FISH OIL DISRUPTS B CELL PLASMA MEMBRANE LATERAL ORGANIZATION AND IMMUNOLOGICAL SYNAPSE FORMATION by Benjamin Drew Rockett November, 2012 Director of Dissertation: Saame Raza Shaikh Biochemistry and Molecular Biology East Carolina University ! Fish oil has immunosuppressive properties that could provide treatment for numerous inflammatory and autoimmune disorders. The primary bioactive components of fish oil, which are the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can exert differential effects on many cell types in the body. A major limitation in the development of fish oil as a therapeutic is the lack of mechanistic understanding of how fish oil exerts its functional effects. One emerging mechanism is that EPA and DHA have unique biophysical properties that could disrupt the lateral organization of plasma membrane lipids and proteins critical for signaling and cell-cell communication, such as lipid rafts and the immunological synapse. In this study, we investigate a potential mechanism for this disruption in B cells, a cell type poorly represented in the field of n-3 PUFA research. The central hypothesis for this study was that fish oil disrupts lateral organization of lipid rafts and suppresses downstream B cell function. Using high and low fat fish oil diets in mice, we demonstrated that fish oil dispersed clustering of B cell lipid rafts on a micron scale, and enhanced membrane molecular order upon cross-linking raft domains. We found that the effects on lipid rafts are primarily driven by DHA and not EPA, and that n-3 PUFAs have a limited influence on non-raft lateral organization. Finally, we show that fish oil suppresses B cell antigen presentation and subsequent CD4 + T cell IL-2 secretion, by disrupting the B cell side of the immunological synapse. Taken together, this work highlights the utility of fish oil, more specifically DHA, as a tool for disrupting plasma membrane lateral organization. We add to the biochemical understanding of how these fatty acids may disrupt various downstream signaling events and cell-cell interactions. It also emphasizes the importance of the plasma membrane as a target for suppressing other cellular functions mediated through lipid raft domains. Finally, these studies add B cells as key targets for suppression of antigen presentation in diseases such as autoimmune disorders. FISH OIL DISRUPTS B CELL PLASMA MEMBRANE LATERAL ORGANIZATION AND IMMUNOLOGICAL SYNAPSE FORMATION A Dissertation Presented To the Faculty of the Department of Biochemistry and Molecular Biochemistry East Carolina University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by Benjamin Drew Rockett November, 2012 © Benjamin Drew Rockett, 2012 FISH OIL DISRUPTS B CELL PLASMA MEMBRANE LATERAL ORGANIZATION AND IMMUNOLOGICAL SYNAPSE FORMATION by Benjamin Drew Rockett APPROVED BY: DIRECTOR OF DISSERTATION: _______________________________________ Saame Raza Shaikh, Ph.D. COMMITTEE MEMBER: _______________________________________ Joseph M. Chalovich, Ph.D. COMMITTEE MEMBER: _______________________________________ Lance C. Bridges, Ph.D. COMMITTEE MEMBER: _______________________________________ Barbara J. Muller-Borer, Ph.D. CHAIR OF THE DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY: _______________________________________ Phillip H. Pekala, Ph.D. DEAN OF THE GRADUATE SCHOOL: _______________________________________ Paul J. Gemperline, Ph.D. ACKNOWLEDGEMENTS ! I would first like to thank my parents, Ron Rockett and Annette Adams, for providing me every opportunity I ever wanted to pursue my dreams. I cannot be more grateful for all you have done to guide me through each stage of me life. I next want to thank my wife, Alexis Rockett. Without you, I would not be where I am today. You give me strength and purpose. To my mentor, Dr. Raz Shaikh, before starting this stage of my career, I never would have imagined where I would end up. I owe much of my success to you. To the my lab-mates Kristen, Mo, Kaitlin, Heather, Mitch, and Mark, all science is a team effort. I could not accomplish this without your hard work and dedication. To my committee, the faculty and staff at ECU, and our collaborators, thank you for the opportunity to work with you, and all the guidance and assistance you have given me. TABLE OF CONTENTS PAGE LIST OF TABLES ............................................................................................... xv LIST OF FIGURES ............................................................................................ xvi LIST OF ABBREVIATIONS ................................................................................ xix CHAPTER 1: INTRODUCTION ......................................................................... 1 Overview ................................................................................................. 1 Historical perspective ................................................................... 1 Biochemistry of n-3 PUFAs .......................................................... 2 Recommended intake of n-3 PUFAs ............................................ 4 n-3 PUFAs and the immune system ............................................. 6 B cells ........................................................................................... 8 Potential mechanisms for n-3 PUFAs .......................................... 10 Lipid rafts ...................................................................................... 11 The immunological synapse ......................................................... 13 Specific aims ........................................................................................... 16 Aim 1: Test the hypothesis that dietary n-3 PUFAs disrupt B cell lipid raft organization and function .......................................... 17 Aim 2: Test the hypothesis that n-3 PUFA diet modified B cells diminish stable immunological synapse formation .................. 17 CHAPTER 2: MEMBRANE RAFT ORGANIZATION IS MORE SENSITIVE TO DISRUPTION BY N-3 PUFA THAN NONRAFT ORGANIZATION IN EL4 AND B CELLS .............................................................................................. 19 Introduction ............................................................................................. 19 PAGE Materials and Methods ............................................................................ 20 Cells ............................................................................................. 20 Fatty acid treatment ..................................................................... 20 Mice, diets, and B cell isolation .................................................... 21 Fatty acid analysis ........................................................................ 23 Column chromatography .............................................................. 23 Staining and imaging with 1,19-dilinoleyl-3,3,39,39- tetramethylin-docarbocyanine perchlorate and 4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- hexadecanoic acid .................................................................. 23 Sample preparation for FRET microscopy ................................... 24 FRET microscopy ......................................................................... 24 Lipid raft staining .......................................................................... 25 Image analysis ............................................................................. 25 Cell growth and apoptosis measurements ................................... 26 Statistical analysis ........................................................................ 26 Results .................................................................................................... 27 EPA and DHA treatment disrupted nonraft organization of EL4 cells ......................................................................................... 27 EPA and DHA treatment increased cell size and growth .............. 30 In vivo administration of n-3 PUFA did not modify nonraft organization but disrupted lipid raft clustering ........................ 35 Disruption of nonraft organization in vitro ..................................... 39 PAGE Nonraft organization was not disrupted by n-3 PUFA in vitro or ex vivo when cell size was unaffected .................................... 40 Lipid raft clustering was sensitive to n-3 PUFA ............................ 42 Implications .................................................................................. 43 CHAPTER 3: HIGH DOSE OF AN N-3 POLYUNSATURATED FATTY ACID DIET LOWERS ACTIVITY OF C57BL/6 MICE ............................................. 45 Introduction ............................................................................................. 45 Experimental Methods ............................................................................ 46 Mice and diets .............................................................................. 46 Metabolic cage studies ................................................................. 46 Results .................................................................................................... 48 Body weight and food consumption ............................................. 48 Metabolic cage studies ................................................................. 48 Discussion ............................................................................................... 50 CHAPTER 4: FISH OIL INCREASES RAFT SIZE AND MEMBRANE ORDER OF B CELLS ACCOMPANIED BY DIFFERENTIAL EFFECTS ON FUNCTION ................................................................................................... 53 Introduction ............................................................................................. 53 Materials and Methods ............................................................................ 55 Mice and diets .............................................................................. 55 Cell isolation and cell culture ........................................................ 57 Isolation of detergent-resistant membranes and analysis ............ 57 2 H NMR spectroscopy .................................................................. 58 PAGE Lipid raft cross-linking and total internal reflection fluorescence imaging ................................................................................... 58 Live cell imaging of DiIC 18 ............................................................ 59 Generalized polarization imaging ................................................. 59 B-cell activation with lipopolysaccharide ...................................... 60 B-cell activation of naive CD4 + T cells ......................................... 60 Statistical analyses ....................................................................... 61 Results .................................................................................................... 62 Body weights, food intake, and cellularity .................................... 62 FO promoted uptake of EPA/DHA into DSMs and DHA into PC of DRMs .................................................................................. 62 FO increased B-cell lipid raft size induced by cross-linking GM1 65 FO increased membrane order upon cross-linking rafts relative to the absence of cross-linking ............................................... 67 EPA and DHA incorporated into DRMs/DSMs and did not increase GM1 surface expression in vitro ............................... 69 DHA, but not EPA, increased membrane order with cross-linking relative to the absence of cross-linking in vitro ....................... 74 FO enhanced B-cell activation in response to LPS stimulation but suppressed B-cell stimulation of naive CD4 + T cells ......... 74 Discussion ............................................................................................... 77 Biochemical measurements revealed some differences between ex vivo and in vitro measurements ......................................... 80 PAGE Imaging demonstrated that FO increased membrane order upon cross-linking ............................................................................ 82 Emerging model by which FO increases raft size ........................ 83 Disruption of rafts with FO was accompanied by differential effects on innate and adaptive B-cell functions ...................... 84 Conclusion .............................................................................................. 86 CHAPTER 5: MHC CLASS II ORGANIZATION ON THE B CELL SIDE OF THE MURINE IMMUNOLOGICAL SYNAPSE IS DISRUPTED BY DIETARY N-3 POLYUNSATURATED FATTY ACIDS ................................... 87 Introduction ............................................................................................. 87 Experimental Procedures ........................................................................ 89 Mice and cells .............................................................................. 89 GC ................................................................................................ 89 Reagents and antibodies ............................................................. 89 TIRF microscopy and analysis ..................................................... 90 FRET microscopy and analysis .................................................... 90 Immunological synapse staining and analysis ............................. 90 Statistics ....................................................................................... 91 Results .................................................................................................... 91 Fish oil increases B cell EPA and DHA levels .............................. 92 Fish oil has no impact on B cell MHC class II organization in the absence of an immunological synapse ................................... 92 PAGE Pharmacological agents disrupt B cell MHC class II organization in the absence of an immunological synapse ......................... 95 Naďve B and naďve T cells form a stable immunological synapse after 30 minutes of conjugation ............................................... 95 Fish oil decreases B cell MHC class II and cognate CD4+ T cell PKC? recruitment to the immunological synapse independent of changes in cell-cell adhesion ......................... 97 Pharmacological agents disrupt B and T cell protein organization in the immunological synapse similar to fish oil . 102 Discussion ............................................................................................... 105 The APC side of the immunological synapse ............................... 106 Fish oil versus pharmacological disruption of the lateral organization of MHC class II in the absence of the synapse .. 107 Fish oil, similar to pharmacological agents, inhibits the ability of B cells to organize MHC class II molecules at the immunological synapse .......................................................... 109 Fish oil suppresses T cell activation by targeting both B and T cells ......................................................................................... 110 CHAPTER 6: CONCLUSIONS .......................................................................... 113 Overview ................................................................................................. 113 The challenge of studying lipid rafts ............................................. 113 DHA, a distinct disrupter of raft clustering .................................... 114 Raft clustering is dispersed on the sub-micron scale ................... 115 PAGE B cells are critical for autoimmune diseases ................................ 116 Precaution for high doses of fish oil ............................................. 119 Conclusion .............................................................................................. 120 REFERENCES .................................................................................................. 121 APPENDIX A: ANIMAL USE PROTOCOL ......................................................... A-1 APPENDIX B: SUPPLEMENTAL FIGURES ...................................................... B-1 LIST OF TABLES PAGE Table 2.1: Composition of experimental diets .................................................... 22 Table 2.2: Fatty acid analysis of B cells isolated from mice fed differing diets .. 36 Table 3.1: Composition of control, low fat, and high fat n-3 PUFA diets ............ 47 Table 4.1: Composition of control and fish oil diets ........................................... 56 Table 4.2: DHA-containing PC displayed increased molecular order in the presence of lipid raft molecules relative to PE ............................................. 66 Table 5.1: Fatty acid analysis of B cells ............................................................. 93 Supplemental Table 2.1: Fatty acid analysis of experimental diets ................... B-1 Supplemental Table 4.1: Analysis of major fatty acids in mouse diets .............. B-6 Supplemental Table 4.2: Cholesterol levels in DRM and DSM fractions from ex vivo and in vitro studies ........................................................................... B-9 LIST OF FIGURES PAGE Figure 1.1: PUFA synthesis ............................................................................... 3 Figure 1.2: The B-T cell immunological synapse ............................................... 15 Figure 2.1: EPA and DHA treatment disrupted nonraft organization of EL4 cells .............................................................................................................. 28 Figure 2.2: EPA and DHA treatment increased EL4 cell size and growth and prevented cell death ..................................................................................... 31 Figure 2.3: EPA and DHA treatment had no effect on nonraft organization when cell size was unchanged .................................................................... 33 Figure 2.4: (n-3) PUFA diets did not modify nonraft organization, but the HF (n-3) PUFA diet disrupted lipid raft clustering ............................................... 37 Figure 3.1: HF n-3 PUFA diet lowered activity ................................................... 49 Figure 3.2: HF n-3 PUFA diet lowered VCO2 and RER .................................... 51 Figure 4.1: FO promoted uptake of EPA/DHA into DSMs and DHA into PC of DRMs ........................................................................................................... 63 Figure 4.2: FO increased the size of rafts induced by CTxB cross-linking ........ 68 Figure 4.3: FO increased B-cell membrane molecular upon cross-linking relative to no cross-linking ........................................................................... 70 Figure 4.4: EPA and DHA treatment targeted DRM and DSM composition of EL4 cells ...................................................................................................... 72 Figure 4.5: DHA, but not EPA, increased membrane molecular order upon cross-linking relative to no cross-linking in vitro ........................................... 75 Figure 4.6: FO increased B-cell CD69 expression and cytokine secretion upon activation with LPS .............................................................................. 78 PAGE Figure 4.7: FO suppressed B-cell stimulated IL-2 secretion from naive CD4 + T cells ........................................................................................................... 79 Figure 5.1: Fish oil diet has no effect on B cell MHC class II lateral organization prior to synapse formation ....................................................... 94 Figure 5.2: Cholesterol depletion and actin disruption reveal differential effects on MHC class II clustering ................................................................ 96 Figure 5.3: Localization of B and T cell molecules at the immunological synapse as a function of conjugation time ................................................... 98 Figure 5.4: Fish oil disrupts the B-T cell immunological synapse ...................... 100 Figure 5.5: Pharmacological agents disrupt the B-T cell immunological synapse ........................................................................................................ 103 Figure 5.6: Fish oil suppresses CD4+ T cell activation by targeting both B and T cells ........................................................................................................... 111 Figure 6.1: DHA disrupts lipid raft clustering and the immunological synapse .. 117 Supplemental Figure 2.1: C16-Bodipy imaging studies .................................... B-2 Supplemental Figure 2.2: MHC class I molecules were randomly distributed on EL4 cells ................................................................................................. B-3 Supplemental Figure 2.3: (n-3) PUFA diets did not increase FAST-DiI accumulation but HF (n-3) PUFA diet lowered C16-Bodipy uptake ............. B-4 Supplemental Figure 2.4: HF (n-3) PUFA diet disrupted lipid raft clustering ..... B-5 Supplemental Figure 4.1: FO increased spleen size ......................................... B-7 Supplemental Figure 4.2: Safflower oil-enriched diet did not increase lipid raft size ............................................................................................................... B-8 PAGE Supplemental Figure 4.3: FO had no impact on non-raft organization as measured by uptake of DiIC 18 ...................................................................... B-10 Supplemental Figure 4.4: EPA and DHA treatment had no effect on GM1 surface levels ............................................................................................... B-11 Supplemental Figure 5.1: Fish oil disrupts the B-T cell immunological synapse ........................................................................................................ B-12 Supplemental Figure 5.2: Pharmacological agents disrupt the B-T cell immunological synapse ................................................................................ B-13 LIST OF ABBREVIATIONS AA Arachidonic acid ALA ?-Linolenic acid APC Antigen presenting cell BSA Bovine serum albumin c-SMAC Central supramolecular activation cluster C16-Bodipy 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- hexadecanoic acid CD Control diet CTx-B cholera toxin subunit B DC Dendritic cell DHA Docosahexaenoic acid DiIC 18 1,1’-dioctadecyl-3,3,3?,3?-tetramthylindocarbocyanine perchlorate DRM Detergent-resistant membrane DSM Detergent-soluble membrane E Energy transfer ELA Elaidic acid EPA Eicosapentaenoic acid FAST-DiI 1,1?-Dilinoleyl-3,3,3?,3?-tetramethylindocarbocyanine perchlorate FFA Free fatty acid FO Fish oil fPALM Fluorescence Photoactivation Localization Microscopy FRET Förster resonance energy transfer GC Gas chromatography GP Generalized polarization GPI Glycosylphosphatidylinositol GPR G-protein coupled receptor HF High-fat I Intensity IFN Interferon IL Interleukin LF Low-fat LPS Lipopolysaccharide MHC Major histocompatibility complex M?CD Methyl-beta-cyclodextrin OA Oleic acid OVA 323–339 Chicken ovalbumin 323–339 p-SMAC Peripheral supramolecular activation cluster PC Phosphatidylcholine PE Phosphatidylethanolamine PPAR Proliferator-activated receptor PUFA Polyunsaturated fatty acid RER Respiratory exchange ratio SFA Saturated fatty acid SM Sphingomyelin SMAC Supramolecular activation cluster STED Stimulated emission depletion STORM Stochastic optical reconstruction microscopy TCR T cell receptor TG Triglycerides Th1 T helper 1 Th2 T helper 2 TIRF Total internal reflection fluorescence TLR Toll-like receptor TNF Tumor necrosis factor VCO 2 Maximal CO 2 consumption VO 2 Maximal O 2 consumption CHAPTER 1: INTRODUCTION Overview ! n-3 polyunsaturated fatty acids (PUFAs), also known as omega 3 fatty acids, are important bioactive molecules with many potential health benefits (1). In particular, consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the major n-3 PUFA bioactive components of fish oil, have been established as regulators of immune function (2). However, a major roadblock to widespread clinical utility of fish oil is the lack of clear understanding of the mechanisms for its beneficial effects. This dissertation addresses that limitation by investigating how incorporation of EPA and DHA into the plasma membrane modifies B cell function. Historical perspective ! Research on n-3 PUFAs originated in the 1970s, when scientists began studying Greenland’s Inuit populations due to their incredibly low incidence of ischemic heart disease compared to the rest of the world (3). What they discovered was the Inuit population consumed a unique diet that led to decreased plasma lipid concentrations (4). Unlike most industrialized civilizations, this diet consisted of very high levels of n-3 PUFAs from fish and other seafoods. The dietary n-3 PUFA levels amounted to around 7% of total daily energy (5, 6). Some modern Japanese populations also have elevated n-3 PUFA intakes, especially when compared to the western diet, though not to the degree of the Greenland Inuits. The Japanese consume roughly 1-2% of daily energy from n-3 PUFA, which also correlates to moderate decrease in total mortality (7, 8). ! Not until the 1990s and the early 2000s did increased awareness of the additional possible health benefits of consuming n-3 PUFAs become widespread. n-3 PUFAs have been shown to be beneficial in a number of conditions such as cardiovascular disease (9-13), cancer (14-16), various inflammatory disorders (17-20), and cognitive health (21-23). A link between many of these conditions is the immunosuppressive properties of n-3 PUFAs (24). Therefore, this dissertation is focused on unveiling a mechanism for how fish oil suppresses immune cell function. Biochemistry of n-3 PUFAs ! The unique structure of n-3 PUFAs is distinguished by multiple double bonds starting 3 carbons from the methyl end of the acyl chain. Similarly, n-6 PUFAs, a structurally related group of fatty acids, are distinguished by multiple double bonds starting 6 carbons from the methyl end. Animal tissues, unlike plants, are unable to synthesize either group of these fatty acids due to an inability to insert double bonds further than 9 carbons from the carboxyl end of the acyl chain (25). Once consumed through the diet, ?