Arts and Sciences, Irving K. Barber School of (Okanagan)
DSpace
UBCO
Winkler, John McCauley
Master of Science - MSc
University of British Columbia
Dietary n-6 polyunsaturated fatty acids (n-6 PUFA) like linoleic acid (LA) are linked to inflammatory diseases. It is believed that arachidonic acid (ARA), generated from LA is primarily responsible for such inflammation. However, studies show that dietary LA does not raise ARA in vivo and chronic disease patients often demonstrate low serum ARA. Thus, I hypothesized that LA, the parent n-6 PUFA causes inflammation independent of ARA. First, I show that adding LA activates RAW 264.7 macrophages (MФs) which demonstrates increased mitochondrial respiration, glucose uptake, cholesterol storage and reactive oxygen species (ROS) production compared to oleic acid (OA), a monounsaturated fatty acid (MUFA). Interestingly though, feeding a diet rich in LA (corn oil, CO; 54% LA) or OA (olive oil, OO; 54% OA) for six weeks in mice did not alter the inflammatory state of macrophages (PMФs). However, when I fed these diets to ELOVL5 +/- mice, which were deficient in an enzyme that allows the conversion of LA to ARA and therefore has higher LA, the accumulation of LA in PMФs demonstrated proinflammatory effects. Taken together, I conclude that LA accumulation in the short-term does not activate macrophages but long-term accumulation of LA mediated by congenital ELOVL deficiency does. In addition, feeding a MUFA diet did not counteract the activation of macrophages or higher LA levels in ELOVL5 ⁺/⁻ mice. Thus, I conclude that dietary MUFA cannot overcome a genetic deficiency of ELOVL5, indicating the critical role of this enzyme in maintaining PUFA levels in macrophages. Thus, my thesis clarifies the crucial role of ELOVL5 in the PUFA bioconversion pathway and its central importance in regulating the pro-inflammatory properties of n-6 PUFA. In the current milieu of high LA in our diet, activation of ELOVL5 and clearance of LA could be used as a therapeutic target against inflammation.
THE INFLUENCE OF DIETARY N-6 POLYUNSATURATED FATS ON THE INFLAMMATORY STATE OF MACROPHAGES by John McCauley Winkler B.Sc., Lakehead University, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) July 2019 © John McCauley Winkler, 2019 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: The influence of dietary n-6 polyunsaturated fats on the inflammatory state of macrophages submitted by John McCauley Winkler in partial fulfillment of the requirements for the degree of Master of Science Examining Committee: Dr. Sanjoy Ghosh, Irving K. Barber School of Arts and Sciences Supervisor Dr. John Klironomos, Barber School of Arts and Sciences Supervisory Committee Member Dr. Isaac Li, Barber School of Arts and Sciences Supervisory Committee Member Dr. Jonathan Little, Barber School of Arts and Sciences University Examiner iii Abstract Dietary n-6 polyunsaturated fatty acids (n-6 PUFA) like linoleic acid (LA) are linked to inflammatory diseases. It is believed that arachidonic acid (ARA), generated from LA is primarily responsible for such inflammation. However, studies show that dietary LA does not raise ARA in vivo and chronic disease patients often demonstrate low serum ARA. Thus, I hypothesized that LA, the parent n-6 PUFA causes inflammation independent of ARA. First, I show that adding LA activates RAW 264.7 macrophages (MФs) which demonstrates increased mitochondrial respiration, glucose uptake, cholesterol storage and reactive oxygen species (ROS) production compared to oleic acid (OA), a monounsaturated fatty acid (MUFA). Interestingly though, feeding a diet rich in LA (corn oil, CO; 54% LA) or OA (olive oil, OO; 54% OA) for six weeks in mice did not alter the inflammatory state of macrophages (PMФs). However, when I fed these diets to ELOVL5 +/- mice, which were deficient in an enzyme that allows the conversion of LA to ARA and therefore has higher LA, the accumulation of LA in PMФs demonstrated proinflammatory effects. Taken together, I conclude that LA accumulation in the short-term does not activate macrophages but long-term accumulation of LA mediated by congenital ELOVL deficiency does. In addition, feeding a MUFA diet did not counteract the activation of macrophages or higher LA levels in ELOVL5 +/- mice. Thus, I conclude that dietary MUFA cannot overcome a genetic deficiency of ELOVL5, indicating the critical role of this enzyme in maintaining PUFA levels in macrophages. Thus, my thesis clarifies the crucial role of ELOVL5 in the PUFA bioconversion pathway and its central importance in regulating the pro-inflammatory properties of n-6 PUFA. In the current milieu of high LA in our diet, activation of ELOVL5 and clearance of LA could be used as a therapeutic target against inflammation. iv Lay Summary Canadian food recommendations have led to over consumption of omega-6 (n-6) PUFAs in our diet. Since these recommendations were introduced an increase in inflammatory diseases have been seen in the western world. Such correlation is concerning because the influence of n-6 PUFA consumption on the immune system remains unclear. My thesis sheds light on one main question in the field; whether LA (the parent n-6 PUFA) or its downstream constituent arachidonic acid (ARA) activate inflammatory responses in macrophages (MФs). By diminishing the conversion of LA to ARA in mice, I show LA can activate MФs and lead to a pro-inflammatory state in mice. The mechanism is unknown, but my data supports that LA itself, but not the oxidation products of LA are responsible. Since MФs are the cornerstone of the immune system, my thesis indicates LA consumption should be re-considered in hopes to reduce inflammatory diseases. v Preface The design of this Master’s research program belongs in part to Dr. Sanjoy Ghosh in collaboration with John Winkler. Regarding chapter 2: All handling of macrophages and experimental analyses were performed by me, John Winkler. In addition, animal care, diet preparation and feeding were completed by me, in accompaniment with Jiayu Ye a Ph.D. candidate in Dr. Ghosh’s lab. Cellular images of lipid storage presented in Figures 2, 3 and 4 were gathered with assistance from Jacqueline Barnett a Master’s student under Dr. Deanna Gibson. Animal testing in chapter 2 was approved by the UBC animal ethics board under the experimental protocol A13-0305. A version of Chapter 2 is prepared and ready for submission to a journal. The potential publication is titled “Intrinsic LA bio-accumulation supersedes dietary LA in activating pro-inflammatory macrophages” with authors John Winkler, Jiayu Ye, Jacqueline Barnett and Sanjoy Ghosh. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiii Dedication ................................................................................................................................... xiv Chapter 1: Introduction ................................................................................................................1 1.1 The diet dilemma ............................................................................................................ 1 1.2 Fatty acids ....................................................................................................................... 2 1.2.1 Chronic inflammatory disease: an n-6 PUFA link ...................................................... 3 1.2.2 Omega-6 bio-conversion ............................................................................................. 5 1.2.3 Lipid inflammatory mediators .................................................................................... 8 1.2.4 Discrepancies in historical thinking ............................................................................ 8 1.3 Oxidative stress ............................................................................................................... 9 1.3.1 ROS formation ............................................................................................................ 9 1.3.2 Lipid (per)oxidation .................................................................................................. 11 1.4 Macrophages ................................................................................................................. 12 1.4.1 Resident macrophages .............................................................................................. 13 vii 1.4.2 RAW Macrophages ................................................................................................... 14 1.4.3 Macrophage activation state ..................................................................................... 15 1.4.3.1 Classical activation ........................................................................................... 15 1.4.3.2 Alternative activation ........................................................................................ 16 1.4.4 Role of macrophages in atherosclerosis .................................................................... 16 1.4.5 Dietary influence on macrophages............................................................................ 17 1.4.5.1 Saturated fatty acids and MФs .......................................................................... 18 1.4.5.2 Polyunsaturated fatty acids effect on MФs ....................................................... 19 1.4.5.2.1 Linoleic acid and MФs ................................................................................ 20 1.4.5.2.2 Arachidonic acid and MФs ......................................................................... 21 1.5 Hypothesis and aims ..................................................................................................... 22 Chapter 2: Intrinsic LA bio-accumulation supersedes dietary LA in activating inflammatory macrophages ........................................................................................................24 2.1 Overview ....................................................................................................................... 24 2.2 Materials and methods .................................................................................................. 25 2.2.1 Animal model and diet .............................................................................................. 25 2.2.2 Peritoneal macrophage isolation and treatments....................................................... 27 2.2.3 Serum cytokine analysis ........................................................................................... 27 2.2.4 In vitro cell model and treatments ............................................................................ 27 2.2.5 Reactive oxygen species assay.................................................................................. 28 2.2.6 Succinate dehydrogenase activity assay ................................................................... 29 2.2.7 Resazurin assay ......................................................................................................... 29 2.2.8 Aldehyde dehydrogenase activity assay ................................................................... 29 viii 2.2.9 Immuno dot blot ........................................................................................................ 30 2.2.10 Lipid droplet assay ................................................................................................ 30 2.2.11 Glucose uptake ...................................................................................................... 30 2.2.12 Gas Chromatography Analysis ............................................................................. 31 2.2.13 Statistical analysis ................................................................................................. 