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University of California, San Diego UNIVERSITY OF CALIFORNIA, SAN DIEGO A Lipidomic Perspective on Inflammatory Macrophage Eicosanoid Signaling A Thesis submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemistry by Paul Christopher Norris Committee in charge: Professor Edward A. Dennis, Chair Professor Pieter C. Dorrestein Professor Partho Ghosh Professor Christopher K. Glass Professor Michael J. Sailor 2013 The Dissertation of Paul Christopher Norris is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2013 iii DEDICATION To my parents, Darrell and Kathy, for always allowing me to think (and choose) for myself. iv TABLE OF CONTENTS Signature page ............................................................................................................................ iii Dedication .................................................................................................................................. iv Table of contents ......................................................................................................................... v List of symbols and abbreviations ........................................................................................... viii List of figures ............................................................................................................................. xi List of tables ............................................................................................................................. xiv Acknowledgements ................................................................................................................... xv Curriculum Vita ...................................................................................................................... xvii Abstract of the Dissertation ...................................................................................................... xx Chapter 1. Introduction To Macrophage Biology And Eicosanoid Signaling ................... 1 1.A Introduction ............................................................................................................ 2 1.B Macrophages .......................................................................................................... 3 1.B.1 Macrophage origins and phenotypic variability ..................................... 3 1.B.1.a What is a macrophage? .......................................................... 3 1.B.1.b Haematopoiesis and macrophage lineages ............................. 4 1.B.1.c Monocytes .............................................................................. 6 1.B.2 Functions of the macrophage ................................................................. 7 1.B.2.a The “janitor” role: phagocytosis ............................................ 7 1.B.2.b The “sentinel” role: responding to danger signals ................. 7 1.B.2.c Activation states: M1 vs. M2 ................................................. 8 1.B.2.d Wound healing ..................................................................... 11 1.B.2.e Eicosanoid metabolism in macrophages .............................. 12 1.B.3 Primary and immortalized macrophages for ex vivo studies ................ 12 1.B.4 Macrophages in health and disease ...................................................... 14 1.B.4.a Effects of macrophage ablation ............................................ 14 1.B.4.b Role in atherosclerosis: macrophage foam cells .................. 15 1.B.4.c Role in insulin resistance: Type-2 diabetes .......................... 16 1.B.4.d Macrophages and cancer ...................................................... 16 1.C Eicosanoids .......................................................................................................... 17 1.C.1 Major classes of eicosanoids and other oxidized fatty acids ................ 17 1.C.1.a Cyclooxygenase metabolites ................................................ 17 1.C.1.b 5-Lipoxygenase metabolites ................................................ 20 1.C.1.c 8-, 12-, and 15-Lipoxygenase metabolites ........................... 23 1.C.1.d Cytochrome P450 metabolites ............................................. 25 v 1.C.1.e Non-enzymatic AA metabolites: isoprostanes and racemic HETEs .................................................................... 27 1.C.1.f Nitrated fatty acids ................................................................ 29 1.C.1.g Esterified eicosanoids .......................................................... 29 1.C.2 Cellular biosynthetic control of eicosanoids and related species ......... 30 1.C.2.a Phospholipase A2 isoforms ................................................... 30 1.C.2.b Functional enzyme coupling ................................................ 32 1.C.2.c Transcellular eicosanoid metabolism ................................... 34 1.C.3 Eicosanoids and therapeutic intervention ............................................. 36 1.D Discussion ........................................................................................................... 38 1.E Acknowledgments .............................................................................................. 39 Chapter 2. Specificity of Eicosanoid Production Depends on the TLR-4 Stimulated Macrophage Phenotype .................................................................................................... 40 2.A Abstract ................................................................................................................ 41 2.B Introduction .......................................................................................................... 42 2.C Results .................................................................................................................. 45 2.C.1 Eicosanoid production in TLR-4 stimulated macrophages .................. 45 2.C.2 Induced COX-2 expression controls total prostanoid production in different macrophage cell types ..................................... 51 2.C.3 Terminal prostanoid synthase expression ............................................ 53 2.C.4 Enzyme competition for PGH2 differs between cells with and without PGIS ................................................................ 55 2.D Discussion ............................................................................................................ 59 2.D.1 Lipidomic analysis of different macrophage phenotypes .................... 59 2.D.2 Macrophages share a singular mechanism of COX-2 metabolism ...... 60 2.D.3 Correlating enzyme expression with metabolite profiles .................... 61 2.D.4 Primary cells vs. cell lines and the advantage of their differences ...... 64 2.E Experimental Procedures ...................................................................................... 66 2.F Acknowledgments ................................................................................................ 70 Chapter 3. Omega-3 Fatty Acids Cause Dramatic Changes in TLR4 and Purinergic Eicosanoid Signaling ...................................................................................... 71 3.A Abstract ................................................................................................................ 72 3.B Introduction .......................................................................................................... 73 3.C Results .................................................................................................................. 75 3.C.1 Effects of PUFA supplementation on membrane composition ............ 75 3.C.2 Analysis of PUFA release after supplementation ................................ 77 3.C.3 Effects of PUFA supplementation on TLR4 eicosanoid metabolism .. 82 3.C.4 Effects of PUFA supplementation on purinergic stimulated eicosanoid metabolism ......................................................................... 87 vi 3.C.5 Effects of 22-carbon fatty acid supplementation on eicosanoid metabolism ......................................................................... 89 3.D Discussion ............................................................................................................ 95 3.D.1 Fatty acid elongation and membrane incorporation ............................ 95 3.D.2 PLA2 activity on different membrane PUFA compositions ................ 95 3.D.3 Inhibition of COX-1 and COX-2 ......................................................... 96 3.D.4 AA, EPA, and DHA supplementation shift AA metabolism to higher 5-LOX vs. COX-1 .................................................................... 97 3.D.5 Importance of considering AdA and DPA in understanding ω3 metabolism ..................................................................................... 98 3.E Experimental methods .......................................................................................... 99 3.F Acknowledgments .............................................................................................. 103 Chapter 4. Macrophages Produce Lipoxins During Inflammasome Activation ........... 104 4.A Abstract .............................................................................................................. 105 4.B Introduction ........................................................................................................ 106 4.C Results ...............................................................................................................
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