SELENOPROTEINS MODIFY OXYLIPID S FROM LINOLEIC ACID IN MACROPHAGES By Sarah Asheley Peek A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Comparative Medicine and Integrative Biology Master of Science 2014 ABSTRACT SELENOPROTEINS MODIFY OXYLIPID S FROM LINOLEIC ACID IN MACROPHAGES By Sarah Asheley Peek I nflammatory diseases are characterized by uncontrolled inflammation and remain the leading cause of death in humans. Selenium ( Se ) is an essential nutrient in the mammalian diet and its bioactivities are critical for optimum immune function. Se exhibits immune - modulatory effects through antioxidant - functioning selenoproteins that can exert control over oxidative tone of cells and the expression of pro - inflammatory mediators. Oxylipids are among the more potent, redox - regulated inflammatory mediators that orchestrate the deg ree and duratio n of inflammation . Whereas previous works show Se - deficiency results in enhanced pro - inflammatory , arachidonic acid - derived oxylipid synthesis by macrophages, there is a need to define how antioxidant selenoprotein activity might control the balance betwee n pro - and anti - inflammatory oxylipid biosynthesis. Therefore the objective of this work was to investigate the role of decreased selenoprotein activity in modulating the production of biologically active oxylipids from macrophage s. Reduced selenoprotein activity increased free radical s , enhanced inflammatory cytokine expression, and decreased LA - derived oxylipids from both in vivo and in vitro macrophages. When these oxylipids were added to in vitro macrophages subjected to a pro - ox idant challenge , inflammatory TNF production was abrogated, suggesting an anti - inflammatory action for these LA - derived oxylipids. Future studies shou ld focus on which antioxidant selenoproteins have an impact on oxylipid biosynthesis and the mechanisms behind their effect in order to help prevent pathologies associated with uncontrolled inflammation . iii I dedicate this thesis to my husband Joshua, my parents Linda, Thomas and Peggy, Colleen and John, and the countless family and friends for their continuous love and support of my educational and professional ambitions . iv ACKNOWLEDGEMENTS I would like to acknowledge my research advisor , Dr. Lorraine Sordillo , and guidance committee: Dr. Jenifer Fenton, Dr. Narayanan Parameswaran, and Dr. Gavin Reid for their thoughtful guidance and direction throughout my graduate education . I would also like to thank my other mentors , Dr. Nettavia Curry and Njia Lawrence - Porter , for their faithful support and positive i nfluences , helping to encourage me through graduate school and beyond . Finally, I would like to thank the all members of the Meadow Brook Laboratory especially Chris Corl, Jeffrey Gandy, William Raphael , and Valerie Ryman for their support and assistance with this project. v TABLE OF CONTENTS LIST OF TABLES v ii LIST OF FIGURES vi ii KEY TO ABBREVIATIONS CHAPTER 1 Regulation of Inflammation by Selenium and Selenoproteins: Impact on Oxylipid Biosynthesis Abs tract .. . 2 Introduction . . 3 Selenium: An Essential Micronutrient with Anti - inflammatory Properties 4 Selenium and Inflammatory Diseases Selenium Functions as an Antioxidant through the Activity of Sel enoproteins Role of Selenoproteins in Cellular Redox Signaling . Can Se and Selenoproteins Impact Inflammation is Thr ough Oxylipid Biosynthesis? 8 Regulation of Inflammation by Oxylipids ... 8 Selenium and Oxylipid Profiles ... ... 10 Antioxidant - dependent Regulation of Oxylipid Biosynthesis ... . 1 1 Redox - Regulation of Oxylipid Biosynthesis 13 Se Can Affect Oxylipid Biosynthesis in Cancer Models . .... .14 ... 15 - types: Endothelial Cells .. 16 Impact of Se on Oxylipids in Specific Cell - types: Leukocyte Function .. Conclusions .. 20 Acknowledgement s . 22 CHAPTER 2 Reduced Selenoprotein Activity Alters the Production of Oxidized Lipid Metabolites from 23 Abstract .. . 2 4 Introductio n .. Materials & Met hods . Mice & Macrophage Samples ... Quantitative Real - Time PCR (qPCR) Solid Phase Lipid Extractions & Liquid Chromatography - Mass Spectrometry vi (LC - MS) RAW 264.7 Macrophage Culture & Oxylipid Stimulation Cytometric Bead Array Statistical Analyses . Results .. Selenopro tein Expression from In V ivo Macrophages . Selenoprotein Activity Modifies Inflammatory Gene Expression in PEM . .34 M Mice & RAW 264.7 Macrophages . Oxylipid Treatment .. ..35 Discussio n .. Conclusion ... 43 Acknowledgements APPENDIX 45 BIBLIOGRAPHY ... 70 vii LIST OF TABLES Table 1: Summary of mammalian selenoproteins with characterized functions 46 Table 2: The impact of Se and Selenoproteins on Oxylipid Biosynthesis 47 Table 3: Pearson Correlations for Oxylipids Produced by Cont n=5 54 viii LIST OF FIGURES Figure 1: .. 48 Figure 2: Se metabolism from different dietary sources ... ... 49 Figure 3: General reaction mechanisms for antioxidant GPxs and TrxRs ... 50 Figure 4: Oxylipid Biosynthesis Pathways .. 51 Oxylipid Biosynthesis Pathways . . 52 Figure 6: Selenoprotein Knockout in Murine Macrophages . .. 55 Figure 7: Oxylipid Biosynthetic Enzyme Expression in Macrophages .. 56 Figure 8: Inflammatory Cytokine Expression by Macrophages 57 Figure 9: Oxylipid 58 Figure 10: Oxylipid B iosynthesis from RAW 264.7 M acrophages 60 Figure 11: ROS Production Following Pro - 62 Figure 12: Effect of Oxylipid Stimulation on RAW 264.7 Macrophage ...................... ........................................................................... ..............................63 Figure 13 : Glutathione peroxidase 1 activity from ma crophages cultured with various 64 Figure 14 : Reactive oxygen species production by RAW 264.7 macrophages cultured in 5% (A) ix Figure 15 : Total fatty acid analysis of arachidonic (A) or linoleic (B) a cid from murine peritoneal macropha Figure 16: Effect of LA - derived Oxylipid Stimulation on RAW 264.7 Macrophage Production x KEY TO ABBREVIATIONS AA: Arachidonic acid COX: Cyclooxygenase DHA: Docosahexaenoic acid EPA: Eicosapentaenoic acid FAHP: Fatty acid hydroperoxide GSH: Glutathione GSSG: Glutathione disulfide GPx: Glutathione peroxidase H 2 O 2 : Hydrogen peroxide HETE: Hydroxyeicosatetraenoic acid HPE TE: Hydroperoxyeicosatetraenoic acid HODE: Hydroxyoctadecadienoic acid HPODE: Hydroperoxyoctadecadienoic acid IsoP: Isoprostane LA: Linoleic acid LOX: Lipoxygenase LT: Leukotriene LXA: Lipoxin MaR: Maresin oxoETE: oxo - eicosatetraenoic acid oxoODE: oxo - octa decadienoic acid PG: Prostaglandin xi PD: Protectin ROS: Reactive oxygen species RNS: Reactive nitrogen species Rv: Resolvin Se: Selenium Sec: Selenocysteine Trx: Thioredoxin TrxR: Thioredoxin Reductase Tx: Thromboxane 1 CHAPTER 1 Regulation of Inflammation by Selenium and Selenoproteins: Impact on Oxylipid Biosynthesis S.A. Peek 1 , B.A. Carlson 2 , L.M. Sordillo 1 1 College of Veterinary Medicine, Michigan State University, East Lansing MI, 48824 2 Section on the Molecular Biology of Selenium, Laboratory of Cancer Prevention, National Cancer Institute, NIH, Bethesda, MD 20892, USA Published in JOURNAL OF NUTRITIONAL SCIENCE, May 2013 , Regulation of Inflammation by Selenium and Selenoproteins: Impact on Eicosanoid Biosynthesis, by S.A. Mattmiller, B. A. Carlson , and L. M. Sordillo . Copyright form found in appendix , page 69 . 2 A bstract Uncontrolled inflammation is a contributing factor to many leading causes of human morbidity and mortality including atherosclerosis, cancer, and diabetes. Selenium ( Se ) is an essential nutrient in the mammalian diet that has some anti - inflammatory properties and, at sufficient amounts in the diet, was shown to be protective in various inflammatory - based disease models. More recently, Se was shown to alter the expression of oxylipid s that orchestrate the initiation, magnitude, and resolution of inflammation. Many of the health benefits of Se are thought to be due to antioxidant and redox - regulating properties of certain selenoproteins. This review will discuss the existin g evidence that supports the concept that optimal Se intake can mitigate dysfunctional inflammatory responses, in part, through the regulation of oxylipid metabolism. The ability of selenoproteins to alter the biosynthesis of oxylipid s by reducing oxidati ve stress and/or by modifying redox regulated signaling pathways also will be discussed. Based on the current literature, however, it is clear that more research is necessary to uncover the specific beneficial mechanisms behind the anti - inflammatory proper ties of selenoproteins and other Se - metabolites, especially as related to oxylipid biosynthesis. A better understanding of the mechanisms involved in Se - mediated regulation of host inflammatory responses may lead to the development of dietary intervention strategies that take optimal advantage of its biological potency. Key Words: Selenium, selenoproteins, oxylipid biosynthesis, inflammation 3 Introduction Uncontrolled inflammatory responses can contribute to the pathogenesis of many health disorders. Dy sfunctional or uncontrolled inflammation can be characterized as a chronic low - grade inflammation such as that observed in diabetes, obesity, and atherosclerosis ( 1 , 2 ) . Alternatively, uncontrolled inflammation also may manifest as an exacerbated acute inflammation as observed in disea ses such as sepsis and mastitis ( 3 ) . Oxylipid s are a class of lipid mediators that constitute one of the several pathways that regu late the inflammatory response and are biosynthesized by many cell - types including endothelial cells and leukocytes . During uncontrolled inflammation, a combination of the over - production of pro - inflammatory oxylipid s and a diminished synthesis of anti - inf lammatory oxylipid s can contribute to an improper and incomplete resolution process. Current non - steroidal anti - inflammatory drug therapies that target specific enzymes involved in oxylipid biosynthesis have limited efficacy in controlling some inflammator y - based diseases and can cause adverse side effects in both humans and veterinary species ( 4 ) . Therefore, there is a growing interest to identify alternate therapeutic strategies to regulate uncontrolled inflammation through dietary intervention. The potential of optimizing host inflammatory responses by modifying Se dietary intake was explored in several inflammatory - based disease models such as cancer ( 5 ) , cardiovascular disease ( 6 ) , mastitis ( 7 ) , and osteoporosis ( 8 ) . Although Se nutritional status was often associated with the magnitude and duration of inflammation, the underlying beneficial mechanisms ascribed to this micronutrient are not fully described. The aim of th is review is to assess how the antioxidant and redox - regulating properties of certain selenoproteins can contribute to the beneficial properties of Se - nutrition in controlling inflammatory - based diseases. The ability of selenoproteins to regulate oxylipid biosynthetic pathways in both in whole animal models of disease and in individual cell - 4 types will be critically evaluated as potential anti - inflammatory mechanisms resulting from optimal Se intake. A greater understanding of the factors that can regulate the delicate balance between the initiation and resolution of inflammatory responses is needed in order to help diminish the morbidity and mortality associated with the pathology of inflammatory - based diseases. Selenium: An Essential Micronutrient with Anti - inflammatory Properties Selenium and Inflammatory Diseases. Se was once considered a toxin when livestock and poultry suffered from alkali disease after consuming grass containing 10 - 20ppm Se. Subsequent stu dies confirmed the potential for Se poisoning when laboratory rodents were supplemented with 5 - 15ppm of dietary Se displayed varying degrees of pathology ( 9 ) . In contrast, others found that Se - deficiency (diets containing less than 0.1ppm Se) caused diseases such as white muscle disease in cattle and lambs ( 10 ) and Keshan disease in humans ( 11 ) . Based on these earlier studies, Se is now understood to be an essential micronutrient in the mammalian diet and our knowledge of its metabolism (Figure 2) and beneficial functions has grown immensely. Current recommendations indicate the upper tolerable intake of Se is between 90 - intake between 30 - ( 12 ) and 0.4 mg/kg body weight in rodents ( 13 ) . In a review and meta - analysis of the literature, Huang et al. ( 14 ) found that supplementation with Se (between 500 - critically ill patients decreased mortality rates associated wi th sepsis. Additionally, women with normal pregnancies exhibited significantly higher blood Se concentrations compared to women with preeclampsia, the leading cause of perinatal and maternal mortality globally ( 15 ) . In a exhibited decreased colonic tissue necrosis ( 16 ) . It is important to note, however, that not all 5 clinical trials involving Se supplementation improved health outcomes in a significant way. Recently published results from The Selenium and Vitamin E Cancer Prevention Trial or a period between 7 - 12 years, did not prevent diseases such as prostate, lung, or colon cancers and there were no significant differences in cardiovascular events or diabetes between treatment groups in men ( 17 ) . Based on these equivocal findings, it is now clear that more research is required to better understand the underlying mechanisms of design nutritional intervention strategies that yield more consistent and positive results across a range of human health disorders. Selenium Functions as an Antioxidant through the Activity of Selenoprotein s. Although the importance of Se to health is not fully understood, one well - characterized function of Se is its ability to mitigate oxidative stress through antioxidant functioning selenoproteins (Table 1), including the well - studied glutathione peroxidas es ( GPx ) and thioredoxin reductases ( TrxR ) families ( 18 , 19 ) . Oxidative stress occurs when the production of free radicals, including reactive oxygen species ( ROS ), reactive ni trogen species ( RNS ), oxidized proteins, and oxidized ( 20 ) . The GPx and TrxR selenoproteins contain a selenocysteine in their active site making them suitable for oxidation/reduction reactions (Figure 3). Whereas GPx1 can reduce ROS in the cytoplasm, GPx4 has the ability to reduce fatty acid hydroperoxides ( FAHP ) and phospholipid hydroperoxides within cellular membranes (Figure 3a) ( 21 , 22 ) . A longer, alternative transcript of GPx4 also was localized to mitochondrial membranes ( 23 ) and shown to maintain ATP production during oxidative stress which could have implications on cellular activity and function during disease ( 24 ) . Thioredoxin ( Trx ) reduces a variety of radicals including li pid 6 hydroperoxides, protein thiols, and ROS/RNS. Oxidized Trx is then restored to its reduced form by TrxR selenoproteins (Figure 3b). Selenoproteins W, K, and P ( Sepw1, Selk, Sepp1 ) also were suggested to have antioxidant capabilities, but mechanisms are less understood ( 25 , 26 ) . Oxidative stress is a contributing factor in inflammatory disease pathologies including atherosclerosis ( 27 ) , diabetes ( 28 ) , and mastitis ( 29 ) among others. There is ample evidence to indicate that selenoproteins can interrupt disease pathogenesis through antioxidant - dependent mechanisms. Numerous studies in humans, food - animal species, and rodent models demonstrated a negative correlation between measures of selenoprotein activity and