31 2.3 Results ........................................................................................................................... 31 2.3.1 LA drives a M1 ‘classical’ inflammatory state in macrophages compared to OA. .. 31 2.3.2 Elovl5 deficiency results in significant LA accumulation and a proinflammatory environment in mice. ............................................................................................................ 34 2.3.3 Olive oil diet fails to reduce ROS production caused by reduced n-6 PUFA bio-conversion. ............................................................................................................................ 36 2.3.4 CO feeding does not increase cholesterol uptake in PMФs whereas direct LA incubation does. .................................................................................................................... 37 2.3.5 OXLAMS contrast the effect of LA incubation ex vivo and in vitro. ....................... 39 2.3.6 ALDH2 activity and 4-HNE may not regulate LA induced ROS production in vitro. ................................................................................................................................... 40 2.4 Discussion ..................................................................................................................... 43 2.5 Summary ....................................................................................................................... 47 Chapter 3: Conclusion .................................................................................................................49 3.1 Significance of findings ................................................................................................ 49 3.2 Limitations of research ................................................................................................. 49 3.3 Main conclusions .......................................................................................................... 50 References .....................................................................................................................................51 ix Appendices ....................................................................................................................................65 Appendix A ............................................................................................................................... 65 A.1 Dietary information ................................................................................................... 65 A.2 Cytokine expression .................................................................................................. 66 x List of Tables Table 2.1 Nutrient composition of experimental diets.................................................................. 26 Table A1.1 Fatty acid profile of CO and OO diets ....................................................................... 65 Table A2.1 Macronutrient composition of diets ........................................................................... 66 xi List of Figures Figure 1.1 Removal of reactive oxygen species (ROS) in the mitochondria matrix ...................... 7 Figure 1.2 n-6 PUFA bio-conversion from LA to ARA ............................................................... 10 Figure 1.3 Influence of dietary fatty acids on macrophage activation state ................................. 18 Figure 2.1 LA incubation initiates classical activation and increases fat storage in RAW .......... 33 Figure 2.2 ELOVL 5 deficiency leads to LA accumulation and a proinflammatory environment....................................................................................................................................................... 35 Figure 2.3 ELOVL 5 induces ROS production in PMOs under a high fat diet ............................ 38 Figure 2.4 OXLAMS impair macrophage ROS production and cholesterol uptake .................... 40 Figure 2.5 4-HNE does not regulate LA induced ROS production .............................................. 42 Figure A2.1 Expression of macrophage secreted cytokines ......................................................... 66 xii List of Abbreviations 4-HNE…………..4-hydroxynonenal 9-HODE………...9-hydroxy-octadecadienoic acid 13-HODE……….13-hydroxy-octadecadienoic acid ALDH…………..Aldehyde dehydrogenase ARA…………….Arachidonic acid CAD…………….Coronary Artery Disease CO………………Corn Oil CVD…………….Cardiovascular Disease ELOVL5………..Elongase 5 FADS……………Fatty Acid Desaturase FC……………….Free Cholesterol GLA…………….Gamma Linolenic Acid LA………………Linoleic Acid NADPH………...Nicotinamide adenine dinucleotide phosphate PMФ……………Peritoneal Macrophage OA………………Oleic Acid OO………………Olive Oil ROS……………..Reactive oxygen species SDH……………..Succinate dehydrogenase WT………………Wild type xiii Acknowledgements It is with resounding gratitude I thank my friends, family and girlfriend for the continued love and support throughout my Masters. Without the emotional and educational help from my parents, siblings and the incredible people I have met at UBC, I would not be the person I am today. The people who have influenced me are too numerous to name, but a special thanks to my colleagues Jiayu Ye, Lisa Renaud, Amy Botta and Svetlana Simtchouk for teaching me all the skills I know. Cheers to all the graduate students of ASC 374 (past and present), thanks for making UBCO feel like a home! Thank you to my supervisor Dr. Sanjoy Ghosh. Thank you for opening my eyes to the world of research, guiding me along this task and providing me with countless opportunities to grow and learn. I would also like to thank my committee members Dr. John Klironomos and Dr. Isaac Li for always providing me with sage advice and ensuring my work is relevant and translatable. Lastly, without the financial support of the Natural Science and Engineering Research Council of Canada (NSERC) and the University of British Columbia, my research would not be possible. xiv Dedication This thesis is dedicated to my family. My parents Paul and Mary Lynn Winkler, my siblings Patrick, Andrew and Elizabeth and my pup, Murphy. 1 Chapter 1: Introduction 1.1 The diet dilemma Currently, there is no consensus on which dietary fat an individual should consume to live a healthy life. Research in the field is not only contradictory but also insufficient. Such uncertainty has led to poor decisions concerning diet and has impeded the creation of appropriate regulations and recommendations for the public. The diet dilemma began in the 1960s. Research in North America during this time linked saturated fatty acids (SFAs) to increased cholesterol rich low-density lipoproteins (LDL) when compared to polyunsaturated and monounsaturated fatty acid (PUFA, MUFA) rich diets (McNamara 2000). As a result, saturated fat was associated with poor cardiac health because cholesterol had been implicated in heart disease several years before (Keys 1970). This conclusion by researcher Ancel Keys, led Health Canada and other regulatory agencies around the world to create dietary guidelines that discouraged the intake of saturated fat (Wong et al. 2015). In 1980, Canadians started eating plant-based oils, rich in omega-6 polyunsaturated fatty acids (n-6 PUFAs) like corn oil and sunflower oils as a substitute for SFAs in the diet and also started increasing their sugar intake (“Food Statistics: Tables” n.d.). Despite the switch from a primarily saturated fat diet to an n-6 PUFA diet, cardiovascular disease (CVD) rates have not reduced in the Western world. In fact, 1.6 million Canadians continue to suffer from CVD with numbers predicted to increase by 2020 (Go et al. 2013). This projection is not only limited to CVD but is also true for other chronic inflammatory diseases (CIDs), like diabetes and colitis (Kaplan et al. 2018). CIDs are currently a major health and economic concern in Canada, showcasing that our change in diet has not succeeded. 2 Food Statistics declares Canadians on average consumed 26 grams of polyunsaturated fats a day in 2009, a 42% increase compared to 1981. Although n-6 PUFAs are essential in our diet, only ~1% of energy is required for nutritional and developmental needs (P. M. Kris-Etherton et al. 2000). Canadians now consume an astonishing ~7% energy from linoleic acid (LA) and other n-6 PUFAs (Madden, Garrioch, and Holub 2009; Bartsch, Nair, and Owen 1999). This increase in LA consumption may be concerning as recent studies link high n-6 PUFA intake to oxidative damage and inflammation in organs, including the heart (Wu and Cederbaum 2003; Awada et al. 2012; Aragno et al. 2008). This thesis will highlight the concerns regarding Canadian dietary patterns and provide evidence for how n-6 PUFAs can cause inflammation. 1.2 Fatty acids Fatty acids are amphiphilic molecules, composed of a hydrophobic carbon chain and a hydrophilic carboxyl head. Fatty acids differ on the size of the carbon chain and the amount of hydrogen atoms present. Based on the latter difference, known as the saturation level, fatty acids can be categorized into 3 families. These categories include: saturated, monounsaturated and polyunsaturated fatty acids (Berg et al. 2002). Firstly, saturated fatty acids (SFAs), like the name implies, are saturated with protons, and void of any double bonds in the carbon chain. SFAs are important in maintaining membrane stability and energy production; they are obtained in our diet mostly from animal products (Sofi et al. 2008). SFAs in the diet are primarily palmitic and stearic acids (PA, SA). Palmitic acid and stearic acid have chains that are 16 and 18 carbons long, respectively, contain no double bonds and are denoted as C16:0 and C18:0. Canadians consume roughly 10% of their daily energy from SFAs. Palmitic acid is consumed most, as it comprises about 50-60% of total fats in meat and dairy products (Carta et al. 2017). PA is also the most common SFA found in the human body (Carta et al. 2017). 3 Second, monounsaturated fats (MUFAs) contain a single double bond in their carbon chain and have been found to reduce low-density lipoproteins (LDL) and the risk of cardiovascular disease (McNamara 2000; Sofi et al. 2008). Within our diet, we obtain MUFAs from sources like olive oil, which contains oleic acid (OA). Oleic acid is denoted in literature as n-9 C18:1. n-9 refers to omega-9 and represents the position of the double bond in relation to the terminal methyl carbon, in this case, on the ninth (9th) carbon. Last, polyunsaturated fatty acids (PUFA) contain two or more double bonds within the carbon chain. Unlike the other fatty acids, they are essential in our diet. Animals do not synthesize these fatty acids however need them for multiple functions like eliciting immune responses, brain development and maintaining membrane fluidity (Berg et al. 2002; Burr 2012). The essential PUFAs are split up into two groups: omega-3 (n-3) and omega-6 (n-6) PUFAs, with the nomenclature being most recent from IUPAC (IUPAC 1977). n-3 PUFAs are found in foods like fish and flaxseeds. Fish contain the n-3 PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and plants contain the parent n-3 PUFA, alpha linoleic acid (ALA) (Penny M. Kris-Etherton, Harris, and Appel 2002). n-6 PUFAs, on the other hand, are found mostly in plant material and come in the form of linoleic acid (LA). Among common oils consumed in Canada, corn, sunflower and safflower oils are especially high in the n-6 PUFA, LA. 1.2.1 Chronic inflammatory disease: an n-6 PUFA link Chronic inflammatory diseases (CIDs) are characterized by consistent low-grade activation of immune cells in an area. Many of the conditions that plague the Western World are CIDs. Diabetes, CVD and inflammatory bowel disease (IBD) all have inflammation that leads to the progression of symptoms (Donath and Shoelson 2011, 2; Danesh et al. 2000). 