Åsa Brunnström

Oxylipin Profiling of the Hypersensitive Response in Arabidopsis thaliana FORMATION OF A NOVEL OXO-PHYTODIENOIC ACID-CONTAINING GALACTOLIPID, ARABIDOPSIDE E

Oxidation products of unsaturated fatty acids, collectively known as oxylipins, function as signaling molecules in plants during development, wounding, and insect and pathogen attack. Certain oxylipins are also known to have direct cytotoxic effects on pathogens. We used inducible expression of bacterial avirulence proteins in planta to study the involvement of oxylipins in race-specific defense against bacterial pathogens. We demonstrate that recognition of the Pseudomonas syringae avirulence protein AvrRpm1 induces 9- and 13-lipoxygenase-dependent oxylipin synthesis in Arabidopsis thaliana. The major oxylipins accumulated were jasmonic acid, 12-oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. The majority of the newly formed oxylipins (>90%) was found to be esterified to glycerolipids, whereby 12-oxo-phytodienoic acid and dinor-oxo-phytodienoic acid were found to be esterified to a novel galactolipid. The structure of the substance was determined as a monogalactosyldiacylglycerol containing two 12-oxo-phytodienoic acids and one dinor-oxo-phytodienoic acid acyl chain and was given the trivial name arabidopside E. This substance accumulated to surprisingly high levels, 7-8% of total lipid content, and was shown to inhibit growth of a bacterial pathogen in vitro. Arabidopside E was formed also after recognition of the avirulence protein AvrRpt2, suggesting that this could be a conserved feature of defense reactions against bacterial pathogens. In conclusion, the data presented suggest a role of enzymatically formed oxylipins, especially the octadecanoids and arabidopside E in race-specific resistance against bacterial pathogens.

Eoxins are proinflammatory arachidonic acid metabolites produced via the 15-lipoxygenase-1 pathway in human eosinophils and mast cells

Human eosinophils contain abundant amounts of 15-lipoxygenase (LO)-1. The biological role of 15-LO-1 in humans, however, is unclear. Incubation of eosinophils with arachidonic acid led to formation of a product with a UV absorbance maximum at 282 nm and shorter retention time than leukotriene (LT)C4 in reverse-phase HPLC. Analysis with positive-ion electrospray tandem MS identified this eosinophil metabolite as 14,15-LTC4. This metabolite could be metabolized to 14,15-LTD4 and 14,15-LTE4 in eosinophils. Because eosinophils are such an abundant source of these metabolites and to avoid confusion with 5-LO-derived LTs, we suggest the names eoxin (EX)C4, -D4, and -E4 instead of 14,15-LTC4, -D4, and -E4, respectively. Cord blood-derived mast cells and surgically removed nasal polyps from allergic subjects also produced EXC4. Incubation of eosinophils with arachidonic acid favored the production of EXC4, whereas challenge with calcium ionophore led to exclusive formation of LTC4. Eosinophils produced EXC4 after challenge with the proinflammatory agents LTC4, prostaglandin D2, and IL-5, demonstrating that EXC4 can be synthesized from the endogenous pool of arachidonic acid. EXs induced increased permeability of endothelial cell monolayer in vitro, indicating that EXs can modulate and enhance vascular permeability, a hallmark of inflammation. In this model system, EXs were 100 times more potent than histamine and almost as potent as LTC4 and LTD4. Taken together, this article describes the formation of proinflammatory EXs, in particular in human eosinophils but also in human mast cells and nasal polyps.

Oxo-Phytodienoic Acid-Containing Galactolipids in Arabidopsis : Jasmonate Signaling Dependence