disease severity due to oxidative stress ( 30 - 32 ) . Direct evidence of the importance of selenoproteins in mitigating oxidative stress was demonstrated in transgenic studies where overexpression of GPx4 significantly reduced lipid peroxidation in atherosclerosis and ischemia/reperfusion mouse models ( 33 , 34 ) . Several in vitro studies also demonstrated that TrxR1 and selenoprotein P could directly reduce the lipid hydroperoxide, 15 - HPETE, to its correspondi ng hydroxyl ( 15 - HETE ) ( 35 - 37 ) , thus having implications in reducing atherosclerotic lesion formation as a consequence of oxidative stress ( 38 ) . Collectively, these studies support the contention that optimally functioning antioxidant selenoproteins may be crucial for reduci ng excess free radicals accumulation and preventing oxidative tissue damage during acute or chronic inflammation. Role of Selenoproteins in Cellular Redox Signaling. Another way in which selenoproteins may protect against immunopathology associated with u ncontrolled inflammatory responses is through redox - regulation of inflammatory signaling. The redox state of cells or tissues can be defined as the ratio of oxidized and reduced forms of specific redox couples ( 39 ) . Some redox couples relevant to inflammation include NADP+: NADPH, glutathione disulfide ( GSSG ): 2 glutathiones ( GSH ), and oxidized thioredoxin ( Trx(SS) ): reduced thioredoxin 7 ( Trx(SH)2 ). Thioredoxin and glutathione redox couples function with the help of TrxR and GPx selenoproteins, respectively. Into et al. found that GSH was capable of modifying nitrosylated forms of the myeloid differentiation factor 88 ( MyD88 ) adaptor protein which enhanced signaling through the toll - like receptor ( TLR4 ) pathway during acute inflammation and resulted in altered IL - 8 and IL - 6 expression ( 40 ) . Mitogen - activated protein kinase ( MAPK ) signaling also can be affected by redox tone. Apoptosis signal - regulating kinase 1 ( ASK - 1 ) is a MAPK intermediate that activates downstream pro - inflammatory and pro - apoptotic signaling cascades ( 41 , 42 ) . Mammalian Trx is a direct inhibitor of ASK - 1 kinase activity and a negative regul ator of ASK - 1 - dependent gene expression ( 41 ) . The interaction between ASK - 1 and Trx was found to be highly dependent on redox status since oxidation of Trx by ROS results in ASK - 1 activation. In contrast, the reduced Trx blocked ASK - 1 dependent signalin g indicating a protective role of selenoproteins in regulation of apoptosis during oxidative stress ( 43 ) . redox regulated at several levels. Vunta et al. report ed an association between increased pro - macrophages were cultured in Se - deficient media that contained only 6 pmoles/ml of Se when compared to cells cultures with 2 n moles/ml of Se ( 44 , 45 ) . Decreased plasma Se (0.37 ± 0.05 ( 46 ) and decreased selenoprotein synthesis ( 47 ) in HIV patients was associated with enhanced oxidative stress - promoted HIV viral transcription. In the cytoplasm, ROS - mediated facilitated through activation of protein kinase A ( PKAc ) ( 48 ) , and overexpression of Trx caused a decrease in ROS - mediated ( 49 ) - binding by reducing 8 ( 50 ) . Hirota et al. ( 51 ) showed that reduced Trx is primarily found withi n the cytoplasm of cells; but upon oxidant stimulation, Trx migrates to the nucleus to - DNA binding. These few examples demonstrate how selenoproteins can both positively and negatively control cell signaling depending on the inflammatory pathw ay and/or cellular location. Overall, Se nutrition and selenoprotein activity have the potential to improve inflammatory response outcomes in several ways including combating oxidative stress in cells/tissues and through the redox - regulation of inflammator y signaling pathways that lead to cytokine/chemokine production. However, another potentially important but less studied mechanism underlying the health benefits of Se may involve the biosynthesis of bioactive lipid mediators that include the oxylipid s (F igure 1). Can Se and Selenoproteins Impact Inflammation Through Oxylipid Biosynthesis? Regulation of Inflammation by Oxylipid s. Oxylipid s are a class of lipid mediators that contribute to the orchestration of inflammatory responses. Oxylipid s are synthesized from polyunsaturated fatty acid substrates primarily found in the cellular membrane including the omega - 6 arachidonic ( AA ) and linoleic ( LA ) or the omega - 3 eicosapentaenoic ( EPA ) and docosahexaenoic ( DHA ) acids ( 52 ) . These fatty acid substrates are oxidized non - enzymatically by free radicals or through different enzymatic pathways including the cyclooxygenases ( COX ), lipoxygenases ( LOX ), and cytochrome P450 pathways to produce both pro - inflammatory and resolving oxylipid s (Figure 4). N on - enzymatic oxidation of AA produces the isoprostane series of prostaglandin - like oxylipid s. These lipid mediators have been characterized as biomarkers for oxidative stress ( 53 ) . As such, they have been quantified in models of inflammatory disease, like atherosclerosis , to identify relationships between disease progression and oxidative damage ( 54 ) . In addition to the isoprostanes, non - enzymatic oxidation of AA or LA can also produce 9 hydroperoxide metabolites HPETEs or HPODEs respectively that are enhanced during oxidative stress ( 55 ) . Two isoforms of COX enzymes are involved in the enzymatic oxidation pathways. Whereas COX1 is constitutively expressed in cells, COX2 expression is inducible during inflammation ( 56 , 57 ) . COXs catalyze the oxidation of omega - 6 AA to prostaglandin ( PG ) PGG 2 and PGH 2 ( 58 ) . From PGH 2 , downstre am PG synthases produce PGE 2 , PGD 2 , PGI 2 , PGF , among others. Alternatively, thromboxane (TX) synthases convert PGH 2 to TXA 2 , and TXB 2 . Similar to the COX family, there are several isoforms of LOX involved in the enzymatic oxidation of fatty acids. For example, 5LOX catalyzes the oxidation of omega - 6 AA to 5 - hydroperoxyeicosatetraenoic acid ( 5 - HPETE ) which can be further metabolized to produce leukotrienes ( LT ). Both 15LOX - 1 (12LOX in mice) and 15LOX - 2 (8LOX in mice) oxidize AA to 12/15 - hydroperoxyeicos atetraenoic acid ( 12/15 - HPETE ) ( 59 ) . More recent studies have led to the discovery of anti - inflammatory lipox ins ( LX ) that are produced from the metabolism of 12/15HPETE intermediates by the 5LOX pathway ( 60 ) . Likewise, 12/15LOX - 1 can oxidize the omega - 6 LA into 9 - hydroperoxy - 10E, 12Z - octadecadienoic acid ( 9 - HPODE ) and 13S - hydroperoxy - 9Z, 11E - octadecadienoic acid ( 13 - HPODE ) ( 61 ) . Hydroperoxides can then be reduced to form hydroxyl intermediates (HETEs and HODEs) and further dehydrogenated to form ketone intermediates (oxoETEs and oxoODEs) ( 62 ) . Omega - 3 fatty acids also can be oxidized by COXs and LOXs to produce oxylipid s with more anti - inflammatory or resolving propert ies ( 52 ) . EPA is metabolized by 5LOX and modified forms of COX2 to produce E - series resolvins ( Rv ) ( 63 ) , whereas 12/15LOX converts DHA to the D - series Rvs, protectins ( PD1 ), and the macrophage - specific maresin ( MaR1 ). During uncontrolled inflammation, a combination of exacerbated production of pro - inflammatory oxylipid s and diminished production of anti - 10 inflammatory oxylipid s prevents full resolution and restoration of homeostasis ( 64 ) . Therefore, the balance between production of pro - and anti - inflammatory oxylipid s is one factor that determines the inflammatory phenotype of a cell/surrounding microenvironment ( 65 ) . Oxylipid abundance and timing of their production are crucial to successfully initiate and resolve inflammation. Oxylipid biosynthesis is regulated at several levels and both Se and selenoproteins have been stu died in the context of: 1) altering oxylipid profiles as a function of manipulating dietary Se, 2) feedback - loops from other oxylipid s, 3) chemically reducing lipid hydroperoxides, and 4) modifying expression and activity of COX/LOX enzymes (Table 2 and F igure 5). However, research has just begun to uncover the underlying mechanisms of how Se can influence oxylipid biosynthesis at each level of regulation. Selenium and Oxylipid Profiles. Previous studies have documented how dietary Se impacts the biosynthesis of oxylipid s in several different species. Following 2 years of decreased ratio of urinary 11 - de hydro TXB 2 /2,3 dinor 6 - keto PGF . Increased ratios of TXB 2 /6 - keto PGF are an indicative biomarker for thrombosis and atherosclerosis ( 66 ) . Previous research by Meydani ( 67 ) then Haberland et al. ( 68 ) confirmed that adequate Se intake 2 /PGF following short term (2mo) and long term (8 generations) of dietary modulation, respectively. In dairy cattle with mastitis, Se - suffici ent diets (0.05 mg Se/kg) were associated with decreased pro - inflammatory TXB 2 , PGE 2 , and LTB 4 oxylipid production and secretion in milk compared to cows with deficient Se intake after 1yr of dietary interventions ( 69 ) . Taken together, these 11 results indicate that dietary Se could potentially diminish pro - inflammatory oxylipid biosynthesis during inflammatory diseases. Se can also alter feedback loops involved with oxylipid biosynthesis. One example was reported on the positive feedback loop involving the ability of 15 - deoxy - PGJ 2 ( 15d - PGJ 2 ) to perpetuate anti - inflammatory oxylipid production by enhancing the expression of its upstream synthesis enzyme in macrophages. Compared to Se - deficiency (6 pmoles/ml of Se from media FBS compared to cells supplemented with 250 nM) , culturing murine macrophages with Se to maximize GPx activity enhanced 15d - PGJ 2 production; 15d - PGJ 2 once activated, enhance d H - PGDS expression. H - PGDS converts PGH 2 to PGD 2 , which is an upstream metabolite of 15d - PGJ 2 ( 70 ) . Thus, depending on the level of regulation, Se could potentially dampen pro - inflammatory oxylipid biosynthesis and enhance more anti - inflammatory oxylipid production; however more research is needed to determine t he specific mechanisms involved at different levels of regulation of oxylipid biosynthesis and which selenoproteins could have an effect. Antioxidant - dependent Regulation of Oxylipid Biosynthesis. There is evidence that certain selenoproteins are at least partially responsible for the ability of Se to modify oxylipid biosynthesis. A direct cause and effect relationship between GPx4 and LT production in cancer cells was previously explored by Imai et al. ( 71 ) . At the metabolite level, GPx4 overexpression was shown to reduce FAHPs from the 5LOX pathway (5 - HPETE to 5 - HETE), thus preventing the production of LTB4 and C4 in the leukemia cell line ( 71 ) . The proposed mechanism was the antioxidant capabilities of GPx4 and the ability to reduce FAHP to hydroxyl - derivatives. Others 12 found that GPx4 reduced 15 - HPETE to 15 - HETE and preincubation endothelial cells with GPx4 could prevent peroxide formation ( 72 ) . Both TrxR and Sepp1 also were shown to have lipid hydryperoxidase activity for 15 - HPETE, thus supporting the contention that these selenoproteins can function as antioxidant enzymes against highly reactive hydroperoxy intermediates formed during oxylipid metabolism ( 37 , 73 ) . Collectively, these studies suggest that selenoproteins have an important role in protecting cells against oxidative damage caused by lipid hydroperoxides found in the oxylipid network. Individual selenoproteins also can modify oxylipid biosynthesis thro ugh controlling the activity of COX/LOX enzymes. Walther et al. described how the Se - containing compound ebselen inhibited 15LOX activity by altering the oxidation status of the active - site iron molecule ( 74 ) . The activation of COX enzymes also requires oxidation of their active sit e heme iron to form a tyrosyl - radical that is then capable of oxidizing AA and other fatty acid substrates ( 75 ) . GPx1 can inhibit COX enzyme activity by chemically reducing hydroperoxides that could otherwise activate enzymatic oxidation ( 76 ) . An abundance of oxylipid metabolites and other radicals, however, can also inhibit the activity of oxylipid enzymes through what is known as ( 77 ) , prostaglandin I synthase ( 78 ) , and TXAS ( 79 ) . A decrease in COX activity was described in human endothe lial cells due to a buildup of peroxides during diminished GPx1 activity ( 80 ) . These findings suggest that cellular levels of FAHP are critical in COX enzyme activity; both an excess of FAHP or absence of these radicals can result in COX inhibition. This is interesting because GPx - mediated reduction of FAHP could have different effects on COX or LOX activity depending on the accumulation of FAHP. FAHP generated by the 15LOX pathway were shown to be affected by another selenoprotein in vitro . Sepp1, a selenoprotein present in plasma , was shown to chemically reduce 15 - HPETE into 15 - 13 HETE ( 37 ) . Additionally, Sepp1 decreased the production of free radicals following stimulation with 15 - HPETE in vitro ( 37 ) . This study highlighted the antioxidant properties of the plasma selenoprotein, Sepp1, which could have significant implications in preventing oxidative stress associated with vascular inflammatory diseases, such as atherosclerosis. Redox - Regulation of O xylipid Biosynthesis. Another way that Se can affect oxylipid profiles is through the redox - regulation of oxylipid enzyme expression. Pretreating chondrocytes with physiological levels of Se - - induced gene e xpression of COX2 and consequent synthesis of PGE 2 ( 8 ) . Hwang et al. showed in mice, COX2 expression in a model of colon cancer ( 81 ) . Addition of various supraphysiological doses of Se (250 - - 29 cells dampened ERK signaling following stimulation with a tumor promoting agent, 12 - O - tetradecanoylphorbol - 13 - acetate (TPA), and incre ased MAPK signaling; both of which decreased COX2 expression ( 81 ) . In another model, prostate activity, which is another pathway known to control COX2 expression ( 82 ) . As described earlier, the redox control of these signaling pathways can occur at several signaling intermediates. Collectively, these studies support the concept that Se can decrease COX2 expression, at least in part, through the regulation of various redox - dependent signaling pathways. More research is needed, however, to characterize cause and effect relationships identifying where spe cific selenoproteins could be regulating COX2 expression through other redox - regulated signaling pathways. 14 Se Can Affect Oxylipid Biosynthesis in Cancer Models. Inflammatory pathways can play an important role in cancer development through regulation of cell proliferation and migration ( 83 ) . For example, oxylipid s can play an important role in tumorgenesis by regulating apoptosis and proliferation of cancer cells ( 84 , 85 ) and Se may exert anti - cancerous properties through the manipulation of oxylipid signaling. For example, Ghosh et al. reported th at supplementation with various Se doses (0 - prostate cancer cells but not of normal PrEC prostate cells ( 86 ) . Additionally, they noted that stimulation of LNCaP with 5LOX - derived oxylipid s, 5 - HETE and 5 - apoptotic effect and enhanced growth of cancerous cells; thus indicating that 5LOX - derived oxylipid s may p lay a role in promoting cancerous cell growth in prostate cancer ( 86 ) . Other researchers explored the relationship betwee n specific selenoproteins and oxylipid regulation in models of colon cancer. In GPx2 - silenced HT - 29 colon cancer cells, an increase in COX2 and mPGES - 1 enzyme expression with a concomitant increase in PGE 2 production was reported ( 5 ) . The authors proposed that GP x2 disrupted the positive feedback - loop of PGE 2 - dependent expression of COX2, representing a unique role specific for GPx2 in the colon cancer model ( 5 ) . This same feedback - loop also was studied in the context of GPx4 and a fibrosarcoma cancer model. In L29 fi brosarcoma tumor cells, overexpression of GPx4 prevented tumor growth, decreased COX2 expression, PGE 2 production, and abrogated PGE 2 - dependent COX2 expression ( 87 ) . These studies provide examples in cancer models that the redox - regulating properties of certain selenoprote ins could decrease pro - inflammatory oxylipid production and reduce inflammatory - dependent tumor progression. 