lipid mediators were significantly increased at 1 and 4h after TBI with return to basal levels by 24h. Anti-inflammatory lipid mediators remained significantly increased across all three time points, indicating an elevated and sustained anti-inflammatory response following TBI.

The double-edged role of 12/15-lipoxygenase during inflammation and immunity.

12/15-Lipoxygenase (12/15-LOX) mediates the enzymatic oxidation of polyunsaturated fatty acids, thereby contributing to the generation of various bioactive lipid mediators. Although 12/15-LOX has been implicated in the pathogenesis of multiple chronic inflammatory diseases, its physiologic functions seem to include potent immune modulatory properties that physiologically contribute to the resolution of inflammation and the clearance of inflammation-associated tissue damage. This review aims to give a comprehensive overview about our current knowledge on the role of this enzyme during the regulation of inflammation and immunity. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.

Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and 39%, respectively) yet has kinetics comparably fast versus those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 µM(-1) s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates (e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted LoxA over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilm mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the host airway epithelium, suggesting a role for LoxA in mediating bacterium-host interactions during colonization.

An integrated expression phenotype mapping approach defines common variants in LEP, ALOX15 and CAPNS1 associated with induction of IL-6.

Interleukin-6 (IL-6) is an important modulator of inflammation and immunity whose dysregulation is associated with a number of disease states. There is evidence of significant heritability in inter-individual variation in IL6 gene expression but the genetic variants responsible for this remain to be defined. We adopted a combined approach of mapping protein and expression quantitative trait loci in peripheral blood mononuclear cells using high-density single-nucleotide polymorphism (SNP) typing for approximately 2000 loci implicated in cardiovascular, metabolic and inflammatory syndromes to show that common SNP markers and haplotypes of LEP (encoding leptin) associate with a 1.7- to 2-fold higher level of lipopolysaccharide (LPS)-induced IL-6 expression. We subsequently demonstrate that basal leptin expression significantly correlates with LPS-induced IL-6 expression and that the same variants at LEP which associate with IL-6 expression are also major determinants of leptin expression in these cells. We find that variation involving two other genomic regions, CAPNS1 (encoding calpain small subunit 1) and ALOX15 (encoding arachidonate 15-lipoxygenase), show significant association with IL-6 expression. Although this may be a subset of all such trans-acting effects, we find that the same ALOX15 variants are associated with induced expression of tumour necrosis factor and IL-1beta consistent with a broader role in acute inflammation for ALOX15. This study provides evidence of novel genetic determinants of IL-6 production with implications for understanding susceptibility to inflammatory disease processes and insight into cross talk between metabolic and inflammatory pathways. It also provides proof of concept for use of an integrated expression phenotype mapping approach.

Monocyte 15-lipoxygenase gene expression requires ERK1/2 MAPK activity.

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cδ as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13-mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cδ pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2-dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies characterize an important regulatory role for ERK1/2 in mediating IL-13-induced monocyte 15-LO expression via the transcription factors Egr-1 and CREB.

12/15-Lipoxygenase Regulates IL-33-Induced Eosinophilic Airway Inflammation in Mice.

Dysregulated fatty acid metabolism is clinically associated with eosinophilic allergic diseases, including severe asthma and chronic rhinosinusitis. This study aimed to demonstrate the role of 12/15-lipoxygenase (12/15-LOX) in interleukin (IL)-33-induced eosinophilic airway inflammation; to this end, we used 12/15-LOX-deficient mice, which displayed augmented IL-33-induced lung inflammation, characterized by an increased number of infiltrated eosinophils and group 2 innate lymphoid cells (ILC2s) in the airway. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics revealed that the levels of a series of 12/15-LOX-derived metabolites were significantly decreased, and application of 14(S)-hydroxy docosahexaenoic acid (HDoHE), a major 12/15-LOX-derived product, suppressed IL-33-mediated eosinophilic inflammation in 12/15-LOX-deficient mice. Using bioactive lipid screening, we found that 14(S)-HDoHE and 10(S),17(S)-diHDoHE markedly attenuated ILC2 proliferation and cytokine production at micromolar concentration in vitro. In addition, maresin 1 (MaR1) and resolvin D1 (RvD1), 12/15-LOX-derived specialized proresolving mediators (SPMs), inhibited cytokine production of ILC2s at nanomolar concentration. These findings demonstrate the protective role of endogenous 12/15-LOX-derived lipid mediators in controlling ILC2-mediated eosinophilic airway inflammation and related diseases. Thus, 12/15-LOX-derived lipid mediators may represent a potential therapeutic strategy for ameliorating airway inflammation-associated conditions.