4 Reducing the incidence of CIDs can be challenging. First, chronic inflammation is extremely hard to detect; the amount of cytokine production, cell proliferation as well as cell infiltration is substantially less than acute bacterial infection and are thus sub-clinical (Locke et al. 2007) (Pahwa and Jialal 2018). Second, chronic inflammation is even harder to treat (Fabbri et al. 2008), as the cause of it is essentially unknown. One current approach to tackle CIDs is treating symptoms, like poor insulin signaling or hypertension (Rendell 2004; López-Sendón et al. 2004). Another approach is to suppress immune signaling. This technique is utilized in IBD patients, where cytokine expression is reduced, however, such treatment is associated with its own risks, like higher rates of infection and even cancers (Hügle, Speth, and Haas 2017; Scheinfeld 2004). A potentially better method to tackle CIDs may involve understanding the cause of inflammation, so it can be addressed in hopes to prevent disease. One causative factor of chronic inflammation is diet (C. K. Roberts and Barnard 2005) along with many others like smoking, exercise, stress and air pollution (Pahwa and Jialal 2018). When looking at epidemiological and investigatory studies regarding diet, some indicate that n-6 PUFA consumption is associated with CIDs. In Australia, men with a recent coronary event were given different dietary interventions in hopes to avoid another incident. One dietary group had a 15% reduction in saturated fat and a subsequent increase in LA, as a substitute. The results indicate that death from all causes, CVD and coronary heart disease significantly increased in the group given LA as a substitute when compared to the control group (Ramsden et al. 2013). In another study, the inflammatory effect of LA is showcased in IBD. Participants who consume LA in the highest quartile have the largest risk of developing ulcerative colitis (IBD in EPIC Study Investigators et al. 2009). These studies are solidified by Tobias Pischon and 5 colleagues in 2003, who show that high LA consumption along with low n-3 PUFA intake may elevate markers of inflammation like sTNF-R in the serum (Pischon et al. 2003). Even though n-6 PUFAs are correlated with chronic diseases, the acceptance of this is not universal because of conflicting data. For example, diets high in LA have also been associated with low C-reactive protein (Virtanen et al. 2018). C-reactive protein activates the complement system and is generally accepted as an indicator of chronic inflammation (Mold, Gewurz, and Du Clos 1999; Luan and Yao 2018). In addition, some metanalyses indicate that n-6 PUFAs have no influence on CIDs, neither increasing nor decreasing things like CVD (Chowdhury et al. 2014). Nonetheless, there is substantial evidence in mouse models that associate n-6 PUFAs, like LA, with inflammation and chronic disease (Ghosh et al. 2004) Due to the inconsistency, it is apparent that more research with appropriate controls and conditions are needed to elucidate if n-6 PUFAs are linked to CID’s in humans. To understand how n-6 PUFAs may be associated with immune response the regulation and conversion of them in the body might give us answers. 1.2.2 Omega-6 bio-conversion LA is the parent n-6 PUFA, meaning all other n-6 PUFAs in the body are made from it. When LA reaches the liver it is enzymatically converted to longer and more unsaturated n-6 PUFAs in the endoplasmic reticulum (Sprecher 2002). It is important to note that this conversion is not only reserved for liver, but happens in most tissues, as needed (J.-Y. Hwang et al. 2018) (Fagerberg et al. 2014). Conversion of LA (18:2) begins with the enzyme delta 6 fatty acid desaturase 2 (FADS2). FADS2 utilizes NADPH to add a double bond to the 6th carbon of LA, creating gamma-linolenic acid (GLA; 18:3) (Tosi et al. 2014). GLA is found in our diet via borage and 6 primrose oils (Gil 2002). Next, GLA is converted to dihomo-gamma-linolenic acid (D-GLA; 20:3) via Elongase 5 (ELOVL5). ELOVL5 adds two carbons with the cofactor acetyl-CoA to the delta carbon of GLA (Tosi et al. 2014). ELOVL5 will be further highlighted in this thesis as manipulation of it results in attenuated LA conversion. There is no large source of D-GLA in our food, as it is a rare fatty acid (Umeda-Sawada et al. 2006). Finally, for the purpose of this thesis, the last important step in LA conversion is the desaturation of D-GLA to arachidonic acid (ARA; 20:4) by the enzyme FADS1 (Tosi et al. 2014). In reality, ARA can further be elongated and desaturated by the same enzymes and finally oxidized to docosapentaenoic acid (DPA; 22:5) in the mitochondria (Tosi et al. 2014). A scheme of the conversion of LA to ARA is highlighted in Figure 1.2. Since LA is the parent n-6 PUFA, the composition of downstream n-6 PUFAs rely on its dietary consumption to some extent (O’Neill, Caldwell, and Meng 1977). Hence, the amount of LA in our diet is critically important, along with the expression of enzymes which regulate its conversion. The enzymes found in this pathway are controlled mainly by the transcription factor called sterol regulatory element-binding protein 1 (SERBP1). SERBP1 is activated upon the absence of sterols and FAs (Espenshade and Hughes 2007). This pathway is shown to exhibit a negative feedback loop, as long chain PUFAs can inhibit SREBP-1 abundance (Shikama et al. 2015) and thus the conversion of LA. 7 Figure 1.1 n-6 PUFA bio-conversion from LA to ARA. Blue boxes represent fatty acids. Grey boxes represent different enzymes (FADS=Fatty acid desaturase) (Elovl= Elongase of very long FAs). LA is Oxidized (Ox.) to 9-HODE, 13-HODE and 4-HNE. ARA is enzymatically converted to immune mediators via cycloxygenase (COX) and lipoxygenase (LOX). 8 1.2.3 Lipid inflammatory mediators n-6 PUFAs have diverse roles within the mammalian system - one of them being a precursor for lipid inflammatory mediators. ARA is a well-known precursor for pro-inflammatory eicosanoids (prostaglandins and leukotrienes), however, these mediators can be formed from D-GLA as well (Levin et al. 2002). Eicosanoids are produced enzymatically after some immune stimulus. Leukotrienes are a result of 15/5-lipoxygenase (15/5-LOX) placing a hydroxyl group at either the 5th or 15th carbon on ARA (Bäck et al. 2011). Leukotrienes have pleiotropic effects, but in general are useful in creating immediate and acute inflammatory responses. For example, they function as bronchoconstrictors, vasoconstrictors, assist in vascular permeability and act as a chemoattractant for leukocytes (Samuelsson 1983). Prostaglandins are a result of cycloxygenase (COX) action on ARA. Prostaglandins have similar functions to leukotrienes like vasoconstriction and chemotaxis but they also assist in activating adaptive immune responses, platelet aggregation, and pain responses (Funk 2001). It is important to note that there are also numerous leukotrienes that counter or resolve pro-inflammatory responses. These are known as anti-inflammatory leukotrienes and are formed mostly by n-3 PUFAs, such as EPA (Calder 2010). 1.2.4 Discrepancies in historical thinking Since LA intake is correlated with chronic inflammatory disorders (see 1.1.1), the rationale suggested by some is that ARA and its eicosanoid metabolites are accountable (Zaman et al. 2010). Although it is well established that ARA metabolites have immune functions, this historical viewpoint on the diet-inflammation dilemma is primitive. This is because research shows that ARA is a strictly regulated fatty acid and its concentration in membranes is generally 9 static (Leslie 2004). In fact, studies show that a high intake of LA does not increase ARA concentrations in blood or in tissue (Rett and Whelan 2011; Greupner et al. 2018; O’Neill, Caldwell, and Meng 1977). Instead, some new research points to LA itself as an immune modulator. LA levels rather than ARA are elevated in children with eczema who also show lower expression of the enzyme elongase 5 (ELOVL5) (Chisaguano et al. 2013). ELOVL5 is a principal protein in the bio-conversion of n-6 PUFAs (discussed earlier), specifically elongating gamma-linolenic (18:3) to dihomo-gamma-linolenic acid (20:3). Deficiency in this enzyme may be attributed to inflammatory disorders because of the resulting increase in LA concentrations when attenuated (Moon, Hammer, and Horton 2009). This information, along with the knowledge of ARA control systems, insinuates that the link between n-6 PUFAs and immune response remains unanswered. 1.3 Oxidative stress Another explanation for the inflammatory action of LA and n-6 PUFAs is their vulnerability to oxidization. PUFAs are susceptible to oxidation by reactive oxygen species (ROS), a result of their electron rich double bonds (Murphy 2009). The metabolites from PUFA oxidation are extremely toxic and have been linked to inflammatory conditions. 1.3.1 ROS formation Mitochondria are a major source of ROS - a consequence of their role in adenine triphosphate (ATP) production. During oxidative phosphorylation, oxygen (O2) accepts single electrons utilized in the electron transport chain. This action is beneficial in that it maintains the electrochemical gradient of the cell, ensuring homeostasis (P. D. Ray, Huang, and Tsuji 2012). The uptake of single electrons by oxygen, however, forms a toxic molecule known as superoxide 10 (O2-) (Murphy 2009). In homeostatic cells, O2- is easily managed. The enzyme superoxide dismutase (SOD) readily transfers superoxide back to O2, yielding hydrogen peroxide (H2O2) as a by-product. H2O2 itself has oxidative properties as well, thus it must also be neutralized. To do this, another endogenous antioxidant in the mitochondria is utilized. In this mechanism glutathione peroxidase (GPx) catalyzes the transfer of electrons from the molecule glutathione (GSH) to H2O2. The transfer of electrons from GSH forms water (H2O) and an oxidized version of glutathione (GSSG) (Berg et al. 2002) (see Figure 1.1). Catalase is another enzyme that neutralizes H2O2, but is not shown in the schematic below (Kehrer, Robertson, and Smith 2010). When the above-mentioned enzymatic mechanisms fail or the production of O2- exceeds threshold, accumulation of ROS occurs. Accumulation of ROS leads to deleterious interactions with essential proteins, lipids and DNA. Such interactions cause DNA breaks, aberrant proteins and lipid peroxidation, which all inhibit normal cellular function (Murphy 2009). Figure 1.2 Removal of reactive oxygen species (ROS) in the mitochondria matrix. Black Arrows outline the normal physiological progression of ROS, red arrows indicate when ROS exceeds threshold. Abbreviations – SOD: Superoxide Dismutase; H2O2: Hydrogen Peroxide; GPX1: Glutathione Peroxidase 1; GSH: Glutathione; GR: Glutathione Reductase; GSSG: Oxidized Glutathione Mitochondria 11 1.3.2 Lipid (per)oxidation Lipid peroxidation occurs when O2- and H2O2 attack membrane lipids creating a reactive OOH group on the hydrocarbon chain (Schneider et al. 2001). PUFAs like LA are extremely susceptible to this manipulation because of their multiple unsaturated areas. Double bonds are rich in electrons and attract reactive oxygen. When LA or other n-6 PUFAs undergo peroxidation metabolites are formed; 4-hydroxynonenal (4-HNE) being one. 4-HNE has been associated with high ROS, tissue damage and inflammatory conditions in individuals with CVD and fibrosis (Lee et al. 2012; Guo et al. 2013). Oxidation of PUFAs occur in the same process, but the OOH group is converted by GPx to a hydroxyl (OH) functional group. This reduction produces oxidative LA metabolites (OXLAMS) namely, 9 and 13-hydroxy-octadecadienoic acid (9-HODE and 13-HODE).These molecules, 9 and 13-HODE, are bioactive and are associated with inflammation and chronic diseases as well (Hattori et al. 2008). Most research into OXLAMs is regarding their link to CVD. The most abundant oxidized lipids found in arterial plaques 9 and 13-HODE are (Spiteller 1998). As a result, they have been associated with atherosclerosis and have been shown to change macrophage signaling and metabolic processing through peroxisome proliferator-activated receptor (PPAR) activation (Nagy et al. 1998; Tontonoz et al. 1998). Macrophages are implicated in the formation of atherosclerotic plaques by ingesting oxidized cholesterol. The role of macrophages in CIDs and their reliance on metabolism and diet will be discussed below. 