15 Oxylipid Biosynthesis in Cardiovascular Disease Models. Atherosclerosis is another inflammatory - based disease that remains the leading cause of death in the developed world ( 1 ) . As such, an interest is growing in understanding how Se may be beneficial in cardiovascular disease models. Oxidative stress plays a significant role in the etiology of cardiovascular lesion development by promoting the production of oxidized lipoproteins ( oxLDL ) and lipids such as the non - enzymatically oxidized oxylipid s, prostaglandin - like F2 isoprostanes ( F2 - IsoP ) ( 54 ) . These radicals (oxLDL in particular) are recognized and internalized by circulating monocytes which initiates foam cell development and macrophage infiltration into blood vessels ( 88 ) . The lipid hydroperoxide scavenging GPx4 was overexpressed in a mouse model of atherosclerosis (ApoE - / - mice) which resulted in decreased overall atheroscle rotic lesion development ( 33 ) . The mech anisms behind the protective effect of GPx4 in accumulation of hydroperoxide radicals and diminish oxidative stress. In support of this theory, both F2 - IsoP pro duction and accumulation of intercellular and secreted hydroperoxides were significantly decreased in GPx4 overexpressing mouse aortic endothelial cells compared to atherosclerotic cells ( 33 ) . When mitochondrial GPx4 was overexpressed in a mouse ischemia/reperfusion model, researchers documente d significantly increased cardiac function and decreased lipid peroxidation ( 34 ) . In another atherosclerosis mod el, ApoE - / - and GPx1 double knockout mice exhibited significantly increased atherosclerotic lesion development suggesting that GPx1 may also play a role in disease progression ( 89 ) . Taken together, these data suggest that of GPxs could be a potential therapeutic target during heart disease due to their antioxidant properties and their capability to reduce lipid hydroperoxides and other radicals to less reactive lipid alcohols. 16 In addition to the antioxidant properties of selenoproteins, other possible mechanisms to Paniker et al. explored the impact of fatty acid substrate availabil ity and downstream oxylipid enzymatic expression ( 90 ) supplementation for 30d in isoproterenol - induced myocardial infarction in rats decreased LOX activity, leukotriene A4 hydrolase ( LTA4H ) expression, and LTB 4 production in monocytes ( 90 ) . Se - supplementation also decreased the amount of non - esterifie d fatty acids ( NEFA ) in the heart which can serve as substrates for LOX enzymatic pathways. The expression of LTA4H was diminished and resulted in decreased LTB 4 concentrations. By diminishing the expression of LTA4H, the intermediate lipid metabolite LTA 4 , is prevented from being metabolized to the more pro - inflammatory oxylipid , LTB 4 , and preserved for the biosynthesis of resolving oxylipid s, such as LXA 4 . Although the mechanism behind the decrease in LTA4H in Se - treated animals was not explored, evidence suggests that specific enzymatic pathways are potential target for Se - mediated treatment of uncontrolled inflammation. The current findings support the concept that antioxidant selenoproteins could play a role in controlling both non - enzymatic and COX/LOX - mediated oxidation of lipid mediators during cardiovascular disease. Further research is needed, however, to determine which antioxidant selenoproteins are most critical for regulating oxylipid biosynthesis and lipid peroxide - mediated disease progression . Oxylipid s in Specific Cell - types: Endothelial Cells. Since many different cell - types function in concert during inflammation, studies have focused on characterizing the effects of Se on single cell cultures to determine their role in infl ammatory disease. Endothelial 17 cells are an important component of the immune system. They are the barrier between the blood and tissue, regulate immune cell trafficking, and have been the focus of a number of studies on Se - nutrition and oxylipid biosynthes is. Confirmation that selenoprotein expression within endothelial cells are essential to survival was demonstrated when targeted knock out of selenoproteins in murine endothelial cells resulted in embryonic death due to hemorrhaging and erythrocyte immatur ity ( 91 ) . The ability of Se to reduce lipid radicals accumulation in endothelial cells was explored in early studies by Cao et al. ( 92 ) . Se - deficient bovine aortic endothelial cells ( BAEC ) cultured in the presence of only 0.01ppm Se were characterized by a significant decrease in GPx1 activity with a con comitant increases in 15 - HPETE and TXB 2 compared to cells supplemente d with 10 ng/ml sodium selenite ( 92 ) . The same group then explored the association between diminished Se - status of endothelial cells and the ability of 15 - HPETE to elicit signs of oxidative stress ( 36 ) , enhanced adhesion molecule expression ( 93 ) , higher rates of apoptosis ( 94 ) , and dampened expression PGI 2 ( 95 ) . Collectively, these studies support the concept that the antioxidant ab ility of selenoproteins are necessary to mitigate the pro - inflammatory effects of 15 - HPETE and reduce endothelial cell death as a consequence of oxidative stress. Evidence also supports a direct effect of TrxR in controlling oxidative stress and inflammat ion in vascular endothelial cells. Trigona et al. examined the role that TrxR activity may have on the differential regulation of the antioxidant enzyme heme - oxygenase (HO - 1) in 15 - HPETE challenged endothelial cells ( 36 ) . Silencing TrxR expression and activity prevented the compensatory increase in HO - 1 when endothelial cells were stimulated with 15 - HPETE. Additional experiments demonstrated that HO - 1 induction was dependent on the TrxR redox activity since restoring intracellular levels of reduced Trx was sufficient to increase HO - 1 expression when endothelial cells were cultured in Se - deficient media (less than 0 .1ppm Se) 18 ( 36 ) . This area requires more attention in future research , especially in the context of 15LOX activity and redox - regulation of signaling that controls 15LOX - derived metabolite formation as there are some conflicting reports of the role of this pathway in disease progression. Whereas some researchers have found t hat enhancing 15LOX enzyme activity leads to resolving oxylipid production ( 65 ) , others have found enhanced pro - inflammatory effects ( 96 ) . It will be necessary to identify how selenoproteins, such as TrxR1, affect the balance of pro - and anti - inflammatory oxylipid s as a function of 15LOX activity in en dothelial cells to better understand their role in inflammatory responses. Impact of Se on Oxylipid s in Specific Cell - types: Leukocyte Function. Lymphocytes are critical responders to inflammatory stimuli. They play a major role in inflammatory - based diseases including cardiovascular disease by producing chemoattractants such as macrophage chemoattractant protein - 1 ( MCP - 1 ) to enhance macrophage infiltration ( 97 ) . Lymphocytes are also important sources of oxylipid s and were studied in the context of Se - nutrition. One group found significant dec reases in oxylipid production from lymphocytes obtained from rats fed a Se deficient diet containing only <0.05 mg Se/kg ( 98 ) . The underlying mechanism behind the decrease in oxylipid biosynthe sis was proposed to be that Se - deficient lymphocytes had significantly diminished phospholipase D activation which is responsible for liberating fatty acid substrates from cellular membranes. Future studies should focus on determining how antioxidant sele noproteins can specifically affect the expression and activity of phospholipases, potentially through redox regulation, and how this may affect the oxylipid s produced during inflammation. Macrophages are especially crucial in pathogen recognition and orchestration of inflammation. Since macrophages synthesize copious amount of ROS to aid in pathogen 19 destruction, they rely on selenoprotein antioxidants to reduce excess radicals that have the potential to cause self - damage ( 99 ) . Ma crophages were acknowledged as a key cell - type in the early development of atherosclerosis because they are responsible for recognizing and ingesting oxLDL ( 88 ) . Macrophages were the focus of several reports characterizing oxylipid regulation as a function of Se - status. Prabhu et al. were interested in exploring the relationship between Se - nutrition and the pro - infl ( 100 ) . These investigators described an association between enhanced NF B activity in macrophages cultured in media containing only 6 pmoles/ml of Se when compared to compared to cells supplemented with 2 nmoles/ml of sodium selenite ( 100 ) . Additional studies proved that a significant increase in COX2 enzyme expression during Se - ( 101 ) . In contrast, Se - supplementation (20 - COX2 expression through the TLR4 pathway ( 102 ) . In microglial cells (macrophages specific to the central nervous system and brain), pretreating cells with Se - containing compounds (0 - decreased LPS - in 2 production ( 103 ) . Collectively, these studies suggest that Se, through the activity of antioxidant selenoproteins, could mediate oxylipid biosynthesis by cont - dependent COX2 expression. Other signaling pathways also may be involved in regulating COX2 expression and the subsequent metabolism of lipids through this pathway. For example, LPS - stimulated macrophages cultured in Se supplemented media (0 - induced expression of COX2 and TNF by inhibition of the MAPK signaling pathway ( 45 ) . Addi tional experiments demonstrated that mice maintained on a Se - deficient diet had significant increases in LPS - mediated infiltration of lung macrophages when compared to animals maintained on a Se adequate diet ( 45 ) . One way that Se status was suggested to alter macrophage 20 inflammatory properties was through changes in the profile of COX - derived oxylipid s. Macrophages cultured in Se supplemented media demonstrated a time - dependent increase in the production of 15d - PGJ 2 which is an endogenous inhibitor of activation ( 44 ) . Recently, repo rts showed that downstream oxylipid synthase enzymes also are affected by selenoproteins ( 70 ) . Se - of H - - PGJ 2 and 15d - PGJ 2 production. These effects where mediated by selenoproteins as confirmed by silencing selenoprotein expression through selenophosphate synthatase 2 in macrophages. On the other hand, microsomal - PGE 2 synthase ( m - PGES ) and TXAS were decreased during Se - supp lementation ( 70 ) . Together, these studies have begun to dem onstrate the association between antioxidant selenoproteins and different levels of oxylipid regulation in macrophages through several different mechanisms including modification of signaling (i.e. MAPK) to affect COX/LOX expression , manipulating dow nstream oxylipid synthase expression, altering the production of specific oxylipid s, and disrupting oxylipid feedback - loops. However, more research is warranted to determine which specific selenoproteins are responsible for these effects in order to gain a better understanding of where in oxylipid cascade that Se nutritional intervention may be possible. Conclusions Uncontrolled inflammation, governed in part by oxylipid s, is recognized to play a prominent role in the major life - threatening diseases of the developed world. Although the beneficial anti - inflammatory properties of Se have been appreciated for many years, the underlying mechanisms of action are not fully und erstood. There is ample evidence to suggest that optimal Se nutrition can combat uncontrolled inflammation, at least in part, because of the 21 antioxidant and redox - regulating capabilities of selenoproteins. Considerably less is known, however, about the s pecific selenoproteins that are responsible for these regulatory mechanisms and dynamic changes in their activity that occur during inflammatory processes. More recently, there is a growing body of evidence that further highlights the importance of selen oprotein - dependent regulation of oxylipid biosynthesis in controlling inflammatory responses. Antioxidant selenoproteins can reduce FAHP and lipid radicals directly, affecting oxylipid stability as well as phospholipase and COX/LOX activity. Certain selen oproteins also can regulate cellular redox tone which has implications on cell signaling through MAPK pathways, all of which can control expression of COX/LOX enzymes. A major gap in the existing literature, however, is knowledge of how specific selenoproteins can modify oxylipid networks in such a way as to switch from a pro - inflammatory to resolution state and thereby mitigate uncontrolled inflammatory responses that lead to disease pathogenesis. With the advent of new lipidomic analytical techn iques ( 104 ) , it should now be possible to conduct more detailed investigations of how specific selenoproteins, acting individually or in concert with others, can alter the global expression of oxylipid relevant to specific disease models. Genomic - based approaches al so will be necessary to evaluate the differential expression of selenoproteins in various tissues and how selenoprotein activity can affect oxylipid biosynthesis in different cells in involved in the inflammatory response. Some of the equivocal findings f rom existing clinical studies involving Se nutritional status can be attributed to the lack of information that links dietary intakes of Se - rich foods with tissue levels of selenoproteins that are need ed to modify specific inflammatory - regulating biologica l responses. More precise details of how selenoproteins can modify oxylipid metabolism may not only identify relevant therapeutic targets, but also provide accurate biomarkers for assessing optimal Se intake. A better 22 understanding of the mechanisms invol ved in Se - mediated regulation of host inflammatory responses will lead to more efficient and consistent nutritional intervention strategies than what has been achieved to date. Acknowledgements This work was supported, in part, by the Agriculture and F ood Research Initiative Competitive Grants Program numbers 2011 - 67015 - 30179 and 2012 - 67011 - 19944 from the United States Department of Agriculture National Institute for Food and Agriculture and by an endowment from the Matilda R. Wilson Fund (Detroit, MI). Authors contributed as follows: S. Mattmiller prepared and wrote manuscript, B. Carlson and L. Sordillo contributed to writing and editing the manuscript. All authors proof - read and approved the final draft. There is no conflict of interest for the presen t article. 