Alpha-linolenic acid enhances the phagocytic and secretory functions of alternatively activated macrophages in part via changes to the oxylipin profile.

Alternatively activated macrophages are innate immune cells that contribute to resolution of inflammation and maintenance of homeostasis. Modulation of available fatty acid sources is thought to affect cellular physiology through a variety of mechanisms, including through alterations to the profile of oxygenated free fatty acid metabolites, called oxylipins, produced in a cell type specific manner. Here, we investigated how treatment with the plant-sourced omega-3 fatty acid α-linolenic acid (ALA) affects the oxylipin profile and functional capacity of a cell culture model of human alternatively activated (M2a-like) macrophages. In a targeted but unbiased screen, ALA enhanced the production of oxylipins from all polyunsaturated fatty acid (PUFA) precursors, with oxylipins derived from ALA being enhanced the most. Consistently, ALA treatment enhanced the expression of both cytoplasmic and calcium-independent phospholipase A2. At a functional level, ALA treatment increased phagocytic activity and altered production of the chemokine MCP-1 by M2a-like cells in a manner dependent on the time of treatment. ALA treatment during polarization increased MCP-1 secretion, which was sensitive to pharmacological inhibition of 15-LOX-1 by ML351. Thus, ALA modulates the phenotype of alternatively activated macrophages, likely through its own LOX-derived oxylipins and/or through general modulation of oxylipin biosynthesis. These effects likely contribute to the overall anti-inflammatory benefit observed with ALA supplementation.

Protective activities of distinct omega-3 enriched oils are linked to their ability to upregulate specialized pro-resolving mediators.

Clinical studies using a range of omega-3 supplements have yielded conflicting results on their efficacy to control inflammation. Omega-3 fatty acids are substrate for the formation of potent immune-protective mediators, termed as specialized pro-resolving mediators (SPM). Herein, we investigated whether observed differences in the potencies of distinct omega-3 supplements were linked with their ability to upregulate SPM formation. Using lipid mediator profiling we found that four commercially available supplements conferred a unique SPM signature profile to human macrophages, with the overall increases in SPM concentrations being different between the four supplements. These increases in SPM concentrations were linked with an upregulation of macrophage phagocytosis and a decreased uptake of oxidized low-density lipoproteins. Pharmacological inhibition of two key SPM biosynthetic enzymes 5-Lipoxygenase or 15-Lipoxygenase reversed the macrophage-directed actions of each of the omega-3 supplements. Furthermore, administration of the two supplements that most potently upregulated macrophage SPM formation and reprogrammed their responses in vitro, to APOE-/- mice fed a western diet, increased plasma SPM concentrations and reduced vascular inflammation. Together these findings support the utility of SPM as potential prognostic markers in determining the utility of a given supplement to regulate macrophage responses and inflammation.

15-lipoxygenase metabolites play an important role in the development of a T-helper type 1 allergic inflammation induced by double-stranded RNA.