-linolenic acid (ALA), 18:3 (n-3), or linoleic acid, 18:2 (n-6), can be converted into the longer chain PUFAs through a series of elongation and desaturation reactions (Figure 1.1). Retroconversion or ?-oxidation of DHA can also generate EPA and docosapentaenoic acid (26). However, since ALA is at the top of this pathway, this makes ALA and linoleic acid essential dietary fatty acids. There are two major metabolic fates for ALA when consumed in the diet: 1) ?-oxidation for use as a fuel source, 2) conversion into longer chain n-3 PUFAs for a variety of uses. Some studies suggest that most is used for ?-oxidation (27, 28). In addition, the conversion of ALA to longer-chain n-3 PUFA is very inefficient, although the rate is slightly higher in pre-menopausal women (29). For instance, studies of plasma lipid levels in healthy individuals found that only 0.2% of radio-labeled ALA was able to be converted into EPA, whereas 63% of 2 3Figure 1.1: PUFA synthesis 18:2 (n-6) Linoleic acid 18:3 (n-6) ?-Linolenic acid 20:4 (n-6) Arachidonic acid (AA) 22:5 (n-6) Docosapenaenoic acid ?6 desaturase elongase ?5 desaturase 20:3 (n-6) Dihomo-?-linolenic acid elongase 22:4 (n-6) Adrenic acid 24:4 (n-6) Tetracosatetraenoic acid 24:5 (n-6) Tetracosapentaenoic acid elongase ?6 desaturase ?-oxidation 18:3 (n-3) ?-Linolenic acid (ALA) 18:4 (n-3) Stearidonic acid 20:5 (n-3) Eicosapentaenoic acid (EPA) 22:6 (n-3) Docosahexaenoic acid (DHA) 20:4 (n-3) Eicosatetraenoic acid 22:5 (n-3) Docosapentaenoic acid 24:5 (n-3) Tetracosapentaenoic acid 24:6 (n-3) Tetracosahexaenoic acid n-3 PUFAsn-6 PUFAs Figure 1.1: Polyunsaturated fatty acid (PUFA) synthesis. n-6 PUFA (left) and n-3 PUFA (right) elongation pathways. Each step in either pathway uses the same enzymes. EPA was able to be converted into DHA (30). This finding suggested that direct dietary consumption of EPA/DHA may be more beneficial for studying the effects of EPA/DHA than consumption of ALA alone. On the other hand, additional evidence suggests this limitation could be tissue dependent (31). ! An additional limitation of longer chain n-3 PUFA synthesis from ALA is the competition between the synthesis pathways of n-3 versus n-6 PUFAs. This competition is due to the fact that they share the elongase and desaturase enzymes for this process (32-34). Furthermore, animals are not able to convert an n-3 PUFA into an n-6 PUFA or vice versa. However, the C. elegans gene, fat-1, was identified for this process (35). Consequently, this discovery led to development of a transgenic mouse line where the fat-1 gene was expressed, resulting in abundant n-3 PUFA and reduced n-6 PUFA levels, without a dietary n-3 PUFA supply (36). Recommended intake of n-3 PUFAs ! According to the American Heart Association, World Health Organization, and European Food Safety Agency, n-3 PUFAs should be added to the daily diet for good overall health and to prevent many diseases (37). The daily dietary recommended level of n-3 PUFAs has continually evolved, increasing as more information about their beneficial effects is gathered. Currently these organizations recommend healthy individuals consume on average 1 - 2.5 grams per day of total n-3 PUFA, including 200 to 600 mg per day of EPA/DHA (37-40). Due to the limited conversion rate described above, and the evidence of the necessity of DHA for overall health, there has been a push to recognize DHA in addition to ALA as an essential dietary fatty acid (41, 42). These recommended levels can be achieved through a minimum of 2 servings of fish 4 per week, with one being oily fish (tuna, mackerel, salmon, or sardine). Consumption of fish does not come without some risks. Some contaminants in fish are methylmercury, dioxins, polychlorinated biphenyls, and organic pollutants from industrial processes. Advancement in dietary enrichment of milk, eggs, meat and other products with n-3 PUFAs provides many new alternatives to fish consumption alone (43). In fact, market research by Packaged Facts predicts the n-3 PUFA food and beverage market, excluding fish, will become a $7 billion commercial industry by 2015 (44). ! As supplements or food additives, n-3 PUFAs exist as two major forms, triglycerides or ethyl esters. The n-3 PUFAs present in fish or other oils are mostly in the triglyceride form. The triglyceride form seems to show increased efficiency in being absorbed through dietary intake compared to the ethyl ester form (45, 46). In both forms, the fatty acids are absorbed in the small intestine, cleaved by lipases, and reassembled into triglycerides. However, in the ethyl ester form, absorption and re- synthesis is delayed, perhaps making this form less effective as a clinical treatment (47, 48). Ethyl ester n-3 PUFAs can be re-esterified into a glycerol backbone during manufacturing, but this is usually not done because of the added costs. However, all forms seem to have similar abilities in treating hypertriglyceridemia (49, 50). ! Purified fish oil as ethyl esters has been clinically developed to lower elevated triglyceride levels with a drug called Lovaza. This is the only use for n-3 PUFAs that is clinically approved in the United States by the FDA, although the mechanism of action is still unclear. Four grams per day of EPA and DHA is prescribed for severe hypertriglyceridemia (?500 mg/dL). This treatment represents approximately 465 mg of EPA and 375 mg of DHA per 1 g capsule of ethyl ester fatty acids. Lovaza is thought to 5 work by reducing triglyceride production and liver secretion, in addition to removing excess triglycerides from the blood (51). Both the types of n-3 PUFA and the dose used in many other therapeutic applications have varied widely, making it very difficult to make specific clinical recommendations for further applications (52). Fully elucidating the mechanisms remains a major theme of n-3 PUFA research. Specifically, this dissertation addresses that limitation by investigating how a specially formulated fish oil diet disrupts plasma membrane lateral organization and immunological function of B cells. n-3 PUFAs and the immune system ! n-3 PUFAs have been extensively studied with regards to targeting immune cell function (53). Although there are many differential findings, common themes are that n-3 PUFAs, specifically EPA/DHA, are immune system regulators. They assist in resolution of inflammation and act as immunosuppressants by regulating immune cell function (2, 54, 55). The immune system, however, is layered in complexity, and n-3 PUFAs are thought to play a role in modifying many of its components. Immune cells are produced in bone marrow from multipotent progenitor cells, or hematopoietic stem cells, and migrate to the thymus, spleen, and lymph nodes, as well as many other organs. Multipotent hematopoietic stem cells further differentiate into either lymphoid progenitor or myeloid progenitor cells (56). Lymphoid progenitor cells will become B and T lymphocytes or natural killer cells (56). Myeloid progenitor cells develop to become monocytes, macrophages, basophils, neutrophils, erythrocytes, mast cells, and dendritic cells (56). These cell types make up the major cells of the immune system. The immune system can be broken down into two primary types of immune responses, which are the 6 innate response and the adaptive response. ! Innate function is evolutionarily conserved and is the first line of defense against invading pathogens and other infectious agents. Inflammation plays a critical role in this process as a protective response to an injury or infection. The vascular and immune systems work together to respond to invasion of pathogens, irritants, or damaged cells by increasing blood flow and cell permeability to the affected area (57). Inflammation can be characterized as either acute or chronic. Resolving acute inflammation promptly is critical for overall health, as chronic inflammation is associated with a variety of diseases such as atherosclerosis, rheumatoid arthritis, metabolic syndrome, and some types of cancer (58). Cytokine release by damaged cells recruits immune cells. Phagocytic immune cells, such as macrophages, dendritic cells, and neutrophils, are recruited to the area to engulf and eliminate pathogens that could cause further infection. Some pathogens are recognized by pattern recognition receptors such as the Toll-like receptors (TLRs) (59). Phagocytized foreign pathogens are degraded, processed and various proteins are presented to cells of the adaptive immune system. This is a critical link between the two types of immune responses (60). ! The adaptive immune response can also be broken down into two parts: humoral and cellular. Humoral immunity is mediated primarily by B cell antibody secretion. Cell- mediated immunity involves T cells activated by antigen presenting cells (APCs). The primary APCs are dendritic cells, but B cells and macrophages can also serve this function. Cell-mediated immunity involves direct contact through immunological synapses, described in detail below. Unlike the innate immune system, the adaptive immune system is designed to protect against many foreign contagions, and not just 7 specific evolutionarily conserved patterns. However, the two types of immune responses often work together to clear pathogens and maintain health. ! A number of studies have shown that n-3 PUFAs can be immunosuppressive by inhibiting proliferation of lymphocytes in both rodents and humans (24, 61-64). However, in some studies with human patients, conjugated linoleic acid was found to have limited results (65, 66). There is also evidence that cytokine secretion can be inhibited by n-3 PUFA dietary supplementation. For example, secretion of pro-inflammatory cytokines IL (interleukin)-6 and TNF (Tumor necrosis factor)-? by macrophages was inhibited in rats (67). In dendritic cells, TNF-? and IL-12 cytokine secretion was diminished by n-3 PUFAs (68). n-3 PUFAs have also been shown to suppress APCs ability to present antigen (69, 70). In addition, they have been shown to inhibit CD4 + T cell signaling and function (71, 72). Other studies suggest that the response between cell types may not be universally suppressive (73). However, B cells are a cell type poorly represented in the field of n-3 PUFA research. Therefore, a major focus of this work is on how n-3 PUFAs modify B cell function. B cells ! B cells are continually generated throughout the lifespan of most mammals. In the fetus, B cells differentiate in the liver. After birth, B cell generation moves to the bone marrow where they must undergo a differentiation and maturation process before becoming functioning members of the immune system (74). B cells leave the bone marrow and migrate through the periphery and into the lymphoid tissues of the lymph nodes and spleen. Currently there are ten known B cell–specific cell surface markers that have been identified: CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, and 8 CD79a,b (75). The two major mature B cell subtypes are B-1 and B-2. B-1 cells are self- renewing and long-lived. They are crucial for innate immune function in defense against encapsulated bacterial infections (76, 77). B-1 cells produce natural antibodies, which are secreted before an infection occurs. B-2 cells are the major adaptive immune response B cell type. There are two major splenic subpopulations, marginal zone and follicular cells. Marginal zone B cells mature from transitional type 1 and transitional type 2 precursors (78, 79) Marginal zone B cells are non-circulating and are also critical for defense against blood-borne encapsulated bacterial pathogens (79). Upon encountering an antigen, naďve mature follicular B cells process and present that antigen to cognate helper T cells in the periphery of a lymphoid follicle. The activated B cells become short-lived antibody secreting plasma cells, or migrate to a germinal center to rapidly proliferate and differentiate. In the germinal center, antibody affinity is refined through a process known as affinity maturation (80). The differentiated B cells then become long-lasting memory B cells that can rapidly proliferate and differentiate into plasma cells upon a second encounter with the same antigen (81). ! While the primary role of B cells is antibody production, they are also recognized as important APCs for the initiation of immune responses (82-85). B cell MHC class II antigen presentation is critical for many autoimmune diseases like rheumatoid arthritis, lupus, allograft rejection, and chronic allergic lung disease (86-89). Suppression of B cell antigen presentation is a potential target for treatment of these disorders. For example, rituximab, a chimeric monoclonal antibody against CD20, suppresses B cell antigen presentation, which has some clinical benefits, especially in rheumatoid arthritis (90-93). B cells can also play a role in cytokine production, tumor immunity, cell 9 regulation, antiviral immunity, and wound healing (75, 94). Potential mechanisms for n-3 PUFAs ! Consumption of n-3 PUFAs can affect nearly every cell in the body. In doing so, there are a variety of mechanisms that n-3 PUFAs can target. They can directly bind to receptors and modify gene regulation. For instance, n-3 PUFAs have been shown to bind peroxisome proliferator-activated receptors (PPARs) and liver x receptors (95, 96). In addition, there has been a recent discovery of a specific n-3 PUFA receptor, the G- protein coupled receptor (GPR) 120 (97, 98). These studies with knockout mice demonstrated that GPR120 signaling plays a major role in obesity, inflammation, insulin resistance, and other symptoms of metabolic syndrome. ! n-3 and n-6 PUFAs also serve as precursors for pro- and anti-inflammatory metabolites. The discovery of these metabolites has established that the resolution of inflammation is not a passive process, but requires a variety of metabolites to activate inflammatory pathways, followed by further synthesis of pro-resolving mediators to resolve the inflammation once the process is complete (99-101). These pro- inflammatory molecules are primarily the n-6 PUFA arachidonic acid (AA) derived eicosanoids, made up of leukotrienes, prostaglandins, thromboxanes, and prostacyclins. However, EPA competes for enzyme activity with AA, limiting the AA production pathway and promoting alternative EPA derived eicosanoid production (102). The eicosanoids derived from EPA have weaker pro-inflammatory bioactivity than AA derived products (103). EPA and DHA also serve as precursors for the potent inflammation-resolving molecules known as E or D series resolvins, protectins/ neuroprotectins and, more recently, maresins. (104). After an inflammatory response, 10 these resolvins/protectins promote resolution of inflammation through removal of leukocytes (104). ! n-3 PUFAs also have unique structural properties. They can be esterified at the sn-2 position of phospholipids. As a phospholipid constituent, it has been shown that DHA can cause significant alteration to fluidity and permeability, as well as surrounding membrane protein function (105). Molecular dynamic simulations have shown that DHA acyl chains have an unusually high degree of conformational flexibility in membranes compared to other acyl chains (106). It is therefore proposed that DHA modifies plasma membrane order by disrupting hydrogen bonding between surrounding acyl chains and cholesterol (105). This results in an incompatibility between the rigid structure of cholesterol and DHA. In addition, DHA is thought to form DHA rich domains that are segregated from cholesterol rich membrane domains (107). These cholesterol rich membrane domains, known as lipid rafts, have critical functions for many cellular processes. Defining how dietary n-3 PUFAs disrupt lateral organization of lipid rafts in B cells is one of the primary aims for this dissertation. Lipid rafts ! Our understanding of the complexities of the plasma membrane has advanced greatly since the establishment of the fluid mosaic model in the 1970‘s by Singer and Nicolson (108). We now have evidence for non-random associations of proteins and lipids in functional membrane domains of various sizes and functions (109). Lipid rafts are membrane domains of recent importance (110). Highly ordered in nature, they are defined as sphingolipid and cholesterol-rich domains. They are heterogeneous in composition but small in size, roughly 10 - 200 nm in diameter (111). Functionally, lipid 11 rafts are thought to compartmentalize important cell signaling events by serving as platforms for protein clustering (110, 112, 113). ! Despite extensive characterization of lipid raft components and properties, there remains controversy associated with the existence of in vivo lipid raft domains (114, 115). Much of this controversy lies in the methods used to isolate or image these membrane domains (116-118). Many of the early studies relied on insolubility in mild detergent followed by density centrifugation, but this method is now highly debated because of the potential for artifacts (119). Furthermore, detergent methods homogenize lipid raft components and give no information about how they were distributed in an intact plasma membrane. ! Progress in fluorescence microscopy has allowed for limited visualization of these elusive domains by fluorescently tagging raft-associated proteins identified by the detergent extraction studies (120, 121). One such raft constituent, the ganglioside GM-1, binds the non-lethal subunit of cholera toxin (CTx-B) and is commonly used for imaging lipid rafts (122). This protein is readily available as a kit, with a variety of fluorescent proteins. The CTx-B proteins are then cross-linked by antibodies to aggregate larger lipid raft clusters that are now visible on the micron scale. However, a limitation to this application is that the CTx-B/antibody kit induces raft formation, and the natural in vivo state may not be represented by this method. Other studies use quantitative microscopy techniques such as Förster resonance energy transfer (FRET) to obtain nanoscale spatial information about components of rafts, and attempt to visualize these domains with minimal disruption. FRET can detect changes in protein distribution less than 10 nm apart, the size scale in which lipid raft protein clustering 12 occurs. FRET measures non-radiative energy transfer from a donor fluorophore on one protein to that of an acceptor on another closeby protein (117). One such study found evidence for highly dynamic nanoclusters of GPI-linked proteins using FRET (123). Further advances in super resolution microscopy like stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM), and fluorescence photoactivation localization microscopy (fPALM) have revealed dynamic nanoscale lipodomains in living cells (124). Recently, advanced single-molecule fluorescent tracking methods were used to study the lateral organization of glycosylphosphatidylinositol-anchored (GPI) proteins in rafts (125). Suzuki et al. found homodimer formation of GPI proteins may be a critical mechanism for raft assembly (125). Further advances in studying lipid rafts will enhance understanding of numerous cellular processes because of the diversity of signaling pathways that utilize lipid rafts. ! As mentioned above, DHA has a low affinity for cholesterol due to steric incompatibility (126). This property makes DHA, and likewise other n-3 PUFAs, a potential disrupter of lipid rafts. Subsequently it has been shown that n-3 PUFAs do affect cell signaling by altering lipid raft domains (127-129). However, most n-3 PUFAs actually end up in non-raft domains. Therefore, there are also implications to protein organization in non-raft domains (130). This dissertation further investigates the mechanism of how dietary n-3 PUFAs disrupt plasma membrane lateral organization in B cells. The immunological synapse ! In APCs such as B cells, direct cell-cell communication and subsequent activation can occur through contact between complementary regions of the plasma 13 membrane on B and T cells. This contact region is defined as the immunological synapse, and its formation is thought to be dependent on lipid rafts (131). APCs use major histocompatibility complex (MHC) proteins on the plasma membrane surface to present antigens to T cells. The antigen-loaded complex is recognized and bound by the T cell receptor (TCR). This contact, along with a host of other proteins, constitutes the immunological synapse. The immunological synapse can be further defined as a supramolecular activation cluster (SMAC), which consists of central, peripheral, and distal regions. The complex is actively assembled and matures over time (132). Initially, MHC class II, found on B cells, is bound to TCR and gradually clustered to the central SMAC (c-SMAC), along with other proteins such as protein kinase C? (PKC?), CD2, CD4, CD8, CD28, Lck, and Fyn (132). Cytoskeletal elements such as talin and actin surround this central region in the peripheral SMAC (p-SMAC), driving other proteins toward the c-SMAC (133). The functional anatomy of some of the elements involved in this contact region is depicted in Figure 1.2. ! At high antigen levels synapse formation initiates very quickly and maturation occurs over as much as 6 hours (134). First the APC and T cell scan the surface to find complementary TCR and MHC class II molecules, respectively. In order to check antigen specificity, T cells also undergo a form of kinetic proofreading, similar to mechanisms in DNA synthesis (135). During this process, phosphorylation events pause signal transduction after antigen recognition to discriminate self and foreign antigens (135). This greatly enhances the receptor's ability to discriminate between a foreign antigen and self-antigens and ensures incorrect signals are not processed. When antigen levels are low or TCR affinity to antigen is low, many TCRs are triggered 14 15 Figure 1.2: The B-T cell immunological synapse. Shown in blue (top) is the B side of the immunological synapse. In green (bottom) is the T cell side. B proteins MHC class II and CD80, as well as T cell proteins TCR, CD28, and CD4 cluster in the cSMAC shown in yellow. Adhesion molecules, ICAM1 and LFA-1, cluster in the pSMAC, which surrounds the cSMAC. B cell T cell cSMAC MHC class II TCR pSMAC ICAM1 CD80 LFA-1 CD4CD28 ICAM1 LFA-1 pSMACcSMAC Figure 1.2 sequentially through a process called serial triggering to resolve the lower affinity (136). Once the antigen is recognized and verified, calcium signaling and synapse assembly begins immediately. After initial APC contact with a T cell, signaling can begin at as early as 45 seconds. However, full maturation and T cell activation requires SMAC formation (137). As the synapse matures over the course of 5-60 minutes, SMAC formation and segregation occurs. Some evidence shows that although dendritic cells are professional APCs, full synapse maturation does not occur in these cells (138). Finally, after a few hours when signaling is complete, the TCR internalizes and the synapse is dissolved. ! The immunological synapse is a potential target for n-3 PUFAs as a mechanism for their immunosuppressive properties (71, 139). Dietary n-3 PUFAs are known to suppress recruitment of PKC?, as well as other structural proteins, to the site of the immunological synapse in CD4 + T cells by increasing lipid raft formation (140). Dietary n-3 PUFAs also limit mitochondrial translocation to the immunological synapse in T cells, an early step in T cell activation (141). Additionally, studies in other immune cell types yield similar lipid raft disruption and impaired function (142, 143). Much less is known, however, about B cells. B cells have a critical function in antibody production, but, like dendritic cells, they also are involved in activating T cells through the immunological synapse. Therefore, the goal of this dissertation is to describe how EPA and/or DHA can target lipid rafts in B cells, thereby disrupting antigen presentation. Specific aims ! n-3 PUFAs are the primary bioactive components of fish oil. Consumption of fish oil can elicit immunosuppressive effects, as well as many other potential health benefits. However, a limitation in the clinical utility of n-3 PUFAs is the lack of understanding of 16 how they exert their effects. To address this limitation, we investigated a potential mechanism for how n-3 PUFAs suppress B cell immune function. The central hypothesis was that n-3 PUFAs disrupt lipid rafts, as well as the direct contact of plasma membrane regions between B cells and T cells in the immunological synapse, thereby suppressing B cell function. This study serves to aid in potential treatment for chronic inflammatory and autoimmune disorders where immunosuppression of B cell antigen presentation by n-3 PUFAs would be beneficial (86-89). To address the central hypothesis, the specific aims are: Aim 1: Test the hypothesis that dietary n-3 PUFAs disrupt B cell lipid raft organization and function. ! Proteins important for B cell function localize to lipid rafts where they can cluster for signal transduction. Disruption of protein and lipid lateral organization in lipid rafts could lead to modification of B cell function. To investigate the hypothesis that dietary n-3 PUFAs disrupt lipid raft organization, we first identified how n-3 PUFAs incorporate into the plasma membrane. We then determined the types of phospholipids into which dietary n-3 PUFAs incorporate. Using various fluorescent probes, we determined how high and low fat n-3 PUFA diets modified raft and non-raft biophysical properties. Finally, we measured the effect of fish oil on micron and nanoscale clustering of lipid raft localized functional proteins vital to immunological synapse formation. Aim 2: Test the hypothesis that n-3 PUFA diet modified B cells diminish stable immunological synapse formation. ! The immunological synapse is structurally organized into cSMAC- and pSMAC- containing antigen receptors and adhesion molecules. Disruption of this structure in T 17 cells by n-3 PUFAs has been shown. Here we tested the functional consequences of dietary modifications on B cell plasma membranes by n-3 PUFAs. In doing so, we tested the hypothesis that B cells from n-3 PUFA fed mice would inhibit the formation of the immunological synapse, and therefore suppress T cell activation. We first measured the effect n-3 PUFAs have on the kinetics of initial immunological synapse or conjugate formation. We then directly imaged the immunological synapse using confocal microscopy to determine how n-3 PUFA dietary modification of B cells modified lateral organization of key T cell proteins. Finally, we determined the functional outcomes of dietary B cell modification on CD4 + T cell activation efficiency. ! Taken together, this study defines a mechanism through which n-3 PUFAs modify B cell function and regulate immunological synapse formation. In addition, we characterized various n-3 PUFA rich diets and some of the metabolic implications of consuming very high doses of these unique fatty acids. We also further established that relevant dietary doses of fish oil can modify lipid raft organization in B cells as well as immunological synapse formation. This study will aid in the clinical application of n-3 PUFAs as immunosuppressants. 