The jasmonate family of phytohormones, as represented by 12-oxo-phytodienoic acid (OPDA), dinor-phytodienoic acid (dn-OPDA), and jasmonic acid in Arabidopsis (Arabidopsis thaliana), has been implicated in a vast array of different developmental processes and stress responses. Recent reports indicate that OPDA and dn-OPDA occur not only as free acids in Arabidopsis, but also as esters with complex lipids, so-called arabidopsides. Recently, we showed that recognition of the two bacterial effector proteins AvrRpm1 and AvrRpt2 induced high levels of a molecule consisting of two OPDAs and one dn-OPDA esterified to a monogalactosyl diacylglycerol moiety, named arabidopside E. In this study, we demonstrate that the synthesis of arabidopsides is mainly independent of the prokaryotic lipid biosynthesis pathway in the chloroplast, and, in addition to what previously has been reported, arabidopside E as well as an all-OPDA analog, arabidopside G, described here accumulated during the hypersensitive response and in response to wounding. We also show that different signaling pathways lead to the formation of arabidopsides during the hypersensitive response and the wounding response, respectively. However, the formation of arabidopsides during both responses is dependent on an intact jasmonate signaling pathway. Additionally, we report inhibition of growth of the fungal necrotrophic pathogen Botrytis cinerea and in planta release of free jasmonates in a time frame that overlaps with the observed reduction of arabidopside levels. Thus, arabidopsides may have a dual function: as antipathogenic substances and as storage compounds that allow the slow release of free jasmonates.

15-Lipoxygenase-1 induces expression and release of chemokines in cultured human lung epithelial cells

15-Lipoxygenase-1 (15-LOX-1) has been proposed to be involved in various physiological and pathophysiological activities such as inflammation, atherosclerosis, cell maturation, and tumorigenesis. Asthma and chronic obstructive pulmonary disease are associated with increased expression of 15-LOX-1 in bronchial epithelial cells, but the potential functions of 15-LOX-1 in airway epithelial cells have not been well clarified. To study the function of 15-LOX-1 in bronchial epithelial cells, we ectopically expressed 15-LOX-1 in the human lung epithelial cell line A549. We found that overexpression of 15-LOX-1 in A549 cells leads to increased release of the chemokines MIP-1alpha, RANTES, and IP-10, and thereby to increased recruitment of immature dendritic cells, mast cells, and activated T cells. These results suggest that an increased expression and activity of 15-LOX-1 in lung epithelial cells is a proinflammatory event in the pathogenesis of asthma and other inflammatory lung disorders.

Hodgkin Reed-Sternberg cells express 15-lipoxygenase-1 and are putative producers of eoxins in vivo

Classical Hodgkin lymphoma has unique clinical and pathological features and tumour tissue is characterized by a minority of malignant Hodgkin Reed–Sternberg cells surrounded by inflammatory cells. In the present study, we report that the Hodgkin lymphoma‐derived cell line L1236 has high expression of 15‐lipoxygenase‐1 and that these cells readily convert arachidonic acid to eoxin C4, eoxin D4 and eoxin E4. These mediators were only recently discovered in human eosinophils and mast cells and found to be potent proinflammatory mediators. Western blot and immunocytochemistry analyses of L1236 cells demonstrated that 15‐lipoxygenase‐1 was present mainly in the cytosol and that the enzyme translocated to the membrane upon calcium challenge. By immunohistochemistry of Hodgkin lymphoma tumour tissue, 15‐lipoxygenase‐1 was found to be expressed in primary Hodgkin Reed–Sternberg cells in 17 of 20 (85%) investigated biopsies. The enzyme 15‐lipoxygenase‐1, however, was not expressed in any of 10 biopsies representing nine different subtypes of non‐Hodgkin lymphoma. In essence, the expression of 15‐lipoxygenase‐1 and the putative formation of eoxins by Hodgkin Reed–Sternberg cells in vivo are likely to contribute to the inflammatory features of Hodgkin lymphoma. These findings may have important diagnostic and therapeutic implications in Hodgkin lymphoma. Furthermore, the discovery of the high 15‐lipoxygenase‐1 activity in L1236 cells demonstrates that this cell line comprises a useful model system to study the chemical and biological roles of 15‐lipoxygenase‐1.

The influence of aspirin on release of eoxin C4, leukotriene C4 and 15-HETE, in eosinophilic granulocytes isolated from patients with asthma.