BACKGROUND: We recently demonstrated that the T-helper type 1 (Th1) immune response plays an important role in the development of non-eosinophilic inflammation induced by airway exposure of an allergen plus double-stranded RNA (dsRNA). However, the role of lipoxygenase (LO) metabolites in the development of Th1 inflammation is poorly understood. OBJECTIVE: To evaluate the role of LO metabolites in the development of Th1 inflammation induced by sensitization with an allergen plus dsRNA. METHODS: A Th2-allergic inflammation mouse model was created by an intraperitoneal injection of lipopolysaccharide-depleted ovalbumin (OVA, 75 microg) and alum (2 mg) twice, and the Th1 model was created by intranasal application of OVA (75 microg) and synthetic dsRNA [10 microg of poly(I : C)] four times, followed by an intranasal challenge with 50 microg of OVA four times. The role of LO metabolites was evaluated using two approaches: a transgenic approach using 5-LO(-/-) and 15-LO(-/-) mice, and a pharmacological approach using inhibitors of cysteinyl leucotriene receptor-1 (cysLTR1), LTB4 receptor (BLT1), and 15-LO. RESULTS: We found that the Th1-allergic inflammation induced by OVA+dsRNA sensitization was similar between 5-LO(-/-) and wild-type (WT) control mice, although Th2 inflammation induced by sensitization with OVA+alum was reduced in the former group. In addition, dsRNA-induced Th1 allergic inflammation, which is associated with down-regulation of 15-hydroxyeicosateraenoic acids production, was not affected by treatment with cysLTR1 or BLT1 inhibitors, whereas it was significantly lower in 12/15-LO(-/-) mice compared with WT control mice. Moreover, dsRNA-induced allergic inflammation and the recruitment of T cells following an allergen challenge were significantly inhibited by treatment with a specific 15-LO inhibitor (PD146176). CONCLUSION: 15-LO metabolites appear to be important mediators in the development of Th1-allergic inflammation induced by sensitization with an allergen plus dsRNA. Our findings suggest that the 15-LO pathway is a novel therapeutic target for the treatment of virus-associated asthma characterized by Th1 inflammation.

A dual role of 12/15-lipoxygenase in LPS-induced acute renal inflammation and injury.

Recent studies suggest a potential role of bioactive lipids in acute kidney injury induced by lipopolysaccharide (LPS). The current study was designed to determine the profiling activities of various polyunsaturated fatty acid (PUFA) metabolizing enzymes, including lipoxygenases (LO), cyclooxygenase, and cytochrome P450 in the plasma of LPS-injected mice using LC-MS. Heat map analysis revealed that out of 126 bioactive lipids screened, only the 12/15-LO metabolite, 12-HETE, had a significant (2.24 ±â€¯0.4) fold increase relative to control (P = 0.0001) after Bonferroni Correction (BCF α = 0.003). We then determined the role of the 12/15-LO in LPS-induced acute kidney injury using genetic and pharmacological approaches. Treatment of LPS injected mice with the 12/15-LO inhibitor, baicalein, significantly reduced levels of renal injury and inflammation markers including urinary thiobarbituric acid reactive substance (TBARs), urinary monocyte chemoattractant protein-1 (MCP-1), renal interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Similarly, knocking-out of 12/15-LO reduced levels of renal inflammation and injury markers elicited by LPS injection. Next, we tested whether exogenous supplementation with docosahexaenoic acid (DHA) as a substrate would divert the role of 12/15-LO from being pro-inflammatory to anti-inflammatory via increased production of the anti-inflammatory metabolite. DHA treatment restored the decreased in plasma level of resolvin D2 (RvD2) and reduced renal injury in LPS-injected mice whereas DHA treatment failed to provide any synergistic effects in reducing renal injury in LPS injected 12/15-LO knock-out mice. The ability of RvD2 to protect kidney against LPS-induced renal injury was further confirmed by exogenous RvD2 which significantly reduced the elevation in renal injury in LPS injected mice. These data suggest a double-edged sword role of 12/15-LO in LPS-induced acute renal inflammation and injury, depending on the type of substrate available for its activity.

Inflammation and immune regulation by 12/15-lipoxygenases.