12 1.4 Macrophages Macrophages (MФs) are the cornerstone of the mammalian innate immune system. They are one of the first immune cells to identify infection and cellular damage and are responsible for recruiting leukocytes to the site of injury and signal for repair and resolution (Zhang and Mosser 2008). MФs commence inflammation by first identifying and ingesting material expressing pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) via membrane receptors called toll-like receptors (TLRs). PAMPs and DAMPs are unique sequences that are present on either bacterial components or damaged cells, respectively (Zhang and Mosser 2008). MФs produce inflammatory mediators upon the recognition of PAMPs and DAMPs. These mediators can be either cytokines or chemokines, but both help with the inflammatory process (Arango Duque and Descoteaux 2014). Cytokines are proteins responsible for activating nearby immune cells that did not interact with the initial PAMP or DAMP. They can have autocrine, paracrine and endocrine capabilities. The main cytokines produced by MФs include: interferon gamma (IFN-y), tumor necrosis factor alpha (TNF-α), interleukin one alpha and beta (IL-1α/β), IL-10, as well as transforming growth factor beta (TGF-β). These cytokines all have various effects on immune cells. For example, TNF-α is recognized by several different immune cells and initiates the activation of NFkB (a transcription factor). In MФs, one result of TNF-α driven NFkB activation is to increase the transcription of inducible nitric oxide synthase (iNOS). Inducible NOS is responsible for producing the nitric oxide that kills foreign pathogens (Magez et al. 2007). In a general sense, TNF-α is considered a proinflammatory cytokine. TGF-β on the other hand is sometimes refered to as an anti-inflammatory cytokine. It can activate nearby fibroblast cells. TGF-β release from MФs results in the proliferation of fibroblasts and the 13 production of collagen and other proteins that are essential in wound healing (Clark et al. 1997; Cavaillon 1994). MФs also synthesize chemokines and these molecules help recruit circulating monocytes and other immune cells to the area of infection or damage. MФs can produce many chemokines upon activation, these include monocyte chemoattractant protein-1 (MCP-1), CCL5/RANTES (regulated upon activation normal T-cell expressed and secreted), Keratinocyte chemoattractant (KC), and IL-8 (Mantovani et al. 2004). Each of these have individual and redundant functions in attracting immune cells to the area of damage or infection (Arango Duque and Descoteaux 2014). 1.4.1 Resident macrophages Resident macrophages are pre-differentiated MФs that are found in tissues like the liver, peritoneal cavity, lungs and the heart (Epelman et al. 2014). They constantly sample their environment and help initiate the appropriate immune response to bacterial infection and/or cellular damage (Epelman et al. 2014). Originally thought to be a distinct population of cells, resident MФs are actually a dynamic population of proinflammatory and anti-inflammatory MФs (Davies et al. 2013). For instance, peritoneal macrophages (PMФs) can originate from embryonic stem cells capable of self-replication called large peritoneal macrophages (LPMs). They can also originate from circulating monocytes, which are recruited to the cavity via chemokines - these are called small peritoneal macrophages (SPMs) (Cassado, D’Império Lima, and Bortoluci 2015). Peritoneal macrophages have been extensively used in research for several decades (Abrass et al. 1985). They are isolated from the peritoneal cavity which is a fluid filled membrane that encapsulates the abdominal organs (A. Ray and Dittel 2010). Since PMФs have a 14 dynamic population, analyzing them can act as a window into the inflammatory state of mice (Ghosn et al. 2010). For example, PMФs shift to a pro-inflammatory phenotype (SPMs) during peritonitis, which is easily detectable by flow cytometry and by investigating their metabolism (Accarias et al. 2016). In addition, PMФs are a great research tool because they send inflammatory mediators into the blood for cellular recruitment and the activation of other immune cells (Chen et al. 2010). Due to their central role in immune function, peritoneal macrophages have been selected as markers for inflammation. 1.4.2 RAW Macrophages Another essential cell in developing inflammatory experiments is the ‘macrophage like’ RAW 264.7 cell line. RAW cells are an immortal macrophage cell from BALB/c mice (Raschke et al. 1978). These cells are invaluable for investigating the role of MФs in disease (Moore et al. 2001; Takahashi et al. 2001) and have been used to understand the mechanism of macrophages in several different pathologies. Interpreting data from RAW MФs comes with several caveats. Since these cells are immortal, many researchers have questioned their stability and comparability among laboratories and growth conditions. This is because increasing the passage number of immortal cell lines can induce genetic shift, which may alter the cellular response to media and stimuli conditions (Geraghty et al. 2014). A recent study, however, has investigated the stability of RAW macrophages cultured in high glucose media with 10% FBS supplementation. They show that between the passage numbers (10 to 30), the phenotypic and functional characteristics (nitric oxide production) of the RAW MФ cells remains steady (Taciak et al. 2018). As a result, in this thesis, RAW MФs were grown in the same conditions and discarded after passage number 30 to ensure reproducibility. 15 1.4.3 Macrophage activation state MФs have two main functions, initiating inflammation and/or resolving it. How the MФ is stimulated or its metabolic state determines whether it is pro-inflammatory or anti-inflammatory. In an oversimplified characterization, pro-inflammatory MФs are referred to as M1 macrophages which are classically activated. In contrast, anti-inflammatory MФs, which are responsible for resolving inflammation are considered M2 macrophages and are thought to be alternatively activated. 1.4.3.1 Classical activation Classical activation occurs, in general, when a stimulus induces in a pro-inflammatory state in MФ. The most studied M1 stimulus is lipopolysaccharide (LPS). LPS is produced from gram-negative bacteria and found at high levels during infections. MФs recognize and bind to this molecule via TLR-4 and CD-14. The binding of LPS to TLR-4 induces an intracellular cascade, leading to the engulfment of bacteria and the subsequent production of a phagolysosome (Zhang and Mosser 2008). Phagolysosomes are a fusion between a phagosome and a lysosome. They are highly acidic (pH < 5) and have high levels of ROS to kill bacteria and digest proteins (Mindell 2012). During engulfment of cellular debris and bacteria, expression of NADPH oxidase and nitric oxide synthetase increase and produce the required ROS (Mindell 2012). There are several stimuli that classically activate MФs (Bianchi 2007), so another way to characterize pro-inflammatory MФs is by their metabolic state (Galván-Peña and O’Neill 2014). One indicator of a classical M1 MФ is the utilization of glycolysis for ATP production. In a pro-inflammatory state, MФs increase their glycolytic cycle and reduce the function of the electron transport chain (ETC) (Yamashita et al. 2014; Tawakol et al. 2012). This drives glucose uptake 16 and also increases succinate levels in the cell via the Krebs cycle (E. Mills and O’Neill 2014). Succinate is shunted to the enzyme succinate dehydrogenase (SDH) where oxidation of succinate increases mitochondrial ROS (Evanna L. Mills et al. 2016). Mitochondrial ROS production drives the inflammatory phenotype of MФs and when inhibited reduces the function of the MФs. Interestingly, the amount of succinate is shown to increase in inflammatory disorders and inhibitors of SDH are currently being tested to reduce inflammation (Lampropoulou et al. 2016). 1.4.3.2 Alternative activation Alternative activation is necessary for the reduction of inflammation (Porcheray et al. 2005). MФs stimulated in this sense are termed M2 macrophages and can be characterized by their metabolic state and the mediators they release. The main stimulus for alternative activation is IL-4, which initiates a metabolic shift in the cell (Martinez, Helming, and Gordon 2009). M2 MФs utilize fatty acid oxidation for most of their energy production and reduce glycolysis, which helps reduce ROS (Galván-Peña and O’Neill 2014). M2 MФs are important for tissue repair and releasing IL-10, which reduces the inflammatory action of other immune cells (Schreiber et al. 2009). IL-10 works by 1.4.4 Role of macrophages in atherosclerosis MФs play an important role in the progression of chronic inflammatory disorders. Dysfunction and/or dysregulation of MФs can lead to serious medical consequences like fibrosis, atherosclerosis, inflammatory bowel disease and diabetes (Lech and Anders 2013) (Luca and Olefsky 2008; Rogler et al. 1997). In atherosclerosis, MФs are responsible for the formation of plaque in arterial walls. This process reduces blood flow, causes blockages and ultimately leads to stroke or organ failure (Gloviczki Monika L. et al. 2010) (Van der Donckt et al. 2015). MФs aid in atherogenesis when recruited to the intima (space between endothelial and muscle cells) of 17 arteries walls (Bobryshev et al. 2016). When there, pro-inflammatory MФs express scavenger receptor proteins such as CD-36 and begin sequestering fat (Stewart et al. 2010). Scavenger receptors recognize and ingest cholesterol such as oxidized low-density lipoproteins (oxLDL) and store it in the ER, becoming fat laden foam cells (Bobryshev et al. 2016). Foam cells are stationary cells that form the building blocks of arterial plaques. Unfortunately, foam cells eventually undergo apoptosis and lead to a brittle, necrotic, and pro-inflammatory plaque (Gonzalez and Trigatti 2017). This is a detrimental cycle that drives more inflammation and MФ recruitment, leading to either plaque growth or breakage. 1.4.5 Dietary influence on macrophages It has been suggested that MФs are crucial in understanding the link between diet and inflammation (Dall’Asta et al. 2012). This is because the inflammatory state of a MФ is dependent on its metabolic programming (shown above 1.4.3). For example, MUFA intake is known to increase M2 MФ populations and reduce COX and iNOS expression in the cells (Camell and Smith 2013). Here, the dietary impact of MUFAs produce an anti-inflammatory environment, which might explain why Mediterranean diets are associated with low CIDs (Trichopoulou et al. 2003). While MUFAs are generally accepted to alternatively activate MФs, the effect of SFAs and PUFAs is more controversial and complex. In an oversimplified summary, the effect of each FA category on MФ state is highlighted in Figure 1.3. 