23 CHAPTER 2 Reduced Selenoprotein Activity Alters the Production of Oxidized Lipid Metabolites from Arachidonic and Linoleic Acid in Murine Macrophages S.A. Peek 1 , B.A. Carlson 2 , J.C. Gandy 1 , and L.M. Sordillo 1 1 College of Veterinary Medicine, Michigan State University, East Lansing MI, 48824 2 Molecular Biology of Selenium Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Submitted to JOURNAL OF NUTRITIONAL BIOCHEMISTRY , Nov 2013 24 Abstract Uncontrolled inflammation is an underlying etiology for multiple diseases and m acrophages orchestrate inflammation largely through the production of oxidized fatty acids known as oxylipids. Previous studies showed that selenium ( Se ) status altered the expression of oxylipids and magnitude of inflammatory responses. Although selenoproteins are thought to mediate many of the biological effects of Se, the direct effect of s elenoproteins on the production of oxylipids is unknown. Therefore, the role of decreased selenoprotein activity in modulating the production of biologically active oxylipids from macrophages was investigated . Thioglycollate - elicited peritoneal macrophages were collected from wild - type and myeloid - cell specific selenoprotein knockout mice to analyze oxylipid production by LC/MS as well as oxylipid biosynthetic enzyme and inflammatory marker gene expression by qPCR. Decrease d selenoprotein activity resulted in the accumulation of reactive oxygen species, enhanced COX and LOX expression, and decreased oxylipids with known anti - inflammatory properties such as arachidonic acid - derived l ipoxin A 4 ( LXA 4 ) , and linoleic acid - derived 9 - oxo - octadecadienoic acid ( 9 - oxoODE ). Treat ing RAW 264.7 macrophages with LXA 4 or 9 - oxoODE diminished oxidant - induced macrophage inflammatory response as indicated by decreased production of TNF . The results show for the first time that selenoproteins are important for the balanced biosynthesis of pro - and anti - inflammatory oxylipids during inflammation . A better understanding of the Se - dependent control mechanisms governing oxylipid biosynthesis may uncover nutritional intervention strategies to counter act the harmful effects of uncontrolled inflammation due to oxylipids. Key Words: selenium, selenoproteins, macrophage, oxylipids, eicosanoids 25 Introduction Inflammation is an essential compone nt of innate immune defenses that works to eliminate infectio us microbes and other causes of tissue damage . Optimal inflammatory events should be robust enough to destroy pathogens, but resolve quickly to restore normal organ function and eliminate the source of injury ( 65 ) . Uncontrolled inflammation , however, can contribute significantly to several disease pathologies by c ausing tissue damage. Uncontrolled inflammation can be defined as either an exacerbated acute inflammation, such as that seen during sepsis ( 19 ) ; or a chronic, low - grade inflammation, such as that observed duri ng atherosclerosis ( 64 ) . Therefore, tight regulation and timing of inflammatory events are crucial to ef fectively eliminate the insult and prevent host damage ( 65 ) . Macrophages are pivotal in orchestrating and resolving inflammation . They produce reactive oxygen species ( ROS ) to phagocytize pathogens and secrete cytokines to control immune cell diapedesis and promote tissue remodeling ( 107 ) . In addition, macrophages are a major source of oxidized lipid mediators, such as the linolei c acid ( LA ) - derived oxidized LA metabolites or the arachidonic acid ( AA ) - derived eicosanoids, collectively called oxylipids. Oxylipids such as fatty acid hydroperoxides ( FAHP ) from LA and AA ( HPODEs and HPET Es respectively) can be produced enzymatically by cyclooxygenase s ( COX ) 1 and 2 and lipoxygenase s ( LOX ) enzym es ( 52 ) . These hydroperoxides are also formed by non - enzymatic oxidation by free radicals, making them suitable markers of oxidative stress ( 108 ) . FA HP from LA and AA can be chemically reduced to hydroxyls ( HODEs and HETEs ) and these hydroxyls can undergo dehydrogenation to produce ketone derivatives ( oxoODEs and oxoETEs ) ( 108 ) . Oxylipid biosynthesis can be regulated by enzymatic expression and activity, oxida tive tone of the cell/tissue, and feedback from other oxylipids; all of which must be tightly controlle d. 26 Unregulated oxylipid biosynthesis can contribute significantly to inflammatory - based disease pathologies ( 64 ) . Furthermore, some hydroxyl metabolites, 15 - HETE, 12 - HETE, 9 - HODE and 13 - HODE , were shown to be pro - or anti - inflammatory in atherosclerosis, arthritis, and cancer models ( 109 - 111 ) . Therefore, it will be necessary to understand how the synthesis of AA and LA - derived oxylipids and their inflammatory properties are regulated in different disease models. Selenium ( Se ) is an essential nutrie nt in the mammalian diet that has anti - inflammatory properties in cancer, cardiovascular, mastitis and other inflammatory disease models ( 19 , 30 ) . Se can affect oxylipid biosynthesis in several ways. In murine macrophages, supplementation with Se - compounds decreased prostaglandin E 2 ( PGE 2 ) production by diminishing protein expressio n of COX2 ( 101 , 103 ) . I n bovine endothelial cells , Se - deficiency increased the ratio of AA - derived hydroperoxide : hydroxyl , 15 - HPETE :15 - HETE, and 15 - HPETE exhibited pro - inflammatory effects by inhibiting the synthesis of prostacyclin ( PGI 2 ) ( 95 ) . Overexpression of 15 - LOX in Se - deficient endothelial cells resulted in increased production of 15 - HPETE and expression of the intercellular adhesion molecule, ICAM - 1 ( 93 ) . I n rat aortas , Se - deficiency significantly decreased production of the LA hydroxyl, 9 - HODE and a downstream product of PGI 2 , 6 - keto PGF 1 , which can have implications on endothelial cell function ( 112 ) . I n contrast, i ncreased L A - derived hydroxyl 13 - HODE resulted from both Se - deficiency and free radical insult in Jurkat T - cells ( 113 ) . Whereas much of the previous research characterizes oxylipid biosynthesis as a function of dietary Se, much less is known on how other Se - metabolites , such as selenoproteins, affect the oxylipid signaling network s. 27 Se is incorporate d into selenoproteins via the Se - containing amino acid selenocysteine (Sec) , that is biosynthesized on its tRNA, Sec tRNA [Ser]Sec , which in turn reads codon ensur ing proper Sec insertion into protein ( 114 ) . The antioxidant functioning glutathione peroxidases ( GPx ) and thioredoxin reductases ( TrxR ) are the most well - characterized selenoproteins and are expressed within macrophages ( 11 5 ) . In cancer models, manipulation of GPx2 or GPx4 resulted in altered COX2 expression and PGE 2 production ( 5 , 87 ) . In macrophages specifically, research regarding on oxylipid biosynthesis focuses largely on specific AA - derived oxylipids such as prostaglandins ( 44 , 70 , 116 ) . However, LA - derived oxylipids are also essential in promoting and diminishing inflammation associated with disease ( 117 ) . Furthermore, antioxidant functioning GPx and TrxR can directly regulate both AA and LA - derived oxylipid production, such as HPETEs and HPODEs, because these FAHP can be synthesized by free radicals during conditions of oxidant stress. Since the oxylipid signaling network is complex , there is a need to charac terize the effect of selenoprotein activity on macrophage - derived , biologically active oxylipids that affect inflammation in order to uncover specific mechanisms behind potential anti - inflammatory properties. Therefore, the hypothesis of this study wa s that decreased macrophage selenoprotein activity reduces the biosynthesis of oxylipids with anti - inflammatory properties. Materials & Methods Mice & Macrophage Samples In vivo selenoprotein - status was manipulated in a murine model using a conditional knockout of the selenocysteine tRNA gene ( Trsp ) driven by the Cre - recombinase system . C57BL/6 mice carrying a floxed Trsp gene were generated as described previously and served as 28 control mice ( 99 ) . Briefly, control mice were mated with a transgenic C57BL/6 line carrying the Lysozyme - M - Cre trans gene from the Jackson Laboratory to generate Trsp knockout mice ( M ). This knockout system was driven by the lysozyme M promoter which restricted Trsp knockout to myeloid - derived cells including macrophages. All animals were maintained according to IAC UC - approved protocols and in accordance with the National Institutes of Health institutional guidelines. P eritoneal fluid, and peritoneal exudate macrophages ( PEM ) were isolated and prepared as described previously ( 99 ) . Detection of decreased Sec tRNA [Ser]Sec gene expression was measured in vivo using n orthern blot to compare levels of Sec tRNA to control Ser tRNA in wild - type and selenoprotein knockout M murine macrophages . Protein expression was quantified by Western blot. Macrophage - 75 Se for 24h, electrophoresed on gels, stained with Coomassie Brilliant Blue, and exposed to a Phosphor Imager as described ( 99 ) . Free radical production was also observed from ex vivo PEM by flow cytometry using carboxy - H 2 DCFDA (Life Technologies, Grand Island, NY ) as a fluorescent indicator of ROS. Quantitative Real - Time PCR (qPCR) Total RNA was isolated from in vivo murine PEM using Trizol (Invitrogen, Carlsbad, CA) and 1 µg of total RNA was reverse transcribed using an iScript cDNA Synthesis Kit (Bio - Rad, Hercules, CA), according to the manufacturer's instructions. All primers used in the present study were derived from the Mus musculus genome (GenBank). Each sample was amplified using Taqman PreAmp Kit (Applied Biosystems). Qu antitative real - time PCR ( qPCR ) 29 was carried out in a 7500 Fast Real - Time PCR system (Applied Biosystems) using pre - designed TaqMan minor groove binding probes from Applied Biosystems. The PCR was per formed in triplicate using a 2 0 µL reaction mixture per w ell, containing 10 µL of TaqMan Gene Expression PCR Master Mix (2x, Applied Biosystems), 1 µL of (20x) TaqMan Gene Expression Assa y Mix (Applied Biosystems), 5 µL of amplified cDNA, and the balance was nuclease - free water. Targeted genes were amplified with the reaction mixture described above. Pre - designed (20x) Taqman Gene Expression Assays for murine beta - glucuronidase ( GUSB ), glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH 2 microglobulin ( B2M ) from Applied Biosystems were used as reference genes . Each PCR plate included a non - template control to ensure no contamination was present. A non - RT control was run to ensure genomic DNA was not being amplified. The thermal cycling conditions for 2 - step PCR were us ed: stage 1 enzyme activ ation, 5 0 ° C for 2 min; stage 2, 95 ° C for 10 min ; stage 3, 95 ° C for 15 s; stage 4, 60 ° C for 1 min with 40 replications through stages 3 and 4 . Quantification was carried out with the relative quantification method ( 118 ) . The abundance of target genes, normalized to the average of the 3 reference genes and relative to a calibrator, are calculated by 2 Ct , where C t is the cycle number at which the fluorescence signal of the product crosses an arbitrary threshold set with exponential phase of the PCR and Ct = (Ct target gene unknown sample Ct average of 3 endogenous control genes unknown sample ) (Ct target gene calibrator sample Ct average of 3 endogenous control genes calibrator sample ). Averaged abundance of target genes in control PEM was used as the calibrator sample for all subsequent samples. Solid Phase Lipid Extractions & Liquid Chromatography - Mass Spectrometry (LC - MS) 30 Macrophage cell pellets, peritoneal fluid , and RAW 264.7 macrophage media supernatant sampl es were collected as follows. Cell pellets were first suspended in 600 1x PBS and sonicated. All s antioxidant/reducing agent that reduced hydroperoxides to their corresponding hy droxyls containing ethylenediaminetetraacetic acid ( EDTA ), butylhydroxy toluene ( BHT) , triphenylphosphine ( TPP Samples contain 200ul of the following deuterated oxylipids (0.1 ng/µl, 20 ng tota l): LTB 4 - d4 , TxB 2 - d4 , PGF - d4 , PGE 2 - d4 ,PGD 2 - d4 , 13(S) - HODE - d4 , 6 - keto PGF - d4 , 13 - oxoODE - d3 , 9 - oxoODE - d3 , 12(S) - HETE - d8 , 15(S) - HETE - d8 , 8 - iso - PGF - d4 (Cayman Chemical, Ann Arbor, MI). Samples received 60% total v/v of methanol ( MeOH ), and were kept at - 80 o C for 30 min to precipitate protein. Samples were then centrifuged at 4000 xG for 30 min at 4 o C for peritoneal fluid and media supernatants or 14,000 xG for 15 min for cell pellets . Lipids were isolated from the samples by solid phase extraction using a Phenomenex Strata - X 33u Polymeric Reverse Phase Column 200 mg/6mL (8B - S100 - FCH, Phenomenex, Torrance, CA) for cell pellets or an Oasis HLB 12cc(500 mg) LP Extraction Cartridge (186000116, Waters, Milford, MA) for peritoneal fluid and media supernatant . Columns were first conditioned with 6 mL MeOH then 6 mL water. Samples were diluted to 10% v/v MeOH with water then run through the column, washed with 40% MeOH, dried, and eluted from the columns i n MeOH/acetonitrile (50:50 v/v). Samples were then dried in a Sevant SVD121P SpeedVac (Thermo Scientific, Waltham, MA), suspended in acetonitrile/water/formic acid (37:63:0.02 v/v/v) , and centrifuged at 14,000 xG for 30 min prior to analyzing by LC - MS. 31 Oxylipids were analyzed using two distinct LC - MS methods. Both utilized reverse - phase LC on a Waters ACQUITY UPLC ® BEH C18 1.7µm column (2.1 X 100mm) at a flow rate of 0.6 ml/min at 35 o C and a quadrupole mass spectrometer (Waters ACQUITY SQD H - Class) in electrospray negative ionization mode. The electrospray v oltage was - 3 kV and the turbo ion spray source temperature was 450 o C. Nitrogen was used as the drying gas. For each method, 10 µl samples were injected in triplicates. An isocratic mobile phase consisting of Aceton itrile:Water:0 .1% Formic acid (35:55: 10; v/v/v) with an analysis time of 15 min was used to analyze 8 - iso PGF , LTB 4 , PGE 2 , PGD 2 , Lipoxin A 4 , PGF , TxB 2 , 6 - keto PGF , Resolvin D 1 , and Resolvin D 2 . The second method utilized an isocratic mobile phase of Acetonitrile:Methanol:Water:0.1%Form ic acid (47.4:15.8:26.8:10; v/v/v/v) and an analysis time of 10 min to analyze 9(S) - HODE, 13(S) - HODE, 15 - OxoETE, 5 - OxoETE, 5(S) - HETE, 11(S) - HETE 12(S) - HETE, 15(S) - HETE, 9 - oxoODE, 13 - oxoODE, 7(S) - Maresin1 ( MaR1 ), Protectin D 1 ( PD 1 ), and LTD 4 . Oxylipids w ere identified in samples by matching their deprotonated (i.e., [M - H] - ) m/z values and LC retention times with those of a pure standard. Quantitative Oxylipid Analysis was performed with Waters Empower 2 software. A linear calibration curve with 5 point s (r 2 > .99) was generated for each oxylipid with standards and internal standards purchased from Cayman Chemical (Ann Arbor, MI). The curves range from 0.002 ng to 2.38 ng/µl . Empower 2 identifies the sample peak by matching its retention time with the s tandard. A response is calculated for each matched peak by dividing the sample concentration of the internal standard for each matched peak. The amount for each samp le is 32 calculated using the response peak, injection volume, and DNA concentration of the sample to yield n g of oxylipid/ng of DNA which is expressed in figures as a fold change to control samples . DNA was quantified using Quant - iT DNA Assay Kit, Broad Rang e according to ( Life Technologies ). RAW 264.7 Macrophage Culture & Oxylipid Stimulation Murine - derived RAW 264.7 macrophages were cultured in Se - sufficient ( +Se ) media or Se - deficient ( - Se ) media to model decreased selenoprotein - activity in vitro . E nzyme activity assays (GPx and TrxR) and ROS accumulation were calculated to confirm diminished selenoprotein activity and increased ROS from Se samples as described previously by our group ( 36 , 92 ) . Macrophages were obtained from t he ATCC (TIB - 71; ATCC, Manassas, VA). Briefly, cells were grown in a T75 culture flask at 37°C with 5% CO 2 . Once cells reached 75 to 90% confluence, they were split and transferred into a T225 culture flask either with or without Se. Before all experiments acid free albumin for 24 h as previously described ( 119 ) to mimic the lipid content of mouse PEM. The macrophages were cultured in RPMI 1640 medium (17 - 105 - CV; Cellgro, Manassas, VA) containing 5% fetal bovine serum (FBS), antibiotics and antimycotics (100 U/mL), L - glutamine (300 mg/mL), and sodium selenite (0.1 µM , for +Se media ). To determine the inflammatory effect of certain oxylipids that were significantly macrophages were stimulated with the LA - derived ketone, 9 - oxoODE , or the AA - derived lipid hydroperoxide, 15 - HPETE or lipoxin A 4 ( LXA 4 ) . RAW 264.7 macrophages were seeded into 100 mm dishes , incubated overnight , and stimulated with a - 1, Cayman) for 1 h then LXA 4 or 9 - oxoODE ( 100 nM for 2 h , 33 Cayman Chemical) , or with 15 - for 2 h , synthesized by our group as previously described ( 93 ) ). Ethanol served as the vehicle did not exceed 0.1% of culture medium . Pro - and anti - inflammatory cytokine production was quantified using a cytometric bead array as described below to characterize the inflammatory response of the macrophages. Cellular viability was measured using the CellTiter - Glo Luminescent Cell Viability Assay according to manufactures instructions (Promega, Madison, WI). Cytometric Bead Array Mouse i nflammation multi - plex bead assay kits w ere instructions to quantify the production of inflammatory mediators in RAW 264.7 macrophage samples (552364, BD Biosciences, San Jose, CA). Cytokine production was quantified as pg of cytokine per ng of DNA and expressed in figures as fold change to controls . DNA was quantified using Quant - iT DNA Assay Kit, Broad Range ( Life Technolog ies ) according to instructions. Statistical Analyses All statistical analyses were conducted using SAS, version 9.1.2 for Windows (SAS Institute Inc., Cary, NC). Pearson correlation coefficients were computed to determine relationships betwe activity. The effect of selenoprotein - status or other treatments in macrophages on the relative abundance of mRNA, protein expression, cytokine production and oxylipid synthesis were tested by the MIXED procedure of SAS (using mouse as the random effect) - test for RAW 264.7 macrophage data 34 compare least squares means and data are reported as least squares mea ns ± standard error of the means. Significant differences were declared at p Results Selenopro tein Expression from In V ivo Macrophages. N orthern blot analysis was used to compare Sec tRNA to control Ser tRNA in wild - type and selenoprotein conditi on knockout M murine macrophages ( 99 ) . A substantial decrease of Sec tRNA was found in M when compared to control mice. To confirm that no selenoproteins were being synthesized, proteins were labeled with 75 Se and electro phoresed on an SDS gel (Figure 6 ) ( 99 ) . When compared to control macrophages, M had consi derably decreased selenoprotein expression. ROS accumulation was examined previously as a functional consequence of reducing antioxidant selenoprotein activity. Notably increased production of ROS was observed in M macrophages compared to controls ( 99 ) . Selenoprotein Activity Modifies Inflammatory Gene Expression in PEM. Gene expression of oxylipid biosynthetic enzymes and inflammatory cytokines from in vivo PEM is outlined in Figure s 7 and 8 , respectively. A significant increase in gene expression of the COX2, 15 - LOX1, 15 - LOX2, and 5 - LOX oxylipi d biosynthetic enzymes (Figure 7 ) was observed in M macrophages compared to control PEM. Macrophages from M mice exhibited a significant increase in the mRNA expression of the pro - inflammatory cytokine TNF , and a cadherin that can mediate macrophage migration during inflammation CHD11 35 (Figure 8 ) . There was no difference in the expression of monocyte chemotactic protein - 1 ( MCP - 1 ) or IL - (Figure 8 ). M Mice & RAW 264.7 Macrophages. O xylipid s derived fro m LA or AA were quantified in peritoneal fluid as outlined in Figure 9 and from media supernatant of RAW 264.7 macrophages in Figure 10 . There was very little oxylipid accumulation found within the cell, most oxylipids where present in the peritoneal fluid and media supernatant. In the peritoneal fluid, when compared to controls, M mice had significantly diminished production of LXA 4 ( p < 0.05 ) , as well as the AA - derived hydroxyl 12 - HETE ( p=0.06 ) (Figure 9 a - b ). LA - derived oxylipids including: fatty acid hydroxyl 9 - HODE ( p=0. 13 ), and fatty acid ketone 9 - oxoODE ( p < 0.05 ) were also diminished in M peritoneal fluid compared to controls (Figure 9 c ) . In RAW 264.7 macrophage media supernatant, oxylipids that were significantly decreased in Se compared to +Se control cells are outlined in Figure 10 and included : TxB 2 , PGF (Figure 10 a), AA - derived hydroxyls , 15 - HETE, 11 - HETE and 12 - HETE (F igure 10 b), LA - derived hydroxyls , 13 - HODE and 9 - HODE, LA - derived ketones 13 - oxoODE and 9 - oxoODE (Figure 10 c). Oxylipids that did not change as a function of selenoprotein activity included: LTB 4 , PGE 2 , PGD 2 , RvD1, RvD2, 15 - OxoETE, 5 - OxoETE, 5 - HETE, MaR1, P D 1 , and LTD 4 (data not shown). Oxylipid Treatment Pearson correlations between oxylipid production and TNF expression as a function of selenoprotein activity are outlined in Table 3 . Significant correlations were found for several 36 oxylipids and TNF from Se supplemented macrophage samples that expressed selenoproteins activity. Oxylipids derived from LA; 13 - HODE, 9 - HODE, and 9 - oxoDE were all negatively correlated with TNF . Conversel y, the AA - derived oxylipid, 15 - HETE, was positively correlated with TNF expression in the Se supplemented macrophage samples. In order to get a better understanding of how oxylipids might affect the inflammatory response of macrophages during oxidative st ress, RAW 264.7 macrophages were cultured either without Se or stimulated with SIN - 1 to elicit ROS production (Figure 11 ). The RAW 264.7 macrophages cultured under these pro - oxidant conditions were then treated with LXA 4 or 9 - oxoODE to determine if enhanc ed TNF production could be reduced to the amounts observed in the Se supplemented cells. Alternatively, the Se supplemented cells were treated with the pro - oxidant oxylipids, 15 - HPETE , to determine the effect on TNF . Compared to +Se macrophages, LXA 4 and 9 - - HPETE signif icantly increased ) . Cellular viability did not change as a function of any treatment groups (data not shown). Discussion This study is the first to directly link loss of sel enoproteins activity to changes in macrophage oxylipids biosynthesis that can impact inflammatory phenotype. A macrophage - specific selenoprotein knock out model ( M ) was used to investigate the direct effects of altered selenoproteins activity on oxylipids biosynthesis. As previously confirmed by Carlson et al. ( 99 ) , M macrophages exhibit significantly decreased Sec tRNA gene expression, selenoprotein expression, and increased accumulati on of ROS. This pro - oxidant phenotype is 37 consistent with other studies that investigated effects of Se deficiency in bovine endothelial cells, rodent lymphocytes and macrophages, and whole animal murine models ( 44 , 92 , 99 ) . Using this M model, the impact that ablated selenoproteins activity has on macrophage inflammatory markers relevant to acute and chronic inflammation were characterized. E xpression of a cadherin (CHD11) i n M macrophages was increased in this study and is an indicator of inflammatory response. Cadherins are a family of transmembrane proteins that play an important role in cell adhesion and the maintenance of tissue architecture. CHD11 expression is a lso known to increase in inflamed synovial fluid and selective down regulation of CHD11 significantly reduced joint inflammation in experimental arthritis ( 120 ) . Earlier reports using bone marrow - derived macrophages ( BMDM ) obtained from M mice also found an increased expression of CHD11 ( 99 ) . Thus, the increased expression of CHD11 by peritoneal macrophages obtained from M mice can function as a marker of inflammation during diminished selenoprotein activity. This study also reported a significant increase in the pro - inflammatory mediator TNF from M macrophages. Increased TNF as a consequence of decreased Se in murine macrophages was formerly established ( 45 ) . Additionally, TNF expression was used as a marker of inflammatory diseases such as atherosclerosis ( 121 ) and mastitis ( 122 ) . However, TNF was not increased in BMDM when selenoprotein expression was decreased, which suggests that macrophages in different stages of maturity and location will have different inflammatory responses during decreased selenoprotein activity ( 99 ) . The increased expression of both CHD11 and TNF in the peritoneal exudate obtained from M 38 mice, however, suggests that reduced selenoprotein activity results in enhance pro - inflammatory phenotype. Since e nzymatic oxidation of fatty acids by COX and LOX enzymes is a significant source of oxylipids , the gene expression of these enzymes as a function of selenoprotein activity was assessed . This study showed that M macrophages had significantly increased COX2, 15 - LOX1, and 15 - LOX2 gene expression compared to contro ls. These findings are of significant interest since this is the first direct evidence that selenoproteins are involved in regulating these oxidizing enzymes. Alterations in overall Se status were previously linked with modifications of these enzymatic pa thways. For example, e nhanced expression of COX2 was well documented in Se - deficient macrophages in several previous studies ( 45 , 100 , 101 ) . A suggested mechanism behind increased COX2 expression during Se - deficiency involved the redox - sensitive y Zamamiri - Davis et al. ( 101 ) Se - deficiency. However, less is known about how Se and selenoproteins specifically may affect 15 - LOX expressio n . Previous studies did show that the enzyme activity of purified 15 - LOX was decreased with increasing concentrations of the seleno - organic compound ebselen ( 74 ) . The proposed mechanism involved is that Se could be interacting with the oxidation status of the active iron in 15 - LOX; thus reducing its activity. During decreased selenoprotein expression in this study, increased expression of both 15 - LOX - 1, and 15 - LOX - 2 was found. Further research would be necessary to determine how selenoprotein s such as GPx and TrxR could specifically affect transcriptional and post - transcriptional regulation of expression and activity of COXs, 39 LOXs , and cytochrome P450 ( CYP450 ) enzymes that are involved in oxylipid production in macrophages . After showing that COX/LOX expression and free radicals were increased as a function of decreased selenoprotein expression, a targeted lipidomic profile of oxylipids implicated in the regulation of inflammation during disease was characterized . Oxylipids derived from LA and AA were specifically included in the profile s ince both are found in significant quantities in human Western diet s and are major components of cellular membranes ( 123 ) . No difference in the production of several well - characterized pro - inflammatory oxylipids w as observed in peritoneal fluid of M mice , including PGE 2 , PGD 2 , LTB 4 , and 5 - HETE . These results were surprising since p rior studies described a decrease in macrophage - derived PGE 2 following Se - supplementation ( 124 ) . A possible explanation for these disparate finding s may be due to the analytical method used for oxylipid detection. Whereas earlier reports quantified oxylipids by e nzyme immunoassays ( 124 ) , the current study is among the first to document changes in oxylipid production as a function of selenoprotein activity in murine macrophages using LC/MS which is capable of yielding a higher degree of specificity . More specific analysis of oxylipid biosynthesis is becomin g increasingly important due to the low production and unstable nature of many oxylipids. Another possible explanation for reported differences may be due to the method used to induce oxylipids biosynthesis. For example , previous studies documented increa sed PGE 2 production using a TLR4 - mediated inflammatory response induced by lipopolysaccharide ( LPS ) activation in RAW 264.7 macrophages , while the present study characterized peritoneal thioglycollate - elicited macrophages and RAW 264.7 macrophages exposed to pro - oxidant challenge ( 101 , 124 ) . In bovine endothelial cells, however, Se - deficiency significantly altered 40 productions of PGE 2 , TxB 2 , and 5 - HETE wit hout LPS induction ( 92 ) . Taken together, these results suggest that differing oxylipid responses depend on cell - type and the specific inflammatory model studied. This study did report for the first time, however, that M selenoprotein knockout mice have a significant decrease in several LA - and AA - derived oxylipids. Both LXA 4 and 9 o xoODE production was significantly reduced in peritoneal fluid of M mice when compared to controls. The appearance of LX is thought to signal the resolution of inflammation and plays an important role in controlling the pathogenesis of inflammatory - based diseases. For example, LXA 4 was shown to diminish macrophage pro - in flammatory cytokine production and prevent the development of atherosclerosis ( 64 ) . Similarly, the LA - derived ketone 9oxoODE was also associated with anti - inflammatory functions. This ketone is formed through the reduction of 9 - HPODE to the hydroxyl 9 - HODE which is then oxidized through the actions of a dehydrogenase to form 9 - oxoODE. The LA - derived hydroxyls (9 - HODE and 13 - HODE) and their oxidized ketones (9 - oxoODE and 13 - oxoODE) are natural ligands for PPAR signaling that can inhibit inflammation by supp ressing NF B activation ( 125 ) . On the other hand, LA - derived hydroxyls and ketones were found to increase inflammatory pain in the spinal cord by activating pain receptors ( 126 ) . Whereas only 9oxoODE was decreased in the peritoneal fluid of M mice, macrophages cultured in Se deficient media exhibite d significant decreases in all of these LA - derived metabolites suggesting that selenoprotein activity maybe a critical part of their metabolic pathway. 