12/15-Lipoxygenases (12/15-LOX) are members of the LOX family, which are expressed in mammals by monocytes and macrophages following induction by the T helper type 2 cytokines, interleukins-4 and -13. They oxygenate free polyenoic fatty acids but also ester lipids and even complex lipid-protein assemblies such as biomembranes and lipoproteins. The primary oxidation products are either reduced by glutathione peroxidases to corresponding hydroxy derivatives or metabolized into secondary oxidized lipids including leukotrienes, lipoxins and hepoxilins, which act as lipid mediators. Examination of knockout and transgenic animals revealed important roles for 12/15-LOX in inflammatory diseases, including atherosclerosis, cancer, osteoporosis, angiotension II-dependent hypertension and diabetes. In vitro studies suggested 12/15-LOX products as coactivators of peroxisomal proliferator activating-receptors (PPAR), regulators of cytokine generation, and modulators of gene expression related to inflammation resolution. Despite much work in this area, the biochemical mechanisms by which 12/15-LOX regulates physiological and pathological immune cell function are not fully understood. This review will summarize the biochemistry and tissue expression of 12/15-LOX and will describe the current knowledge regarding its immunobiology and regulation of inflammation.

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.

Reduction of isoforms of 15-lipoxygenase (15-LOX)-1 and 15-LOX-2 in human breast cancer.

15-lipoxygenase (15-LOX) belongs to the structurally and functionally related nonheme iron dioxygenases family. It has two isoforms, type-1 (leukocyte type) and type-2 (epidermis type) and converts arachidonic acid to eicosanoids including the anti-cancer 13-HODE. In the current study, we investigate the expression of both isoforms of 15-LOX in human breast cancer (n=120) and normal mammary tissues (n=32), using immunohistochemistry and quantitative analysis of the gene transcripts. Both 15-LOX-1 and 15-LOX-2 were found in normal mammary epithelial cells and in vascular endothelial cells. The staining of both 15-LOX-1 and 15-LOX-2 was markedly weaker in breast cancer cells. Using quantitative analysis, it was found that the 15-LOX-1 and 15-LOX-2:CK19 ratios were lower in breast tumour tissues, compared with normal tissues (P=0.05 and P=0.035, respectively). Although no significant correlation was seen between either isoforms and nodal status and tumour grade, significantly lower ratio of 15-LOX-2:CK19 was seen in late stage breast tumours. Both 15-LOX-2 and 15-LOX-1 were found to be at significantly lower levels in tumours from patients who developed metastasis (P=0.0018 for 15-LOX-2 and P=0.031 for 15-LOX-1, compared with patients who remained disease free), and in patients who died of breast cancer related causes (P=0.043 and P=0.020 vs disease-free group, for 15-LOX-2 and 15-LOX-1, respectively). It was also demonstrated that ER-positive tumours had significantly lower levels of 15-LOX-2, but not 15-LOX-1, compared with ER-negative tumours (P=0.031). Finally, the study has shown that the 15LOX1:15LOX2 ratio had a strong value in predicting clinical outcome. Patients who developed metastasis, local recurrence and died of breast cancer had significantly lower ratio compared with those who remained disease free (P=0.0057, P=0.0075, P=0.0091, respectively). In conclusion, the current study reports aberrant expression of both isoforms of 15-LOX, 15-LOX-1 and 15-LOX-2, in human breast cancer. The reduction is correlated with the disease progression of breast cancer and a poor clinical outcome. The study has also reported a link between 15-LOX-2 and oestrogen receptor status in breast tumours. Both isoforms of 15-lipoxygenase have a tumour suppressing role in breast cancer.

Hypoxia increases LDL oxidation and expression of 15-lipoxygenase-2 in human macrophages.