18 Figure 1.3 Influence of fatty acids on macrophage activation state. Graphic representation is an over-simplified summary of the literature regarding macrophage – fat interaction. SFA= saturated fatty acid. MUFA= monounsaturated fatty acid. N-3 PUFA = omega-3 polyunsaturated fatty acid. N-6 PUFA= omega-6 polyunsaturated fatty acids. Ф = macrophage. 1.4.5.1 Saturated fatty acids and MФs Research has connected the intake of SFAs to the classical activation of MФs (Lesna et al. 2008; Suganami Takayoshi et al. 2007). This research indicates that high SFA intake can stimulate MФs to increase production of pro-inflammatory chemokines and cytokines in animals (Robblee et al. 2016). In addition to this, in vitro studies suggest that SFAs can activate TLR-2 and TLR-4 cascades in the macrophage. This leads to the release of TNF-a and other inflammatory mediators by M1 style MФs (Snodgrass et al. 2013) (D. H. Hwang, Kim, and Lee 2016; Suganami, Nishida, and Ogawa 2005). Such data implies that SFAs act as agonists for TLR receptors, however a recent study by Lancaster and colleagues (2018) denies such idea (Lancaster et al. 2018). Their results show that SFAs do not bind directly to TLRs but rather 19 initiate a pro-inflammatory response in MФs by some other process that still relies on the TLR cascade. While current evidence suggests SFAs modulate macrophage state, it is important to note that some discrepancies arise when reviewing the literature. Studies that investigate the interaction between SFAs and MФs sometimes fail to address or even control for n-6 PUFAs. For example, in the Lancaster et al. (2018) study, discussed above, the dietary information was not published. This is concerning as mouse food often contains proportionally high n-6 PUFA levels (“LabDiet 5053” n.d.), which can influence the interpretation of data. Additionally, a study that suggests SFAs regulate macrophage activity fails to control for LA in their diets (Enos et al. 2013). As a result, they neglect to conclude that their diet containing the lowest amount of n-6 PUFAs significantly reduce macrophage infiltration, TLR-2 expression and TNF-α secretion when compared to other diets (Enos et al. 2013). Hence, research that suggests SFA can activate MФs, may be complicated by n-6 PUFAs. 1.4.5.2 Polyunsaturated fatty acids effect on MФs When discussing the influence of PUFA on MФ state, specificity is critical, as both n-6 and n-3 PUFAs have differential outcomes. For example, it is well established that n-3 PUFAs like DHA and EPA reduce the inflammatory action of MФs by increasing the production of resolvins and by inhibiting TLR signaling (H.-Q. Liu et al. 2013; De Boer et al. 2015; Singer et al. 2008). On the other hand, n-6 PUFAs are generally thought to activate pro-inflammatory MФs. Since n-6 PUFAs are considered pro-inflammatory, I will highlight the known effects of both LA and ARA on MФs. Understanding the effect of these FAs on MФs may provide potential targets to prevent CIDs. 20 1.4.5.2.1 Linoleic acid and MФs In vitro and ex vivo studies are necessary to investigate the specific effect of LA on MФs. These methods allow for the removal of in vivo processes like bio-conversion which complicate dietary findings. Literature regarding the direct effect of LA on MФ state is sparse, but it suggests that LA can be pro-inflammatory. Back in 1981, LA was shown by (S. Lynn, Mukherjee, and A. Freeman 1981) to initiate a chemoattractant effect in macrophages. When introduced into the lungs of mice, LA leads to increased numbers of alveolar MФs and to the secretion of chemokines, when compared to other injected FAs. This finding is complemented by research in 1998 that showed LA elevates IL-1 and IL-6 production in peritoneal macrophages (Grimble and Tappia 1998). More recently, research shows that LA can increase ROS levels in MФs and subsequently decrease the amount of antioxidants present in the cell, both hallmarks of classical activation (Rybicka et al. 2011). Additional studies have shown that LA also influences cholesterol uptake and lipid storage in MФs (Mauerer, Ebert, and Langmann 2009). LA in combination with high glucose, significantly decreases the expression of cholesterol efflux pumps compared to SFAs, which results in higher cholesterol accumulation and foam cell production. This research challenges the notion that SFAs contribute to CAD and reiterates the importance of controlling all FAs when doing dietary interventions studies. Clearly, evidence regarding the effect of LA on MФs is limited, but initial studies suggest it can modulate their inflammatory state. 21 1.4.5.2.2 Arachidonic acid and MФs As stated earlier, ARA is known to be proinflammatory because it is required for the formation of eicosanoids (Samuelsson 1983). When MФs are stimulated via TLRs, they produce large levels of eicosanoids from membrane-bound ARA (Norris and Dennis 2014). Although this is the main effect of ARA, it is important to note ARA itself can exert an effect independent of these metabolites. Research into this interaction is limited but nonetheless important when talking about our diet (Shen et al. 2018). In vitro evidence indicates that when MФs are exposed to free ARA, lipid storage increases along with stress proteins, P38 and JNK (Shen et al. 2018; Guijas et al. 2012). In addition, ARA can also increase ROS production in MФs similar to LA (Pompeia, Cury-Boaventura, and Curi 2003). Although it may seem ARA behaves like LA, some research suggests that this is inaccurate. In a study by (Wang et al. 2009) ARA can actually reduce MCP-1, IL-6 and TNF-a secretion in human macrophage cells, similar to omega-3 FAs. In the same study, LA showed elevated chemokine and cytokine expression in MФs similar to SFA treated cells. Although ARA may be anti-inflammatory in some cases, dietary studies indicate that foods supplemented with ARA are still pro-inflammatory (Walter et al. 2019). ARA can elevate prostaglandin production as well as TNF-a expression in alveolar MФs after LPS exposure when put in milk (Walter et al. 2019). Both ARA and LA potentially initiate inflammatory responses in MФs. This information illustrates why n-6 PUFA linked inflammation is hard to interpret (Ramsden et al. 2013). It is important to note though, that the above studies implicate free ARA in inflammation, which is not the same as LA being bio-converted to ARA in vivo. Nonetheless, the central theme of this thesis is to ask, ‘What species is responsible for inflammation in a high LA diet as seen in the 22 Western world?’ This question will always remain a factor in dietary interpretations as long as n-6 PUFA conversion goes uncontrolled for in studies. Hence, in my study, I attempt to answer this question using a mouse model with deficient enzymatic conversion of LA to ARA. 1.5 Hypothesis and aims Considering the above information, a link between n-6 PUFAs and inflammation clearly exists. How n-6 PUFAs establish this effect, however, is a topic of debate as well as their effect on macrophages. This thesis aims to answer one main question, whether LA (highly consumed in our diet) exerts a pro-inflammatory effect in PMФs or if downstream n-6 PUFAs (ARA) play a substantial role. Since ARA concentration is tightly regulated and shown to remain static during high LA consumption (Rett and Whelan 2011), I have the following null hypothesis: The parent n-6 PUFA, linoleic acid, does not induce a proinflammatory state in macrophages independent of downstream n-6 PUFAs. To understand this question, two main approaches will be taken. First aim: Understand how direct LA exposure effects the metabolic/activation state of RAW 264.7 macrophages in vitro. Second aim: Elucidate the activation state of peritoneal macrophages after a 6-week high LA diet in both wild type and ELOVL5 deficient mice. By investigating ELOVL5 deficient mice, the impact of downstream and upstream n-6 PUFAs on inflammation will be understood. In addition, identifying the metabolic state and main 23 features of an activated MФ will help clarify the effect of LA on MФs and also indicate if cells are in a pro-inflammatory or anti-inflammatory state. 24 Chapter 2: Intrinsic LA bio-accumulation supersedes dietary LA in activating inflammatory macrophages 2.1 Overview Dietary n-6 PUFAs have been linked to chronic inflammatory disease, like CVD and type 2 diabetes (Hodge et al. 2007) (Beam et al. 2015) (Ghosh et al. 2004). A rationale for this inflammatory action suggests that over consumption of LA (18:2) in our diet can lead to arachidonic acid (ARA; 20:4) production and increased eicosanoid levels (James, Gibson, and Cleland 2000). Although this rationale is logical, it lacks scientific evidence. Mostly, research shows that the concentration of ARA in the blood as well as in the tissues of animals remain steady despite high LA intake (Rett and Whelan 2011; Greupner et al. 2018). Thus, the cause of n-6 PUFA inflammation is unknown, which ultimately reduces the ability to implement appropriate regulations to reduce its incidence. My thesis is designed to understand whether n-6 PUFA induced inflammation is mediated through ARA or LA. To answer the above question, I will first determine if exposing RAW macrophages to LA can induce a pro-inflammatory state. Oleic acid (OA) a common MUFA will be used as a FA control for these experiments. After in vitro investigations, PMФs from WT and ELOVL5 deficient mice will be analyzed after an iso-caloric diet rich in PUFA (corn oil) or MUFA (olive oil). ELOVL5 deficiency leads to the reduction of LA conversion to ARA (Chisaguano et al. 2013) (see Figure 2.1) (Moon, Hammer, and Horton 2009) and will help determine if this process is responsible for inflammation. Since ARA levels are tightly regulated and remain static under high LA intake (Rett and Whelan 2011), it is expected ELOVL5 deficiency will lead to 25 accumulation of LA and to pro-inflammatory markers in PMФs. In addition, I will investigate how RAW 264.7 MФs respond to oxidative LA metabolites (OXLAMS) in hopes to elucidate how LA might activate MФs. 2.2 Materials and methods 2.2.1 Animal model and diet All animal protocols were approved by the UBC’s Animal Care Committee under protocol A13-3050. Elongase 5 heterozygous deficient mice (ELOVL5+/-) were purchased from KOMP repository (University of California, USA) and kept at 26°C on a 12-h light/dark cycle with free access to food and water. Wild type littermates and ELOVL5+/- mice aged 3-4 months old were randomly assigned to either a) Olive oil (control; MUFA) diet or b) Corn oil (n-6 PUFA) diet. Corn oil (CO) diet consisted of 20% w/w addition of corn oil and Olive oil (OO) diets consisted of 20% w/w addition on top of a basal mouse diet mix (BioServ AIN-76A, New Jersey, USA). In total, both diets contained 43% energy from fat. The diets were made fresh once a week and gas chromatography was completed to ensure proper fatty acid profile. The carbohydrate and protein contents were 42% and 15% of energy respectively, indicating both diets were iso-caloric and iso-nitrogenous (see Table A1.2). The nutrient composition of these diets is depicted in Table 2.1 and percentage of long chain FAs are shown in Table A1.1. All diets were fed ad libitum for 6 weeks. At the end of the experiment, mice were euthanized with isoflurane (Fresenius Kabi) followed by cervical dislocation. In addition to the experimental diet, mice were fed a standard mouse laboratory diet for 3-4 months before starting the above-mentioned dietary regime (PicoLab 