41 Free radical - mediated oxylipid metabolism could prove to be important in macrophages with altered seleno protein activity since the primary selenoproteins expressed in macrophages include the antioxidant functioning GPx and TrxR ( 99 ) . In bovine endothelial cells, increased ROS during Se - deficiency resulted in enhanced production of the hydroperoxide, 15 - HPETE, whereas Se - sufficient cells had increa sed production of the reduced hydroxyl, 15 - HETE ( 93 ) . In rat aortas, Se - deficiency resulted in significantly diminish ed GPx activity and 9 - HODE production ( 112 ) . Our data is consistent with these previous reports in that we also found that increased ROS during diminished selenoprotein activity coincided with decreased 9 - HODE an d 15 - HETE. As described in the extraction procedure to measure oxylipids, the addition of an antioxidant and reducing agent to inhibit autoxidation prevented us from measuring the highly unstable lipid hydroperoxides derived from AA and LA. Therefore, more research would be needed to characterize AA - and LA - derived FAHP during decreased selenoprotein activity. Additionally, it will be important to determine the role each oxylipid plays during inflammation and disease as a function of selenoprotein activity in both different cell - types and disease models. To obtain a better insight into how oxylipids that are produced as a consequence of selenoprotein expression might affect inflammation, TNF expression was correlated with the production of oxylipids . Interestingly, several LA - derived oxylipids that were increased in control macrophages, including the hydroxyls 13 - HODE and 9 - HODE, and the 9 - oxoODE ketone, where all negatively correlated with TNF expression. These results suggest the increased productio ns of these LA oxylipids during sufficient selenoprotein activity may exert anti - inflammatory effects. Conversely, 15 - HETE production was positively correlated with TNF that expressed selenoprotein activity. Previously, our group 42 explored the production and effect of 15 - HPETE and 15 - HETE in bovine endothelial cells ( 93 ) . When +Se endothelial cel ls were stimulated with 15 - HPETE, adhesion molecule expression increased to values statistically the same as Se cells, while 15 - HETE had no effect ( 93 ) . Since 15 - HETE was decreased in macrophages that expressed selenoprotein activity in our model, the present results may suggest that the activity of the upstream hydroperoxide, 15 - HPETE , may be pro - inflammatory in macrophages . Furthermore, because the oxylipid extraction method used in this study reduced all hydroperoxides to their corresponding hydroxyl forms, more research is needed to specifically quantify lipid hydroperoxides in macrophages as a function of sele noprotein expression . To further explore the impact of altered oxylipids biosynthesis on macrophage inflammatory phenotype, RAW 264.7 macrophages cultured under pro - oxidant conditions were stimulated with LXA 4 , 9 - oxoODE, or 15 - HPETE . After addition of LXA 4 or 9 - oxoODE , there was a significant decrease in the production of TNF Unlike LXA 4 , which was previously ( 64 ) , this is the first study to quantify TNF stimulation with 9 - oxoODE . Previously, o xidation product s of linoleic acid, including 9 - oxoODE, were found to be significant components of atherosclerotic plaques , although there was no correlation between these oxylipids and symptomatic vs. asymptomatic patients ( 127 ) . Interestingly, 9 - oxoODE also serves as an agonist of PPAR and can bind with greater affinity than the hydroxyl HODE metabolites of LA ( 125 ) . The current results suggest an anti - inflammatory role for 9 - oxoODE in macrophages. Oppositely, w hen 15 - HPETE was added to macrophage s, there was a significant increase in the production of TNF which was p reviously shown in endothelial cells ( 93 ) . These results suggest that the balance of 43 15 - HETE and 15 - HPETE production is critical in regulating inflammation compared to the addition of the hydroperoxide metabolite alone. Future studies should aim at identifying the significance in the balance of hydroperoxide to hydroxyl production and how this ratio could affect the infl ammatory response. Overall, our study demonstrated that selenoproteins play a role in macrophage - derived oxylipid biosynthesis. Further studies are warranted to determine the mechanisms by which selenoproteins regulate inflammation through oxylipid product ion such as their capacity to: (1) reduce FAHP, (2) mitigate oxidative tone, and (3) regulate COX/LOX en zymatic expression and activity . Conclusion Since Se was shown to play a beneficial role in inflammation, and the anti - inflammatory properties of Se are thought to occur though selenoproteins, it was important to characterize how selenoprotein activity affects the oxylipid network in the context of the inflammatory response . A dequate Se - intake to maximize selenoprotein activity is associated with decrease d production of pro - inflammatory, AA - derived oxylipids in macrophages ( 8 , 101 ) . In the current study , for the first time, oxylipid biosynthesis was characterized in murine macrophages as a function of selenoprotein activity directly , focusing on oxylipids derived from AA and LA that mediate inflammation. Furthermore, this study examined how the inflammatory response was altered as a consequence of specific oxylipids. Overall, selenoprotein - status had a significant effect on hydroxyl and ketone oxylipids metabolized from AA and LA . Additionall y, some of these oxylipids have the potential to mediate the inflammatory response of macrophages during pro - oxidant challenge. It is important to study how oxylipids from AA and LA are affected by selenoproteins, because both AA and LA make up a significa nt portion of the human diet, are predominate in cellular membranes, and have been implicated in inflammatory diseases. 44 Moreover, free radical - mediated oxidation of AA and LA could play a significant role in controlling inflammation during oxidative stress , which could potentially be mediated by selenoproteins . Future studies are needed to uncover how these oxylipids are produced (i.e. by enzymes and/or by free radical oxidation), which selenoproteins have an effect, and what affect these oxylipids have on macrophage - derived inflammatory responses as a function of selenoprotein activity. Acknowledgements and Lori Bramble for their assistance in running the Cytometric Bead Array for quantification of inflammatory cytokines. Authors would also like to thank Drs. Dan Jones and Scott Smith at the Michigan State University Research Technology Support Facility Mass Spectrometry Core for their assistance developing a method to quantitate fatty acids in cellular samples by GC/MS. This work was supported, in part, by the Agricultu re and Food Research Initiative Competitive Grants Program numbers 2011 - 67015 - 30179 and 2012 - 67011 - 19944 from the United States Department of Agriculture National Institute for Food and Agriculture and by an endowment from the Matilda R. Wilson Fund (Detro it, MI). 45 APPENDIX 46 Table 1. Summary of mammalian selenoproteins with characterized functions. Selenoprotein Proposed Function GPx: 1,2,3,4,6* TrxR: 1,2,3 Antioxidant/Modify Redox Tone Sepw1, Selk, Sepp1 Antioxidant SelR Reduction of Methyl Sulphoxy Groups Sepp1 Se Transport in Blood Sephs2 Selenoprotein Synthesis Sep15, Selm, Seln, Sels Involved in Misfolded Protein Response in the ER SelH Redox Sensitive DNA - Binding Protein SelI Phospholipid Synthesis Sepn1 Calcium Signaling in the ER DIO1,2,3 Thyroid Hormone Synthesis Sel O, V Unknown Function Adapted from ( 31 , 105 , 106 ) * GPx6 contains a Sec in humans and a Cys in rodents. 47 Table 2. The impact of Se and Selenoproteins on Oxylipid Biosynthesis. Se - metabolite Outcome Resulting from Sufficient Levels of Se - metabolite Level of Oxylipid Regulation Selenium Substrate Selenium - PDGS Enzyme Expression - 1 4 H Enzyme Activity GPx4 Oxylipid Production Selenium 2 :6keto - PGF ratio 2 4 2 , PGF GPx1,4 Reduces HPETEs to HETEs GPx1,4 Reduces HPODEs to HODEs 48 Impact on the Regulation of Inflammation. Some of the several ways in which inflammation is mediated include: 1) signaling through the - kinase, and PPAR , 2) cellular redox tone, 3) the production of inflammatory mediators such as cytokines, and chemokines, 4) oxidative stress, and 5) oxylipid biosynthesis. Selenium was shown to affect each of these regulators and this review will focus s impact on the production of oxylipids. 49 Figure 2. Se metabolism from different dietary sources. Dietary intake sources of Se include the inorganic selenate and selenite (depicted in the right stars); whereas organic sources (depicted in the left s tars) are obtained from animal and plant sources that provide Se in the form of selenocysteine, selenomethionine, and Se - methylselenocysteine (Se - methyl - Sec). Inorganic forms of Se are reduced by TrxR and Trx or converted to selenodiglutathione (GS - Se - SG) by GSSG, reduced by glutathione reductase to glutathioselenol, then converted to hydrogen selenide (H 2 Se) in a reaction with GSSG. Selenoproteins are broken down by lyases to form H2Se in intestinal enterocytes. H 2 Se can then be converted into selenophosph ate by selenophosphate synthase and selenocysteine by selenocysteine synthase for incorporation of Sec into selenoproteins. Hydrogen selenide can also be converted into methylated metabolites by methyltransferases which are primarily excreted through exhal ation, urine and feces. 50 A) B) Figure 3. General reaction mechanisms for antioxidant GPxs and TrxRs. A) GPxs catalyze the chemical reduction of lipid or hydrogen peroxides to respective alcohols and water by glutathione (GSH) which forms glutath ione disulfide (GSSG). Glutathione reductase (GSR) catalyzes the reduction of GSSG back to GSH in the presence of NADPH. B) Oxidized protein disulfides and other free radicals are reduced to their corresponding thiols by thioredoxin (Trx). TrxR then cataly zes the reduction of oxidized Trx in the presence of NADPH. 51 Figure 4. Oxylipid Biosynthesis Pathways. Omega - 3 and omega - 6 fatty acids are released from the cellular membrane by phospholipase enzymes. Long - chain, polyunsaturated fatty acids (PUFAs) are oxidized either non - enzymatically by free radicals or by COX1/2, 15LOX, and 5LOX enzymes to produce oxylipid signaling metabolites. Isoprostanes; F 2 IsoP, prostaglandins; PG, thromboxanes; TX, resolvin E/D series; Rv, lipoxins E/D series; LX, protectin ; PD, Maresin; MaR, leukotrienes; LT. 52 A) B) Interaction with Oxylipid Biosynthesis Pathways. 53 A) Selenium and selenoproteins interfere with oxylipid feedback loops. While GPx1 and 4 can reduce fatty acid hydroperoxi des (FAHP) to decrease COX2 activity, a buildup of FAHP, when GPx activity is lacking, can also inhibit COX2. GPx2 and 4 diminish PGE 2 - dependent expression of COX2. Se enhances 15d - PGJ 2 signaling enhances H - PGD S, which synthesizes PGD2, an upstream metabolite of 15d - PGJ 2 . B) and AP - 1 and expression of COX/LOX and other inflammatory mediators such as TNF and MCP - 1. GPxs can alter the redox state of the MyD88 adaptor protein, when MyD88 is denitrosylated by GPx with GSH, signaling is enhanced. ROS - can be dampened when antioxidant selenoproteins are present to scavenge ROS. T he MAP - kinases can also be affected; ROS - mediated oxidation of Trx causes its dissociation from ASK - 1 kinase, enhancing signaling activity. In the nucleus, Trx can reduce oxidized Cys residues on 54 Table 3. Pe arson Correlations for Oxylipids Produced by Control or M Knockout Mice and TNF , n=5. N.S. 1 Not significant, p>0.05 . Selenoprotein Activity Oxylipid Metabolite Control LXA 4 R 0.69050 p N.S 1 9 - HODE R - 0.9709 p 0.0291 9 - oxoODE R - 0.9623 p 0.0377 13 - HODE R - 0.988 p 0.012 13 - oxoODE R - 0.8361 p N.S 1 15 - HETE R 0.96408 p 0.0359 M LXA 4 R 0.20771 p N.S 1 9 - HODE R - 0.3264 p N.S 1 9 - oxoODE R - 0.8985 p N.S 1 13 - HODE R 0.33796 p N.S 1 13 - oxoODE R 0.67470 p N.S 1 15 - HETE R - 0.8627 p N.S 1 55 Figure 6. Selenoprotein Knockout in Murine Macrophages. Peritoneal elicited macrophages (PEM) were labeled with radioactive 75 Se and electrophoresed. The left panel depicts Coomassie Brilliant Blue staining (CBB) which served as the loading control, and the right panel depicts the labeled selenoproteins. L anes 1 and 2 represent control mice with ample selenoprotein expression while lane 3 represents a M knockout mouse lacking selenoprotein expression in macrophages. 56 Figure 7. Oxylipid Biosynthetic Enzyme Expression in Macrophages. In vivo PEM were collected from control and M knockout mice. Cells were collected for gene expression COX1, COX2, 15 - LOX1, 15 - LOX2, and 5 - LOX. Quantification was carried out with the 2 - relative quantification method [ 26 ] . Averaged abundance of target genes for control samples was used as the calibrator sample for all subsequent sampl es, * Significance p < 0.05 , n=4 . 57 Figure 8. Inflammatory Cytokine Expression by Macrophages. In vivo PEM were collected from control and M knockout mice. Cells were collected for gene expression of IL - - . Quantification was carried out with the 2 - relative quantification method [ 26 ] . Averaged abundance of target genes for c ontrol samples was used as the calibrator sample for all subsequent samples, * Significance p < 0.05 , n=4 . 58 A) B ) C) Figure 9. Oxylipid Biosynthesis in the Absence of Selenoproteins . 59 Oxylipid production is represented from in vivo peritoneal fluid from control ( white bar ) or selenoprotein knockout ( M , filled bars ). ( A) Production of AA - derived prostaglandins and lipoxin A 4 . (B) Production of AA - derived 11 - HETE, 12 - HETE, 15 - HETE, and 15 - oxoETE. (C ) Production of LA - derived 13 - HODE, 13 - oxoODE, 9 - HODE, and 9 - oxoODE . Oxylipids are expressed as ng of oxylipid metabolite per mL total peritoneal fluid and depicted as a fold over the con trol mouse samples. * Significance p < 0.05 compared to control mice, n=5 . 60 A) B) C) Figure 10. Oxylipid B iosynthesis from RAW 264.7 M acrophages. 61 Oxylipid production is represented from media supernatants of +Se ( white bar ) or Se ( filled bars ). ( A) Production of AA - derived prostaglandins and thromboxane. (B) Production of AA - derived 11 - HETE, 12 - HETE, 15 - HETE, and 15 - oxoETE. (C ) Production of LA - derived 13 - HODE, 13 - oxoODE, 9 - HODE, and 9 - oxoODE . Production is expressed as ng of oxyl ipid metabolite per ng DNA and depicted as a fold over the +Se samples . * Significance p < 0.05 compared to +Se controls , n= 4 . 62 Figure 11. ROS Production Following Pro - oxidant Challenge. Flow cytometric analysis of ROS production from +Se (white bar), - Se (grey bar), and +Se stimulated with SIN - redox - sensitive fluorescence dye, H 2 - DCFDA (Life Technologies). 63 Figure 12. Effect of Oxylipid Stimulation on RAW 264.7 Macrophage Production . When macrophages were stimulated with 2 0 15 - HPETE for 2 h, production of TNF increased compared to control s . When macrophages were stimulated with 100 nM of either LXA 4 or 9 - oxoODE for 2 h, TNF decrease d compared to controls. Vehicle represents ethanol without any oxylipid. Production of TNF was quantified as pg of cytokine per ng DNA and expressed as a fold over control cells . DNA was quantified using Quant - i T DNA Assay Kit, Broad Range. * Significance p < 0.05 compared to controls, n=4. 64 Figure 13 : Glutathione peroxidase 1 (GPx1) activity from RAW 264.7 macrophages cultured with various doses of selenium. RAW 264.7 cells were seeded in 6 - well plates and cultured for 3d with various doses of Se provided as sodium selenite. Following 3 d culture, cells were harvested an analyzed for GPx1 activity as described in the Materials & Methods section of Chapter 2. 65 A) B) Figure 14 . Reactive oxygen species production by RAW 264.7 macrophages cultured in 5% (A) or 10% (B) FBS , n=4 . RAW 264.7 macrophages were cultured in 5% or 10% FBS, stained with the redox - sensitive dye, H 2 - DCFDA for 30min, and analyzed for ROS production using flow cytometry. (A) A significant increase in ROS production is observed from Se compared to +Se macrophages cultured in 5% FBS, whereas (B) there is no difference when macrophages are cultured in 10% FBS. * Significance p < 0.05. 