18 CHAPTER 2: MEMBRANE RAFT ORGANIZATION IS MORE SENSITIVE TO DISRUPTION BY N-3 PUFA THAN NONRAFT ORGANIZATION IN EL4 AND B CELLS 1 Introduction ! EPA and DHA, the bioactive n-3 PUFA of fish oil, are increasingly available and consumed by the general public as over-the-counter supplements (43, 144). Clinically, EPA and DHA have applications for the prevention and/or treatment of some metabolic diseases (50, 145-147); in addition, they have potential utility for treating the symptoms associated with inflammatory and autoimmune disorders (2, 54, 148). However, one major limitation of further developing these fatty acids for clinical use is an incomplete understanding of their targets and molecular mechanisms. ! An emerging mechanism of the action of n-3 PUFA, due to their unique molecular structure, is modification of plasma membrane lipid rafts (149), which are sphingolipid- cholesterol enriched domains that compartmentalize signaling proteins (124). We recently discovered an n-3 PUFA disrupted lipid raft clustering of EL4 cells (150). The data raised a new question, i.e. could n-3 PUFA also disrupt the organization of nonraft domains. These membrane domains are broadly defined as those regions that are not enriched in sphingolipids and cholesterol that also compartmentalize specific proteins (e.g. MHC class I, TLR4, etc.) (151). There were 2 reasons to hypothesize n-3 PUFA would disrupt nonraft organization. First, experiments using model membranes demonstrated DHA acyl chains, due to their structural incompatibility with cholesterol, primarily incorporated into nonrafts to enhance nonraft formation (130, 152, 153). 1 This research was originally published in The Journal of Nutrition. Rockett, B. D., Franklin, A., Harris, M., Teague, H., Rockett, A., & Shaikh, S. R. Membrane raft organization is more sensitive to disruption by (n-3) PUFA than nonraft organization in EL4 and B Cells. J Nutr. 2011; 141:1041–1048. © the American Society for Nutrition. Second, biochemical detergent extraction studies showed a large fraction (up to 70%) of EPA and DHA localized into nonrafts (150, 154-156). Thus, these studies suggest that a major role of n-3 PUFA acyl chains is to modify nonraft domain organization. ! The first objective of this study was to extend our previous work by determining if EPA and DHA treatment disrupted nonraft organization of EL4 cells. The second objective was to translate the findings on EL4 cells by testing the impact of dietary n-3 PUFA on both nonraft and lipid raft organization in an animal model. To address our objectives, we relied on quantitative imaging methods of confocal and FRET microscopy. Application of these methods to the study of n-3 PUFA and membrane domains advances the field by overcoming the use of cold detergent extraction as a primary method of studying how n-3 PUFA modify membrane domains. Although detergent resistance has great predictive value, the detergent can induce artifacts (157-159). Furthermore, the biochemical detergent method does not report on the effects of n-3 PUFA on the appropriate length scales on which membrane domains form (124). Therefore, we used more appropriate imaging methods to address the effects of n-3 PUFA on membrane domain organization. Materials and Methods Cells ! EL4 cells were maintained in RPMI 1640–1× (Mediatech) with 10% heat- inactivated defined FBS (Hyclone), 2 mmol/L L-glutamine (Mediatech), and 1% penicillin/streptomycin (Mediatech) at 37°C in a 5% CO 2 incubator. The lipid composition of the FBS was as previously reported (150). Fatty acid treatment 20 ! A total of 9–10 × 10 5 EL4 cells was treated for 15.5 h with 25 µmol/L FFA (free fatty acid, Nu-Check Prep) complexed to fatty acid-free BSA (Bovine serum albumin, Roche Biochemicals) in serum-free medium as previously described (150). For select experiments, EL4 cells were treated in the presence of 10% FBS. The rationale for selecting the fatty acid concentration and time of treatment was to be consistent with our previous study on EL4 cells and membrane domains (150). Oleic (OA) and AA were tested to rule out general effects of fatty acid treatment and to ensure specificity of EPA and DHA. Mice, diets, and B cell isolation ! Male C57BL/6 mice (Charles River), aged 4–6 wk (~18 g), were fed the following diets (Harlan-Teklad) for 3 wk: a purified control diet (CD) (5% fat wt:wt), a n-6 PUFA diet (20% fat wt:wt), a low-fat (LF) n-3 PUFA diet (5% fat wt:wt), or a high fat (HF) n-3 PUFA diet (20% fat wt:wt) (Table 2.1) (160, 161). For the CD and LF n-3 PUFA diets, ~13% of the total energy was from fat. For the HF n-6 and n-3 PUFA diets, ~41% of the total energy was from fat. For the LF n-3 PUFA diet, 3.3% of the total energy was from ?-linolenic acid, 1% from EPA, and 0.6% from DHA (160). For the HF n-3 PUFA diet, 10.5% of the total energy was from ?-linolenic acid, 3% from EPA, and 2% from DHA (160). The diets were analyzed for their fatty acid composition (Supplemental Table 2.1). For B cell isolation, mice were killed using CO 2 inhalation after 3 wk of feeding. Naďve B220 + B cells (>90% purity) from splenocytes were purified with negative selection (Miltenyi Biotec) as previously described (160). All experiments with mice fulfilled guidelines established by the East Carolina University for euthanasia and humane treatment. 21 Table 2.1: Composition of experimental diets Ingredients (g/kg) CD HF (n-6) PUFA LF (n-3) PUFA HF (n-3) PUFA Casein 185.0 220.0 185.0 220.0 L-Cystine 2.5 3.0 2.5 3.0 Corn starch 370.0 173.9 370.0 173.9 Maltodextrin 140.0 140.0 140.0 140.0 Sucrose 150.0 150.0 150.0 150.0 Cellulose (fiber) 50.0 50.0 50.0 50.0 Flaxseed oil 0.0 0.0 23.1 92.5 Fish oil (Menhaden) 0.0 0.0 23.1 92.5 Soybean oil 50.0 30.0 3.75 15.0 Safflower oil 0.0 125.0 0.0 0.0 Hydrogenated coconut oil 0.0 45.0 0.0 0.0 Mineral mix, AIN-93M 2 35.0 42.0 35.0 42.0 Vitamin mix, AIN-93 2 15.0 18.0 15.0 18.0 Choline bitartate 2.5 3.0 2.5 3.0 TBHQ 0.02 0.06 0.02 0.06 C57BL/6 mice were fed for 3 weeks CD, HF (n-6) PUFA, LF and HF (n-3) PUFA diets. Composition of mineral and vitamin formulas are as reported in (161). 22 Fatty acid analysis ! For analysis of diets, ?0.01–0.05 g of the diet pellets was homogenized using a Dounce homogenizer prior to extraction. Total lipids were extracted and analyzed relative to standards (Restek) from the differing cell types or diets with gas chromatography (GC) using our previously published protocol (150, 160). Areas of identified peaks were summed and each peak area is expressed as the percentage of total peak area for a given treatment (150, 160). Column chromatography ! To assess incorporation of EPA and DHA into neutral lipid, FFA, and polar lipid fractions, 2 × 10 6 EL4 cells were treated with fatty acids spiked with 185 kBq [ 14 C]EPA or [ 14 C]DHA (American Radiolabeled Chemicals). Extracted lipids were separated into different lipid classes using aminopropyl beads (Sigma) loaded in a Pasteur pipette with a fiberglass plug and elution with HPLC-grade organic solvents (Fisher Scientific) as previously described (160, 162). Staining and imaging with 1,19-dilinoleyl-3,3,39,39-tetramethylin-docarbocyanine perchlorate and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- hexadecanoic acid ! A total of 2 × 10 6 cells was washed twice with cold PBS, stained with 0.66 mg/L of the nonraft probe 1,1?-dilinoleyl-3,3,3?,3?-tetramethylindocarbocyanine perchlorate (FAST-DiI; Invitrogen) or 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- hexadecanoic acid (C16-Bodipy; Invitrogen) for 5 min on ice followed by 2 washes with cold PBS (163, 164). Cells were fixed in 4% paraformaldehyde (Fisher Scientific) for 1 h, washed twice, placed in Vitrotubes (Fiber Optic Center), and mounted onto slides 23 with nail polish. All imaging studies, described below, relied on a Zeiss LSM510 confocal microscope using a 100× oil objective. Pinhole, detector gain, and laser settings were kept constant between samples for a given experiment. For some experiments, images were acquired as z-stacks. Sample preparation for FRET microscopy ! MHC class I molecules were used as the probe to measure nanometer scale molecular proximity, because these molecules are localized to nonrafts and are established to serve as excellent reporters for measuring changes in membrane organization (165-167). Cells were stained with varying concentrations of Cy3 (donor) and Cy5 (acceptor) separately labeled M1/42.3.9.8 anti-MHC class I antibodies (BioXCell). Fluorophores were conjugated to the antibody using a standard fluorophore conjugation kit (GE Healthcare). Specificity of the antibody was confirmed by using isotype controls (BioXCell) (150). Total antibody levels were held constant at 0.02 mg/ sample using unlabeled antibody as donor:acceptor ratios were varied from 1:1, 1:2, and 1:3 in 1 × 10 ?4 L PBS (168, 169). Cells were stained for 15 min on ice, washed twice with PBS, fixed, washed, and mounted to slides as described above. A positive control M1/42.3.9.8 antibody was generated by simultaneously conjugating Cy3 and Cy5 fluorophores, yielding a dual labeled antibody to ensure high FRET. Negative control samples were labeled with donor fluorophore only to check for donor channel bleaching during acceptor fluorophore bleaching and acceptor fluorophore only to optimize bleaching time. FRET microscopy ! FRET was measured in terms of the efficiency of energy transfer (E) from donor 24 (Cy3) to acceptor (Cy5) fluorophores separated by distance r, given by the following equation: E = 1/{1 + (r/Ro) 6 }, where Ro is the Förster radius of the donor and acceptor fluorophore pairs and is 54 Ĺ for Cy3/Cy5 (170, 171). The filter sets were 560- to 615- nm band pass for Cy3, and 650-nm long pass for Cy5 in 2 separate tracks. Excitation relied on 543- and 633-nm lasers, respectively, for Cy3 and Cy5. Eight-bit 512 × 512 pixel images were acquired. Acceptor photobleaching was carried out by iterative scanning of the 633-nm laser for ~2 min. Gain settings were optimized to ensure images were not saturated after photobleaching. Lipid raft staining ! B cells were labeled with cholera toxin subunit B-FITC (Invitrogen) for cross- linking lipid rafts and imaged as previously described (150). Image analysis ! All images were analyzed with NIH ImageJ. Approximately 20–33 cells per treatment per experiment were analyzed as previously described (150). For FAST-DiI and C16-Bodipy studies, differential interference contrast images were routinely used to confirm plasma membrane compared with intracellular staining. For image intensity analysis, images were background subtracted and manually thresholded for regions of interest either inside the cell or on the plasma membrane. The approach was confirmed by selecting several regions of interest manually drawn on the cell surface and inside the cell. Cells were also scored as high intensity if they were in the top one-third of the distribution of fluorescence intensities. ! FRET images were analyzed with FRETcalc v3.0 plug-in for NIH ImageJ (172). Some cells could not be properly registered and were not included in the analysis. 25 Images were registered by manual translation or rotation. All images were background subtracted and smoothed using a 3 × 3 filter. The threshold values for the donor and acceptor images were determined manually. This was required to calculate FRET efficiency of the entire cell by the FRETcalc plug-in (172). ! Images of lipid rafts were background subtracted and raft size was determined in terms of Feret diameter using NIH ImageJ as previously described (150). Cell growth and apoptosis measurements ! Cell growth was determined by counting cells in duplicate or triplicate using a hemacytometer. Dead cells were excluded with Trypan blue (HyClone, Fisher Scientific) staining. Measurements were routinely confirmed by a second person using blinded samples. We verified that this approach gave the same results as a cell proliferation testing kit (GenScript). The advantage of counting cells over the kit was the absolute number of cells could be determined rather than relative changes. Cell survival was measured in terms of Annexin V-Cy5/Sytox Blue (BD Pharmingen) staining with a BD LSR II flow cytometer as previously described (160). Statistical analysis ! Reported values are means ± SEM from several independent experiments. For animal studies, independent experiments were conducted using 1 mouse from each diet group. All statistical analyses were conducted using Excel and GraphPad Prism (GraphPad Software). Parametric statistics were used, because the data were normally distributed. Unequal variances were tested for prior to ANOVA using a Levene’s test. For FRET studies, efficiency values varied between experiments due to variation in the fluorophore to antibody ratios; thus, significance was established against the control 26 using repeated-measures 1-way ANOVA followed by a Dunnett’s t test. For cell growth and apoptosis measurements as a function of time, 2-way ANOVA analysis was used followed by a Bonferroni t test. The 2-way ANOVA used treatment and time as factors and there was no interaction effect. For all other studies, significance was established against the control using a 1-way ANOVA followed by a Dunnett’s t test. P-values < 0.05 were considered significant. Results EPA and DHA treatment disrupted nonraft organization of EL4 cells ! EPA and DHA treatment of EL4 cells increased accumulation of the nonraft probe FAST-DiI in the plasma membrane by ~50–70% relative to the BSA control (Figure 2.1A,B). The OA and AA treatments did not significantly increase FAST-DiI uptake compared with BSA (Figure 2.1B). The intensity of FAST-DiI inside of the cell was not changed by treatment with any of the fatty acids (Figure 2.1B). The increase in FAST-DiI plasma membrane binding with EPA and DHA was also confirmed by scoring the cells for intensity. EPA and DHA treatment resulted in the largest percentage of cells with a high intensity of plasma membrane staining compared with BSA (Figure 2.1C). ! A major concern was that the EPA and DHA treatment exerted a general effect on fluorescent probe uptake. Therefore, we tested the effects of fatty acid treatment on the uptake of a nonspecific probe, C16-Bodipy (173). C16-Bodipy did not merely report on the same subcellular organization as FAST-DiI. Colocalization analysis of z-stacks of EL4 cells costained with FAST-DiI and C16-Bodipy showed the percent colocalization between the 2 probes was ~32%, as measured by Mander’s coefficients (Supplemental Figure 2.1A) (150). Relative to BSA, EPA and DHA treatment did not promote uptake of 27 28 ),*85( $ % & %6$ 2$ $$ (3$ '+$ (&\' &\ %H IR UH $I WH U Figure 2.1 29 Figure 2.1: EPA and DHA treatment disrupted nonraft organization of EL4 cells. (A) Fluorescence images of EL4 cells treated with BSA, OA, AA, EPA, or DHA and stained with FAST-DiI. (B) FAST-DiI intracellular and plasma membrane image intensity. (C) Percentage of cells with high FAST-DiI intensity as a function of treatment. (D) Sample images of BSA-treated cells from acceptor photobleaching FRET. (E) FRET efficiency values for treated EL4 cells. Images are on a rainbow palette to discriminate differences in relative fluorescence intensity. Red and blue values indicate high and low intensity, respectively. Data are means ± SEM, n = 3–4. Data in E are from 60-90 total cells analyzed. Asterisks indicate different from BSA: *P < 0.05, **P < 0.01, ***P < 0.001. Data in A-B were collected by Franklin, A. C16-Bodipy into the plasma membrane or inside the cell (Supplemental Figure 2.1B). OA and AA treatments also had no effect. ! We also determined if EPA and DHA treatment modified nonraft organization on a nanometer scale using FRET microscopy. Positive control experiments showed the acceptor photobleaching approach with Cy3 and Cy5 antibodies increased FRET (Supplemental Figure 2.2A). The distribution of MHC class I molecules on the surface of EL4 cells was then determined as random, clustered, or a mixture of random and clustered by increasing the donor:acceptor fluorophore ratios. FRET efficiency did not increase with increasing donor:acceptor ratios (Supplemental Figure 2.2B), which is the signature pattern of a random distribution of molecules (168, 169). This allowed us to combine FRET acceptor:donor ratios in the subsequent analyses. ! Analysis of FRET efficiency values from FRET images (Figure 2.1D) in every single experiment showed EPA and DHA treatment consistently decreased FRET by 25–30% relative to the BSA control (Figure 2.1E). OA and AA treatments did not lower FRET relative to BSA. The most plausible explanation for an increase in the distance between neighboring MHC I molecules with n-3 PUFA was an increase in cell size, which was investigated next. EPA and DHA treatment increased cell size and growth ! Cell size was measured in terms of forward scatter with flow cytometry (Figure 2.2A). EPA and DHA treatment increased cell size by ~26% relative to BSA after 15.5 h of treatment. OA and AA treatment had no effect. A similar trend was confirmed with microscopy measurements (data not shown). EPA and DHA, in addition to AA, treatment also increased side scatter relative to BSA (data not shown). 30 31 ),*85( $ % & ' Figure 2.2 Figure 2.2: EPA and DHA treatment increased EL4 cell size and growth and prevented cell death. (A) Median forward scatter values for EL4 cells treated with BSA, OA, AA, EPA, or DHA. (B) Cell growth, (C) early apoptosis, and (D) late apoptosis as a function of time for the different treatment groups. Data are means 6 SEM, n = 4. Asterisks indicate different from BSA: *P < 0.05, **P < 0.01, ***P < 0.001. ! The subsequent experiment tested if the increase in cell size related to a change in cell growth (Figure 2.2B). At 15.5 h of treatment, cell number between the BSA control and the differing fatty acids did not differ. After 24 h, EPA and DHA treatment increased the number of viable cells. In contrast, OA and AA treatment did not increase cell growth compared with BSA. We then tested if the increased ability to grow was driven by an ability to prevent cell death. At 15.5 h of treatment, the cells were equally viable when measured for early and late apoptosis (Figure 2.2C,D) (150). After 24 h of treatment, all 3 PUFA (AA, EPA, and DHA) prevented early and late apoptosis relative to the BSA control (Figure 2.2C,D). OA treatment had no effect relative to BSA. EPA and DHA treatment did not modify nonraft organization when cell size was unchanged. ! We determined if preventing the expansion of cell size prevented changes in nonraft organization. Treatment conditions were first optimized to prevent an increase in cell size but still allowed for efficient uptake of the fatty acids. Cells were treated in the presence of 10% FBS, which significantly elevated EPA and DHA levels (Figure 2.3A). Uptake of EPA and DHA into polar lipids did not change relative to treatment in serum free conditions, as measured with radiolabeled fatty acids (Figure 2.3B). Forward scatter values did not differ between EPA and DHA treatment compared with the BSA control (data not shown). Image analysis of cells stained with FAST-DiI (Figure 2.3C) showed no change in fluorescence intensity with EPA- or DHA-treated cells relative to BSA. Similarly, subsequent FRET imaging showed treatment of EL4 cells with n-3 PUFA did not lower FRET relative to BSA. FRET efficiency values were ~10% for BSA-, EPA-, and DHA-treated cells. 32 33 ),*85( % $ & Figure 2.3 34 Figure 2.3: EPA and DHA treatment had no effect on nonraft organization when cell size was unchanged. (A) Total levels of 20:4, 20:5, 22:5, and 22:6 upon treatment of EL4 cells with BSA, EPA, or DHA in serum-containing medium. (B) Incorporation of radiolabeled EPA and DHA into neutral lipids, FFA, and polar lipid fractions in serum- free and serum-containing medium. (C) FAST-DiI intensity of EL4 cells treated with BSA, EPA, or DHA in serum-containing medium. Data are means ± SEM, n = 3. Asterisks indicate different from BSA: ***P < 0.001. Data in B were collected by Shaikh, S. R. and Harris, M. In vivo administration of n-3 PUFA did not modify nonraft organization but disrupted lipid raft clustering ! Body weights of C57BL/6 mice fed the HF (n-6), LF n-3, and HF n-3 PUFA diets did not differ from the CD group after 3 wk (~22 ± 0.3 g). Energy intakes also did not differ between these groups and the CD group (11.4 ± 0.1 kcal/d). GC measurements confirmed uptake of fatty acids from the diet into B cells (Table 2.2). Cell size did not differ between the B cells from the CD group and those from the other groups (data not shown). ! Relative to CD, the HF (n-6), LF n-3, and HF n-3 PUFA diets did not increase FAST-DiI uptake into B cells (Figure 2.4A). Unexpectedly, the HF n-3 PUFA diet decreased plasma membrane and intracellular uptake of C16-Bodipy by ~33% (Figure 2.4B). The LF n-3 and HF n-6 PUFA diets did not affect C16-Bodipy binding. Sample images of FAST-DiI and C16-Bodipy staining are presented in Supplemental Figure 2.3. To verify the conclusion that n-3 PUFA did not disrupt nonrafts, MHC I FRET experiments were conducted with the HF n-3 PUFA diet, because it lowered C16-Bodipy uptake. FRET imaging showed nearly identical FRET efficiency values (~12%) for B cells isolated from the CD and HF n-3 PUFA diet-fed mice. ! Finally, we investigated if the HF n-3 PUFA diet disrupted B cell lipid raft organization. Microscopy images showed the HF n-3 PUFA diet decreased the clustering of lipid rafts (Supplemental Figure 2.4) and increased the Feret diameter of the domains by ~40% (Figure 2.4C) compared with CD. In contrast, the HF n-6 and LF n-3 PUFA diets, relative to CD, did not affect lipid raft organization. ! In this study, we addressed if n-3 PUFA disrupted nonraft organization of EL4 35 Table 2.2: Fatty acid analysis of B cells isolated from mice fed differing diets Fatty Acid CD HF (n-6) PUFA LF (n-3) PUFA HF (n-3) PUFA 14:0 0.9 ± 0.2 0.7 ± 0.1 0.6 ± 0.2 0.8 ± 0.1 16:0 24.9 ± 1.7 23.1 ± 2.0 25.8 ± 0.8 24.6 ± 0.5 16:1 0.8 ± 0.2 0.5 ± 0.1 1.0 ± 0.2 1.1 ± 0.1 18:0 16.5 ± 1.2 18.5 ± 2.3 18.5 ± 4.0 19.4 ± 2.8 18:1 trans 0.7 ± 0.2 0.9 ± 0.6 0.9 ± 0.4 0.5 ± 0.2 18:1 cis 14.2 ± 2.4 9.7 ± 0.6 13.2 ± 0.6 12.4 ± 0.6 18:1 (n-7) 1.9 ± 0.5 0.8 ± 0.2 1.7 ± 0.1 1.7 ± 0.1 18:2 (n-6) 17.6 ± 1.6 16.5 ± 1.3 17.1 ± 2.0 18.5 ± 1.0 18:3 (n-6) 0.5 ± 0.3 1.7 ± 0.7 3.6 ± 0.8** 1.6 ± 0.6 18:3 (n-3) 1.6 ± 0.2 1.1 ± 0.1 1.7 ± 0.2 2.3 ± 0.3 20:1 1.1 ± 0.4 1.4 ± 0.4 0.4 ± 0.1 0.7 ± 0.2 20:2 (n-6) 1.4 ± 0.7 1.6 ± 0.7 0.5 ± 0.3 0.7 ± 0.5 20:3 (n-6) 3.5 ± 1.6 0.5 ± 0.2 0.9 ± 0.3 1.0 ± 0.3 20:3 (n-3) 1.1 ± 0.5 1.4 ± 0.2 0.7 ± 0.2 0.8 ± 0.3 20:4 (n-6) 5.1 ± 1.6 3.5 ± 2.0 3.2 ± 0.4 2.4 ± 0.7 20:5 (n-3) 0.2 ± 0.0 0.4 ± 0.1 2.0 ± 0.2* 3.8 ± 0.6*** 22:2 (n-6) 1.2 ± 0.4 0.4 ± 0.2 0.1 ± 0.0* 0.4 ± 0.1 22:4 (n-6) 1.2 ± 0.2 1.1 ± 0.4 0.5 ± 0.0* 0.5 ± 0.1* 22:5 (n-6) 0.3 ± 0.1 2.3 ± 1.7 0.3 ± 0.1 0.4 ± 0.1 22:5 (n-3) 1.0 ± 0.4 1.2 ± 0.1 2.7 ± 0.7 2.4 ± 0.4 22:6 (n-3) 1.7 ± 0.4 1.5 ± 0.3 3.0 ± 0.4 3.5 ± 0.6* 24:1 0.9 ± 0.2 0.2 ± 0.1* 0.4 ± 0.0 0.4 ± 0.1 ?SFA 44.6 ± 2.3 46.2 ± 5.2 45.6± 4.5 45.8 ± 2.4 ?MUFA 19.8 ± 3.2 13.6 ± 1.3 17.8 ± 0.6 16.9 ± 0.5 ?PUFA (n-6) 30.6 ± 2.5 26.3 ± 3.9 26.5 ± 3.1 25.6 ± 2.0 ?PUFA (n-3) 5.0 ± 0.7 4.3 ± 0.7 10.1 ± 0.9** 11.7 ± 0.8*** ?PUFA 35.6 ± 3.0 30.7 ± 4.6 36.6 ± 4.0 37.3 ± 2.0 n-6/n-3 6.6 ± 1.0 6.1 ± 0.2 2.6 ± 0.1** 2.2 ± 0.2*** Values (% of total) are mean ± SEM, n=4-5. Values below 0.5 are not shown for clarity. Asterisks indicate different from CD, *P<0.05, **P<0.01, ***P<0.001. 