5053, Texas, USA). The energy composition of this diet by macronutrient was 24.7% protein, 13.2% fat and 62.1% 26 carbohydrate. How this diet differs from the experimental diets, can be seen in Table A1.2. This diet was used only to gather results in Figure 2.3F as a low-fat diet control, to show the interaction between ELOVL5 and a high-fat diet. Table 2.1 Nutrient composition of experimental diets. Base diet is AIN-76A powder with either olive oil or corn oil added to it. Nutrient information and AIN-76A powder provided by BioServ (New Jersey, USA). Nutrient Olive oil diet (grams/Kg) Corn oil diet (grams/Kg) Casein 160 160 DL-Methionine 2.4 2.4 Starch 120 120 Sucrose 400 400 Cellulose 40 40 Choline Bitartrate 1.6 1.6 Mineral Mix 28 28 Vitamin Mix 8 8 Corn Oil 40 240 Olive Oil 200 0 Total 1000 1000 27 2.2.2 Peritoneal macrophage isolation and treatments The method for isolating peritoneal MФs was adapted from a protocol described elsewhere (A. Ray and Dittel 2010). In short, mice were anesthetized with isoflurane followed by cervical dislocation. After this, 7ml of PBS plus 3% FBS was injected into the abdominal cavity and massaged to ensure uniform distribution. An incision was made in the abdominal wall and 5 mL of the solution was collected with a pasture pipette. The suspension was placed immediately on ice and centrifuged at 1500rpm for 8 minutes to collect the cells. Cells were re-suspended in DMEM medium and plated in a 6-well plate. Four hours after the wells were washed with PBS to remove non-adherent cells and new media was replaced. The remaining cells were removed with a scraper and 0.025% trypsin and counted using Trypan Blue. Once counted, cells were plated at 50,000 cell/well in DMEM media and allowed to re-adhere for 24hrs. 2.2.3 Serum cytokine analysis Serum from mice was analyzed via a cytokine antibody array by Eve Technologies Corp. (Calgary, AB, Canada). In total, 31 cytokines were measured, and results are expressed as ng/mL of plasma. Only RANTES and KC chemokine expression are presented in this thesis. 2.2.4 In vitro cell model and treatments RAW 264.7 macrophages (ATCC, Manassas, VA, USA)) were plated in a Constar 48-well plate until confluent and treated with either of the following: 3% BSA, 3% BSA-conjugated 0.25mM oleic acid (OA), 3% BSA-conjugated 0.25mM linoleic acid (LA) (from Sigma Aldrich) for 24 hours. In addition, RAW macrophages were also exposed to oxidative by-products of LA such as 4-HNE (20µM), 9-HODE (15µM) and 13 HODE (15µM) (from Cayman Chemical) for 24 hours. Following FA incubation, some cells were subjected to 1µg/mL lipopolysaccharide (LPS) (Eschericha Coli; O55:B5; Sigma Aldrich) stimulation for 6 hours. Cells were grown in 28 Dulbecco’s modified eagles media (DMEM; high glucose; 25mM), supplemented with 10% fetal bovine serum (FBS) and 2% penicillin/streptomycin. In some cases, ALDA-1, an activator of ALDH2 was preincubated in media at 20uM for 4-hrs prior to FA incubation. For in vitro measurements, one trial is equivalent to an experiment done on one plate under the same cellular passage number. All subsequent trials were completed on different passage numbers with either triplicates or duplicates wells. Cells were discarded after passage number 30. 2.2.5 Reactive oxygen species assay Cells were incubated with 10μM dichlorofluorsecein-diacetate (DCF-DA) (Invitrogen, California, USA) at 37°C and 5% CO2 for 30 minutes. DCF-DA is a cell permeable dye that once brought into the cell is cleaved from its diacetate group forming a colourless compound (H2DCF) (Eruslanov and Kusmartsev 2010) . Upon oxidation by several reactive oxygen species (ROS) like hydroxyl radicals, nitric oxide radicals and peroxynitrate H2DCF becomes a fluorescent molecule easily detectable by spectrophotometry (Eruslanov and Kusmartsev 2010). Hydrogen peroxide and superoxide cannot actively oxidize H2DCF, but their concentrations influence the fluorescence of DCF in the cell. After a 30-minute incubation with DCF-DA, several washes with PBS were completed and fluorescence was read at 490 nm excitation and 510/570nm emission by a Glomax multi detection system (Promega; Wisconsin, USA). For thorough detail on the procedure please refer to (Eruslanov and Kusmartsev 2010). ROS data are expressed as relative units. The calculation for relative units is as follows: ROS (Relative to BSA control) = (Well Fluorescence) - (Average Fluorescence of BSA control) 29 2.2.6 Succinate dehydrogenase activity assay The protocol was adapted from (Green and Narahara 1980). SDH activity for each macrophage protein homogenate (1µg/µL) was determined by detecting the conversion of 2 mg/mL p-Iodonitrotetrazolium violet (INT) to a coloured formazan product at 37°C after 20 minutes. The reaction was stopped with 10% trichloroacetic acid and product solubilized in ethyl acetate. Absorbance was recorded at 490 nm in a FLUOstar Omega spectrophotometer (Offenburg, GER). 2.2.7 Resazurin assay A working stock of resazurin was made according to manufacturers’ instructions (Alfa Aesar, Haverhill, MA, USA) and recommended protocol was used. Fluorescence was measured using the Glomax multi detection system (Promega; Wisconsin, USA) at 525 nm excitation and 580/640 nm emission after 1 hour of incubation. Resazurin was used as an indicator of cell viability. Resazurin is known to measure mitochondrial metabolic activity and is reduced in the mitochondria by NADPH or NADH to a colourful compound called resorufin (Czekanska 2011). 2.2.8 Aldehyde dehydrogenase activity assay Aldehyde dehydrogenase (ALDH) is an enzyme group that oxidizes aldehydes to carboxylic acids. In the mitochondria ALDH2 oxidizes 4-HNE to the non-toxic 4-HNA (Zhong and Yin 2015). ALDH activity from whole cell homogenates was analyzed. In the enzymatic reaction 94μg of protein was added to a 96-well Constar plate containing 75 μL of 2.5mM NAD+, 150μL of pyrophosphate buffer (pH 9.5) and 3 μL of 1mM acetaldehyde. ALDH converts NAD+ to NADH while oxidizing acetaldehyde. NADH absorbance is detectable at 340nm and ALDH activity is proportional to the production of NADH with a molar coefficient of 6.22 M-1cm-1 (Belmont-Díaz et al. 2016). 30 2.2.9 Immuno dot blot Immuno dot-blot was used to determine 4-hydroxnoneal (HNE) concentrations in RAW 264.7 macrophage protein homogenates (1µg/µL). In a protocol adapted by Abcam (Vancouver, CA), primary HNE antibody (Abcam) was incubated at 1:1000 in 1x-TBST for 2 hrs followed by anti-goat (1:5000) HRP-linked secondary incubation for 1 hr. For detection electro-chemiluminescent (General Electric) substrate was used and visualized on a Mandel Li-cor unit. Light intensity was quantified by Image J studio software. 2.2.10 Lipid droplet assay Both RAW 264.7 and peritoneal macrophages were co-incubated with 20µg/mL cholesterol (Alfa Aesar) and 0.1mM LA or OA for 24hrs. After incubation cells were exposed to 1X lipidspot 610 lipid droplet stain (Biotium; California, USA) for 1 hour at 37oC in cell media and void of light. Once stained, the Glomax multi detection system (Promega; Wisconsin, USA) was used to measure cell emission in the CY5 detection channel (Ex. 625nm, Em. 660-720nm). Cells were then imaged using MetaMorph software on an Olympus IX81 fluorescent microscope at 10X or 20X magnification. 2.2.11 Glucose uptake The amount of glucose remaining in the media was determined after cellular treatments. OneTouch UltraMini glucose monitor was used to determine glucose concentration. Glucose levels are proportional to the production of gluconic acid by glucose oxidase. Glucose uptake was determined by subtracting the recorded value by the amount of glucose initially in the DMEM media (25mM). 31 2.2.12 Gas Chromatography Analysis Fatty acid methyl esters were analyzed from food and whole-cell PMФ homogenates on a Trace 1300 gas using a Supelco Famewax column (30 m × 0.32 mm inner diameter × 0.5 μm). Extraction procedure was followed according to (Kang and Wang 2005). Each fatty acid is represented by a peak in the Chromeleon 7 software (Thermo Scientific). The area under each peak was used to determine relative quantity of each fatty and the retention time was compared against a 37 long chain fatty acid standard (Sigma) to determine the identity of each fatty acid. Values are expressed as a percent, which is derived from the area of a single fatty acid peak over the total area of peaks. 2.2.13 Statistical analysis Results are expressed as box plots with max and min values or bar blots with mean ± S.E.M show the precision of the mean estimation. Data was analyzed by either an unpaired T-test, a One-Way ANOVA or Two-Way ANOVA with a Dunnet’s/Tukey’s post-test when appropriate. Level of significance was set at p > 0.05. GraphPad Prism (version 6) software was used to conduct statistical analyses and figures. Sample size for all experiments are specified in each figure caption. 2.3 Results 2.3.1 LA drives a M1 ‘classical’ inflammatory state in macrophages compared to OA. To understand if LA has an inflammatory effect I exposed RAW 264.7 MФs to the FA and analyzed classical inflammatory markers. It was found that LA exposure leads to several hallmarks of M1-stimulated immune cells, whereas OA incubation does not (see 1.4.2.1). These markers include elevated ROS production, SDH activity and glucose uptake. ROS production in 32 non-stimulated LA incubated MФs is significantly (p<0.05) higher by roughly 175 fluorescent units compared to BSA (vehicle) (Figure 2.1B). The same trend holds true for LPS stimulated RAW MФs. LA also significantly increases SDH activity and glucose uptake in LPS stimulated cells (Figure 2.1D/F), suggesting glycolysis is upregulated in these cells. Although these markers increase for LA treated cells, there is no difference in cell viability amongst treatments (Figure 2.1E) indicating viability does not play a role in the effect. Upon OA incubation however, no significant difference in ROS, SDH activity and glucose intake were seen compared to BSA vehicles, reserving change only for LA. The effect of exogenous LA on RAW MФs is summarized in (Figure A3.1). 33 Figure 2.1 LA incubation initiates classical activation state and increases fat storage in RAW 264.7 macrophages. A) Cholesterol uptake in RAW 264.7 MФs co-incubated with 0.1mM FA and 20ug/ml FC. (n=4). B) ROS production in high glucose (25mM) DMEM media (10% FBS; 2% Pen/Strep) after 24hrs of FA incubation with or without 6hrs LPS (1ug/mL) stimulation. (n=4-5). C.) Fluorescence microscopy representation of cholesterol uptake after FA/FC co-incubation, 10X images, using LipidSpot stain under the CY5 filter. D) Succinate dehydrogenase (SDH) activity is equivalent to the absorbance of INT formazan produced after 30mins at 480nm. (n=8). E) Resazurin or cell viability of cells. (n=4-5). F) Amount of glucose (mM) utilized by RAW MФs with or without LPS stimulation (n=4-5). Data shown as boxplots with max and min values. * indicates significance (p>0.05) in a One-Way ANOVA with a Dunnet’s post-hoc test or unpaired T-test when appropriate. All experiments (n=4-5). 