Background: The effect of aspirin on the release of key arachidonic acid metabolites in activated eosinophils from subjects with aspirin-intolerant asthma (AIA) has not been investigated previously, despite the characteristic eosinophilia in AIA. Methods: Peripheral blood eosinophils were isolated from four groups of subjects: healthy volunteers (HV; n = 8), mild asthma (MA; n = 8), severe asthma (SA; n = 9) and AIA (n = 7). In the absence or presence of lysine-aspirin, eosinophils were stimulated with arachidonic acid or calcium ionophore to trigger the 15-lipoxygenase-1 (15-LO) and 5-lipoxygenase (5-LO) pathways, respectively. 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and eoxin C4 (EXC4) were measured as 15-LO products and leukotriene (LT)C4 as a product of the 5-LO pathway. Results: Activated eosinophils from patients with SA and AIA produced approximately five times more 15-HETE than eosinophils from HV or MA patients. In the presence of lysine-aspirin, eosinophils from AIA, MA and SA patients generated higher levels of 15-HETE than in the absence of lysine-aspirin. Furthermore, in the presence of lysine-aspirin, formation of EXC4 was also significantly increased in eosinophils from AIA patients, and LTC4 synthesis was increased both in AIA and SA patients. Conclusions: Taken together, this study shows an increased release of the recently discovered lipid mediator EXC4, as well as the main indicator of 15-LO activity, 15-HETE, in activated eosinophils from severe and aspirin-intolerant asthmatics, and also elevated EXC4 and LTC4 formation in eosinophils from AIA patients after cellular activation in the presence of lysine-aspirin. The findings support a pathophysiological role of the 15-LO pathway in SA and AIA.

Metabolism of Anandamide into Eoxamides by 15-Lipoxygenase-1 and Glutathione Transferases

Human 15-lipoxygenase-1 (15-LO-1) can metabolize arachidonic acid (ARA) into pro-inflammatory mediators such as the eoxins, 15-hydroperoxyeicosatetraenoic acid (HPETE), and 15-hydroxyeicosatetraenoyl-phosphatidylethanolamine. We have in this study investigated the formation of various lipid hydroperoxide by either purified 15-LO-1 or in the Hodgkin lymphoma cell line L1236, which contain abundant amount of 15-LO-1. Both purified 15-LO-1 and L1236 cells produced lipid hydroperoxides more efficiently when anandamide (AEA) or 2-arachidonoyl-glycerol ester was used as substrate than with ARA. Furthermore, L1236 cells converted AEA to a novel class of cysteinyl-containing metabolites. Based on RP-HPLC, mass spectrometry and comparison to synthetic products, these metabolites were identified as the ethanolamide of the eoxin (EX) C4 and EXD4. By using the epoxide EXA4-ethanol amide, it was also found that platelets have the capacity to produce the ethanolamide of EXC4 and EXD4. We suggest that the ethanolamides of the eoxins should be referred to as eoxamides, in analogy to the ethanolamides of prostaglandins which are named prostamides. The metabolism of AEA into eoxamides might engender molecules with novel biological effects. Alternatively, it might represent a new mechanism for the termination of AEA signalling.

On the biosynthesis of 15-HETE and eoxin C4 by human airway epithelial cells

Several lines of evidence indicate that 15-lipoxygenase type 1 (15-LO-1) plays a pathophysiological role in asthma. The aim for this study was to investigate the 15-LO-1 expression and activity in primary human airway epithelial cells cultivated on micro-porous filters at air-liquid interface. Incubation of human airway epithelial cells with arachidonic acid led to the formation of 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and exposing the cells to bacteria or physical injury markedly increased their production of 15-HETE. The cells were also found to convert arachidonic acid to eoxin C4 (EXC4). Subcellular fractionation revealed that the conversion of EXA4 to EXC4 was catalyzed by a soluble glutathione transferase (GST). The GST P1-1 enzyme was found to possess the highest activity of the investigated soluble GSTs. Following IL-4 treatment of airway epithelial cells, microarray analysis confirmed high expression of 15-LO-1 and GST P1-1, and immunohistochemical staining of bronchial biopsies revealed co-localization of 15-LO-1 and GST P1-1 in airway epithelial cells. These results indicate that respiratory infection and cell injury may activate the 15-LO pathway in airway epithelial cells. Furthermore, we also demonstrate that airway epithelial cells have the capacity to produce EXC4.