66 A) B) Figure 15 . Total fatty acid analysis of arachidonic (A) or linoleic (B) acid from murine peritoneal macrophages (n=3) or RAW 264.7 macrophages (n=6) . 67 RAW 264.7 fatty acid profiles (following 24hr culture with various doses of AA or LA) were compared to PEM from control mice using GC/MS . (A) AA content of RAW 264.7 ma crophages significantly increased in a dose - dependent manner. Any dose of AA added to RAW 264.7 macrophages significantly increased the AA content compared to control mouse PEM. (B) LA content of RAW 264.7 macrophages significantly increased in a dose - depe ndent manner. RAW significantly matched control mouse PEM. * Significance p < 0.05. 68 Figure 16. Effect of LA - derived Oxylipid Stimulation on RAW 264.7 Macrophage Production . When macrophages were stimulated with 100 nM 13 - oxoODE, 5 M 9 - HODE, or 5 M 13 - HODE for 2 h, TNF decreased compared to controls. Vehicle represents ethanol without any oxylipid and KLA used as positive control. Production of TNF was quantified as pg of cytokine per ng DNA and expressed as a fold over control cells . DNA was quantified using Quant - i T DNA Assay Kit, Broad Range. * Sign ificance p < 0.05 compared to controls, n=3. 69 70 BIBLIOGRAPHY 71 BIBLIOGRAPHY 1. Lloyd - Jones, D., Adams, R., Carnethon, M., De Simone, G., Ferguson, T. B., Flegal, K., Ford, E., Furie, K., Go, A., Greenlund, K., Haase, N., Hailpern, S., Ho, M., Howard, V., Kissela, B., Kittner, S., Lackland, D., Lisabeth, L., Marelli, A., McDermott, M. , Meigs, J., Mozaffarian, D., Nichol, G., O'Donnell, C., Roger, V., Rosamond, W., Sacco, R., Sorlie, P., Stafford, R., Steinberger, J., Thom, T., Wasserthiel - Smoller, S., Wong, N., Wylie - Rosett, J., Hong, Y., and for the American Heart Association Statisti cs Committee and Stroke Statistics, S. (2009) Heart Disease and Stroke Statistics - 2009 Update. Circulation 119 , e21 - e181 2. Boosalis, M. G. (2008) The Role of Selenium in Chronic Disease. Nutrition in Clinical Practice 23 , 152 - 160 3. Sordillo, L. M., Contr eras, G. A., and Aitken, S. L. (2009) Metabolic factors affecting the inflammatory response of periparturient dairy cows. Animal Health Research Reviews 10 , 53 - 63 4. Van der Linden, M. W., Van der Bij, S., Welsing, P., Kuipers, E. J., and Herings, R. M. C. (2009) The balance between severe cardiovascular and gastrointestinal events among users of selective and non - selective non - steroidal anti - inflammatory drugs. Annals of the Rheumatic Diseases 68 , 668 - 673 5. Banning, A., Florian, S., Deubel, S., Thalmann, S., Muller - Schmehl, K., Jacobasch, G., and Brigelius - Flohe, R. (2008) GPx2 Counteracts PGE2 Production by Dampening COX - 2 and mPGES - 1 Expression in Human Colon Cancer Cells. Antioxidants & Redox Signaling 10 , 1491 - 1500 6. Flores - Mateo, G., Navas - Acien, A., Pastor - Barriuso, R., and Guallar, E. (2006) Selenium and coronary heart disease: a meta - analysis. The American Journal of Clinical Nutrition 84 , 762 - 773 7. Aitken, S . L., Karcher, E. L., Rezamand, P., Gandy, J. C., VandeHaar, M. J., Capuco, A. V., and Sordillo, L. M. (2009) Evaluation of antioxidant and proinflammatory gene expression in bovine mammary tissue during the periparturient period. Journal of Dairy Science 92 , 589 - 598 72 8. Cheng, A. W. M., Stabler, T. V., Bolognesi, M., and Kraus, V. B. (2011) Selenomethionine inhibits IL - 1 beta inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) expression in primary human chondrocytes. Osteoarthritis and Carti lage 19 , 118 - 125 9. Moxon, A. L., and Rhian, M. (1943) SELENIUM POISONING. Physiological Reviews 23 , 305 - 337 10. Muth, O., Oldfield, J., Remmert, L., and Schubert, J. R. (1958) Effects of selenium and vitamin E on white muscle disease. Science 128 , 1090 - 10 91 11. Chen, X., Yang, G., Chen, J., Chen, X., Wen, Z., and Ge, K. (1980) Studies on the relations of selenium and Keshan disease. Biological Trace Element Research 2 , 91 - 107 12. Monsen, E. R. (2000) Dietary Reference Intakes for The Antioxidant Nutrients: Vitamin C, Vitamin E, Selenium, and Carotenoids. Journal of the American Dietetic Association 100 , 637 - 640 13. Abdo, K. M. (1994) NTP Technical Report on Toxicity Studies of Sodium Selenate and Sodium Selenite (CAS Nos. 13410 - 01 - 0 and 10102 - 18 - 8) Administ ered in Drinking Water to F344/N Rats and B6C3F1 Mice , US Department of Health and Human Services, Public Health Service, National Institutes of Health 14. Huang, T. - S., Shyu, Y. - C., Chen, H. - Y., Lin, L. - M., Lo, C. - Y., Yuan, S. - S., and Chen, P. - J. (2013) E ffect of Parenteral Selenium Supplementation in Critically Ill Patients: A Systematic Review and Meta - Analysis. PloS one 8 , e54431 15. Ghaemi, S., Forouhari, S., Dabbaghmanesh, M., Sayadi, M., Bakhshayeshkaram, M., Vaziri, F., and Tavana, Z. (2013) A Prosp ective Study of Selenium Concentration and Risk of Preeclampsia in Pregnant Iranian Women: a Nested Case Control Study. Biological Trace Element Research , 1 - 6 16. Tirosh, O., Levy, E., and Reifen, R. (2007) High selenium diet protects against TNBS - induced acute inflammation, mitochondrial dysfunction, and secondary necrosis in rat colon. Nutrition 23 , 878 - 886 17. Lippman Sm, K. E. A. G. P. J., and et al. (2009) Effect of selenium and vitamin e on risk of prostate cancer and other cancers: The selenium and vitamin e cancer prevention trial (select). JAMA: the journal of the American Medical Association 301 , 39 - 51 73 18 . Hamilton, J. W., and Tappel, A. L. (1963) Lipid Antioxidant Activity in Tissues and Proteins of Selenium - fed Animals. The Journal of Nutrition 79 , 493 - 502 19. Sordillo, L. M., and Aitken, S. L. (2009) Impact of oxidative stress on the health and immune function of dairy cattle. Vet Immunol Immunopathol 128 , 104 - 109 20. Sies, H. (1986) Biochemistry of oxidative stress. Angewandte Chemie International Edition in E nglish 25 , 1058 - 1071 21. Kernstock, R. M., and Girotti, A. W. (2008) New strategies for the isolation and activity determination of naturally occurring type - 4 glutathione peroxidase. Protein Expression and Purification 62 , 216 - 222 22. Thomas, J. P., Maiori no, M., Ursini, F., and Girotti, A. W. (1990) Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane - damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides. Journal of Biological Chemi stry 265 , 454 - 461 23. Pushpa - Rekha, T. R., Burdsall, A. L., Oleksa, L. M., Chisolm, G. M., and Driscoll, D. M. (1995) Rat Phospholipid - hydroperoxide Glutathione Peroxidase: cDNA CLONING AND IDENTIFICATION OF MULTIPLE TRANSCRIPTION AND TRANSLATION START SIT ES. Journal of Biological Chemistry 270 , 26993 - 26999 24. Liang, H., Remmen, H. V., Frohlich, V., Lechleiter, J., Richardson, A., and Ran, Q. (2007) Gpx4 protects mitochondrial ATP generation against oxidative damage. Biochemical and Biophysical Research Co mmunications 356 , 893 - 898 25. Fairweather - Tait, S. J., Bao, Y., Broadley, M. R., Collings, R., Ford, D., Hesketh, J. E., and Hurst, R. (2011) Selenium in human health and disease. Antioxidants & Redox Signaling 14 , 1337 - 1383 26. Jeong, D. - w., Kim, T. S., C hung, Y. W., Lee, B. J., and Kim, I. Y. (2002) Selenoprotein W is a glutathione - dependent antioxidant in vivo. FEBS Letters 517 , 225 - 228 27. Praticò, D., Tangirala, R. K., Rader, D. J., Rokach, J., and FitzGerald, G. A. (1998) Vitamin E suppresses isoprost ane generation in vivo and reduces atherosclerosis in ApoE - deficient mice. Nature medicine 4 , 1189 - 1192 74 28. Monnier, L., Mas, E., Ginet, C., Michel, F., Villon, L., Cristol, J. P., and Colette, C. (2006) Activation of oxidative stress by acute glucose fluc tuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA: the journal of the American Medical Association 295 , 1681 - 1687 29. Ranjan, R., Swarup, D., Naresh, R., and Patra, R. (2005) Enhanced erythrocytic lipid peroxides and reduced plasma ascorbic acid, and alteration in blood trace elements level in dairy cows with mastitis. Veterinary research communications 29 , 27 - 34 30. Bell inger, F. P., Raman, A. V., Reeves, M. A., and Berry, M. J. (2009) Regulation and function of selenoproteins in human disease. Biochemical Journal 422 , 11 - 22 31. Lu, J., and Holmgren, A. (2009) Selenoproteins. Journal of Biological Chemistry 284 , 723 - 727 3 2. Sordillo, L. M. (2013) Selenium - dependent regulation of oxidative stress and immunity in periparturient dairy cattle. Veterinary medicine international 2013 , 154045 33. Guo, Z., Ran, Q., Roberts Ii, L. J., Zhou, L., Richardson, A., Sharan, C., Wu, D., a nd Yang, H. (2008) Suppression of atherogenesis by overexpression of glutathione peroxidase - 4 in apolipoprotein E - deficient mice. Free Radical Biology and Medicine 44 , 343 - 352 34. Dabkowski, E. R., Williamson, C. L., and Hollander, J. M. (2008) Mitochondri a - specific transgenic overexpression of phospholipid hydroperoxide glutathione peroxidase (GPx4) attenuates ischemia/reperfusion - associated cardiac dysfunction. Free Radical Biology and Medicine 45 , 855 - 865 35. Björnstedt, M., Hamberg, M., Kumar, S., Xue, J., and Holmgren, A. (1995) Human Thioredoxin Reductase Directly Reduces Lipid Hydroperoxides by NADPH and Selenocystine Strongly Stimulates the Reaction via Catalytically Generated Selenols. Journal of Biological Chemistry 270 , 11761 - 11764 36. Trigona, W. L., Mullarky, I. K., Cao, Y., and Sordillo, L. M. (2006) Thioredoxin reductase regulates the induction of haem oxygenase - 1 expression in aortic endothelial cells. Biochem J 394 , 207 - 216 37. Rock, C., and Moos, P. J. (2010) Selenoprotein P protects cells f rom lipid hydroperoxides generated by 15 - LOX - 1. Prostaglandins, Leukotrienes and Essential Fatty Acids 83 , 203 - 210 75 38. George, J., Afek, A., Shaish, A., Levkovitz, H., Bloom, N., Cyrus, T., Zhao, L., Funk, C. D., Sigal, E., and Harats, D. (2001) 12/15 - Lipo xygenase Gene Disruption Attenuates Atherogenesis in LDL Receptor Deficient Mice. Circulation 104 , 1646 - 1650 39. Schafer, F. Q., and Buettner, G. R. (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutath ione couple. Free Radical Biology and Medicine 30 , 1191 - 1212 40. Into, T., Inomata, M., Nakashima, M., Shibata, K., Hacker, H., and Matsushita, K. (2008) Regulation of MyD88 - Dependent Signaling Events by S Nitrosylation Retards Toll - Like Receptor Signal Tr ansduction and Initiation of Acute - Phase Immune Responses. Mol. Cell. Biol. 28 , 1338 - 1347 41. Al - Gayyar, M. M., Abdelsaid, M. A., Matragoon, S., Pillai, B. A., and El - Remessy, A. B. (2011) Thioredoxin interacting protein is a novel mediator of retinal inflammation and neurotoxicity. British journal of pharmacology 164 , 170 - 180 42. Kataoka, K., Toku tomi, Y., Yamamoto, E., Nakamura, T., Fukuda, M., Dong, Y. F., Ichijo, H., Ogawa, H., and Kim - Mitsuyama, S. (2011) Apoptosis signal - regulating kinase 1 deficiency eliminates cardiovascular injuries induced by high - salt diet. Journal of hypertension 29 , 76 - 84 43. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998) Mammalian thioredoxin is a direct inhibitor of apoptosis signal - regulating kinase (ASK) 1. EMBO J 17 , 2596 - 2606 44. Vunta, H. , Davis, F., Palempalli, U. D., Bhat, D., Arner, R. J., Thompson, J. T., Peterson, D. G., Reddy, C. C., and Prabhu, K. S. (2007) The Anti - inflammatory Effects of Selenium Are Mediated through 15 - Deoxy - 12,14 - prostaglandin J2 in Macrophages. Journal of Biolo gical Chemistry 282 , 17964 - 17973 45. Vunta, H., Belda, B. J., Arner, R. J., Reddy, C. C., Vanden Heuvel, J. P., and Prabhu, K. S. (2008) Selenium attenuates pro - inflammatory gene expression in macrophages. Molecular Nutrition & Food Research 52 , 1316 - 1323 46. Allard, J. P., Aghdassi, E., Chau, J., Salit, I., and Walmsley, S. (1998) Oxidative stress and plasma antioxidant micronutrients in humans with HIV infection. The American Journal of Clinical Nutrition 67 , 143 - 147 76 47. Gladyshev, V. N., Stadtman, T. C., Hatfield, D. L., and Jeang, K. - T. (1999) Levels of major selenoproteins in T cells decrease during HIV infection and low molecular mass selenium compounds increase. Proceedings of the National Academy of Sciences 96 , 835 - 839 48. Jamaluddin, M., Wang, S., Boldogh, I., Tian, B., and Brasier, A. R. (2007) TNF - - induced NF - by an ROS - dependent PKAc pathway. Cellular Signalling 19 , 1419 - 1433 49. Meyer, M., Sch reck, R., and Baeuerle, P. A. (1993) H2O2 and antioxidants have opposite effects on activation of NF - kappa B and AP - 1 in intact cells: AP - 1 as secondary antioxidant - responsive factor. The EMBO journal 12 , 2005 50. Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R. T. (1992) Thioredoxin regulates the DNA binding activity of NF - kappa B by reduction of a disulphide bond involving cysteine 62. Nucleic acids research 20 , 3821 51. Hirota, K., Murata, M., Sachi, Y., Nakamura, H., Takeuchi, J. , Mori, K., and Yodoi, J. (1999) Distinct Roles of Thioredoxin in the Cytoplasm and in the Nucleus: A TWO - STEP MECHANISM OF REDOX REGULATION OF TRANSCRIPTION FACTOR NF - Journal of Biological Chemistry 274 , 27891 - 27897 52. Schmitz, G., and Ecker, J. (200 8) The opposing effects of n - 3 and n - 6 fatty acids. Progress in Lipid Research 47 , 147 - 155 53. Roberts Ii, L. J., and Morrow, J. D. (2000) Measurement of F2 - isoprostanes as an index of oxidative stress in vivo. Free Radical Biology and Medicine 28 , 505 - 513 54. Lakshmi, S., Padmaja, G., Kuppusamy, P., and Kutala, V. K. (2009) Oxidative Stress in Cardiovascular Disease. Indian Journal of Biochemistry & Biophysics 46 , 421 - 440 55. Spiteller, P., and Spiteller, G. (1997) 9 - Hydroxy - 10,12 - octadecadienoic acid (9 - H ODE) and 13 - hydroxy - 9,11 - octadecadienoic acid (13 - HODE): excellent markers for lipid peroxidation. Chemistry and Physics of Lipids 89 , 131 - 139 56. Xie, W. L., Chipman, J. G., Robertson, D. L., Erikson, R. L., and Simmons, D. L. (1991) Expression of a mitog en - responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proceedings of the National Academy of Sciences 88 , 2692 - 2696 77 57. Kurumbail, R. G., Kiefer, J. R., and Marnett, L. J. (2001) Cyclooxygenase enzymes: catalysis and inhibition. Current Opinion in Structural Biology 11 , 752 - 760 58. Dubois, R. N., Abramson, S. B., Crofford, L., Gupta, R. A., Simon, L. S., A. Van De Putte, L. B., and Lipsky, P. E. (1998) Cyclooxygenase in biology and disease. The FASEB Journal 12 , 1063 - 1073 59. Kuh n, H., and Thiele, B. J. (1999) The diversity of the lipoxygenase family: Many sequence data but little information on biological significance. FEBS Letters 449 , 7 - 11 60. Chiang, N., Arita, M., and Serhan, C. N. (2005) Anti - inflammatory circuitry: Lipoxin, aspirin - triggered lipoxins and their receptor ALX. Prostaglandins, Leukotrienes and Essential Fatty Acids 73 , 163 - 177 61. Kuhn, H., and O'Donnell, V. B. (2006) Inflammation and immune regulation by 12/15 - lipoxygenases. Progress in Lipid Research 45 , 334 - 3 56 62. Yuan, Z. X., Rapoport, S. I., Soldin, S. J., Remaley, A. T., Taha, A. Y., Kellom, M., Gu, J., Sampson, M., and Ramsden, C. E. (2012) Identification and profiling of targeted oxidized linoleic acid metabolites in rat plasma by quadrupole time of flig ht mass spectrometry. Biomedical Chromatography 