34 2.3.2 Elovl5 deficiency results in significant LA accumulation and a proinflammatory environment in mice. After in vitro testing, I fed WT and ELOVL5+/- mice a CO or an OO diet. Before looking at the inflammatory environment in these mice I first determined if the genetic model worked. I show that ELOVL5 deficiency results in reduced LA to ARA bio-conversion. On average, there is a 55% increase (p<0.05) in LA in ELOVL5+/- PMФs compared to WT PMФs fed a CO diet (Figure 2.2B). In addition, GLA (18:3) and ARA (20:4) are significantly reduced in ELOVL5+/- mice from the CO diet. OO fed mice show the same trend regarding LA accumulation and ARA production, however, not significantly. Interestingly, GLA decreased in ELOVL5+/- mice too. This was unexpected as ELOVL5 elongates GLA to D-GLA (Figure 1.1), thus reduction in ELOVL5 was predicted to increase GLA too. Along with the accumulation of LA in ELOVL5+/- mice, inflammatory markers were elevated in serum, indicating a proinflammatory environment. RANTES (CCL5) a common chemoattractant for monocytes, eosinophils and T-cells is significantly influenced by ELOVL5+/- genotype (row factor p=0.0013) (Figure 2.2D). In contrast, a common anti-inflammatory chemokine, called keratinocyte chemoattractant (KC), was down regulated roughly 2 times in ELOVL5+/- groups (row factor p=0.021) (Figure 2.2E). It appears the inflammatory profile is specific for cellular recruitment because cytokines TNF-α, IL-1α and IL-10 show no differences among diet and genotype (Figure A2.1). To corroborate the chemoattractant profile, the number of macrophages found in the peritoneal cavity significantly increased by roughly 2 times in ELOVL5+/- mice when compared to WT equivalents (Figure 2.2C). 35 Figure 2.2 ELOVL 5 deficiency leads to LA accumulation and a pro-inflammatory environment. A) Composition of total n-6 PUFAs in PMΦs from CO fed mice, analyzed by gas chromatography. (n=4). B) Composition of total n-6 PUFAs in peritoneal macrophages from OO fed mice, analyzed by gas chromatography (n=3). C) Number of viable isolated PMΦs, determined by trypan blue staining. D) Serum RANTES (CCL5) concentration. (n=3-5). E) Keratinocyte Chemoattractant (KC) concentration in serum. Chemokine levels were determined by Eve Technologies (Alberta, CA) (n=5-6). Data is shown as mean ± S.E.M or boxplots with maximum and minimum values. * indicates significance (p>0.05) in a Two-Way ANOVA with a Tukey’s post-hoc or unpaired T-test when appropriate. D 36 2.3.3 Olive oil diet fails to reduce ROS production caused by reduced n-6 PUFA bio-conversion. After isolation, I determined the ROS levels in stimulated and non-stimulated PMФs in order to understand their activation state. In both conditions (Figure 2.3A/B) the amount of reactive oxygen species (ROS) produced by PMФs was elevated in the ELOVL5+/- genotype. Of note, diet appeared to have no effect on ELOVL5+/- mice. The amount of ROS production in non-stimulated and LPS stimulated cells is similar in both CO and OO diets. These data indicate that OO feeding does not reduce the inflammatory effect of ELOVL5+/-, nor did it show a reduction in WT cells. The fact that OO fails to reduce the ELOVL5 effect is corroborated by RANTES and macrophage accumulation data shown in Figure 2.2. Difference in ROS production is not a factor of cell viability in PMФs (Figure 2.3E) Next, I investigated if the ROS production was a consequence of genotype and not LA accumulation. I did this by comparing WT and ELOVL5+/- PMФs under a standard low-fat mouse pellet diet (PICOLAB 5053). As expected, no difference in ROS is seen between WT and ELOVL5+/- PMФs treated with BSA (control). A significant difference in ROS was only seen once exogenous LA (0.25mM) was added to the ELOVL5 background – showing accumulation of LA may be the culprit for ROS not ELOVL5 deficiency. Interestingly, when exogenous OA (0.25mM) is added, the increase in ROS is not significant compared to BSA controls. It is important to note that this is a different cohort of mice, as a result, this data was only compared to itself and not to the experimental high fat diet findings. 37 2.3.4 CO feeding does not increase cholesterol uptake in PMФs whereas direct LA incubation does. Lipid storage increases in macrophages found in the intima of atherosclerotic arteries (ref). I wanted to determine if dietary fatty acids might play a role in the ability of macrophages to store cholesterol. To do this, PMФs from CO and OO fed WT mice were exposed to free cholesterol to determine the influence of diet on lipid storage. Mice fed a high CO diet for six weeks tended toward more cholesterol uptake but ultimately showed no difference compared to PMФs from OO diets (Figure 2.3C/D). However, LA can still increase cholesterol uptake in macrophages. Lipid storage significantly increases when RAW 264.7 ‘macrophage-like’ cells are directly incubated with free cholesterol and 0.1mM LA (Figure 2.1A). Cholesterol uptake data also solidifies that ELOVL5 enhances the effect of LA. Just 15µM LA can induce higher levels of cholesterol uptake in PMФs (not significant) when combined with ELOVL5 deficiency seen in Figure 2.4A. Although this is not exact physiological conditions, it shows that LA can potentially increase lipid storage in MФs. In addition, it is important to note that although LA shows these trends, OA incubation does not. 38 Figure 2.3 ELOVL 5 deficiency induces ROS production in PMФs under a high LA environment. A) ROS production in high glucose (25mM) DMEM media (10% FBS; 2% Pen/Strep) after 20hrs of no stimulation (n=4-5) or B) lipopolysaccharide (LPS) (1ug/ml) stimulation (n=4-5). C) Free cholesterol (20mg/uL) uptake in PMФs analyzed by lipidspot stain. (n=4). D) Fluorescence microscopy images of PMФs after cholesterol (20mg/uL) exposure, 20X images, under the CY5 filter. E) Resazurin or cell viability of PMΦs. (n=4-5). F) ROS production after 24hrs of LA and OA (0.25mM) incubation in ELOVL5 deficient and WT PMΦs from low fat diet (n=4). Data is shown as boxplots with max and min values. * indicates significance (p>0.05) in a Two-Way ANOVA with a Tukey post-hoc test. 39 2.3.5 OXLAMS contrast the effect of LA incubation ex vivo and in vitro. PMФs and RAW cells were both incubated with OXLAMS to understand if the effect of LA on MФs is mediated through its oxidative byproducts. In short, this idea was not supported by my investigation. To highlight, ELOVL5+/- PMФs incubated with LA show high cholesterol uptake, whereas 9-HODE incubation significantly reduces cholesterol uptake in ELOVL5+/- PMФs and WT cells (Figure 2.4A). Interestingly, 13-HODE does not show this same reduction but it does keep cholesterol uptake levels in ELOVL5 PMФs at baseline. This trend aides the idea that OXLAMS act differently than LA, as does ROS production results. LA increases ROS in PMФs and RAW cells (Figure 2.2A/Figure2.1B) but 9- HODE and 13-HODE juxtapose this trend by reducing ROS (Figure 2.4B). Therefore, it appears OXLAMS do not produce the same effect as LA exposure. 40 Figure 2.4 OXLAMS impair macrophage ROS production and cholesterol uptake. A) Cholesterol uptake in PMФs after coincubation of FC (20ug/ml) with 15uM LA, 9-HODE or 13-HODE (n=3-4). C) Fluorescent microscopy representation of FC uptake at 10X using LipidSpot stain under the CY5 filter. B) ROS production in RAW cells incubated with 15 µM LA, 9-HODE or 13-HODE for 24hrs (n=1, rep=3). * indicates significance (p>0.05) in a Two-Way ANOVA with a Tukey’s post-hoc test. 2.3.6 ALDH2 activity and 4-HNE may not regulate LA induced ROS production in vitro. 4-HNE is another oxidative by-product of LA but is different in size and functionality compared to 9-HODE and 13-HODE. Thus, I needed to ensure the effect of LA is not regulated through this toxic OXLAM. To begin, I determined that the levels of 4-HNE in RAW cells 41 during LA incubation do not significantly increase compared to BSA and OA treated cells (Figure 2.5B). In addition, ALDH2 an enzyme that clears 4-HNE to a less toxic molecule, 4-hydroxynoneoic acid (4-HNA), failed to increase as well (Figure 2.5C). To further determine the role of 4-HNE in MФ ROS production, ALDA-1 was added at 20uM to enhance ALDH2 activity however only total ALDH activity was analyzed due to a lack of isolated protein. If 4-HNE was the culprit for ROS production ALDA-1 should lower ROS. This was not the case however, as pre-incubation of ALDA-1 for 4hrs did not reduce ROS in LA or OA treated cells (Figure 2.5A). Lastly, to solidify the fact that OXLAMS work in a mechanism independent of LA, 4-HNE was incubated with RAW cells for 24hrs and significantly reduced ROS production (Figure 2.5D). 42 Figure 2.5 4-HNE does not regulate LA induced ROS production. A) ROS production in RAW cells with or without 20uM pre-incubation of ALDA-1 for 4hrs. C) Aldehyde dehydrogenase (ALDH) activity measured by the production of NADH/min/mg of protein. B) 4-HNE protein levels determined by immunostaining and ImageJ software. D) ROS production in RAW cells exposed to 10uM 4-HNE for 24hrs with or without LPS (1µg/µl) stimulation. * indicates significance (p>0.05) in a One-Way ANOVA with a Dunnet’s post-hoc test or unpaired T-test when appropriate. All experiments (n=4-5). 43 2.4 Discussion Over consumption of n-6 PUFAs are implicated in the incidence of chronic inflammatory diseases (IBD in EPIC Study Investigators et al. 2009) (Pischon et al. 2003). It is unknown if the parent n-6 PUFA LA exerts an inflammatory effect directly, or if it is a consequence of conversion to downstream PUFAs like ARA (Rett and Whelan 2011; Alvheim et al. 2014). I investigated this question by first treating RAW 264.7 cells with exogenous LA and then by studying a transgenic mouse model deficient in ELOVL5 (Figure 2.2C). This mouse model in combination with in vitro data suggest that LA activates a pro-inflammatory state in MФs, regardless of downstream n-6 PUFAs. RAW 264.7 are an immortal macrophage cell line from BALB/c mice (Raschke et al. 1978). These cells are invaluable for investigating the role of MФs in disease (Moore et al. 2001; Takahashi et al. 2001). I utilized these cells to determine if LA exposure can influence immune function. Shown in Figure 2.1, LA at 0.25mM leads to a pro-inflammatory state in RAW MФs. Cells exposed to LA, show signs of metabolic reprograming that occurs in classically activated MФs. This conclusion is well supported by previous research that indicates pro-inflammatory MФs utilize glycolysis instead of oxidative phosphorylation to drive ROS production and inflammation (Galván-Peña and O’Neill 2014). During this process, glucose intake and succinate levels increase in MФs. This is important because succinate is shunted to succinate dehydrogenase (SDH) which creates an electron gradient across the mitochondrial membrane (E. L. Mills et al. 2016). This gradient drives mitochondrial ROS production and helps initiate and prolong pro-inflammatory signaling in MФs (E. L. Mills et al. 2016). How LA exerts this effect is unknown, but this data indicates LA can modulate MФ state. Unfortunately, in vitro experiments do not consider the complex nature of dietary consumption like the conversion of 44 LA to downstream PUFAs. Hence in vivo studies were completed to fully understand if LA or ARA is responsible for n-6 PUFA related inflammation. PMФs were used as indicators of inflammation during in vivo feeding. These cells reside in both mice and humans and are thought to exist as either small or large peritoneal macrophages (SPMs and LPMs) (Accarias et al. 2016). SPMs are derived from circulating monocytes, and produce large amounts of ROS and secrete the chemokine RANTES (Ghosn et al. 2010; Cassado, D’Império Lima, and Bortoluci 2015). SPMs can sustain inflammation during infections and their numbers increase during such conditions (Ghosn et al. 2010). LPMs on the other hand, are derived from embryonic stem cells and generate less ROS, secrete chemokines TGF-β1 and KC and are considered anti-inflammatory PMФs (Cassado, D’Império Lima, and Bortoluci 2015). In this study, cells isolated from ELOVL5+/- mice in both CO and OO groups exhibit SPM behavior like elevated ROS before and after LPS stimulation (Figure 2.3A/B). To corroborate this phenotype, ELOVL5+/- mice have elevated RANTES (produced by SPMs) and lower KC