36 37 ),*85( $ % & Figure 2.4 38 Figure 2.4: (n-3) PUFA diets did not modify nonraft organization, but the HF (n-3) PUFA diet disrupted lipid raft clustering. (A) FAST-DiI intracellular and plasma membrane image intensity of B cells isolated from mice fed CD, HF (n-6), LF (n-3), and HF (n-3) PUFA diets. (B) C16-Bodipy intracellular and plasma membrane image intensity. Fluorescent values in A and B are normalized to CD, because gain settings were not the same between differing sets of experiments. (C) Feret diameter of lipid rafts. Data are means ± SEM, n = 3–5. Data in C are from 48-143 total cells analyzed per diet. Asterisks indicate different from CD: *P < 0.05, **P < 0.01. Data in A-B were collected by Franklin, A. cells followed by translational studies that determined the impact of n-3 PUFA on nonraft and lipid raft organization ex vivo. The approach relied on quantitative imaging methods to overcome limitations of using detergent extraction (157, 158). As discussed below, the data point to an emerging model in which lipid rafts appears more sensitive to disruption than nonrafts in response to n-3 PUFA intervention in EL4 and primary B cells. Disruption of nonraft organization in vitro ! In serum free conditions, EPA and DHA treatment modified nonraft organization of EL4 cells. There were several possibilities by which EPA or DHA treatment increased FAST-DiI in the plasma membrane. One possibility was uptake of FAST-DiI with n-3 PUFA represented EPA- and/or DHA-rich domains (130, 151). Alternatively, increased FAST-DiI accumulation with EPA or DHA represented a dissolving of rafts. Thus, the area of nonrafts could have increased upon treatment, but these nonrafts were not organizationally distinct domains. To address these 2 possibilities, specific fluorescent probes will have to be designed, synthesized, and extensively tested in the future. ! Some other interpretations of the results with FAST-DiI staining were ruled out. One possibility was BSA, OA, and AA treatment made FAST-DiI staining of the membrane less efficient than EPA or DHA. This was highly unlikely, because we observed similar uptake of C16-Bodipy for most of the treatments. A second possibility was EPA and DHA decreased the microviscosity of the membrane, which allowed for more uptake of the probe (105). Although this was possible, one would then have expected increased internalization of the probe with EPA or DHA treatment compared with BSA; furthermore, increased uptake of C16-Bodipy would have occurred. 39 ! The FRET data with EL4 cells revealed EPA and DHA could modify membrane organization on a nanometer scale. To the best of our knowledge, this was the first study to use FRET imaging to study nanometer scale organization of n-3 PUFA in a cellular system. The data highlight the use of this methodology to determine effects of n-3 PUFA on the appropriate size scale. The FRET studies showed EPA and DHA increased the distance between randomly distributed nonraft MHC class I molecules. The increase in cell size with EPA or DHA treatment did not maintain the relative density of MHC class I surface molecules compared with the BSA control. We previously reported MHC I surface levels on EL4 cells increased by ~30–35% with EPA or DHA treatment (150). Here, we found cell size increased by ~26–30% with EPA or DHA. Given that forward scatter roughly reports on the diameter of the cell and if we assume the cell is a sphere, a ~30% increase in diameter of the cell with EPA and DHA treatment should increase surface area by ~60%. Thus, MHC I surface expression should have increased >30–35% with EPA or DHA treatment to maintain the same density of molecules as the BSA-treated cells. Therefore, as cell size increased, MHC I molecules were pushed further apart. Nonraft organization was not disrupted by n-3 PUFA in vitro or ex vivo when cell size was unaffected ! Mechanistically, we speculate an increase in cell size with EPA and DHA treatment under serum free conditions caused a redistribution in the lateral organization of lipids and proteins. The increase in cell size appeared to be related to the ability of EPA and DHA to promote cell growth and prevent cell death. The growth data were consistent with previous studies that showed EPA and DHA enhanced cell growth in 40 some cell types (174, 175). Although the numbers of EL4 cells were equivalent across the treatment groups at 15.5 h (time point at which studies were conducted), by 24 h, there were differences in cell number. Therefore, the data suggest cells treated with EPA or DHA at 15.5 h grew in size at a faster rate than the BSA-treated cells and were likely poised to divide. It has been reported with primary cells that cell size, measured with forward scatter, increased as cells started to divide, because a homogenous population of cells can have varying thresholds for entry into the cell cycle (176). ! The ex vivo data also showed n-3 PUFA administered to mice at 2 different doses had no effect on nonraft organization. The ex vivo data were consistent with the in vitro studies in which EPA and DHA did not affect cell size but inconsistent with the in vitro studies in which cell size increased. Several possibilities, which may not be mutually exclusive, could explain the discrepancy. First, there was the obvious difference in cell size, as discussed above. Second, n-3 PUFA in vitro were administered as single fatty acids, whereas the diet provided a complex mixture of differing fatty acids. Third, B cells from the mice incorporated much lower levels of n-3 PUFA compared with EL4 cells. However, in vivo levels were more physiologically relevant than those administered in vitro. Indeed, 2 independent imaging experiments showed treatment of EL4 cells in serum free conditions with a lower dose (10 µmol/L EPA or DHA) did not affect FAST-DiI uptake relative to BSA (data not shown). Overall, the in vitro studies using serum free conditions suggest the changes in nonraft organization had limited physiological relevance. Of course, our studies do not rule out the possibility that nonraft organization could be disrupted with n-3 PUFA in other cell types. 41 Lipid raft clustering was sensitive to n-3 PUFA ! Unexpectedly, the HF n-3 PUFA diet lowered uptake of C16-Bodipy in B cells. It was unlikely the lowered uptake was due to a modification in nonraft organization, because we measured no change in FAST-DiI intensity or FRET. One possibility could be that as n-3 PUFA increased raft size, it made the membrane more ordered, which prevented uptake of C16-Bodipy. Overall, it was unclear why this probe was not efficiently recruited into the cell, which we aim to investigate in the future. Nevertheless, the HF n-3 PUFA diet disrupted lipid raft organization, which was highly consistent with our previous data with EL4 cells in which DHA diminished raft clustering (150). The data were also consistent with several studies using in vitro or fat-1 transgenic mouse model systems that showed n-3 PUFA disrupted lipid raft molecular organization (128, 140, 155, 177-179). Specifically, the ex vivo data from this study agreed with an electron microscopy study that demonstrated treatment of HeLa cells with DHA had a more robust effect on cholesterol-dependent raft domains compared with cholesterol- independent nonraft domains of the inner plasma membrane leaflet (180). ! The discovery that the HF n-3 PUFA diet made rafts appear larger was the first visual evidence of n-3 PUFA dietary intervention having an effect on lipid rafts in an animal model. Note that although we interpret the rafts to be larger in size, it is possible that they could represent rafts that are much smaller in size but only seem larger due to increased levels of GM1. Future studies will have to address whether the disruption in raft organization represents the formation of highly ordered raft domains or many small raft domains. We also propose lipid raft clustering was diminished, because it was reported that cholera toxin binds more efficiently to GM1 raft molecules when they are 42 declustered (181). Surprisingly, the LF n-3 PUFA diet did not exert an effect on raft organization. Thus, the levels of n-3 PUFA in this diet may not have been high enough to modify lipid rafts. Future studies will have to determine at what dose n-3 PUFA serve to disrupt rafts. Implications ! Although numerous studies have established that n-3 PUFA modify the global membrane parameter of microviscosity (105), the specific effects of n-3 PUFA on membrane domain organization have generally remained elusive. We show n-3 PUFA specifically target the organization of lipid rafts more than nonrafts, which has relevance toward understanding how these fatty acids disrupt downstream intracellular signaling events or cell-cell communication. The data from this study have implications for several model systems. We present 2 examples for simplicity. A recent study demonstrated DHA treatment prevented dimerization and recruitment of the Toll-like Receptor 4 into lipid rafts (182). Our data suggest DHA could prevent recruitment of the protein into lipid rafts from nonrafts as a consequence of the disruption in the spatial distribution of rafts (which may be occurring on several length scales). As another example, it was proposed that suppression of MDA-MB-231 breast cancer migration with n-3 PUFA was due to an inability of the chemokine receptor CXCR4 to cluster, but the mechanism was not investigated (183). We propose a disruption in lipid rafts could prevent CXCR4 clustering and one approach would be to measure CXCR4 clustering with FRET imaging on a nanometer scale. ! In summary, data from EL4 and primary B cells suggest lipid rafts are far more sensitive to disruption in response to intervention with n-3 PUFA than nonraft domains. 43 Future studies will have to address how n-3 PUFA acyl chains initiate a change in the spatial distribution of lipid rafts in the plasma membrane and how this affects protein clustering and ultimately cellular function. 44 CHAPTER 3: HIGH DOSE OF AN N-3 POLYUNSATURATED FATTY ACID DIET LOWERS ACTIVITY OF C57BL/6 MICE 2 Introduction ! n-3 PUFAs, the bioactive components of fish and flaxseed oil, are routinely consumed by the public as food additives or supplements. They are also recognized to have utility for treating metabolic and inflammatory diseases (2, 50, 51, 144, 146). Generally, dietary studies evaluating n-3 PUFA efficacy in differing model systems have relied on low doses of n-3 PUFAs. With animal studies, investigators have commonly used ?5% (weight/weight) fish or flaxseed oil as intervention, corresponding to approximately 2–6% of total energy as n-3 PUFAs (52). This dose, especially of fish oil, is often selected to model n-3 PUFA intake of Greenland Eskimos that consume n-3 PUFAs in the range of 1–6% of total energy (4, 5). A few studies have tested the effects of fish or flaxseed oils at higher doses with mixed results on functional endpoints (184-186). Overall, very little is known about the effects of high doses of n-3 PUFAs, which could have a unique therapeutic niche or could exert negative side effects, raising potential safety issues for the general public. ! We previously reported long-term administration of high levels of a mixed fish/ flaxseed oil diet to C57BL/6 mice promoted significant body weight gain (160). These findings suggested high doses of n-3 PUFAs increased body weight by lowering activity since we ruled out changes in food consumption (160). Therefore, the objective of this study was to determine if short-term dietary consumption of a high dose of n-3 PUFAs 2 This research was originally published in Prostaglandins, Leukotrienes, and Essential Fatty Acids. Rockett, B. D., M. Harris, & S. R. Shaikh. 2012. High dose of an n-3 polyunsaturated fatty acid diet lowers activity of C57BL/6 mice. Prostaglandins Leukot. Essent. Fatty Acids. 86: 137–140. © Elsevier Ltd. could lower energy expenditure prior to any large differences in body weight. Studies were conducted in comparison to a LF purified mouse CD and a LF n-3 PUFA diet. Experimental methods Mice and diets ! All experiments with mice fulfilled guidelines established by the East Carolina University Brody School of Medicine for euthanasia and humane treatment. Male C57BL/6 mice (?4–6 weeks old) were placed for 3 weeks on two experimental diets, developed in collaboration with Harlan-Laboratories (Madison, WI). Mice were administered either a purified CD, 5% fat by weight, a LF n-3 PUFA diet, or a HF n-3 PUFA diet, 20% fat by weight, as previously described (187). The composition of the diets is listed in Table 3.1. Metabolic cage studies ! Mice were placed in fully automated metabolic cages (TSE Systems) for 4 days to monitor activity during week 2. Mice were placed one per cage and were acclimated for 48 h followed by data collection for 48 h. Airflow through the cages was held constant at 0.5 L/min. 12 h light and dark cycles were maintained with ad libitum access to food and water. Locomotor activity in the metabolic cages was measured by the breaking of 32 infrared laser beams that span each cage in the xy and yz planes. TSE LabMaster software recorded in 20 min intervals each time a series of laser beams were broken by ambulatory, rearing, and running wheel activity. Metabolic activity was measured via indirect calorimetry recording maximal O 2 consumption (VO 2 ) and CO 2 production (VCO 2 ). VO 2 and VCO 2 values were normalized by the software to body weight in kilograms and are reported as ml/h/kg (188). Respiratory exchange ratio 46 Table 3.1: Composition of control, low fat, and high fat n-3 PUFA diets Formula (g/Kg) CD LF n-3 PUFA HF n-3 PUFA Casein 185 185 220 L-Cystine 2.5 2.5 3.0 Corn Starch 370 370 174 Mineral Mix, AIN-93M-MX (94049) 35 35 42 Vitamin Mix, AIN-93-VX (94047) 15 15 18 Choline Bitartrate 2.5 2.5 3.0 TBHQ, antioxidant 0.02 0.02 0.06 Maltodextrin 140 140 140 Sucrose 150 150 150 Cellulose 50 50 50 Soybean Oil 50 3.8 15 Flaxseed Oil - 23.1 92.5 Fish Oil - 23.1 92.5 47 (RER) was calculated as VCO 2 /VO 2 . The data were analyzed for average light and dark activity (188). Significance was established using a two-way ANOVA followed by a Bonferroni Multiple Comparison t test using GraphPad Prism (188). P values less than 0.05 were considered significant. Results Body weight and food consumption ! Mice weighing 16–19 g were placed on diets. Final body weight gain, confirmed with measurements of adipose mass with Echo-MRI, was identical between the CD and the experimental diets (data not shown). Mice on the HF n-3 PUFA diet consumed less food relative to CD; however, there was no difference in the total kcal consumed between the differing diets relative to CD (data not shown). Metabolic cage studies ! The time course of ambulatory, rearing, and running wheel activities are shown in Figure 3.1A–C. These data were used to calculate the average activity for the entire light and dark cycles (Figure 3.1D–F). There was a significant increase in activity during the dark cycle with all of the diets relative to the light cycle for all measurements (Figure 3.1D–F). ! There were no statistical differences between the diets during the light cycle. The HF n-3 PUFA diet lowered average dark cycle ambulatory activity by 62% relative to CD (Figure 3.1D). The LF n-3 PUFA diet had no significant effect compared to CD. Rearing activity was significantly lowered with the HF n-3 PUFA diet by 63% compared to CD (Figure 3.1E). The LF n-3 PUFA diets had no effect on rearing activity relative to CD. For the ambulatory and rearing measurements of activity, there were no significant 48 49 Figure 3.1: HF n-3 PUFA diet lowered activity. (A) Ambulatory, (B) rearing, and (C) running wheel activities as a function of time for mice fed differing diets. Corresponding average light and dark cycle (D) ambulatory, (E) rearing and (F) running wheel activities. Counts represent the number of laser beams broken due to movement in the cage. Data are mean ± SEM, n = 7 mice per diet. Significance during the dark cycle compared to CD is indicated by *P < 0.05, †P < 0.01, ‡P < 0.001. Figure 3.1 Light Dark 0 100 200 300 400 ‡ C o u n t s * Light Dark 0 200 400 600 † C o u n t s Light Dark 0 500 1000 1500 * C o u n t s 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 1000 2000 3000 4000 C o u n t s 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 200 400 600 800 1000 C o u n t s 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 500 1000 1500 2000 C o u n t s A. B. C. F. E. D. Ambulatory Rearing Running wheel 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 200 400 600 800 1000 C o u n t s CD LF n-3 PUFA HF n-3 PUFA 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 200 400 600 800 1000 C o u n t s CD LF n-3 PUFA HF n-3 PUFA 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 200 400 600 800 1000 C o u n t s CD LF n-3 PUFA HF n-3 PUFA Light Dark 0 200 400 600 † C o u n t s CD LF n-3 PUFA HF n-3 PUFA Light Dark 0 200 400 600 † C o u n t s CD LF n-3 PUFA HF n-3 PUFA Light Dark 0 200 400 600 † C o u n t s CD LF n-3 PUFA HF n-3 PUFA differences in activity between the LF n-3 PUFA and HF n-3 PUFA diets. Running wheel activity was lowered with the HF n-3 PUFA diet relative to CD by 87%, and 78% relative to the LF n-3 PUFA (Figure 3.1F). The LF n-3 PUFA diet had no effect on running wheel activity relative to CD. ! The time course of VO 2 , VCO 2 , and RER values is shown in Figure 3.2A–C. These data were then used to calculate the average VO 2 , VCO 2 , and RER for the entire light and dark cycles (Figure 3.2D–F). The average dark cycle VO 2 (Figure 3.2D) and VCO 2 (Figure 3.2E) were significantly higher for all of the diets relative to the light cycle. There were no differences between the diets on VO 2 . The HF n-3 PUFA diet lowered dark cycle VCO 2 by 20% relative to CD and LF n-3 PUFA. RER values were significantly different for all of the diets between the light and dark cycles (Figure 3.2F). During the dark cycle, the HF n-3 PUFA had lower RER values relative to CD and LF n-3 PUFA. Discussion ! We present evidence for the first time that a high dose of n-3 PUFAs lowered mouse activity. The reduction in activity with the HF n-3 PUFA diet was consistent with our previous study demonstrating this diet promoted significant body weight gain in mice (160). The LF n-3 PUFA diet also had a tendency to reduce energy expenditure in some of the measurements. This raises the question of whether mixing fish and flaxseed oils has some potential effect that does not promote body weight loss and perhaps even increase body weight gain after long-term feeding due to a reduction in activity. More studies are needed to address this in addition to determining the effects of other fat sources (i.e. hydrogenated oils, coconut oil) on activity. Overall, the reduction in all three measures of activity (ambulatory, rearing, and running wheel) appeared dose 50 51 Figure 3.2: HF n-3 PUFA diet lowered VCO 2 and RER. (A) VO 2 , (B) VCO 2 , and (C) RER as a function of time for mice fed differing diets for 3 weeks. Corresponding average light and dark cycle (D) VO 2 , (E) VCO 2 , and (F) RER. VO 2 and VCO 2 are mL of gas consumed or expelled per hour normalized to body weight. Data are mean ± SEM, n = 7 mice per diet. Significance during the dark cycle compared to CD is indicated by *P < 0.05, ‡P < 0.001. Light Dark 0.5 0.6 0.7 0.8 0.9 1.0 V C O 2 / V O 2 ‡ Light Dark 0 2000 4000 6000 m L / h r / K g * Light Dark 0 2000 4000 6000 8000 m L / h r / K g 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0.5 0.6 0.7 0.8 0.9 1.0 V C O 2 / V O 2 * 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 2000 4000 6000 8000 m L / h r / K g 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 4000 6000 8000 m L / h r / K g A. B. C. F. E. D. VO 2 VCO 2 RER 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 200 400 600 800 1000 C o u n t s CD LF n-3 PUFA HF n-3 PUFA 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 200 400 600 800 1000 C o u n t s CD LF n-3 PUFA HF n-3 PUFA 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 4:00 AM 0 200 400 600 800 1000 C o u n t s CD LF n-3 PUFA HF n-3 PUFA Light Dark 0 200 400 600 † C o u n t s CD LF n-3 PUFA HF n-3 PUFA Light Dark 0 200 400 600 † C o u n t s CD LF n-3 PUFA HF n-3 PUFA Light Dark 0 200 400 600 † C o u n t s CD LF n-3 PUFA HF n-3 PUFA Figure 3.2 dependent with the n-3 PUFA diets. ! It is difficult to directly compare our data to other studies since there are very few studies in this area, which are not in complete agreement. For instance, an EPA enriched oral food supplement provided to advanced pancreatic cancer patients, who are prone to losing weight, enhanced energy expenditure, and provided some improvement in their metabolism (189). However, in another set of studies, supplementing the diet of healthy males with fish or flaxseed oil had no effect on energy expenditure (190, 191). Clearly, more studies are needed in this area with n-3 PUFAs in both animals and humans. ! Overall, the potential therapeutic value of high n-3 PUFA doses appears very limited. It is interesting that despite lowering activity, the HF n-3 PUFA diet reduced serum triglycerides (data not shown). Therefore, the data suggest high doses of n-3 PUFAs are effective in their accepted role of lowering triglycerides (TG) even when they lower activity. There are a few cases where an increase in body fat mass due to lower activity could have some value. For instance, HIV positive patients can have elevated TGs and high energy expenditure, yet low body weight, which are in part driven by aggressive anti-viral therapy (192, 193). The elevated TGs are a risk factor for cardiovascular disease, which is prevalent in HIV positive patients (194). Perhaps short- term intervention with a high dose of fish/flaxseed oils would assist in lowering TGs and lowering energy expenditure. Of course, extensive functional and mechanistic studies in animals and eventually humans would be required to address at what specific dose, composition of n-3 PUFAs, and duration could these fatty acids be utilized for these cohorts. 