63. Oh, S. F., Dona, M., Fredman, G., Krishnamoorthy, S., Irimia, D., and Serhan, C. N. (2012) Resolvin E2 Formation and Impact in Inflammation Resolution. The Journal of Immunology 188 , 4527 - 4534 64. Merched, A. J., Ko, K., Gotlinger, K. H., Serhan, C. N., and Chan, L. (2008) Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators. The FASEB Journal 22 , 3595 - 3606 65. Serhan, C. N., and Savill , J. (2005) Resolution of inflammation: the beginning programs the end. Nat Immunol 6 , 1191 - 1197 66. Arnaud, J., Bost, M., Vitoux, D., Labarère, J., Galan, P., Faure, H., Hercberg, S., Bordet, J. - C., Roussel, A. - M., and Chappuis, P. (2007) Effect of Low D ose Antioxidant Vitamin and Trace Element Supplementation on the Urinary Concentrations of Thromboxane and Prostacyclin Metabolites. Journal of the American College of Nutrition 26 , 405 - 411 78 67. Meydani, M. (1992) Modulation of the platelet thromboxane A2 a nd aortic prostacyclin synthesis by dietary selenium and vitamin E. Biological Trace Element Research 33 , 79 - 86 68. Haberland, A., Neubert, K., Kruse, I., Behne, D., and Schimke, I. (2001) Consequences of long - term selenium - deficient diet on the prostacycl in and thromboxane release from rat aorta. Biological Trace Element Research 81 , 71 - 78 69. Maddox, J. F., Reddy, C. C., Eberhart, R. J., and Scholz, R. W. (1991) Dietary selenium effects on milk eicosanoid concentration in dairy cows during coliform mastit is. Prostaglandins 42 , 369 - 378 70. Gandhi, U. H., Kaushal, N., Ravindra, K. C., Hegde, S., Nelson, S. M., Narayan, V., Vunta, H., Paulson, R. F., and Prabhu, K. S. (2011) Selenoprotein - dependent upregulation of hematopoietic prostaglandin D2 synthase in ma crophages is mediated through the activation of peroxisome proliferator - activated receptor (PPAR)gamma. Journal of Biological Chemistry 286 , 27471 - 27482 71. Imai, H., Narashima, K., Arai, M., Sakamoto, H., Chiba, N., and Nakagawa, Y. (1998) Suppression of Leukotriene Formation in RBL - 2H3 Cells That Overexpressed Phospholipid Hydroperoxide Glutathione Peroxidase. Journal of Biological Chemistry 273 , 1990 - 1997 72. Schnurr, K., Belkner, J., Ursini, F., Schewe, T., and Kühn, H. (1996) The Selenoenzyme Phospholi pid Hydroperoxide Glutathione Peroxidase Controls the Activity of the 15 - Lipoxygenase with Complex Substrates and Preserves the Specificity of the Oxygenation Products. Journal of Biological Chemistry 271 , 4653 - 4658 73. Bjornstedt, M., Hamberg, M., Kumar, S., Xue, J., and Holmgren, A. (1995) Human thioredoxin reductase directly reduces lipid hydroperoxides by NADPH and selenocystine strongly stimulates the reaction via catalytically generated selenols. The Journal of biological chemistry 270 , 11761 - 11764 74 . Walther, M., Holzhutter, H. - G., Kuban, R. J., Wiesner, R., Rathmann, J., and Kuhn, H. (1999) The Inhibition of Mammalian 15 - Lipoxygenases by the Anti - Inflammatory Drug Ebselen: Dual - Type Mechanism Involving Covalent Linkage and Alteration of the Iron Lig and Sphere. Molecular Pharmacology 56 , 196 - 203 75. Marnett, L. J., Rowlinson, S. W., Goodwin, D. C., Kalgutkar, A. S., and Lanzo, C. A. (1999) Arachidonic Acid Oxygenation by COX - 1 and COX - 2. Journal of Biological Chemistry 274 , 22903 - 22906 79 76. Cook, H., and Lands, W. E. M. (1976) Mechanism for suppression of cellular biosynthesis of prostaglandins. Nature 260 , 630 - 632 77. Smith, W. L., and Lands, W. E. M. (1972) Oxygenation of polyunsaturated fatty acids during prostaglandin biosynthesis by shee p vesicular glands. Biochemistry 11 , 3276 - 3285 78. Wade, M. L., Voelkel, N. F., and Fitzpatrick, F. A. (1995) "Suicide" Inactivation of Prostaglandin I2 Synthase: Characterization of Mechanism - Based Inactivation with Isolated Enzyme and Endothelial Cells. Archives of Biochemistry and Biophysics 321 , 453 - 458 79. Jones, D. A., and Fitzpatrick, F. A. (1990) "Suicide" inactivation of thromboxane A2 synthase. Characteristics of mechanism - based inactivation with isolated enzyme and intact platelets. Journal of Bi ological Chemistry 265 , 20166 - 20171 80. Hampel, G., Watanabe, K., Weksler, B. B., and Jaffe, E. A. (1989) Selenium deficiency inhibits prostacyclin release and enhances production of platelet activating factor by human endothelial cells. Biochimica et Biop hysica Acta (BBA) - Lipids and Lipid Metabolism 1006 , 151 - 158 81. Hwang, J. - T., Kim, Y. M., Surh, Y. - J., Baik, H. W., Lee, S. - K., Ha, J., and Park, O. J. (2006) Selenium Regulates Cyclooxygenase - 2 and Extracellular Signal - Regulated Kinase Signaling Pathway s by Activating AMP - Activated Protein Kinase in Colon Cancer Cells. Cancer Research 66 , 10057 - 10063 82. Pei, Z., Li, H., Guo, Y., Jin, Y., and Lin, D. (2010) Sodium selenite inhibits the expression of VEGF, TGF[beta]1 and IL - 6 induced by LPS in human PC3 c ells via TLR4 - NF - KB signaling blockage. International Immunopharmacology 10 , 50 - 56 83. Coussens, L. M., Zitvogel, L., and Palucka, A. K. (2013) Neutralizing Tumor - Promoting Chronic Inflammation: A Magic Bullet? Science 339 , 286 - 291 84. Sheng, H., Shao, J., Morrow, J. D., Beauchamp, R. D., and DuBois, R. N. (1998) Modulation of Apoptosis and Bcl - 2 Expression by Prostaglandin E2 in Human Colon Cancer Cells. Cancer Research 58 , 362 - 366 85. AVIS, I., HONG, S. H., MARTÍNEZ, A., MOODY, T., CHOI, Y. H., TREPEL, J. , DAS, R., JETT, M., and MULSHINE, J. L. (2001) Five - lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions. The FASEB Journal 15 , 2007 - 2009 80 86. Ghosh, J. (2004) Rapid induction of apoptosis in prostate cancer cells by selenium: reversal by metabolites of arachidonate 5 - lipoxygenase. Biochemical and Biophysical Research Communications 315 , 624 - 635 87. Heirman, I., Ginneberge, D., Brigelius - Flohé, R., Hendrickx, N., Agostinis, P., Brouckaert, P., Rottiers, P., and Grooten, J. (2006) Blocking tumor cell eicosanoid synthesis by GPx4 impedes tumor growth and malignancy. Free Radical Biology and Medicine 40 , 285 - 294 88. Quinn, M. T., Parthasarathy, S., Fong, L. G., and Steinberg, D. (1987) Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proceedings of the National Acade my of Sciences 84 , 2995 - 2998 89. Lewis, P., Stefanovic, N., Pete, J., Calkin, A. C., Giunti, S., Thallas - Bonke, V., Jandeleit - Dahm, K. A., Allen, T. J., Kola, I., Cooper, M. E., and de Haan, J. B. (2007) Lack of the Antioxidant Enzyme Glutathione Peroxidas e - 1 Accelerates Atherosclerosis in Diabetic Apolipoprotein E Deficient Mice. Circulation 115 , 2178 - 2187 90. Panicker, S., Swathy, S., John, F., and M, I. (2012) Impact of Selenium on the Leukotriene B4 Synthesis Pathway during Isoproterenol - Induced Myocard ial Infarction in Experimental Rats. Inflammation 35 , 74 - 80 91. Shrimali, R. K., Weaver, J. A., Miller, G. F., Starost, M. F., Carlson, B. A., Novoselov, S. V., Kumaraswamy, E., Gladyshev, V. N., and Hatfield, D. L. (2007) Selenoprotein expression is essen tial in endothelial cell development and cardiac muscle function. Neuromuscular Disorders 17 , 135 - 142 92. Cao, Y. - Z., Reddy, C. C., and Sordillo, L. M. (2000) Altered eicosanoid biosynthesis in selenium - deficient endothelial cells. Free Radical Biology and Medicine 28 , 381 - 389 93. Sordillo, L. M., Streicher, K. L., Mullarky, I. K., Gandy, J. C., Trigona, W. L., and Corl, C. M. (2008) Selenium inhibits 15 - hydroperoxyoctadecadienoic acid - induced intracellular adhesion molecule expression in aortic endothelial cells. Free Radical Biology and Medicine 44 , 34 - 43 94. Sordillo, L. M., Weaver, J. A., Cao, Y. - Z., Corl, C., Sylte, M. J., and Mullarky, I. K. (2005) Enhanced 15 - HPETE production during oxidant stress induces apoptosis of endothelial cells. Prostaglandins & Other Lipid Mediators 76 , 19 - 34 81 95. Weaver, J. A., Maddox, J. F., Cao, Y. Z., Mullarky, I. K., and Sordillo, L. M. (2001) Increased 15 - HPETE production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells. Free Radical Biology and Medicine 30 , 299 - 308 96. Rydberg, E. K., Krettek, A., Ullstrom, C., Ekstrom, K., Svensson, P. - A., Carlsson, L. M. S., Jonsson - Rylander, A. - C., Hansson, G. I., McPheat, W., Wiklund, O., Ohlsson, B. G., and Hulten, L. M. (2004) Hypoxia In creases LDL Oxidation and Expression of 15 - Lipoxygenase - 2 in Human Macrophages. Arteriosclerosis, Thrombosis, and Vascular Biology 24 , 2040 - 2045 97. Takahashi, K., Takeya, M., and Sakashita, N. (2002) Multifunctional roles of macrophages in the development and progression of atherosclerosis in humans and experimental animals. Medical electron microscopy 35 , 179 - 203 98. Cao, Y. - Z., Weaver, J. A., Chana Reddy, C., and Sordillo, L. M. (2002) Selenium deficiency alters the formation of eicosanoids and signal tr ansduction in rat lymphocytes. Prostaglandins & Other Lipid Mediators 70 , 131 - 143 99. Carlson, B., Yoo, M. - H., Sano, Y., Sengupta, A., Kim, J., Irons, R., Gladyshev, V., Hatfield, D., and Park, J. (2009) Selenoproteins regulate macrophage invasiveness and extracellular matrix - related gene expression. BMC Immunology 10 , 57 100. Prabhu, K. S. , Zamamiri - Davis, F., Stewart, J. B., Thompson, J. T., Sordillo, L. M., and Reddy, C. C. (2002) Selenium deficiency increases the expression of inducible nitric oxide synthase in RAW 264.7 macrophages: role of nuclear factor - kappaB in up - regulation. Bioche mical Journal 366 , 203 - 209 101. Zamamiri - Davis, F., Lu, Y., Thompson, J. T., Prabhu, K. S., Reddy, P. V., Sordillo, L. M., and Reddy, C. C. (2002) Nuclear factor - [kappa]B mediates over - expression of cyclooxygenase - 2 during activation of RAW 264.7 macrophag es in selenium deficiency. Free Radical Biology and Medicine 32 , 890 - 897 102. Youn, H. - S., Lim, H. J., Choi, Y. J., Lee, J. Y., Lee, M. - Y., and Ryu, J. - H. (2008) Selenium suppresses the activation of transcription factor NF - [kappa]B and IRF3 induced by TLR 3 or TLR4 agonists. International Immunopharmacology 8 , 495 - 501 103. Nam, K. N., Koketsu, M., and Lee, E. H. (2008) 5 - Chloroacetyl - 2 - amino - 1,3 - selenazoles attenuate microglial inflammatory responses through NF - [kappa]B inhibition. European Journal of Pharmacology 589 , 53 - 57 82 104. Serhan, C. N., and Chiang, N. (2008) Endogenous pro - resolving and anti - inflammatory lipid mediators: a new pharmacologic genus. British journal of pharmacology 153 Suppl 1 , S200 - 215 105. Heras, I. L., Palomo, M., and Madrid, Y. (2011) Selenoproteins: the key factor in selenium essentiality. State of the art analytical techniques for selenoprotein studies. Analytical and Bioanalytical Chemistry 400 , 1717 - 1727 106. Kryukov, G. V., Castellano, S., Novoselov, S. V., Lobanov, A. V., Zehtab, O., Guigó, R., and Gladyshev, V. N. (2003) Characterization of Mammalian Selenoproteomes. Science 300 , 1439 - 1443 107. Gordon, S., and Taylor, P. R. (2005) Monocyte and macrophage heterogeneity. Nat Rev Immunol 5 , 953 - 964 108. Niki, E. (2009) Lipid peroxidation: Physiological levels and dual biological effects. Free Radical Biology and Medicine 47 , 469 - 484 109. Bolick, D. T., Orr, A. W., Whetzel, A., Srinivasan, S., Hatley, M. E., Schwartz, M. A., and Hedrick, C. C. (2005) 12/15 - Lipoxygenase Regulate s Intercellular Adhesion Molecule - 1 Expression and Monocyte Adhesion to Endothelium Through Activation of RhoA and Nuclear Factor - Arteriosclerosis, Thrombosis, and Vascular Biology 25 , 2301 - 2307 110. Fogh, K., Stender Hansen, E., Herlin, T., Knudsen, V ., Brink Henriksen, T., Ewald, H., Bünger, C., and Kragballe, K. (1989) 15 - Hydroxy - eicosatetraenoic acid (15 - Hete) inhibits carragheenan - induced experimental arthritis and reduces synovial fluid leukotriene B4 (LTB4). Prostaglandins 37 , 213 - 228 111. Yuan, H., Li, M. - Y., Ma, L. T., Hsin, M. K. Y., Mok, T. S. K., Underwood, M. J., and Chen, G. G. (2010) 15 - Lipoxygenases and its metabolites 15(S) - HETE and 13(S) - HODE in the development of non - small cell lung cancer. Thorax 65 , 321 - 326 112. Funk, C. D., Bo ubez, W., and Powell, W. S. (1987) Effects of selenium - deficient diets on the production of prostaglandins and other oxygenated metabolites of arachidonic acid and linoleic acid by rat and rabbit aortae. Biochimica et Biophysica Acta (BBA) - Lipids and Lip id Metabolism 921 , 213 - 220 83 113. Saito, Y., Yoshida, Y., and Niki, E. (2007) Cholesterol is more susceptible to oxidation than linoleates in cultured cells under oxidative stress induced by selenium deficiency and free radicals. FEBS Letters 581 , 4349 - 4354 114. Hatfield, D. L., Carlson, B. A., Xu, X., Mix, H., Gladyshev, V. N., and Kivie, M. (2006) Selenocysteine Incorporation Machinery and the Role of Selenoproteins in Development and Health. In Progress in Nucleic Acid Research and Molecular Biology Vol. V olume 81 pp. 97 - 142, Academic Press 115. McKenzie, R. C., S. Rafferty, T., and Beckett, G. J. (1998) Selenium: an essential element for immune function. Immunology Today 19 , 342 - 345 116. Gairola, C., and Tai, H. - H. (1985) Selective inhibition of leukotrien e B4 biosynthesis in rat pulmonary alveolar macrophages by dietary selenium deficiency. Biochemical and Biophysical Research Communications 132 , 397 - 403 117. Wittwer, J., and Hersberger, M. (2007) The two faces of the 15 - lipoxygenase in atherosclerosis. Pr ostaglandins, Leukotrienes and Essential Fatty Acids 77 , 67 - 77 118. Livak, K. J., and Schmittgen, T. D. (2001) Analysis of Relative Gene Expression Data Using Real - Time Quantitative PCR and the 2 - [Delta][Delta]CT Method. Methods 25 , 402 - 408 119. Contreras, G. A., Raphael, W., Mattmiller, S. A., Gandy, J., and Sordillo, L. M. (2012) Nonesterified fatty acids modify inflammatory response and eicosanoid biosynthesis in bovine endothelial cells. Journal of Dairy Science 95 , 5011 - 5023 120. Lee, D. M., Kiener, H. P., Agarwal, S. K., Noss, E. H., Watts, G. F. M., Chisaka, O., Takeichi, M., and Brenner, M. B. (2007) Cadherin - 11 in Synovial Lining Formation and Pathology in Arthritis. Science 315 , 1006 - 1010 121. Li, A. C., Brown, K. K., Silvestre, M. J., Willson, T. M., Palinski, W., and Glass, C. K. (2000) Peroxisome proliferator - activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor - deficient mice. Journal of Clinical Investigation 106 , 523 - 531 122. Sordillo, L. M., Pighetti, G. M., and Davis, M. R. (1995) Enhanced production of bovine tumor necrosis factor - Veterinary Immunology and Immunopathology 49 , 263 - 270 84 123. Simopoulos, A. P. (2002) The importance of the ratio of omega - 6/omega - 3 essential fatty acids. Biomedicine & Pharmacotherapy 56 , 365 - 379 124. Shin, K. - M., Shen, L., Park, S. J., Jeong, J. - H., and Lee, K. - T. (2009) Bis - (3 - hydroxyphenyl) diselenide inhibits LPS - stimulated iNOS and COX - 2 expression in RAW 264.7 macrophage cells through the NF - kB inactivation. Journal of Pharmacy and Pharmacology 61 , 479 - 486 125. Shiraki, T., Kamiya, N., Shiki, S., Kodama, T. S., Kakizuka, A., and Jingami, H. (2005) - Unsatur ated Ketone Is a Core Moiety of Natural Ligands for Covalent Binding to Peroxisome Proliferator - Journal of Biological Chemistry 280 , 14145 - 14153 126. Patwardhan, A. M., Scotland, P. E., Akopian, A. N., and Hargreaves, K. M. (2009) Fro m the Cover: Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia. PNAS 106 , 18820 - 18824 127. Waddington, E. I., Croft, K. D., Sienuarine, K., Latham, B., and Puddey, I. B. (2003) Fatty acid oxidation products in human atherosclerotic plaque: an analysis of clinical and histopathological correlates. Atherosclerosis 167 , 111 - 120