levels (produced by LPMs) in their serum and have more macrophages in their peritoneal cavity. Although cells from ELOVL5+/- groups resemble pro-inflammatory SPMs it is important to note that flow cytometry was not used to characterize them (Ghosn et al. 2010). It is well established that OO has innate anti-inflammatory properties (Bogani et al. 2007). Geographic regions that consume higher amounts of OO have lower incidences of CIDs and tend to live longer (Trichopoulou et al. 2003). The anti-inflammatory action of this food may be derived from its high oleic acid (OA) content (shown in Figure 2.1) or the number of polyphenols in it (Bogani et al. 2007), (Parthasarathy et al. 1990). With this knowledge, it is interesting that feeding OO had no effect on the inflammatory state of PMФs from ELOVL5+/- mice or compared to CO fed WT mice. It was expected that OO would reduce this phenotype 45 based on literature and in vitro results. One explanation for this outcome may be that the OO diet contained 17% LA (Table A1.1) - an unavoidable outcome based on the basal diet and the olive oil used. 17% LA may be enough to overcome the benefits of OO when compounded with ELOVL5 deficiency (Figure 2.2B). In addition, the effect of increasing extracellular LA (via ELOVL5+/- ) is likely multiplied in MФs as they lack FADS2, and thus the ability to regulate LA internally is reduced (Chapkin, Somers, and Erickson 1988). Feeding mice for 6-weeks has proven acceptable for determining the influence of diet on inflammatory conditions in genetically or chemically pre-disposed mice (Boesch-Saadatmandi et al. 2010), (Ghosh et al. 2004), (Burleigh et al. 2005). However, the effect diet has on inflammation in WT conditions is more challenging to elucidate. To see outcomes of chronic disease in WT mice, researchers have fed mice anywhere between three to 16 months for results (N. W. Roberts et al. 2015; Calligaris et al. 2013). Thus, it is reasonable that WT PMФs isolated from CO and OO groups showed no statistical difference for all measured parameters in Figure 2.2 and 2.3 - even though they differed in LA. To understand the influence of diet on WT MФs, literature suggests a longer feeding regime. Another possible reason for the lack of dietary differences in my results may be linked to the resolution of DCF-DA. DCF-DA is a promiscuous molecule, interacting with H2O2, O2- and NO, a difference in these species may not be detected if minimal or if shifted from one molecule to the other (Kalyanaraman et al. 2012). DCF-DA can also interact with cytochrome-c and potentially propagate ROS production by forming O2-, leading to false positives (Kalyanaraman et al. 2012). Oxidative linoleic acid metabolites (OXLAMS) like 9-HODE, 13-HODE and 4-HNE are all bioactive molecules linked to inflammatory conditions like atherosclerosis, diabetes and CVD (reviewed in (Vangaveti et al. 2016)). Although not measured in these mice, at high 46 concentrations these metabolites can interact with cellular receptors and also form deleterious interactions with proteins and DNA causing dysregulation of cellular processes (X. Liu, Lovell, and Lynn 2006) (Poli et al. 1982). I incubated these metabolites with RAW MФs and measured ROS production to understand if the inflammatory action of LA is regulated through them. Unexpectedly, it was found that ROS decreased below baseline levels while exposed to these molecules (Figure 2.4B and Figure 2.5D). This may indicate that OXLAMS reduce inflammation or more likely, impair normal MФ function. Research into the action of 4-HNE on macrophages reiterates this assumption as 4-HNE is shown to impair phagocytosis, and cytokine signaling under TLR-4 activation (Kim et al. 2009). To solidify this conclusion, MФs were pre-incubated with ALDA-1, a molecule that activates ALDH2 that clears 4-HNE. Based on the above result it was expected then that the addition of ALDA-1 (Figure 2.4) would fail to reduce ROS production caused by LA. And lastly, 4-HNE levels in the macrophages were not significantly upregulated in LA treated cells, again showing OXLAMS act through a different mechanism compared to LA. In future studies, the concentration of OXLAMS in the mice and macrophages will hopefully be determined. A major factor in atherogenesis is the formation of fat laden MФs, called foam cells. When MФs are recruited to the intima of arterial walls after insult, scavenger receptors such as cluster of differentiation 36 (CD-36) are expressed and ultimately initiate plaque formation (Stewart et al. 2010). Theses scavenger receptors recognize circulating cholesterol, especially oxidized low-density lipoproteins (ox-LDL) and ingest them. MФs internalize cholesterol and store it as free cholesterol (FC) in the ER, becoming burdened with fat and form foam cells (Bobryshev et al. 2016). My results indicate that the pro-inflammatory state initiated by LA (Figure 2.1) is translatable to this disease state. MФs co-incubated with FC and LA resulted in 47 substantial fat storage compared to BSA controls. This result is important because atherosclerosis can lead to CVD, which is one of the leading causes of death in Canada (Canada 2017). As expected, OA which is proven to be anti-inflammatory does not show the same increase in cholesterol storage (Parthasarathy et al. 1990). Investigation into cholesterol uptake also solidifies that CO and OO feeding for six weeks is not sufficient to show differences in activation state (Figure 2.3C). It also suggests, again, that OXLAMS contrast the mechanistic action of LA (Figure 2.4A). This is interesting because 9-HODE and 13-HODE are known to act upon the peroxisome proliferator activated receptor-gamma (PPAR-y) which increases CD-36 expression and cholesterol uptake (Nagy et al. 1998). However, this outlook is one-sided as 9-HODE and 13-HODE also increase expression of cholesterol efflux pumps. Even though OXLAMS are found at high levels in atherosclerotic lesions, research has shown that 13-HODE can reduce cholesterol accumulation in RAW MФs by increasing transporters ABCA1, ABCG1 and SR-BI (Kämmerer et al. 2011). Perhaps this why I saw reduced cholesterol accumulation in OXLAM treated cells in Figure 2.4A. Additional studies will have to be conducted to elucidate why these molecules act differently than their parent molecule and why LA initiates a proinflammatory state in MФs 2.5 Summary Data presented in this chapter shows that ELOVL5 deficiency results in the accumulation of LA in PMФs. I suggest that this LA accumulation may be the cause of elevated ROS production in PMФs, RANTES secretion and the recruitment of MФs in the peritoneal cavity of mice. This hypothesis is solidified by initial in vitro data, which shows exogenous LA can 48 activate RAW MФs by increasing glucose uptake, SDH activity, ROS production and cholesterol storage. How LA initiates an inflammatory state in PMФs is still unknown and require further research. In this chapter, I investigated if LA exerts its effects through OXLAMS (9-HODE, 13-HODE and 4-HNE). Unfortunately, these OXLAMS were shown to reduce ROS and cholesterol uptake, contrasting the mode in which LA interacts with MФs. To add to this conclusion, 4-HNE levels were not significantly elevated in LA treated cells, nor did ROS production decrease in cells pre-incubation with ALDA-1. Thus, it appears LA acts independent of OXLAMS and ARA when activating MФs.49 Chapter 3: Conclusion 3.1 Significance of findings The link between n-6 PUFAs and inflammation has recently been accepted among researchers, however, how n-6 PUFAs exert this effect is still unknown. Some evidence suggests that LA is converted to ARA and influences inflammation through lipid mediators. Others indicate ARA concentrations remain static, suggesting LA can activate inflammatory responses. My data obtained from aim 2 helps answers this question. It indicates that when the conversion of LA to downstream n-6 PUFAs is diminished, PMФs (an innate immune cell) are in an activated, pro-inflammatory state. This is significant for a multiplicity of reasons. First, it suggests that LA in our diet is responsible for inflammation, which is solidified by in vitro data from aim 1. Second, this knowledge helps highlight the importance of understanding the biochemical effect of LA on immune cells. Often studies investigate other PUFAs and SFAs as means to understand inflammation and avoid determining the outcome of LA exposure (Karasawa et al. 2018; Xue et al. 2012). My data shows that LA should be a primary concern when looking at the perplexing link between diet and inflammation. And last, it is significant because it adds to the existing literature that LA can independently activate PMФs. This implies that in order to reduce inflammation and inflammatory disease, the consumption of LA should be reconsidered. 3.2 Limitations of research It is important to note that although I tried to make this work as applicable to humans, it was completed in mice. Hence, one limitation of these findings is that they are true for mice and 50 not humans. However, based on the similarity in DNA and immune cell function between these mammals (Reynolds and Haniffa 2015) the extrapolations made in this thesis are justified. Another limitation of this research is the length of feeding, differences were only seen in the mice with a transgenic background during 6-weeks and not in WT mice. While my results answer a significant question, they are only applicable for mice with an ELOVL5+/- background. To apply this knowledge to WT mice, a longer period of feeding (suitable for a Ph.D. project) would suffice. And lastly, peritoneal macrophages are one subset of immune cells in the mammalian body. My work only concludes ELOVL5 deficiency acts on these MФs and not others. In the future, this study can be repeated in human macrophages cell lines to corroborate these findings in humans. 3.3 Main conclusions Based on my work completed in aim 1 and aim 2, I can reject my null hypothesis (stated in 1.5). The main conclusions that have led to this result, are the following: 1. Linoleic acid (LA) can initiate pro-inflammatory responses in PMФs and induce an inflammatory environment in mice regardless of downstream n-6 PUFAs like arachidonic acid (Figure 2.1 and Figure 2.2). 2. RAW 264.7 cells exposed to direct LA show the same pro-inflammatory responses seen in PMФs, with additional elevation in cholesterol uptake, glucose utilization, and SDH activity (Figure 2.3). 51 References Abrass, C. 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LCFA = Long chain fatty acid Fatty acid Olive Oil (OO) diet (% of total LCFAs) Corn Oil (CO) diet (% of total LCFAs) Saturated fatty acids 26.8 20.2 Palmitic Acid 13.5 17.3 Steric Acid 2.9 2.0 Monounsaturated fatty acids 54.7 24.7 Oleic Acid 53.9 24.5 N-3 Polyunsaturated fatty acids 1.3 1.1 a-linolenic Acid 1.2 1.1 N-6 Polyunsaturated fatty acids 17.2 54.0 Linoleic Acid 17.1 53.9 Arachidonic acid 0.01 0.01 Total 100 100 66 Table A1.2 Macronutrient composition of experimental diets. %Kcal = Kilocal/Total Kilocal in 100 grams of food. Kcal = Kilocalories. OO = Olive Oil, CO = Corn Oil. Macronutrient OO Diet (%Kcal) CO Diet (%Kcal) PICO Lab 5053 (%Kcal) Protein 15.3 15.3 24.7 Carbohydrate 42.2 42.2 62.1 Fat 42.5 42.5 13.2 Total %Kcal 100 100 100 A.2 Cytokine expression Figure A2.1 Expression of common macrophage secreted cytokines. Mouse serum analyzed by Eve technologies. OO = Olive Oil, CO = Corn Oil. No difference amongst diets and genotype.
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The influence of dietary n-6 polyunsaturated fats on the inflammatory state of macrophages
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