52 CHAPTER 4: FISH OIL INCREASES RAFT SIZE AND MEMBRANE ORDER OF B CELLS ACCOMPANIED BY DIFFERENTIAL EFFECTS ON FUNCTION 3 Introduction ! Fish oil (FO) enriched in n-3 PUFAs is increasingly hypothesized to have beneficial health effects for treating symptoms for a wide range of diseases associated with chronic inflammation (17, 195, 196). However, several hurdles have prevented the therapeutic use of FO in the clinic, including a limited understanding of the molecular targets and mechanisms of n-3 PUFAs. The proposed mechanisms by which the n-3 PUFAs of FO, EPA and DHA, exert immunosuppressive effects are pleiotropic (197). These include incorporating into membrane phospholipids and modifying plasma membrane microdomain organization, serving as precursors for bioactive lipid molecules, disrupting intracellular signaling, and regulating gene expression (197). Recently, manipulation of lipid raft microdomains with n-3 PUFAs has gained attention because this mode of action is central to many of the downstream effects of n-3 PUFAs (149). ! Several models, which are not in complete agreement, have emerged to explain how n-3 PUFAs disrupt raft molecular organization. One model, based on ex vivo studies of the CD4 + T-cell immunological synapse, is that n-3 PUFAs, relative to controls, enhance the formation of more ordered and possibly larger lipid rafts upon stimulation of the cell (140). This model is the most relevant because it relied on the 3 This research was originally published in The Journal of Lipid Research. Rockett, B. D., H. Teague, M. Harris, M. Melton, J. Williams, S. R. Wassall, & S. R. Shaikh. Fish oil increases raft size and membrane order of B cells accompanied by differential effects on function. J Lipid Res. 2012; 53; 674–685. © the American Society for Biochemistry and Molecular Biology. fat-1 transgenic mouse that has higher endogenous levels of n-3 PUFAs compared with control mice. A second model developed with studies in model membranes is that phosphatidylethanolamines containing DHA largely avoid raft molecules to form their own entities as nonraft domains (198, 199). Finally, a more integrated model, based mostly on in vitro studies, is that n-3 PUFAs incorporate directly into rafts and in some cell types force cholesterol molecules to undergo displacement between rafts and nonrafts (149, 200, 201). Overall, the majority of these models have not relied on physiologically relevant conditions that model human intake of FO. ! Based on studies with CD4 + T cells, macrophages, and splenocytes, disruption of rafts with n-3 PUFAs generally suppresses cellular function (140, 142, 143). Far less is known about the relationship between disruption of lipid rafts with n-3 PUFAs and downstream function in other cell types. In particular, B cells are increasingly recognized to have a role in inflammatory processes and autoimmune diseases (202, 203); however, there is limited understanding about how FO affects B cell membrane organization and function, especially at the animal level. We previously demonstrated that feeding C57BL/6 mice a very high dose of n-3 PUFAs for 3 weeks diminished B cell lipid raft clustering (187). We also showed that mice consuming this very high dose for 14 weeks displayed enhanced B cell activation, which we speculated was due to an increase in body weight (160). However, we did not completely test the effects of a physiologically relevant dose of FO on raft organization, the mode of raft disruption, or its relationship with innate and adaptive B cell function. ! The specific objectives of this study were 1) to determine if administration of a dose of FO, modeling human intake, disrupted lipid raft organization of mouse B cells; 54 2) to address the conflicting models on how n-3 PUFAs disrupted raft molecular organization; and 3) to test the hypothesis that altering raft organization with FO would promote immunosuppressive effects on downstream innate and adaptive B cell function. The approach relied on biochemical and imaging measurements of B cells isolated from mice fed FO, supplemented with in vitro and model membrane studies. The data revealed for the first time that a relevant dose of FO increased B cell raft size and membrane molecular order upon cross-linking rafts, which was accompanied by novel downstream functional effects. Materials and Methods Mice and diets ! Male C57BL/6 mice (Charles River), 4–6 weeks old, were fed for 3 weeks a purified CD or a FO diet (Harlan-Teklad). Both diets consisted of 5% total fat by weight or 13% by total energy. All ingredients were essentially identical except the fat source, which was soybean oil in the CD and menhaden fish oil in the FO diet (Table 4.1). The FO diet also contained food coloring, which we ensured had no effects on the measurements described below. The fatty acid distribution of the diet, measured with GC, was within experimental error to that reported by the vendor (Harlan-Teklad) (Supplementary Table 4.1). Approximately 2% of the total kcal came from EPA and 1.3% from DHA. This level of EPA/DHA corresponded to approximately 4 g of FO consumed by a human on a daily basis, which can be achieved through the diet and is currently in use pharmacologically for treating elevated triglycerides (52). Mice were euthanized using CO 2 inhalation and cervical dislocation followed by isolation of spleens. All of the experiments with mice fulfilled the guidelines established by the East Carolina 55 Table 4.1: Composition of control and fish oil diets. Formula (g/Kg) Control Fish oil Casein 185.0 185.0 L-Cystine 2.5 2.5 Corn Starch 369.98 369.88 Maltodextrin 140.0 140.0 Sucrose 150.0 150.0 Cellulose 50.0 50.0 Soybean Oil 50.0 - Fish Oil - 50.0 Mineral Mix, AIN-93M-MX (94049) 35.0 35.0 Vitamin Mix, AIN-93-VX (94047) 15.0 15.0 Choline Bitartrate 2.5 2.5 TBHQ, antioxidant 0.02 0.02 Yellow food coloring - 0.1 56 University Brody School of Medicine for euthanasia and humane treatment. Cell isolation and cell culture ! B220 + B cells (> 90% purity) were isolated from splenocytes by negative selection (Miltenyi Biotech) as previously described (160). For some experiments, CD4 + T cells (> 85% purity) were purified from B6.Cg-Tg(TcraTcrb)425Cbn/J mice (OT-II) (Jackson Laboratory) that express ?- and ?-chain T-cell receptor and CD4 co-receptor specific for recognizing chicken ovalbumin 323–339 (OVA 323–339 ) presented by H-2 IAb molecules. For in vitro studies, mouse EL4 lymphomas were treated with 25 µM EPA or DHA complexed to BSA for 15.5 h as previously described (150). BSA without complexed fatty acid served as the control (150). Isolation of detergent-resistant membranes and analysis ! A total of 30 × 10 6 B cells or 20 × 10 6 EL4 cells were prepared for detergent extraction and layered on a sucrose gradient as previously described (150). Sucrose gradients were centrifuged at 40,000 rpm for 20 h at 4°C in a swinging bucket SW41Ti rotor (Beckman). Twelve 1 ml fractions were collected. Fractions 3–6 representing detergent-resistant membranes (DRMs) or fractions 9–12 representing detergent- soluble membranes (DSMs) were combined based on their cholesterol content, which was measured with an Amplex Red cholesterol assay kit (Invitrogen) relative to protein levels. Lipids were extracted as described previously (150). Samples were dissolved in chloroform, and select phospholipids were separated with a Shimadzu Prominence HPLC using a 150 × 4.6 mm Luna 5 µm NH2 column (Phenomenex) with a mobile phase gradient of H2O (increasing from 5% to 50%) and acetonitrile (decreasing from 95% to 50%) at 30°C. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), and 57 sphingomyelin (SM) fractions were collected by UV detection at 202 nm, and the acyl chain composition of each fraction was further analyzed by GC as previously shown (150). The rationale for selecting these phospholipids was to address the most abundant lipids of the outer (PC, SM) and inner (PE) leaflets and to allow direct comparison with other studies (128). 2 H NMR spectroscopy ! Sample preparation, NMR spectroscopy, and analysis were as previously described (198). The PC phospholipid (1-[ 2 H 31 ] palmitoyl-2- docosahexaenoylphosphatidylcholine,16:0-22:2PC-d 31 ) used for this study was perdeuterated in the sn-1 chain (custom synthesis, Avanti Polar Lipids), and the samples consisted of aqueous multilamellar dispersions of 50 wt% lipid in 50 mM Tris buffer (pH 7.5). Briefly, lipid mixtures of 16:0-22:6PC-d 31 /SM (1:1) and 16:0-22:6PC-d 31 / SM/cholesterol (1:1:1) were codissolved in chloroform. The solvent was removed, the lipids were hydrated with buffer, and the pH was adjusted. After three lyophilizations with deuterium-depleted water (Sigma), samples were transferred to a 5 mm NMR tube. Stringent precautions were taken to prevent oxidation during sample preparation and data acquisition. NMR experiments were performed on a homebuilt spectrometer operating at 7.05 T using a phase-alternated quadrupolar echo sequence (199, 204). Lipid raft cross-linking and total internal reflection fluorescence imaging ! Cells were fluorescently stained for lipid rafts by cross-linking CTx-B (Invitrogen) and then imaged using total internal reflection fluorescence (TIRF) microscopy. For select experiments, GM1 levels were measured by staining with primary with no secondary antibody for cross-linking. TIRF was performed on an Olympus IX-71 58 microscope with excitation at 488 nm by a 20 mW Sapphire laser and a 60× 1.45NA oil- immersion TIRF objective. Fluorescence emission was filtered through a LF-488/561-A- OMF filter cube (Semrock) and detected by a Hamamatsu ORCA-R2 progressive scan interline CCD camera. For all imaging experiments, 9 or 10 cells per diet were analyzed per experiment, and image analysis was performed as previously described (150). Live cell imaging of DiIC 18 ! To assess changes in nonraft organization, we measured uptake of the nonraft probe 1,1’-dioctadecyl-3,3,3?,3?-tetramthylindocarbocyanine perchlorate (DiIC 18 , Invitrogen) (205). The approach was similar to our previous report where we used FAST-DiI (187). However, FAST-DiI uptake was extremely rapid under live cell imaging conditions; therefore, we relied on DiIC 18 . A total of 1 × 10 6 B cells were washed with RPMI 1640 without phenol red at 37°C and resuspended in 1 ml of the same media and placed into Delta T dishes (Bioptechs Inc.). We added 0.2 µl of a 1 mg/ml stock of DiIC 18 to the dishes and imaged. Live cell, wide-field microscopy was conducted with an Olympus IX-70 microscope using an oil 100× objective and a Hamamatsu CCD camera, equipped with stage and objective heaters (Bioptechs Inc.). Imaging was started at 1 min after addition of the fluorescent probe at 37°C. Images were acquired with MetaMorph software and analyzed with NIH ImageJ. All settings were kept constant between experiments. Generalized polarization imaging ! Generalized polarization (GP) was calculated to determine the molecular order of the B and EL4 cell plasma membrane (206). Cells were stained with 4 µM di-4- ANEPPDHQ (Invitrogen) for 30 min at 37°C, washed twice with PBS, and fixed with 4% 59 paraformaldehyde (207). For some experiments, as an additional control, B cells were treated with 10 µM methyl-?-cyclodextrin (Sigma-Aldrich) for 15 min at 37°C to deplete cholesterol. Imaging was conducted on a Zeiss LSM-510 confocal microscope using a 100x objective with excitation at 488 nm and fluorescence emission detected in two channels: 505–545 nm for ordered regions and 650–750 nm for disordered regions. Quantification of fluorescence intensity (I), after background subtraction for the two channels, was performed using NIH ImageJ. GP was calculated using the formula: GP = (I 505–545 ? (G)I 650–750 )/(I 505–545 + (G)I 650–750 ), where G is the G-factor and I is fluorescence intensity (206). B cell activation with lipopolysaccharide (LPS) ! B cells were stimulated with 1 µg/ml of LPS for 24 h, and the secreted cytokine profiles were measured from supernatants using a Multi-Analyte ELISArray kit (SA Biosciences) as previously demonstrated (160). In addition, B cells were analyzed for surface expression of activation markers using fluorescently labeled anti-MHC class II M5/114.15 (Bio X Cell), anti-CD69 H1.2F3 (BD Biosciences), and anti-CD80 16-10A1 (Bio X Cell) on a BD LSR II flow cytometer. Antibodies for MHC class II and CD80 were conjugated with fluorophores using a kit (GE Healthcare). B cell activation of naive CD4 + T cells ! B cells were peptide loaded with OVA 323–339 (GenScript) at various concentrations in serum-free media for 1 h at 37°C and then washed with fresh media. A total of 1 × 10 5 B cells were then plated with 3 × 10 5 nondiet modified OT-II transgenic T cells, spun quickly, and incubated for 24 h at 37°C in 96-well plates. After 24 h, the supernatant was collected to analyze secreted IFN (interferon)-? and IL-2 with an ELISArray kit (SA 60 Biosciences) following the manufacturer's protocol. The cells were stained with fluorescently labeled anti-CD4-PE (Miltenyi Biotech), anti-CD69-FITC (BD Biosciences), and anti-CD25-PE-Cy7 (BD Biosciences) and analyzed by flow cytometry on a BD LSR II. In all flow cytometry experiments, SYTOX Blue (Invitrogen) was used to discriminate dead cells, and at least 10,000 live events were collected for analyses. Control experiments were routinely conducted in the absence of peptide and with T cells only (no B cells) to ensure T-cell activation was driven by peptide-loaded B cells. Statistical analyses ! All ex vivo and in vitro data are from several independent experiments, with each ex vivo experiment representing one mouse per diet. The only exception was the ex vivo DRM/DSM analysis, in which two mice were pooled per experiment. Data were first analyzed for normality using a Kolmogorov-Smirnov test (GraphPad Prism). For a few select experiments with nonparametric distributions, we relied on paired Wilcoxon t- tests. Pairing was required due to differences in experimental settings between experiments as previously described (187). For the majority of animal experiments, statistical significance was established using an unpaired one-tailed or two-tailed Student's t-test. One-tailed unpaired t-tests were used to analyze the GC data from animals because before experimentation we predicted an increase in n-3 PUFA levels (128). In vitro studies relied on one-way ANOVAs followed by a Dunnett's post hoc t-test (160). P values less than 0.05 were considered significant. The NMR data were not analyzed for statistical significance because they were obtained on model membranes of well defined and controlled composition for which a reproducibility of ±1% applies to multiple acquisitions. This approach is standard for model membrane studies using 61 NMR spectroscopy (198, 199). Results Body weights, food intake, and cellularity ! The FO diet, relative to CD, had no effect on the rate of body weight gain (Supplementary Figure 4.1A) or food intake per day (Supplementary Figure 4.1B). Spleen weight (Supplementary Figure 4.1C) was significantly elevated for mice on the FO diet but did not affect the steady-state number of B cells (data not shown). Given that we recently reported n-3 PUFAs at high doses can lower energy expenditure (208), we verified that the FO diet had no impact on whole body energy expenditure using metabolic cages (data not shown). FO promoted uptake of EPA/DHA into DSMs and DHA into PC of DRMs ! The first objective was to biochemically determine if the physiologically relevant dose of FO manipulated the molecular composition of B cell “raft”-like DRMs. We used detergent extraction followed by HPLC-GC to determine the acyl chain composition of PE, PC, and SM ex vivo (Figure 4.1). The relative proportion of PC, PE, and SM in DRM and DSM fractions was not changed by FO (Figure 4.1A). Relative PC and SM levels were generally higher in DRMs than in DSMs. SM was the smallest fraction of PE/PC/ SM, consistent with studies in other cell types (177). ! In PE, EPA (20:5) and DHA (22:6) did not incorporate into DRMs but were significantly increased in DSMs (Figure 4.1B). FO also increased 22:5 and lowered arachidonic acid (20:4) in PE DSMs (Figure 4.1B). In PC, 20:5 was lowered and 22:6 increased with FO in DRMs (P = 0.06) (Figure 4.1C). In DSMs of PC, 20:5 and 22:6 did not significantly increase (Figure 4.1C). In SM, 20:5 and 22:6 did not change in DRMs 62 63 16:0 18:0 18:1 18:2 20:4 20:5 22:5 22:6 0 10 20 30 40 50 Fatty acid P e r c e n t o f t o t a l * ** *** * p=0.05 16:0 18:0 18:1 18:2 20:4 20:5 22:5 22:6 0 10 20 30 40 50 Fatty acid P e r c e n t o f t o t a l ** *** ** * 16:0 18:0 18:1 18:2 20:4 20:5 22:5 22:6 0 10 20 30 40 50 Fatty acid P e r c e n t o f t o t a l 16:0 18:0 18:1 18:2 20:4 20:5 22:5 22:6 0 10 20 30 40 50 Fatty acid P e r c e n t o f t o t a l p=0.06 p=0.06 16:0 18:0 18:1 18:2 20:4 20:5 22:5 22:6 0 10 20 30 40 50 Fatty acid P e r c e n t o f t o t a l 16:0 18:0 18:1 18:2 20:4 20:5 22:5 22:6 0 10 20 30 40 50 Fatty acid P e r c e n t o f t o t a l PE PC SM 0 20 40 60 80 Phospholipid P e r c e n t o f t o t a l PE PC SM 0 20 40 60 80 Phospholipid P e r c e n t o f t o t a l Phosphatidylcholine C. Phosphatidylethanolamine B. Sphingomyelin D. DRM DSM A. PC PE SM 0 20 40 60 80 Lipid P e r c e n t o f t o t a l Control Fish oil Figure 4.1 64 Figure 4.1: FO promoted uptake of EPA/DHA into DSMs and DHA into PC of DRMs. (A) Relative levels of B-cell PE, PC, and SM of DRM (left) and DSM (right) fractions. Acyl chain composition of B-cell DRM or DSM fractions of (B) PE, (C) PC, and (D) SM. B cells were isolated from mice fed control or FO-enriched diets. Data are means ± SEM (n = 4, with two mice pooled per experiment). Asterisks indicate different from control: *P < 0.05; **P < 0.01; ***P < 0.001. (Figure 4.1D). In DSMs of SM, FO significantly decreased palmitic acid (16:0) and increased stearic acid (18:0), 20:5, 22:5, and 22:6 (Figure 4.1D). Because we did not quantify the exact levels of EPA and DHA, we conducted a few select experiments to approximate the levels EPA and DHA incorporating into DRMs and DSMs (data not shown). On average, ?5–8% of the total n-3 PUFA were localized to DRMs with the remaining amount in DSMs, consistent with another study (209). ! Cholesterol levels in DRM and DSM fractions were not changed with FO (Supplementary Table 4.2). We also analyzed the ratio of DRM to DSM cholesterol based on a very recent study that reported that DHA treatment in vitro increased cholesterol into DSMs when analyzed as a ratio (200). FO had no effect on the ratio (Supplementary Table 4.2). ! Overall, the data were highly consistent with predictions from detergent-free model membrane studies in which PCs containing DHA were found to have higher solubility for cholesterol, and by implication to have more favorable interactions with rafts, than PEs containing DHA (152). We confirmed this by comparing the order of cholesterol on the perdeuterated sn-1 chain on a DHA-containing PC (16:0-22:6PC-d 31 ) vs. a DHA-containing PE (16:0-22:2PE-d 31 ) in mixtures with SM (1:1) and SM/ cholesterol (1:1:1), mimicking rafts, using 2 H NMR spectroscopy (Table 4.2) (198). The increase in order due to cholesterol (reflecting proximity), measured for the PC- containing DHA, was more than twice that for the PE-containing DHA (Table 4.2). This observation was consistent with the ex vivo biochemical data that suggest DHA infiltrated into PCs of DRMs and PEs of DSMs. FO increased B cell lipid raft size induced by cross-linking GM1 65 Table 4.2: DHA-containing PC displayed increased molecular order in the presence of lipid raft molecules relative to PE. Average order parameter values were calculated from 2H NMR spectra collected at 40°C for mixtures of 16:0-22:6PC-d31 with sphingomyelin (1:1), and with sphingomyelin and cholesterol (1:1:1). Values for 16:0-22:6PE-d31 were from previously published data (198). Data were collected by Williams, J. and Wassall, S. R. Lipid mixture Order parameter value Percent increase in order 16:0-22:6PC-d 31 /Sphingomyelin 0.115 68.7% 16:0-22:6PC-d 31 /Sphingomyelin/Cholesterol 0.194 16:0-22:6PE-d 31 /Sphingomyelin 0.150 26.0% 16:0-22:6PE-d 31 /Sphingomyelin/Cholesterol 0.189 66 ! The next objective was to measure changes in raft organization with the FO diet relative to the CD using microscopy. This was essential because biochemical approaches are a useful predictive tool but also have limitations due to the use of detergent (158, 159). TIRF imaging of B cells from FO- and CD-fed mice showed a significant difference in the spatial distribution of GM1 molecules cross-linked with CTx- B (Figure 4.2A). TIRF image analysis, confirmed with confocal image analysis (data not shown), revealed the Feret's diameter was increased by 79% with FO relative to CD (Figure 4.2B). The binding of CTx-B to GM1 in the presence of cross-linking, measured in terms of fluorescence intensity, showed on average a 36% increase with FO, but this failed to reach statistical significance with TIRF analysis (Figure 4.2C). To determine if the propensity for an increase in CTx-B intensity was due to increased surface levels of GM1, the binding of CTx-B in the absence of cross-linking was measured, which revealed a 49% increase in intensity with FO (Figure 4.2D). To ensure the observed effects were FO specific, we also measured raft size and binding of CTx-B with a safflower oil-enriched diet and found no evidence of raft disruption with this diet (Supplementary Figure 4.2). We also ensured the effects were specific for rafts. Changes in nonraft organization were measured in terms of uptake of the nonraft probe DiIC 18 (205). Live cell imaging revealed that DiIC 18 uptake was identical between cells isolated from CD- and FO-fed mice (Supplementary Figure 4.3). The imaging results provided the first evidence that a physiologically relevant dose of FO disrupted raft, but not nonraft, organization. FO increased membrane order upon cross-linking rafts relative to the absence of cross-linking 67 68 Control Fish oil 0.0 0.5 1.0 1.5 Diet F e r e t ' s d i a m e t e r ( µ m ) *** Control Fish oil 0 200 400 600 800 I n t e n s i t y Diet *** Control Fish oil 0 500 1000 1500 2000 Diet I n t e n s i t y A. B. Control Fish oil C. D. Cross-linked GM1 surface expression Figure 4.2 Figure 4.2: FO increased the size of rafts induced by CTxB cross-linking. (A) Sample fluorescence images of B cells isolated from control or FO-fed mice and cross- linked to induce raft clustering. Images are from TIRF microscopy (bar, 5 µm). (B) Lipid raft size in terms of Feret’s diameter for B cells. (C) CTxB fluorescence intensity after cross-linking. (D) GM1 surface levels. Values are means ± SEM (n = 6 from 65 total cells analyzed per diet in A–C; n = 4 from 86-90 cells analyzed per diet in D). Asterisks indicate different from control: ***P < 0.001. ! We subsequently determined if the aforementioned changes with FO on raft organization affected membrane molecular order. This directly addressed two opposing models in which n-3 PUFAs in response to stimulation increase membrane order ex vivo versus a decrease in membrane order in vitro (140, 177). To measure molecular order, we used di-4-ANEPPDHQ, an environmental sensing fluorescent dye that can shift its emission spectra 30 nm depending on the degree of packing in the surrounding lipid chains (Figure 4.3A) (210). As an initial control, B cells were treated with 10 µM methyl- ?