Biosynthesis of 14,15-hepoxilins in human L1236 hodgkin lymphoma cells and eosinophils

Hepoxilins are epoxy alcohols synthesized through the 12-lipoxygenase (12-LO) pathway in animal cells. The epidermis is the principal source of hepoxilins in humans. Here we report on the formation of novel hepoxilin regioisomers formed by the 15-LO pathway in human cells. The Hodgkin lymphoma cell line L1236 possesses high 15-lipoxygenase-1 (15-LO-1) activity and incubation of L1236 cells with arachidonic acid led to the formation of 11(S)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),12(E) eicosatrienoic acid (14,15-HxA3 11(S)) and 13(R)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),11(Z) eicosatrienoic acid (14,15-HxB3 13 (R)). In addition, two hitherto unidentified products were detected and these products were collected and analyzed by positive ion electrospray tandem mass spectrometry. These metabolites were identified as 11(S),15(S)-dihydroxy-14(R)-glutathionyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA3-C) and 11(S),15(S)-dihydroxy-14(R)-cysteinyl-glycyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA3-D). Incubation of L1236 cells with synthetic 14,15-HxA3 11(S) also led to the formation of 14,15-HxA3-C and 14,15-HxA3-D. Several soluble glutathione transferases, in particular GST M1-1 and GST P1-1, were found to catalyze the conversion of 14,15-HxA3 to 14,15-HxA3-C. L1236 cells produced approximately twice as much eoxins as cysteinyl-containing hepoxilins upon stimulation with arachidonic acid. Human eosinophils, nasal polyps and dendritic cells selectively formed 14,15-HxA3 11(S) and 14,15-HxB3 13(R) stereoisomers, but not cysteinyl-containing hepoxilins, after stimulation with arachidonic acid. Furthermore, purified recombinant 15-LO-1 alone catalyzed the conversion of arachidonic acid to 14,15-HxA3 11(S) and 14,15-HxB3 13(R), showing that human 15-LO-1 possesses intrinsic 14,15-hepoxilin synthase activity.

Human 15-lipoxygenase-1 is a regulator of dendritic-cell spreading and podosome formation

Dendritic cells (DCs) involved in proinflammatory immune responses derive mainly from peripheral monocytes, and the cells subsequently mature and migrate into the inflammatory micromilieu. Here we report that suppressing of 15‐lipoxygenase‐1 led to a substantial reduction in DC spreading and podosome formation in vitro. The surface expression of CD83 was significantly lower in both sh‐15‐lipoxygenase‐1 (15‐LOX‐1)‐transduced cells and DCs cultivated in the presence of a novel specific 15‐LOX‐1 inhibitor. The T‐cell response against tetanus‐pulsed DCs was only affected to a minor extent on inhibition of 15‐LOX‐1. In contrast, endocytosis and migration ability of DCs were significantly suppressed on 15‐LOX‐1 inhibition. The expression of 15‐LOX‐1 in DCs was also demonstrated in affected human skin in atopic and contact dermatitis, showing that the enzyme is indeed expressed in inflammatory diseases in vivo. This study demonstrated that inhibiting 15‐LOX‐1 ledtoanimpairedpodosome formation in DCs, and consequently suppressed antigen uptake and migration capacity. These results indicated that 15‐LOX‐1 is a potential target for inhibiting the trafficking of DCs to lymphoid organs and inflamed tissues and decreasing the inflammatory response attenuating symptoms of certain immunologic and inflammatory disorders such as dermatitis.—Han, H., Liang, X., Ekberg, M., Kritikou, J. S., Brunnström, Å., Pelcman, B., Matl, M., Miao, X., Andersson, M., Yuan, X., Schain, F., Parvin, S., Melin, E., Sjåberg, J., Xu, D., Westerberg, L. S., Bjårkholm, M., Claesson, H.‐E. Human 15‐lipoxygenase‐1 is a regulator of dendritic‐cell spreading and podosome formation. FASEB J. 31, 491–504 (2017). www.fasebj.org