OBJECTIVE: Macrophage-mediated oxidation of low-density lipoprotein (LDL) by enzymes, such as the lipoxygenases, is considered of major importance for the formation of oxidized LDL during atherogenesis. Macrophages have been identified in hypoxic areas in atherosclerotic plaques. METHODS AND RESULTS: To investigate the role of hypoxia in macrophage-mediated LDL oxidation, we incubated human monocyte-derived macrophages with LDL under normoxic (21% O2) or hypoxic (0% O2) conditions. The results showed that hypoxic macrophages oxidized LDL to a significantly higher extent than normoxic cells. Interestingly, the mRNA and protein expression of 15-lipoxygenase-2 (15-LOX-2) as well as the activity of this enzyme are elevated in macrophages incubated at hypoxia. Both the unspliced 15-LOX-2 and the spliced variant 15-LOX-2sv-a are found in macrophages. In addition, 15-LOX-2 was identified in carotid plaques in some macrophage-rich areas but was only expressed at low levels in nondiseased arteries. CONCLUSIONS: In summary, these observations show for the first time that 15-LOX-2 is expressed in hypoxic macrophages and in atherosclerotic plaques and suggest that 15-LOX-2 may be one of the factors involved in macrophage-mediated LDL oxidation at hypoxia.

Sonic hedgehog-responsive lipoxygenases and cyclooxygenase-2 modulate Dectin-1-induced inflammatory cytokines.

Immune responses during fungal infections are predominately mediated by 5/15-lipoxygenases (LO)- or cyclooxygenase (COX)-2-catalysed bioactive eicosanoid metabolites like leukotrienes, lipoxins and prostaglandins. Although few host mediators of fungi-triggered eicosanoid production have been established, the molecular mechanism of expression and regulation of 5-LO, 15-LO and COX-2 are not well-defined. Here, we demonstrate that, macrophages infected with representative fungi Candida albicans, Aspergillus flavus or Aspergillus fumigatus or those treated with Curdlan, a selective agonist of pattern recognition receptor for fungi Dectin-1, displays increased expression of 5-LO, 15-LO and COX-2. Interestingly, Dectin-1-responsive Syk pathway activates mTOR-sonic hedgehog (SHH) signaling cascade to stimulate the expression of these lipid metabolizing enzymes. Loss-of-function analysis of the identified intermediaries indicates that while Syk-mTOR-SHH pathway-induced 5-LO and 15-LO suppressed the Dectin-1-responsive pro-inflammatory signature cytokines like TNF-α, IL-1ß and IL-12, Syk-mTOR-SHH-induced COX-2 positively regulated these cytokines. Dectin-1-stimulated IL-6, however, is dependent on 5-LO, 15-LO and COX-2 activity. Together, the current study establishes Dectin-1-arbitrated host mediators that direct the differential regulation of immune responses during fungal infections and thus are potential candidates of therapeutic intervention.

12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

The mechanisms underlying asthmatic airway epithelial injury are not clear. 12/15-lipoxygenase (an ortholog of human 15-LOX-1), which is induced by IL-13, is associated with mitochondrial degradation in reticulocytes at physiological conditions. In this study, we showed that 12/15-LOX expressed in nonepithelial cells caused epithelial injury in asthma pathogenesis. While 12/15-LOX overexpression or IL-13 administration to naïve mice showed airway epithelial injury, 12/15-LOX knockout/knockdown in allergic mice reduced airway epithelial injury. The constitutive expression of 15-LOX-1 in bronchial epithelia of normal human lungs further indicated that epithelial 15-LOX-1 may not cause epithelial injury. 12/15-LOX expression is increased in various inflammatory cells in allergic mice. Though non-epithelial cells such as macrophages or fibroblasts released 12/15-LOX metabolites upon IL-13 induction, bronchial epithelia didn't release. Further 12-S-HETE, arachidonic acid metabolite of 12/15-LOX leads to epithelial injury. These findings suggested 12/15-LOX expressed in non-epithelial cells such as macrophages and fibroblasts leads to bronchial epithelial injury.