-cyclodextrin to remove cholesterol from the membrane. GP significantly decreased upon cholesterol depletion (data not shown). In the absence of cross-linking, FO, compared with CD, significantly decreased the average GP by 25% (Figure 4.3B). This decrease was consistent with data showing that n-3 PUFAs exert a disordering effect on membrane microviscosity in the absence of inducing raft formation (105). Although there was no difference in GP values between CD and FO diets upon cross-linking (Figure 4.3C), GP values for FO were elevated to a greater extent than CD relative to no cross- linking (Figure 4.3D). Analysis of the differences in GP values between cross-linking and no cross-linking showed FO had a greater ordering effect by 65% compared with CD (Figure 4.3D). These results supported the ex vivo model that FO is capable of exerting an ordering effect on the membrane upon stimulation (140). EPA and DHA incorporated into DRMs/DSMs and did not increase GM1 surface expression in vitro ! We previously reported that treatment of EL4 cells with DHA, but not EPA, increased raft size (150). Therefore, we conducted comparative biochemical and imaging studies to determine if the in vivo and in vitro modes of raft disruption were 69 70 Figure 4.3: FO increased B-cell membrane molecular upon cross-linking relative to no cross-linking. (A) Sample merged intensity images of B cells, stained with di-4- ANEPPDHQ, isolated from control or fish oil enriched diets. Ordered (green) and disordered (red) channels are merged to illustrate the ratio between channels. B: Plasma membrane GP values from B cells isolated from control and FO-fed mice without CTxB cross-linking. C: GP analysis of B cells cross-linked with CTxB. D: Change in GP values upon cross-linking CTxB relative to no cross-linking. Values are means ± SEM (n = 4 from 40 total cells analyzed per diet). Asterisks indicate different from control: *P < 0.05; **P < 0.01. Figure 4.3 Control Fish oil 0.0 0.1 0.2 0.3 0.4 Diet G P * Control Fish oil 0.0 0.1 0.2 0.3 0.4 Diet G P No cross-linking B. Cross-linked C. Control A. Fish oil Control Fish Oil 0.00 0.05 0.10 0.15 0.20 ** Diet ? M o l e c u l a r o r d e r D. similar in addition to discriminating differences in bioactivity of EPA vs. DHA. ! Similar to ex vivo measurements, relative PC and SM levels were higher in DRMs than in DSMs and were not affected by EPA or DHA (Figure 4.4A). In PE, EPA treatment increased 20:5 and 22:5 in DRMs. DHA treatment had no significant effect on 22:6 in DRMs of PE (Figure 4.4B). In PE DSMs, EPA and DHA treatment lowered 20:4 and DHA increased 22:6 (Figure 4.4B). In PCs, EPA and DHA treatment did not significantly increase 20:5 or 22:6 in DRMs (Figure 4.4C). In PC DSMs, EPA and DHA treatment, respectively, increased 20:5/22:5 and 22:6 levels. EPA and DHA treatment lowered 18:1 in DRMs and DSMs of PC. In SM, EPA and DHA, respectively, increased 20:5 and 22:6 in DRMs (Figure 4.4D). In DSMs of SM, EPA and DHA lowered 18:1 and, respectively, increased 20:5/22:5 and 22:6. Similar to the ex vivo studies, we did not quantify exact levels of EPA and DHA in DRM/DSM. Based on a few select studies (data not shown), ?30% of the added EPA/DHA was localized to DRMs with the remaining amount in DSMs, consistent with our previous work (150). ! Cholesterol levels in the DRM and DSM fractions were not significantly changed with EPA or DHA treatment (Supplementary Table 4.2). There was a tendency for EPA treatment to lower DRM cholesterol relative to BSA when analyzed as a t-test (P < 0.05). The ratio of cholesterol in DRM to DSM also had a tendency to be lowered with EPA and DHA treatment (P = 0.08) (Supplementary Table 4.2). Finally, we measured GM1 surface levels using microscopy before and after cross-linking and found no effect of EPA or DHA treatment (Supplementary Figure 4.4). These results showed that in vitro treatment with EPA/DHA, consistent with most other in vitro studies, changed DRM composition (128, 154); however, we did not measure an increase in GM1 levels in vitro 71 72 Figure 4.4 PE PC SM 0 20 40 60 80 Phospholipid P e r c e n t o f t o t a l PE PC SM 0 20 40 60 80 Phospholipid P e r c e n t o f t o t a l 16:0 18:0 18:1 20:4 20:5 22:5 22:6 0 20 40 60 Fatty acid P e r c e n t o f t o t a l ** ** ** * *** 16:0 18:0 18:1 20:4 20:5 22:5 22:6 0 20 40 60 Fatty acid P e r c e n t o f t o t a l ** * 16:0 18:0 18:1 20:4 20:5 22:5 22:6 0 20 40 60 Fatty acid P e r c e n t o f t o t a l * * 16:0 18:0 18:1 20:4 20:5 22:5 22:6 0 20 40 60 Fatty acid P e r c e n t o f t o t a l ** * ** ** ** 16:0 18:0 18:1 20:4 20:5 22:5 22:6 0 20 40 60 Fatty acid P e r c e n t o f t o t a l ** * 16:0 18:0 18:1 20:4 20:5 22:5 22:6 0 20 40 60 Fatty acid P e r c e n t o f t o t a l ** * ** Phosphatidylethanolamine B. Sphingomyelin D. DRM DSM A. PC PE SM 0 20 40 60 80 Lipid P e r c e n t o f t o t a l BSA DHA EPA Phosphatidylcholine C. 73 Figure 4.4: EPA and DHA treatment targeted DRM and DSM composition of EL4 cells. (A) Relative levels of PE, PC, and SM of EL4 DRM (left) and DSM (right) fractions. Acyl chain composition of DRM or DSM fractions from (B) PE, (C) PC, and (D) SM. EL4 cells were treated with BSA, 25 µM EPA, or DHA. Values are means ± SEM (n = 4). Asterisks indicate different from control: *P < 0.05; **P < 0.01; ***P < 0.001. with EPA or DHA treatment as observed ex vivo. DHA, but not EPA, increased membrane order with cross-linking relative to the absence of cross-linking in vitro ! We next determined if EPA and DHA treatment increased membrane order as measured ex vivo. In the absence of cross-linking, EPA and DHA, respectively, compared with BSA, decreased GP by 32% and 75% (Figure 4.5A). Upon cross-linking, EPA showed no difference in molecular order relative to BSA; however, DHA treatment lowered GP by 27% (Figure 4.5B). Analysis of the differences in GP values between cross-linking relative to no cross-linking showed DHA, but not EPA, had a greater ordering effect by 60% compared with BSA (Figure 4.5C). These results supported the emerging ex vivo model that an n-3 PUFA exerts an ordering effect upon induction of raft formation (140). FO enhanced B cell activation in response to LPS stimulation but suppressed B cell stimulation of naive CD4 + T cells ! The next set of experiments tested the hypothesis that disruption of lipid raft organization with FO would be accompanied by suppressive effects on downstream innate and adaptive B cell function, as reported for T cells, macrophages, and splenocytes (140, 142, 143). Two different B cell functional studies were conducted. First, we measured the activation of B cells after 24 h of LPS stimulation as a measure of an innate response (Figure 4.6). We found the physiologically relevant dose of FO enhanced B cell activation. The number of B cells activated was the same between the CD and FO diets (data not shown). CD69 surface levels were increased by 22% with FO, whereas MHC class II and CD80 surface expression were not affected by FO 74 75 BSA EPA DHA 0.0 0.1 0.2 0.3 0.4 G P Treatment ** *** Treatment G P BSA EPA DHA 0.0 0.1 0.2 0.3 0.4 0.5 *** Treatment ? M o l e c u l a r o r d e r BSA EPA DHA 0.0 0.1 0.2 0.3 ** No cross-linking A. Cross-linked B. C. Figure 4.5 76 Figure 4.5: DHA, but not EPA, increased membrane molecular order upon cross- linking relative to no cross-linking in vitro. (A) Plasma membrane GP values from EL4 cells treated with BSA, EPA, or DHA without CTxB cross-linking. (B) GP analysis of EL4 cells cross- linked with CTxB. (C) Change in GP values upon cross-linking CTxB relative to no cross-linking. Values are means ± SEM (n = 3 from 36 cells analyzed per treatment). Asterisks indicate different from control: **P < 0.01; ***P < 0.001. (Figure 4.6A). Secretion of IL-6, TNF-?, and IFN-? were increased compared with FO relative to the CD by 43, 87, and 182% (Figure 4.6B). IL-10 secretion was unaffected with the FO diet (Figure 4.6B). ! To determined if the immune enhancing effect of the FO diet on B cells was specific to the innate response or applicable to an adaptive immune response, we tested the ability of B cells isolated from FO-fed mice to stimulate transgenic naive CD4 + T cells that were not from FO-fed mice (Figure 4.7). The concentration of OVA 323–339 peptide loaded into B cells was optimized for maximal activation at 10 ?5 M with no activation at 0 M (data not shown). FO had no impact on the percentage of CD4 + CD69 + CD25 + T cells activated (data not shown). In addition, the surface expression of the activation markers CD69 and CD25 on CD4 + T cells was unchanged with FO (Figure 4.7A). Analysis of secreted cytokines revealed that IFN-? levels were not significantly decreased with FO, but IL-2 secretion was decreased by 42% with the FO diet relative to CD (Figure 4.7B). These results supported the notion that raft disruption with FO was accompanied by changes in B cell function. Discussion ! In this study, we focused on B cells, a cell type underrepresented in experiments with n-3 PUFAs, especially at the animal level. The results demonstrated several significant advancements. First, the data showed that a relevant dose of FO increased the size of B cell rafts. Next, data integrated from animals, cell culture, and model membranes challenged some models on how n-3 PUFAs disrupted raft molecular organization. Third, the data highlighted differences between EPA and DHA on membrane organization. Finally, we discovered that a physiologically relevant dose of 77 78 Figure 4.6: FO increased B-cell CD69 expression and cytokine secretion upon activation with LPS. (A) Median fluorescence intensity for B cells stained with anti- MHC class II, anti-CD80, or anti-CD69 after 24 h of LPS activation. B cells were isolated from mice fed a control or FO diet. (B) Cytokine secretion after 24 h of LPS activation. Values are means ± SEM (n = 6). Asterisks indicate different from control: *P < 0.05; **P < 0.01. Data were collected by Teague, H. Figure 4.6 IL-6 IL-10 TNF? IFN? 0.0 0.2 0.4 0.6 0.8 A b s o r b a n c e * ** * MHC II CD69 CD80 0 2500 5000 7500 10000 12500 M e d i a n I n t e n s i t y Control Fish oil * B. A. 79 Figure 4.7: FO suppressed B-cell stimulated IL-2 secretion from naive CD4 + T cells. A: Median fluorescence intensity of CD4 + T cells stained with anti-CD69 and anti- CD25 after 24 h. CD4 + T cells were stimulated with B cells isolated from mice fed a control or FO diet. B: Cytokine secretion after 24 h of activation. Values are means ± SEM (n = 6). Asterisk indicates different from control: *P < 0.05. Data were collected by Teague, H. Figure 4.7 CD69 CD25 0 2000 4000 6000 8000 M e d i a n I n t e n s i t y Control Fish oil CD25 CD69 0 50 100 150 200 250 300 CD69 CD25 0 2000 4000 6000 8000 M e d i a n I n t e n s i t y Control Fish oil IFN? IL-2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 A b s o r b a n c e * A. B. FO enhanced an innate immune response but suppressed an adaptive response. Therefore, the data also challenged the paradigm that modification of rafts with FO exerts global immunosuppressive effects but overall supported the notion that manipulation of immune cell rafts with FO was accompanied by changes in function. Biochemical measurements revealed some differences between ex vivo and in vitro measurements ! The biochemical data from B cells (Figure 4.1) were generally in agreement with ex vivo studies, with a few exceptions (143, 211). Fan et al. demonstrated biochemically that n-3 PUFAs infiltrated DRMs and DSMs differentially depending on the type of headgroup (155). Their data showed the majority of n-3 PUFAs displayed a preference for inner leaflet lipids such as PEs. We also found greater incorporation of n-3 PUFAs into PEs over PCs. However, Fan et al. reported that n-3 PUFAs incorporated in PE DRMs, whereas we observed the majority of n-3 PUFAs in DSMs of PEs (Figure 4.1) (155). We also found DHA in PC DRMs, whereas they found no n-3 PUFAs in PC DRMs. Finally, we observed an increase in surface levels of GM1, a sphingolipid, whereas the same group reported a decrease in SM in a subsequent study (211). The differences between the studies could be cell specific or due to differences in diet compositions. ! The B cell biochemical data showing DHA incorporated more into DRM PC than PE were consistent with model membrane experiments on lipid mixtures containing raft molecules (SM and cholesterol). They showed a DHA-containing PC underwent a greater increase in molecular order upon adding cholesterol compared with a DHA- containing PE (Table 4.2). These results were in agreement with previous studies to 80 show cholesterol was more soluble in n-3 PUFAs containing PCs than PEs (152). It is also important to note that the DHA-containing PC underwent a substantial increase in molecular order when exposed to cholesterol in the mixture with SM, which was consistent with the increase in membrane molecular order observed ex vivo when comparing cross-linked rafts with the absence of cross-linking with FO (Figure 4.3). Although n-3 PUFA acyl chains are highly disordered, very recent data from Mihailescu et al. revealed that cholesterol could raise the order of a PC-containing DHA (212). Thus, the model membrane data suggest that a PC-containing DHA undergoing an ordering effect upon interaction with cholesterol would allow the PC-containing DHA to favorably incorporate into rafts. ! Our ex vivo and in vitro data did not concur with some in vitro measurements from other labs on cholesterol lateral distribution in DRMs and DSMs. Grimm et al. reported that treatment of SH-SY5Y cells with 100 µM DHA increased the normalized DSM/DRM ratio (200). We did not observe differences in cholesterol levels between DRM and DSMs in B cells or EL4 cells; however, the DRM/DSM ratio tended to be lowered in EL4 cells (Supplementary Table 4.2). More work is clearly needed in this area because a very recent study reported that treatment of hepatocytes with EPA increased raft cholesterol levels, opposite of the findings reported by Grimm and coworkers (201). ! There were a few differences between the B cell biochemical data compared with the in vitro studies using EL4 cells. The first major difference was that GM1 levels were increased ex vivo with FO but not in vitro. The second major difference was that EPA/ DHA did not dramatically infiltrate DRMs ex vivo compared with in vitro. Our in vitro data 81 were in agreement with cell culture studies from other labs (128, 154). For example, Stulnig and coworkers showed that treatment of Jurkat T cells with EPA resulted in significant incorporation into DRMs and DSMs of SM, PC, and PEs (128). We also observed some uptake of n-3 PUFAs into the SM fraction in both cell types, similar to Stulnig's lab; however, it is possible that the SM fraction could contain some small contaminants from other phospholipids, such as phosphatidylinositols and phosphatidylserines. ! Overall, we hypothesize that the differences between our ex vivo and in vitro studies were due to differences in cell type (primary vs. immortal) and methods of lipid delivery (diet vs. exogenous addition of EPA or DHA). Furthermore, the levels of fatty acids (data not shown) and cholesterol (Supplementary Table 4.2) in EL4 cells were much higher than B cells, which was not surprising because EL4 cells are dramatically larger than primary B cells. Based on a few select experiments, we observed that ?30% of the total n-3 PUFAs incorporated into DRMs of EL4 cells, whereas ?5–8% of the total n-3 PUFAs were in DRMs of primary B cells, generally consistent with previous work from our lab and others (150, 209). The significant incorporation of EPA/DHA into DRMs in EL4 cells was not due to the dose of fatty acid in culture because even low levels of EPA or DHA (5–20 µM) showed the same distribution within error into DRMs and DSMs (data not shown). Imaging demonstrated that FO increased membrane order upon cross-linking ! Detergent extraction is a predictive tool but can induce artifacts (158, 159). An alternative method would have been to conduct studies with detergent-free methods to isolate rafts (119); however, this was beyond the scope of the study. We discovered, 82 consistent with other studies, that detergent-free methods required a very large number of cells (>100 × 106) to isolate rafts (93). Therefore, we relied heavily on imaging to determine how FO disrupted raft organization upon CTx-B cross-linking. ! The ex vivo imaging data (Figure 4.2, 4.3) were highly consistent with ex vivo data to show that fat-1 mice, which express high endogenous levels of n-3 PUFAs, displayed increased molecular order within the CD4 + T-cell immunological synapse upon activation with a cognate hybridoma cell relative to nonsynapse regions (140). Similarly, we observed that cross-linking GM1 molecules of B cells from FO-fed mice had a greater increase in molecular order relative to no cross-linking (Figure 4.3). Supporting in vitro studies showed DHA, but not EPA, increased membrane molecular order upon cross-linking relative to no cross-linking (Figure 4.5). These results were novel because very little is known about the differences in bioactivity between EPA and DHA on membrane organization. ! The ex vivo and in vitro imaging data were in agreement with our previous studies. We showed previously that treatment of EL4 cells with DHA, but not EPA, diminished CTx-B-induced clustering of EL4 cells (150). We also discovered administration of a very high dose of a fish/flaxseed oil-enriched diet to mice disrupted B cell raft organization with no impact on nonraft organization (187). The data in this study provide the first evidence for changes in B cell raft organization with a physiologically relevant dose of FO. Emerging model by which FO increases raft size ! We propose the following working model to explain the ex vivo and in vitro data. In the absence of cross-linking, n-3 PUFA incorporation into the membrane, either 83 through the diet or in culture, decreased membrane order due to the highly disordered nature of the n-3 PUFA acyl chains (105). In vivo, FO also enhanced GM1 surface levels, which could be due to targeting of GM1 biosynthesis and/or trafficking to the plasma membrane. Although the GM1 molecules were poised to form rafts in the absence of cross-linking, they were not in a state where large-scale phase separation could be observed (213, 214). ! Upon cross-linking CTx-B, the membrane became more ordered as lipid molecules were forced together and micron-scale domains became visible with FO. We speculate that FO administration resulted in the trapping of n-3 PUFAs into the large- scale raft domains. Cholesterol was not displaced out of the rafts in either model system. Instead, the mixing of cholesterol with n-3 PUFAs had an ordering effect on the membrane, as demonstrated with model membrane studies. In vitro studies demonstrated that the effects on membrane order were driven by DHA rather than by EPA. Overall, our data supported the model that FO can exert an ordering effect as opposed to the other models discussed above (140). Disruption of rafts with FO was accompanied by differential effects on innate and adaptive B cell functions ! The previous work on how FO disrupts rafts and the subsequent impact on immune cell function has centered on CD4 + T cells, macrophages, and splenocytes (140, 142, 143). Generally, manipulation of lipid microdomain organization (which may have utility as a biomarker of n-3 PUFA manipulation) with n-3 PUFAs was reported to be accompanied by immunosuppression. Here we discovered that LPS stimulation of B cells increased cytokine secretion (Figure 4.6). The data were similar to a few studies 84 showing that activation of splenic macrophages with LPS resulted in increased cytokine secretion with FO (73, 215, 216). The data were also in agreement with our previous report showing that very long-term administration of a high–fat, fish/flaxseed oil- enriched diet enhanced activation of B cells (160). We had previously suggested that the increase in B cell activation was due to body weight gain, which we now know is not the driving factor. ! The functional data (Figure 4.6) were not entirely in agreement with some in vitro studies showing that DHA suppressed LPS activation of macrophages and dendritic cells (68, 217, 218). This could be due to aforementioned differences (e.g., cell type, diet vs. in vitro treatment, etc.). Increased proinflammatory cytokine secretion does not allow us to conclude that FO is proinflammatory. It is possible that increased B cell activation could have potential clinical utility, especially in terms of antibody production. A few labs have studied the impact of FO on antibody production with mixed results (219-222). Altogether, these data highlighted the notion that FO is not universally immunosuppressive. ! The antigen presentation data were the first to demonstrate that dietary manipulation of B cells with FO suppressed naive T-cell activation (Figure 4.7). Our data confirmed several in vitro studies that showed EPA and/or DHA treatment of B cells, dendritic cells, and monocytes/macrophages diminished antigen presentation to cognate T cells (162, 223). The data were also consistent with one ex vivo study demonstrating a mixture of antigen-presenting cells (B cells, macrophages, dendritic cells) isolated from a DHA-enriched diet suppressed the proliferation of naive CD4 + T cells from mice fed control diets (224). 85 ! The functional data open the door to a wide range of new targets and mechanisms by which FO differentially affects B cell function. The mechanisms downstream of the plasma membrane by which n-3 PUFAs enhance B cell activation will require studying complex signaling pathways and their impact on gene activation. In the case of the antigen presentation studies, we now for the first time raise the possibility that lipid manipulation of the B cell side of the immunological synapse may be a major contributing factor by which T-cell activation was suppressed with n-3 PUFAs. It will be interesting to determine how manipulation of lipid microdomains can lead to an immune-enhancing effect with LPS and to a suppression of function when interacting with a T cell. It was beyond the scope of this study to address this, but it is something we aim to pursue in order to develop FO as a therapeutic agent for chronic inflammation. Conclusion ! We demonstrated that a physiologically relevant dose of FO increased the size of B cell rafts, GM1 surface expression, and membrane molecular order upon cross-linking CTx-B. Supporting in vitro studies suggested that the effects on membrane order were driven by DHA but not EPA. Furthermore, the B cell data supported an emerging model that increasing raft size with FO was accompanied by changes in innate and adaptive function. Overall, the results established the utility of using a relevant dose of FO for the first time to target B cell microdomain clustering and cellular function. 86 CHAPTER 5: MHC CLASS II ORGANIZATION ON THE B CELL SIDE OF THE MURINE IMMUNOLOGICAL SYNAPSE IS DISRUPTED BY DIETARY N-3 POLYUNSATURATED FATTY ACIDS Introduction ! n-3 PUFAs are unique bioactive molecules with emerging health benefits and clinical applications for treating acute and chronic inflammatory diseases (1). In particular, EPA and DHA, the major n-3 PUFAs of fish oil, are increasingly recognized as regulators of both innate and adaptive immunity (2). EPA and DHA exert their functional effects on immunity at a cellular level through several mechanisms. One central but poorly studied mechanism of these fatty acids is to disrupt the lateral organization of plasma membrane lipid-protein molecules that facilitate efficient intracellular signaling or cell-cell communication (225). In this study, we focused on the mechanistic effects of fish oil on the immunological synapse, a highly organized membrane domain formed between APCs and T cells that is critical for immune responses (131, 133). ! A series of studies over the past decade have established that n-3 fatty acids in fish oil disrupt lipid microdomain composition and protein lateral distribution of naďve CD4 + T cells in order to suppress downstream activation. For example, administration of dietary fish oil to mice suppressed recruitment of T cell PKC? into lipid raft microdomains and subsequent IL-2 secretion when activated with anti-CD3/28 antibodies (211). Likewise, in vitro treatment of Jurkat T cells with EPA diminished recruitment of signaling proteins to the immunological synapse when mixed with untreated antigen loaded APCs (139). Similar results were also reported with ex vivo studies using CD4 + T cells from fat-1 transgenic mice that endogenously produce n-3 PUFAs (140, 141). In contrast, very little is known about the effects of fish oil on the molecular organization of proteins on the APC side of the immunological synapse. ! B cells can serve as APCs in addition to their canonical role as antibody producing cells (84, 85). MHC class II proteins on the surface of B cells present antigens to CD4 + T cells by clustering at the site of the immunological synapse (226). Our lab has previously shown that B cells, isolated from mice fed a physiologically relevant dose of fish oil, suppressed IL-2 secretion from CD4 + T cells compared to a control diet (227). Furthermore, the functional changes were accompanied by diminished clustering of cholera-toxin induced GM1 microdomains with fish oil on the B cell plasma membrane. Our functional data were highly consistent with other studies that show fish oil administration to mice can indirectly suppress naďve CD4 + T cell activation by targeting accessory cells including macrophages, dendritic cells and B cells (72, 224, 228). ! In this study, we tested the hypothesis that dietary fish oil disrupts B cell MHC class II clustering and localization to the immunological synapse, a potential mechanism by which fish oil suppresses CD4 + T cell activation. We studied the impact of fish oil on murine MHC class II lateral organization in the absence and presence of the formation of the immunological synapse using a combination of imaging methods. We compared the magnitude of the effects of fish oil with two well-described pharmacological reagents known to disrupt protein lateral organization (229, 230). We used methyl-beta- cyclodextrin (M?CD) to deplete cellular cholesterol and cytochalasin D to prevent actin polymerization. Our data reveal a novel mechanism of fish oil and show for the first time that dietary lipids manipulated the APC side of the immunological synapse. 88 Experimental Procedures Mice and cells ! Male C57BL/6 mice (Charles River), 4–6 weeks old, were fed for 3 weeks a purified 5% total fat by weight control or fish oil diet (Harlan-Teklad) as previously described (227). The mouse fish oil diet models human intake of approximately 4 grams of fish oil per day, which is in use clinically for treating elevated triglycerides (52). Mice were euthanized using CO 2 inhalation and cervical dislocation. B220 + B cells were isolated from the spleens of the C57BL/6 mice and CD4 + T cells were isolated from OT- II transgenic mice as previously shown (227). All of the experiments with mice fulfilled the guidelines established by the East Carolina University Brody School of Medicine for euthanasia and humane treatment. GC ! Total lipids were extracted from B cells and the fatty acid composition was analyzed by GC as previously described (160). Areas of identified peaks were summed and each peak area is expressed as the percentage of total peak area for each sample. Only the major fatty acids are reported for simplicity. Reagents and antibodies ! M5/114 MHC class II antibody (BioXCell) was conjugated to either Cy3 or Cy5 using a standard fluorophore conjugation kit (GE Healthcare). For some experiments, B cells were treated for 15 min with 10 mM M?CD (Sigma) or 2 µg/mL cytochalasin D (Sigma). ?-FITC-CD3? molecular complex (BD Biosciences), goat-?-PKC? (Santa Cruz), and ?-goat Alexa Fluor 633 (Invitrogen) were used for immunological synapse staining. 89 TIRF microscopy and analysis ! 6.25 x 10 5 B220 + B cells were adhered to glass cover slips (Corning) coated with poly-D-lysine (Sigma) and then fixed with 4% paraformaldehyde (Electron Microscopy Sciences). For cells treated with M?CD and cytochalasin D, cells were first adhered and then treated. Cells were stained with anti-MHC class II antibody and mounted onto slides. TIRF microscopy was preformed as shown in a recent study by our lab (227). TIRF images were acquired at a penetration depth of 100 nanometers. Approximately 10 cells per experiment were analyzed for each treatment or diet using NIH ImageJ (http://rsbweb.nih.gov/ij/). The number of clusters and average cluster size was determined by creating a region of interest within a cell, confirmed by DIC, followed by setting a threshold to analyze particles. FRET microscopy and analysis ! MHC class II FRET was measured in terms of the efficiency of energy transfer from donor (Cy3) to acceptor (Cy5) fluorophores as previously described (187). Briefly, B cells were fixed with 4% paraformaldehyde and then stained with separately labeled Cy3 and Cy5 MHC class II antibodies. Cells were imaged on a Zeiss LSM 510 before and after photobleaching the acceptor fluorophore. All images were analyzed using NIH ImageJ and the FRET calc plugin for acceptor photobleaching FRET (172). Positive control images were generated using a single antibody dual labeled with both donor and acceptor. Negative control images were generated to optimize photobleaching time by using only single colors. Immunological synapse staining and analysis ! To form immunological synapses, B cells were first incubated with 10 -5 M OVA 323– 90 339 peptide (GenScript) for 1 hour at 37°C. Peptide loaded B cells from C57BL/6 mice (either from diet modified mice or treated with pharmacological agents) and CD4 + T cells from OT-II transgenic mice were then mixed (1:2) and briefly spun down on a bench-top centrifuge. Cell-cell conjugates were subsequently transferred to poly-D-lysine coated coverslips and incubated at 37°C for 30 min. Additional time points were tested for select experiments. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (Sigma) and blocked overnight in PBS with 2% fatty acid free BSA at 4°C. Cells were stained for key immunological synapse markers. ! Images were acquired on a Zeiss LSM 510 or an Olympus IX-81 FV1000 confocal microscope. Scoring of protein patching at the immunological synapse was performed as described by others (140, 231, 232). The cells were scored by BDR and SRS. The percentage of B cells forming B-T conjugates was also calculated from the same set of images. Statistics ! Reported values are means ± SEM from several independent experiments. All statistical analyses were calculated using GraphPad Prism (GraphPad Software). Select experiments for measuring the recruitment of molecules into the immunological synapse were performed blinded to ensure lack of bias. All the data sets were verified for normality. For experiments when two treatments were performed compared to a control, significance was determined by a one-way ANOVA followed by Dunnett’s post-hoc t test. For all other experiments, significance was established by a two-tailed t test. P-values < 0.05 were considered significant. Results 91 Fish oil increases B cell EPA and DHA levels ! Prior to initiating studies on MHC class II organization, we first ensured that the fish oil diet increased murine B cell levels of EPA and DHA. GC analysis revealed the fish oil diet significantly increased 16:1 levels by 75% compared to the control. EPA, 20:5 n-3, and its elongation production 22:5 n-3 were both increased relative to the control by 760% and 260%, respectively (Table 5.1). DHA, 22:6 n-3, levels increased by 66% with the fish oil diet compared to the control. This resulted in a 124% increase of total n-3 PUFA fatty acid levels with the fish oil diet. Conversely, there was a 36% decrease in 20:4 (n-6) and a 27% decrease in total n-6 PUFA fatty acid levels with the fish oil diet compared to the control (Table 5.1). The n-6/n-3 ratio was significantly decreased from 4.1 for the control diet to 1.5 for the fish oil diet (Table 5.1). These data are in agreement with our previous findings to show fish oil increases total B cell n-3 PUFA levels at the expense of n-6 fatty acids (227). Fish oil has no impact on B cell MHC class II organization in the absence of an immunological synapse ! MHC class II is expressed in clusters on the plasma membrane surface of B cells (233, 234). Therefore, we addressed if dietary fish oil disrupts B cell MHC class II IAb clustering prior to the formation of the immunological synapse. Using TIRF microscopy, we visualized MHC class II protein clustering on the plasma membrane surface (Figure 5.1A). B cells isolated from mice fed the fish oil diet showed no change in average cluster quantity compared to the control (Figure 5.1B). We also measured MHC class II protein distribution on the nanoscale using acceptor photobleaching FRET microscopy. Again, there was no significant difference in FRET efficiency of MHC class II molecules 92 Table 5.1: Fatty acid analysis of B cells Fatty Acid Control Fish Oil 14:0 0.6 ± 0.2 1.0 ± 0.2 16:0 26.9 ± 0.9 28.2 ± 0.9 16:1 1.2 ± 0.1 2.1 ± 0.2*** 18:0 26.2 ± 3.8 24.9 ± 2.6 18:1 trans 0.5 ± 0.2 0.6 ± 0.2 18:1 cis 11.8 ± 1.8 10.9 ± 1.5 18:2 (n-6) 13.4 ± 2.8 8.3 ± 2.5 18:3 (n-6) 1.3 ± 0.4 4.3 ± 2.3 18:3 (n-3) 1.2 ± 0.4 0.9 ± 0.3 20:4 (n-6) 10.6 ± 1.0 6.7 ± 0.4** 20:5 (n-3) 0.5 ± 0.4 4.3 ± 0.6*** 22:5 (n-3) 0.8 ± 0.1 2.9 ± 0.3*** 22:6 (n-3) 3.5 ± 0.4 5.8 ± 0.7* ?SFA 53.7 ± 3.9 54.0 ± 3.2 ?MUFA 13.5 ± 1.9 13.5 ± 1.6 ?PUFA (n-6) 25.2 ± 2.0 18.3 ± 2.3* ?PUFA (n-3) 6.2 ± 0.4 13.9 ± 1.4*** n-6/n-3 4.1 ± 0.3 1.5 ± 0.4*** Fatty acids were extracted from the diets and analyzed with gas chromatography. Values are the percentage of total fatty acids. Values less than 0.5 are omitted for clarity. Asterisks indicate different from control: *P < 0.05; **P < 0.01; ***P < 0.001. 93 94 Figure 5.1: Fish oil diet has no effect on B cell MHC class II lateral organization prior to synapse formation. A) Representative TIRF images of MHC class II molecules on B cells isolated from mice fed control (left) or fish oil (right) diets. Scale bar represents 5 microns. B) Quantification of the number of B cell MHC class II clusters in a given region of interest from control (white) and fish oil (black) fed mice. C) FRET efficiency for MHC class II molecules from control (white) and fish oil (black). Values in B are means ± SEM (n = 5 with 110 - 111 cells analyzed per diet). Values in C are means ± SEM (n = 4 with 39-40 cells analyzed per diet). Data in A-B were collected by Melton, M. Figure 5.1 Control Fish oil 0 5 10 15 Diet N u m b e r o f c l u s t e r s Control Fish oil 0 10 20 30 40 Diet F R E T C. B. Control Fish oil A. on the B cell surface between the fish oil diet compared to the control (Figure 5.1C). There was also no change in the surface expression of MHC class II molecules on B cells from the fish oil diet as measured with microscopy and flow cytometry (data not shown). Pharmacological agents disrupt B cell MHC class II organization in the absence of an immunological synapse ! We next tested the sensitivity of MHC class II organization by treating B cells with pharmacological agents known to disrupt protein organization (233, 235-237). We again used TIRF microscopy to visualize MHC class II protein clustering. Figure 5.2A shows representative images of B cell MHC class II clustering from control cells and those treated with M?CD or cytochalasin D. The number of MHC class II clusters was decreased by 42% with M?CD treatment (Figure 5.2B). In contrast, cytochalasin D treatment had no effect on the average number of clusters per region of interest. On the nanometer scale, M?CD treatment did not modify FRET efficiency (Figure 5.2C). However, cytochalasin D treatment of B cells decreased FRET efficiency between MHC class II molecules by 20% compared to the control. Treatment of B cells with M?CD or cytochalasin D was not associated with any change in the surface expression of MHC class II molecules (data not shown). Taken together, the micron and nanoscale measurements revealed that the B cell plasma membrane MHC class II organization was sensitive to changes in lateral organization with pharmacological agents, albeit in different ways. Naďve B and naďve T cells form a stable immunological synapse after 30 minutes of conjugation 95 96 Figure 5.2 M?CD Cytochalasin D 0 5 10 15 N u m b e r o f c l u s t e r s Treatment Control Treatment *** M?CD Cytochalasin D 0 5 10 15 20 25 F R E T Treatment Control Treatment * Control M?CD Cytochalasin D A. B. C. Figure 5.2: Cholesterol depletion and actin disruption reveal differential effects on MHC class II clustering. A) Representative TIRF images of MHC class II from control, M?CD, or cytochalasin D treated B cells. Scale bar represents 5 microns. B) Quantification of the number of MHC class II clusters in a region of interest from TIRF microscopy for control (white) and treatment (black). C) FRET efficiency for MHC class II molecules from control (white) and treatment (black). Values are means ± SEM (n = 3 with 30 - 50 cells analyzed per treatment). Asterisks indicate different from control: *P < 0.05; ***P < 0.001. Data in A-B were collected by Melton, M. ! The next goal was to determine the effect of fish oil and pharmacological agents on MHC class II lateral organization in the immunological synapse. Due to the novelty of investigating murine naďve B-naďve T synapse formation, the kinetics of synapse formation within this model system were first determined. We measured the patching of key proteins at the immunological synapse as a function of conjugation time. Representative images were acquired for B cell MHC class II and T cell CD3? and PKC? over time (Figure 5.3A). There was a 90% increase in MHC class II patching after 30 minutes, as well as a 100% increase at 45 minutes of conjugation compared to 5 minutes (Figure 5.3B). CD3? patching peaked at 30 minutes, increasing by 250% compared to 5 minutes (Figure 5.3C). PKC? patching plateaued at 30-45 minutes, increasing by 100% compared to 5 minutes (Figure 5.3C). Therefore, we selected 30 minutes of conjugation time for the next set of experiments since this time point provided the highest percentage of patching for all markers tested. Fish oil decreases B cell MHC class II and cognate CD4 + T cell PKC? recruitment to the immunological synapse independent of changes in cell-cell adhesion ! We next tested the hypothesis that in the immunological synapse, fish oil would disrupt MHC class II recruitment. B cells from mice fed fish oil or control diets were loaded with peptide and mixed with CD4 + T cells to stimulate formation of immunological synapses. Representative images of MHC class II, CD3? and PKC? staining are shown in Figure 5.4A. MHC class II patching in the synapse was decreased by 15% with the fish oil diet compared to the control (Figure 5.4B). In addition, FRET microscopy revealed a trend (P=0.07) in lowering FRET efficiency for MHC class II molecules (Figure 5.4C). 97 98 5 15 30 45 0 20 40 60 80 100 Time (min) % P a t c h i n g CD3? PKC? ** ** ** Time (min) % P a t c h i n g 5 15 30 45 0 20 40 60 80 100 ** ** MHC class II MHC class II A. 5 m i n 1 5 m i n 3 0 m i n PKC?CD3?DIC T B. B T B T B C. 4 5 m i n T B Figure 5.3 99 Figure 5.3: Localization of B and T cell molecules at the immunological synapse as a function of conjugation time. A) Representative DIC (left) and immunofluorescent confocal images of MHC class II (yellow), CD3? (green), and PKC? (red) at the immunological synapse after 5, 15, 30, and 45 min of incubation. Scale bar represents 5 microns. B) Quantification of the percentage of conjugated B and T cells displaying patching of MHC class II at the immunological synapse over time. C) Percent patching of CD3? (grey) and PKC? (black) at the immunological synapse over time. Values are means ± SEM (n = 3 - 4 with 42 - 63 cells analyzed per diet). Asterisks indicate different from 5 min: **P < 0.01. 100 CD3? PKC? 0 20 40 60 80 100 % P a t c h i n g T cell markers Control Fish oil *** OVA % C o n j u g a t e s - Peptide + Peptide 0 20 40 60 Control Fish oil Control Fish oil 0 20 40 60 80 100 Diet % P a t c h i n g * Control Fish oil 0 5 10 15 F R E T Diet P=0.07 MHC class II A. B. C o n t r o l F i s h o i l PKC?CD3?DIC C. B D. T B T E. Figure 5.4 101 Figure 5.4: Fish oil disrupts the B-T cell immunological synapse. A) Representative DIC (left) and immunofluorescent confocal images of MHC class II (yellow), CD3? (green), and PKC? (red) at the immunological synapse. B cells were isolated from mice fed a control (top) or fish oil (bottom) diet. Scale bar represents 5 microns. B) Percent patching of MHC class II at the immunological synapse for B cells from control or fish oil fed mice. C) FRET efficiency for MHC class II molecules at the site of the immunological synapse from control and fish oil fed mice. D) Percent patching of CD3? and PKC? at the immunological synapse. The B cells are from control or fish oil fed mice and T cells are from OT-II transgenic mice fed a standard chow. E) Percentage of B cells from control or fish oil fed mice that conjugate with CD4 + T cells. Values in B, D - E are means ± SEM (n = 4 with 129 - 146 cells analyzed per diet). Values in C are means ± SEM (n = 4 with 35 cells analyzed per diet). Asterisks indicate different from control: *P < 0.05; ***P < 0.001. ! To address the impact of the change in MHC class II organization with fish oil on the T cell side, we measured patching of CD3? and PKC? at the immunological synapse. CD3? organization was unmodified by the fish oil diet. However, PKC? patching decreased 27% with fish oil (Figure 5.4D). We confirmed the percentage patching data by also measuring the fluorescent intensity values of the staining for each marker inside the synapse region compared to the non-synapse region (Supplemental Figure 5.1 A,C). We then computed the ratio of the staining synapse/non-synapse (Supplemental Figure 5.1B,D). These changes in protein localization were not a consequence of decreased conjugate formation, as there was no significant difference in the percentage of B cells forming conjugates with T cells (Figure 5.4E). ! Given that previous studies have shown that EPA and/or DHA treatment can suppress cell-cell adhesion in vitro, we performed a static cell adhesion assay on B cells from the control and fish oil fed mice (139, 162). The ability of B cells to bind immunological synapse adhesion proteins VCAM-1, ICAM-1, ADAM28, and the extracellular matrix protein fibronectin was not modified by the fish oil diet (data not shown). Overall, these data show fish oil can modify MHC class II lateral organization and indirectly manipulate PKC? lateral organization in the synapse. Pharmacological agents disrupt B and T cell protein organization in the immunological synapse similar to fish oil ! Finally, we compared the effects of fish oil on the formation of the immunological synapse with M?CD and cytochalasin D treatment to B cells. Representative immunofluorescence images of MHC class II, CD3?, and PKC? are shown in Figure 5.5A. M?CD treatment of B cells decreased MHC class II patching by 23% (Figure 102 103 Control M?CD Cytochalasin D 0 20 40 60 80 % P a t c h i n g Treatment * P=0.06 Control M?CD Cytochalasin D 0 10 20 30 40 Treatment % C o n j u g a t e s Treatment F R E T Control M?CD Cytochalasin D 0 5 10 15 20 25 *** CD3? PKC? 0 20 40 60 80 100 % P a t c h i n g T cell markers Control m?cd Cytochalasin D* ** *** MHC class II A. B. C o n t r o l M ? C D C y t o c h a l a s i n D PKC?CD3?DIC C. T D. B T B T B E. CD3? PKC? 0 20 40 60 80 100 % P a t c h i n g T cell markers Control M?CD Cytochalasin D* ** *** Figure 5.5 104 Figure 5.5: Pharmacological agents disrupt the B-T cell immunological synapse. A) Representative DIC (left) and immunofluorescent confocal images of MHC class II (yellow), CD3? (green), and PKC? (red) at the immunological synapse from control (top), M?CD (middle), or cytochalasin D (bottom) treated B cells. Scale bar represents 5 microns. B) Percent patching of MHC class II at the immunological synapse for control (white), M?CD (grey), or cytochalasin D (black) treated B cells. C) FRET efficiency for MHC class II molecules from control, M?CD, or cytochalasin D treated B cells. D) Percent patching of CD3? and PKC? at the immunological synapse for control, M?CD, or cytochalasin D treated B cells. E) Percentage of control, M?CD, or cytochalasin D treated B cells that conjugate with CD4 + T cells. Values in B, D-E are means ± SEM (n = 3 with 117 - 166 cells analyzed per treatment). Values in C are means ± SEM (n = 3 with 30 cells analyzed per treatment). Asterisks indicate different from control: *P < 0.05; **P < 0.01; ***P < 0.001. 5.5B). Cytochalasin D treatment showed a trend in the reduction in MHC class II patching (P=0.06) (Figure 5.5B). We next measured FRET efficiency of MHC class II molecules at the synapse. Interestingly, despite lower localization at the synapse, M?CD treatment increased MHC class II FRET efficiency by 174% compared to the control (Figure 5.5C). Cytochalasin D treatment did not impact FRET efficiency at the synapse (Figure 5.5C). ! Similar to fish oil, treatment of B cells with M?CD and cytochalasin D also diminished protein patching on the T cell side of the immunological synapse. CD3? patching was unchanged by M?CD treatment (Figure 5.5D). However, cytochalasin D treatment of B cells decreased CD3? patching by 95% compared to control (Figure 5.5D). M?CD and cytochalasin D treatment both reduced patching of PKC? at the synapse by 20% and 26% respectively (Figure 5.5D). We also confirmed the percentage patching data here also by measuring the fluorescent intensity values of the staining for each marker inside the synapse region compared to the non-synapse region (Supplemental Figure 5.2 A,C). We then computed the ratio of the staining synapse/ non-synapse (Supplemental Figure 5.2 B,D). Although protein localization was disrupted, the percent of cells that formed conjugates was not modified by either treatment (Figure 5.5E). These data reveal that pharmacological treatment of B cells disrupts the organization of immunological synapse proteins on both the B and T cell side of the immunological synapse, similar to the fish oil diet. Discussion ! In this study we establish that fish oil-mediated disruption of B cell MHC class II organization at the immunological synapse can translate into an effect on T-cell 105 membrane organization. Our data show for the first time that fish oil can target the APC side of the immunological synapse in a manner similar to that reported for the effects of fish oil on T cells (139-141, 211). This work significantly expands our understanding of how fish oil, compared to other pharmacological agents, can target the molecular organization of B cells. Furthermore, our data establish a role for dietary lipids on the APC side of the synapse. The APC side of the immunological synapse ! Although the literature is replete with investigations on how fish oil modulates the organization of the synapse, the work-to-date has focused on the T cell side and far less is known about the molecular organization of the APC side, in particular the B cell component (238). Our findings underscore the impact of manipulating specifically B cells through dietary means on the formation of the immunological synapse. Our rationale for focusing on B cells was driven by our previous data to show that administration of fish oil to mice suppressed B cell mediated activation of naďve T cells. We did pursue select studies with CD11c + dendritic cells (DC) and discovered that activated DCs did not affect on naďve T cell activation; furthermore, fish oil had no effect on DC lipid-protein microdomain organization (data not shown, manuscript in review). ! Peptide loaded clusters of MHC class II complexes are critical for initiating T cell activation (233). Even a single MHC–peptide complex can trigger a Ca 2+ signal and a mature synapse can be formed when approximately 10 complexes are present (239). However, what is unclear is how the underlying lipid composition controls MHC class II organization and its impact on the formation of the synapse on the T cell side. To date, a few other studies have previously suggested that membrane lipids have a role in 106 disrupting the APC side of the synapse (162, 240). For example, Fooksman et al. showed that the concentration of PI(4,5)P 2 at the synapse regulated APC function (240). Sequestration of PI(4,5)P2 upon transfection with specific constructs in mouse and human B cells prevented the APCs from effective recognition by cognate T cells. These effects were likely tied to changes in the organization of the cytoskeleton. Recently, myosin 1c was identified to be co-localized with MHC class II molecules and recruited on the B cell side of the synapse (241). Our data demonstrate that dietary composition influences the APC side of the synapse, and indirectly alters the T cell side by targeting MHC class II. ! Some studies suggest that naďve B cells do not efficiently activate naďve T cells or do so only under certain circumstances (242, 243). Due to this limitation, we felt that it was imperative to establish the time course of naďve B-naďve T cell synapse formation. In our model system, we found the maximum amount of MHC class II and PKC? localized at the synapse after 30 minutes of conjugation. Watson et al. also found maximal PKC? localization at 30 minutes with naďve APCs and naďve T cells in a similar transgenic system with BALB/c mice (231). However, the population of APCs used in their study was splenocytes and not purified B cells. Another study also reported the formation of a mature synapse using human naďve B and naďve T cells, but the kinetics were not reported (138). Fish oil versus pharmacological disruption of the lateral organization of MHC class II in the absence of the synapse ! MHC class II organization is critical for immunological synapse formation and its function is dependent on the residing lipid environment (244). MHC class II molecules 107 reside in lipid raft-like domains to enhance antigen presentation to T cells (245, 246). Some have even proposed that newly synthesized MHC class II molecules are trafficked to lipid raft domains prior to antigen loading (247). Since we previously demonstrated that fish oil diminished B cell lipid microdomain clustering induced by cholera toxin cross-linking (227), we expected MHC class II organization would be disrupted with n-3 fatty acids (in the absence of a synapse). However, contrary to our expectation, our data showed no effect of fish oil on the lateral organization of MHC class II molecules prior to the formation of an immunological synapse. Perhaps, we were unable to detect subtle changes considering that a fraction of MHC class II molecules are pre-clustered on the B cell surface (248). However, another study showed that primary B cells displayed MHC class II clustering upon exposure to antigen (234). Our data support this notion, as we observed no changes in MHC class II lateral organization in the absence of antigen. ! Given that little is known about the molecular organization of the APC side of the synapse, we compared the effects of fish oil to known disrupters of membrane organization. Treatment of B cells with pharmacological agents provided varying results on MHC class II organization. Previously, Vrljic et al. showed that removal of cholesterol with M?CD decreased MHC class II diffusion (235). Our TIRF data appear to be in agreement with this study. Removing cholesterol from the plasma membrane generates solid-like domains (containing lipids with a high melting temperature) (235). This process slows protein diffusion and limits the number of protein clusters dispersed on the plasma membrane. The actin cytoskeleton also serves a role in clustering or corralling molecules on the plasma membrane surface. It was previously demonstrated 108 that cytochalasin D treatment of B cells did not change the lateral organization of MHC class II molecules (233). Umemura et al. explains this by showing that MHC class II actually undergoes hop diffusion in the plasma membrane (249). Our data suggest that the cytoskeleton may impact nanoscale spatial distribution of MHC class II molecules but may not completely remodel the surface organization observed at the micron scale. Taken together, our data show that in the absence of the synapse, fish oil and pharmacological agents have distinct effects on B cell MHC class II organization. Fish oil, similar to pharmacological agents, inhibits the ability of B cells to organize MHC class II molecules at the immunological synapse ! Although we previously reported B cells isolated from mice fed a fish oil diet suppressed T cell IL-2 secretion (227), we did not investigate the underlying mechanism at the level of the immunological synapse. Our data suggest the downstream functional effects reported previously by our lab and others are driven, in part, by an inability of MHC class II molecules to recruit to the synapse and thereby prevent recruitment of PKC?, which is an early marker for stable formation of an immunological synapse (132). ! We did not observe an effect of fish oil on B cell adhesion or surface expression of MHC class II (data not shown). It was previously reported that human B lymphoblasts treated with DHA in vitro could evade lysis by alloreactive human CD8 + T cells as a result of an inability to conjugate (162). Similarly, Stulnig and co-workers reported that EPA treatment of T cells could suppress the formation of the synapse by diminishing APC-T cell conjugation (128). Furthermore, fish oil supplementation in humans was shown to decrease the expression of adhesion molecules and MHC class II levels in monocytes, which prevented antigen presentation to autologous lymphocytes (70, 250). 109 Therefore, we conducted extensive studies on B cell adhesion to known ligands (data not shown) and found no effect of fish oil. Furthermore, we conducted select studies in which we treated immortal Raji B cells with n-3 fatty acids and indeed discovered that cell adhesion with Jurkat T cells was suppressed in vitro (data not shown). Thus, we reconcile our data with those of others based on differences between treatment in cell culture and administration of dietary fish oil to animals. ! We relied on pharmacological agents to disrupt B cells to compare with the effects of fish oil. Although there were differential effects on MHC class II clustering prior to synapse formation, both M?CD treatment and dietary manipulation of B cells resulted in diminished localization of MHC class II and PKC? at the immunological synapse. This finding is consistent with evidence that treatment of either B or T cells with M?CD results in inhibition of T cell activation (251-253). Likewise, cytoskeletal disruption of B or T cells similarly results in inhibition of T cell activation (252, 254, 255). Although we did not find a statistically significant reduction in MHC class II localization to the synapse with cytochalasin D treatment, we did observe a trend (P=0.06). Pharmacological disruption of membrane organization appears to have a similar functional impact on synapse protein localization to that of fish oil. Fish oil suppresses T cell activation by targeting both B and T cells ! Figure 5.6 shows how fish oil impacts both B and T cells during the formation of the immunological synapse. Shown in red are results from this study (Figure 5.6). Fish oil modifies the composition of the B cell plasma membrane and directly suppresses MHC class II localization and thereby prevents PKC? recruitment into the immunological synapse (Figure 5.6). Shown in blue are results from previous studies 110 111 Figure 5.6: Fish oil suppresses CD4 + T cell activation by targeting both B and T cells. MHC class II proteins localize to the immunological synapse on the surface of B cells (top) to present antigen to T cells (bottom). CD3? and PKC? localize to the immunological synapse in response to antigen stimulation by the APC, ultimately promoting downstream cytokine secretion. The cumulative work from this study and those of others show fish oil can block the process of CD4 + T cell activation by targeting molecular organization of both B cells (shown in red) and T cells (shown in blue). B cell T cell Synapse MHC class II MHC class II PKC? PKC? Synapse TCR complex TCR complex IL-2 secretion X X X X X X Effects of fish oil on T cells Effects of fish oil on B cells Thursday, September 27, 12 Figure 5.6 from other labs focusing on the impact of n-3 fatty acids on T cells (Figure 5.6) (139, 140). For instance, Kim et al. show that fat-1 mouse CD4 + T cells have reduced PKC? localization to the site of CD3/CD28 binding (140). The same group later showed that mitochondrial localization and subsequent calcium signaling was also impaired by n-3 fatty acids in the same model system (141). Combined with our previous findings of reduced T cell activation by B cells and those of others on the T cell side, we propose that fish oil exerts very similar effects on the both sides of the synapse to suppress T cell activation (Figure 5.6) (139-141). ! In summary, our data demonstrate a novel mechanism to explain how fish oil suppresses T cell activation. We specifically demonstrate that fish oil can target MHC class II organization in the synapse, which has an impact on the T cell side. Furthermore, this is the first evidence demonstrating that the molecular organization of the APC side of the immunological synapse is sensitive to dietary fish oil lipids. This work significantly expands our understanding of how fish oil, modeling current clinical use, can exert immunosuppressive effects. 112 CHAPTER 6: CONCLUSIONS Overview ! Using various high and low fat n-3 PUFA mouse diet models, we have established how n-3 PUFAs can target lipid-protein organization and thereby B cell antigen presentation. In doing so, we have advanced the field of n-3 PUFAs in a number of areas. We demonstrated that a physiologically relevant dose of fish oil dispersed clustering of B cell lipid rafts on a micron scale, and enhanced membrane molecular order upon cross-linking CTx-B. We found that the effects on lipid rafts are primarily driven by DHA and not EPA, and that n-3 PUFAs have a limited influence on non-raft lateral organization. We also demonstrate reduced activity when consuming very high doses of n-3 PUFAs. Finally, we show that fish oil suppresses B cell antigen presentation and subsequent CD4 + T cell IL-2 secretion, by disrupting the B cell side of the immunological synapse. While we have not solved all the mechanisms for how fish oil can exert immunosuppressive effects on B cells, we have demonstrated that plasma membrane lateral organization plays a critical role in this process. The challenge of studying lipid rafts ! A major limitation for studying lipid rafts is the lack of clear evidence for the specific structure and function of these membrane domains (114). Detergent extraction has some success as a predictive tool for which proteins may be localized to lipid raft domains, but this method is also very prone to inducing artifacts because of the harsh nature of detergents (158, 159). In Chapter 4 we used detergent extraction and HPLC/ GC methods to determine the alteration in phospholipid acyl chain profile upon fish oil consumption. However, in Chapters 2, 4 and 5, we also relied heavily on a combination of imaging techniques, such as FRET, TIRF, and generalized polarization, to support our findings from the lipid extraction and biochemical techniques. Mass spectrometry and other advances in lipidomics will give additional details about novel acylation of lipids or proteins by EPA/DHA. These added resources would provide tremendous benefit for finding new targets that may provide better insight into how fish oil disrupts lipid raft clustering. Here our studies provided clear visual evidence that fish oil disrupts plasma membrane domains of B cells, and we have gained some insight into how this disruption occurs. DHA, a distinct disrupter of raft clustering! ! EPA and DHA do not behave identically in targeting the plasma membrane. In Figure 4.5, we show that DHA has a much more dramatic effect on plasma membrane molecular order than EPA. We also have previously shown that treatment of EL4 cells with DHA, but not EPA, disrupted lipid raft clustering (150). The Fenton lab has since confirmed this finding in animals through specific mouse diet models (manuscript in preparation). One explanation for this may be that, when DHA is incorporated into PCs, it is twice as likely as EPA to localize in a raft-like domain (256). Inside a raft domain, without a stimulus to preserve the molecular packing and order, the highly dynamic biophysical properties of DHA would displace cholesterol and proteins. However, most DHA and EPA incorporate into PEs, which localize primarily on the inner leaflet of the plasma membrane in non-raft like domains (Figure 4.1). Yet, as we have shown in Chapter 2, non-raft proteins are not as sensitive to disrupted clustering as raft proteins. These findings establish DHA as a potent disrupter of lipid raft clustering, and could 114 possibly yield functional differences in antigen presentation, compared to EPA, which should be explored in the future. Raft clustering is dispersed on the sub-micron scale ! One of the significant questions we set out to answer was what effect the increased visible raft size has on molecular order and the nanoscale raft environment. Lipid rafts are defined as highly ordered in nature, yet n-3 PUFAs, DHA most of all, are highly disordered in nature, having a high degree of conformational mobility (105, 116). In Chapter 4, we used generalized polarization measurements to test molecular order with a unique florescent probe di-4-ANEPPDHQ. This probe is environmentally sensitive and displays an increased charge separation when excited in polar solvents (257, 258). The charge separation yields an increased dipole moment resulting in a red shift in its fluorescent emission. The calculation of membrane order is dependent on the change in water molecule penetration into the plasma membrane (259). Without a stimulus such as CTx-B cross-linking or immunological synapse formation, molecular order was decreased in B cells from the fish oil diet (Figure 4.3). Since solvent penetration is directly dependent on membrane packing order with this probe, it seems unlikely that this decrease in order was an artifact of just solvent penetration. Once a stimulus like the immunological synapse forms, DHA-PCs, unlike EPA-PCs, can interact with cholesterol, allowing for recovery in molecular order (256). This is exactly what we found in Figure 4.3. The change in order before and after cross-linking was significantly elevated with fish oil. ! In Chapter 5 we demonstrate that although synapse formation occurs, the proteins vital for signaling have been displaced from the site of the immunological 115 synapse. MHC class II shows a trend toward nanoscale disrupted clustering, and a significant decrease in synapse localization (Figure 5.4). Although we did not measure molecular order at the synapse in B cells, there is evidence for n-3 PUFAs to actually enhance molecular order at the T cell side of the immunological synapse (140). Taken together, we propose a model to describe how DHA disrupts lipid raft clustering by creating a network of disrupted nanoscale size raft pieces, which DHA-PCs separate (Figure 6.1A). Disrupting raft clustering also frees GM1 clustering, which would allow for more binding of CTx-B (181). These pieces are now more easily extensively cross- linked generating the increased visualized size. In a model of the immunological synapse, which has raft-like properties, a similar process would occur (Figure 6.2B). EPA and DHA-PEs have little affect on synapse organization. On the other hand, DHA- PC in the synapse recruits cholesterol and slows MHC class II recruitment, disrupting synapse organization and, ultimately, T cell activation. B cells are critical for autoimmune diseases ! We focused our studies on B cells, a cell type underrepresented in experiments with n-3 PUFAs, especially at the animal level. What we discovered in Chapter 5 was B cells are targetable in antigen presentation critical for subsequent CD4 + T cell activation. This is an important finding for autoimmune diseases such as rheumatoid arthritis, lupus, allograft rejection, and chronic allergic lung disease, where directed targeting of B cell antigen presentation would have clinical utility. In addition, n-3 PUFAs have been shown to promote a T helper 2 (Th2) response instead of a T helper 1 (Th1) response (260-264). Th1 cells produce pro-inflammatory cytokines IL-2, IFN-?, and TNF-?, while Th2 cells produce anti-inflammatory cytokines IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 116 117 Fish oil B cells Th1 T cell response Beneficial impact: Rheumatoid arthritis Allograft rejection Inflammation Th2 T cell response Negative impact: Allergies Lung inflammation ? Figure 6.1 Lipid raft declustering B cell Clustered raft non-raft 2.Rafts are declustered non-raft 1.DHA-PC infiltrates rafts 3.Crosslinking yields micron- scale increased raft size declustered raft non-raft non-raft B cell T cell Immunological synapse 4.MHC class II 5.Activation 3.Cholesterol 1.EPA/DHA PE 2.DHA PC A. B. C. 118 Figure 6.1: DHA disrupts lipid raft clustering and the immunological synapse. A) DHA-PC (red) can localize into clustered lipid rafts (yellow). This declusters the raft and when cross-linked the micron scale size appears larger. B) In B cells EPA/DHA can incorporate into the plasma membrane as PEs, but localize outside of the immunological synapse (yellow). DHA incorporated as PC can localize in the synapse along with cholesterol. This displaces signaling proteins such as MHC class II for the site of the immunological synapse, diminishing T cell activation. C) Overview of potential positive and negative affect of fish oil consumption on cell mediated and humoral immune response. (265). Th2 cells are protective in diseases such as rheumatoid arthritis, type 1 diabetes, and multiple sclerosis, whereas Th1 cells are pathogenic (266). However, promoting Th2 immunity may not be universally beneficial. For example, in the case of lung inflammation disorders like allergic asthma, a Th2 response enhances progression of the disease (267-269). Though not fully investigated, this study demonstrates that fish oil in B cells may contribute to Th2/Th1 as evidenced by the reduced IL-2 secretion though disrupted antigen presentation (Figure 4.7B). While there are many important potential pathways to which fish oil can contribute, the implications of membrane disruption could lead to breakthroughs in other disease models. In Figure 6.1C is an outline describing these implications. A shift from Th1 to Th2 would decrease cell mediated immune response and increase the humoral immune response. This would not be universally beneficial for all diseases. In addition, studies looking at the other functions of B cells such as antibody production will further determine the utility of fish oil supplementation. Precaution for high doses of fish oil ! Once the mechanisms are better established, moving fish oil into the clinic will still have other considerations. One of these is the dosing necessary for clinical efficacy. The dose of fish oil used in many other studies varies widely, and dosing remains one of the larger challenges for strengthening our understanding of how fish oil exerts its effects (52). Our novel findings of body weight gain and energy expenditure reductions shown in Chapter 3 raise questions that must be addressed for proper clinical and dietary recommendations. Although fish oil is a source of EPA and DHA, these fatty acids only make up a modest percentage of the total fatty acid profile of fish oil. Analysis 119 of our fish oil diets shows a full fatty acid profile (Supplemental Table 2.1). In addition, there are some additional contaminants from consuming fish or fish oil including: methylmercury, dioxins, polychlorinated biphenyls, and organic pollutants from industrial processing (270). Monitoring the metabolic implications and dietary formulation for fish oil supplementation should be done in combination with any clinical studies moving forward. Conclusion ! 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JAMA 296, 1885–1899. 156 APPENDIX A: ANIMAL USE PROTOCOL A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9 A-10 A-11 A-12 A-13 A-14 A-15 A-16 A-17 A-18 A-19 A-20 A-21 A-22 A-23 A-24 A-25 A-26 A-27 A-28 A-29 A-30 A-31 A-32 A-33 A-34 A-35 A-36 APPENDIX B: SUPPLEMENTAL TABLES AND FIGURES Supplemental Table 2.1: Fatty acid analysis of experimental diets Fatty Acid CD LF (n-3) PUFA HF (n-3) PUFA HF (n-6) PUFA 14:0 0.5 3.1 3.7 4.6 16:0 12.2 14.6 13.6 10.0 16:1 0.1 4.3 4.9 0.1 18:0 6.9 8.7 7.2 7.5 18:1 trans 0.5 1.5 0.9 0.5 18:1 cis 20.5 16.0 15.7 15.5 18:2 (n-6) 48.0 13.6 12.1 58.3 18:3 (n-3) 6.5 24.1 26.9 1.4 20:5 (n-3) 0.0 4.4 5.0 0.1 22:2 (n-6) 0.2 0.7 0.6 0.0 22:5 (n-3) 0.1 0.8 0.8 0.0 22:6 (n-3) 0.0 3.3 3.5 0.1 ?SFA 20.4 27.0 25.0 22.6 ?MUFA 23.0 24.3 24.0 17.1 ?PUFA (n-3) 6.6 32.6 36.2 1.5 ?PUFA (n-6) 50.0 16.1 14.8 58.7 Fatty acids were extracted from the diets and analyzed with gas chromatography. Values are the percentage of total fatty acids. Values less than 0.5 are omitted for clarity. B-2 Supplemental Figure 2.1: C16-Bodipy imaging studies. (A) EL4 cells were co- stained with FAST-DiI and C16-Bodipy. The images are from a single z-stack used to calculate percent co- localization. The data are representative of 2 separate experiments. Co-localization is indicated by orange in the merge. (B) Average C16- Bodipy intracellular and plasma membrane image intensity for EL4 cells treated overnight with BSA, OA, AA, EPA, and DHA. Data are means ± SEM, n= 4. )$67'L, &%RGLS\ 0HUJH 6XSSOHPHQWDO)LJXUH&%RGLS\LPDJLQJVWXGLHV $ (/FHOOVZHUHFRVWDLQHGZLWK )$67'L,DQG&%RGLS\7KHLPDJHVDUHIURPDVLQJOH]VWDFNXVHGWRFDOFXODWHSHUFHQWFR ORFDOL]DWLRQ7KHGDWDDUHUHSUHVHQWDWLYHRIVHSDUDWHH[SHULPHQWV&RORFDOL]DWLRQLVLQGLFDWHG E\RUDQJHLQWKHPHUJH % $YHUDJH&%RGLS\LQWUDFHOOXODUDQGSODVPDPHPEUDQHLPDJH LQWHQVLW\IRU(/FHOOVWUHDWHGRYHUQLJKWZLWK%6$2$$$(3$DQG'+$'DWDDUHPHDQV? 6(0Q  2QOLQH6XSSRUWLQJ0DWHULDO $ % Supplemental Figure 2.1 B-3 %&\ &\ %H IR UH $I WH U $ 6XSSOHPHQWDO)LJXUH0+&FODVV,PROHFXOHVZHUHUDQGRPO\GLVWULEXWHGRQ(/FHOOV $ 3RVLWLYHFRQWUROEHIRUHDQGDIWHUSKRWREOHDFKLQJLPDJHVRI(/FHOOVVWDLQHGZLWK&\ GRQRU  DQG&\ DFFHSWRU ODEHOHGDQWLERGLHVDJDLQVW0+&,)5(7HIżFLHQF\LQFUHDVHGE\a % 6DPSOHGDWDIURPDVLQJOHH[SHULPHQWWRVKRZ%6$RU(3$WUHDWPHQWGLGQRWPRGLż\0+&, FOXVWHULQJVLQFHHIżFLHQF\YDOXHVGLGQRWLQFUHDVHZLWKLQFUHDVLQJGRQRUDFFHSWRUUDWLR+RZHYHU QRWH(3$WUHDWPHQWORZHUHG)5(7YDOXHVUHODWLYHWR%6$ 2QOLQH6XSSRUWLQJ0DWHULDO Supplemental Figure 2.2 Supplemental Figure 2.2: MHC class I molecules were randomly distributed on EL4 cells. (A) Positive control before and after photobleaching images of EL4 cells stained with Cy3 (donor) and Cy5 (acceptor) labeled antibodies against MHC I. FRET efficiency increased by ~80%. (B) Sample data from a single experiment to show BSA or EPA treatment did not modifiy MHC I clustering since efficiency values did not increase with increasing donor:acceptor ratio. However, note EPA treatment lowered FRET values relative to BSA. B-4 Supplemental Figure 2.3: (n-3) PUFA diets did not increase FAST-DiI accumulation but HF (n-3) PUFA diet lowered C16-Bodipy uptake. (A) Fluorescent images of B cells isolated from C57BL/6 mice fed CD, HF (n-6), LF or HF (n-3) PUFA diets and stained with FAST-DiI. (B) Fluorescent C16-Bodipy images of B cells isolated from mice fed differing diets. Images are on a rainbow palette to discriminate differences in relative fluorescence intensity. Red and blue values, respectively, indicate high and low fluorescence intensity. 2QOLQH6XSSRUWLQJ0DWHULDO 6XSSOHPHQWDO)LJXUH Q 38)$GLHWVGLGQRWLQFUHDVH)$67'L,DFFXPXODWLRQEXW +) Q 38)$GLHWORZHUHG&%RGLS\XSWDNH $ )OXRUHVFHQWLPDJHVRI%FHOOVLVRODWHG IURP&%/PLFHIHG&'+) Q /)RU+) Q 38)$GLHWVDQGVWDLQHGZLWK)$67'L, %  )OXRUHVFHQW&%RGLS\LPDJHVRI%FHOOVLVRODWHGIURPPLFHIHGGLIIHULQJGLHWV,PDJHVDUHRQD UDLQERZSDOHWWHWRGLVFULPLQDWHGLIIHUHQFHVLQUHODWLYHŔXRUHVFHQFHLQWHQVLW\5HGDQGEOXHYDOXHV UHVSHFWLYHO\LQGLFDWHKLJKDQGORZŔXRUHVFHQFHLQWHQVLW\ $ &' )$ 67 ' L, +) Q 38)$ /) Q 38)$ +) Q 38)$ &  %R GLS \ % Supplemental Figure 2.3 B-5 Supplemental Figure 2.4: HF (n-3) PUFA diet disrupted lipid raft clustering. Fluorescent images of B cells isolated from C57BL/6 mice fed CD, HF (n-6), LF or HF (n-3) PUFA diets and stained for lipid rafts. &' +) Q 38)$ /) Q 38)$ +) Q 38)$ 2QOLQH6XSSRUWLQJ0DWHULDO 6XSSOHPHQWDO)LJXUH+) Q 38)$GLHWGLVUXSWHGOLSLGUDIWFOXVWHULQJ)OXRUHVFHQW LPDJHVRI%FHOOVLVRODWHGIURP&%/PLFHIHG&'+) Q /)RU+) Q 38)$GLHWVDQG VWDLQHGIRUOLSLGUDIWV Supplemental Figure 2.4 Supplemental Table 4.1: Analysis of major fatty acids in mouse diets. Fatty Acid Control Fish oil 14:0 0.2 6.7 16:0 11.7 24.6 16:1 0.1 13.1 18:0 5.2 6.3 18:1 trans 0.1 0.1 18:1 cis 21.6 12.6 18:2 (n-6) 53.7 3.1 18:3 (n-6) 0.3 0.6 18:3 (n-3) 6.9 1.7 20:4 (n-6) 0.0 1.3 20:5 (n-3) 0.0 15.7 22:5 (n-3) 0.0 0.3 22:6 (n-3) 0.0 11.2 ?SFA 17.2 37.6 ?MUFA 21.9 25.8 ?(n-6) PUFA 54.0 5.5 ?(n-3) PUFA 6.9 28.8 B-6 B-7 Supplemental Figure 4.1 0 1 2 3 16 18 20 22 24 26 B o d y w e i g h t ( g ) Week Control Fish oil Control Fish oil 0.00 0.02 0.04 0.06 0.08 0.10 Diet S p l e e n ( g ) ** Control Fish oil 0 1 2 3 4 Diet F o o d c o n s u m e d ( g / d a y ) A. B. C. Supplemental Figure 4.1: FO increased spleen size. (A) Body weights as a function of time for mice fed a control or FO diet. (B) Average daily food consumption. (C) Spleen weight after 3 weeks. Values are means ± SEM, n = 20-30 mice. Asterisks indicate different from control: **P< 0.01. B-8 Control Safflower oil 0.0 0.2 0.4 0.6 0.8 1.0 Diet F e r e t ' s d i a m e t e r ( µ m ) Control Safflower oil 0 500 1000 1500 2000 Diet I n t e n s i t y A. B. Control Safflower oil C. Supplemental Figure 4.2 Supplemental Figure 4.2: Safflower oil-enriched diet did not increase lipid raft size. (A) TIRF microscopy images of B cell lipid rafts, induced upon cross-linking, from mice fed a control or safflower-enriched diet for 3 weeks. (B) Quantification of lipid raft size. (C) CTxB fluorescence intensity after cross-linking. Values are means ± SEM, n = 4. Supplemental Table 4.2: Cholesterol levels in DRM and DSM fractions from ex vivo and in vitro studies. Mice were fed for 3 weeks a control or FO diet; EL4 cells were treated overnight with BSA, EPA, and DHA, as described in the Methods. Cholesterol values are fluorescence per µg of protein. Values are mean ± SEM from 4-5 independent experiments. P values are for the DRM/DSM ratio. DRM DSM DRM/DSM p value Control 419.8 ± 285.5 66.9 ± 6.1 7.1 ± 5.3 Fish Oil 305.6 ± 91.6 68.1 ± 3.5 4.6 ± 1.5 0.66 BSA 4962.6 ± 988.0 190.2 ± 31.0 27.2 ± 4.9 EPA 2390.2 ± 395.4 207.1 ± 25.9 12.7 ± 2.9 0.08 DHA 3425.4 ± 1490.9 204.5 ± 21.5 15.5 ± 5.7 0.08 B-9 B-10 Supplemental Figure 4.3: FO had no impact on non-raft organization as measured by uptake of DiIC 18 . (A) Sample images of B cells from control and FO fed mice stained with DiIC 18 . (B) Quantification of DiIC 18 intensity. Values are means ± SEM, n = 3-4. Control Fish oil 0 200 400 600 800 1000 I n t e n s i t y Diet A. B. Control Fish oil Supplemental Figure 4.3 B-11 Supplemental Figure 4.4: EPA and DHA treatment had no effect on GM1 surface levels. GM1 surface levels (A) before and (B) after cross-linking rafts with CTxB upon treatment of EL4 cells with EPA or DHA relative to BSA.   Values are means ± SEM, n = 4. BSA EPA DHA 0 500 1000 1500 I n t e n s i t y Treatment BSA EPA DHA 0 200 400 600 800 1000 I n t e n s i t y Treatment A. B. Supplemental Figure 4.4 B-12 A r b i t r a r y u n i t s MHC class II MHC class II 0 200 400 600 800 Control Fish oil ** SynapseNon-synapse A r b i t r a r y u n i t s CD3? PKC? CD3? PKC? 0 500 1000 1500 2000 Control Fish oil ** SynapseNon-synapse Supplemental Figure 5.1 Supplemental Figure 5.1: Fish oil disrupts the B-T cell immunological synapse. A) Fluorescent intensity of MHC class II staining outside (Cell) and inside (Synapse) the immunological synapse for B cells from control (white) or fish oil (black) fed mice. B) Ratio of fluorescent intensity Synapse/Cell values for MHC class II staining. C) Fluorescent intensity of CD3? and PKC? staining outside (Cell) and inside (Synapse) the immunological synapse for B cells from control or fish oil fed mice. D) Ratio of fluorescent intensity Synapse/Cell values for CD3? and PKC? staining. Values are means ± SEM (n = 4 with 66 - 69 cells analyzed per diet). Asterisks indicate different from control: *P < 0.05; **P < 0.01. Control Fish oil 0 1 2 3 R a t i o Diet * CD3? PKC? 0 1 2 3 4 R a t i o T cell markers Control Fish oil * A. B. C. D. B-13 CD3? PKC? CD3? PKC? 0 500 1000 1500 2000 2500 A r b i t r a r y u n i t s Control M?CD Cytochalasin D SynapseNon-synapse * *** ** *** MHC class II MHC class II 0 200 400 600 800 1000 A r b i t r a r y u n i t s Control M?CD Cytochalasin D SynapseNon-synapse * ** CD3? PKC? 0 1 2 3 4 5 T cell markers R a t i o Control M?CD Cytochalasin D Control MBCD Cytochalasin D 0 1 2 3 4 Treatment R a t i o ** ** Supplemental Figure 5.1 Supplemental Figure 5.2: Pharmacological agents disrupt the B-T cell immunological synapse. A) Fluorescent intensity of MHC class II staining outside (Cell) and inside (Synapse) the immunological synapse for B cells from control (white) or M?CD (grey) and cytochalasin D (black) treatment. B) Ratio of fluorescent intensity Synapse/Cell values for MHC class II staining. C) Fluorescent intensity of CD3? and PKC? staining outside (Cell) and inside (Synapse) the immunological synapse. D) Ratio of fluorescent intensity Synapse/Cell values for CD3? and PKC? staining. Values are means ± SEM (n = 4 with 66 - 69 cells analyzed per diet). Asterisks indicate different from control: *P < 0.05; **P < 0.01. A. B. C. D.