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Constitutive Variation in Hepatic Expression of Porcine Innate Immune Genes Associated with Novel Promoter Polymorphisms By Heindrich Nicolaas Snyman A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph In partial fulfilment of the requirements for the degree of Doctor of Veterinary Science Guelph, Ontario, Canada © Heindrich Nicolaas Snyman, November, 2013 ABSTRACT CONSTITUTIVE VARIATION IN HEPATIC EXPRESSION OF PORCINE INNATE IMMUNE GENES ASSOCIATED WITH NOVEL PROMOTER POLYMORPHISMS Heindrich N. Snyman Advisor University of Guelph, 2013 Dr. Brandon Lillie Infectious diseases are an important factor limiting production, growth performance, economics and animal welfare in the global swine industry. Numerous pathogenic, environmental and host genetic factors contribute to infectious disease susceptibility, including defects in the innate immune system. The secreted, membrane-bound and intracellular proteins of the innate immune system are critical components in the defense against infectious pathogens, especially in young pigs. Previous studies have identified a number of single nucleotide polymorphisms (SNPs) in the innate immune genes of pigs, some of which are more frequent in pigs with economically important infectious diseases such as enteritis, serositis and pneumonia. Additional novel genetic differences (SNPs, insertions, deletions or other genetic variations) that alter the expression and/or function of important resistance-conferring innate immune proteins could, together with previously identified markers, form the basis for future development of genetic selection strategies aimed at increasing innate disease resistance in pigs. In this thesis, genome-wide expression analysis and DNA sequencing were used to screen for polymorphisms that were associated with decreased or increased expression of selected innate immune genes in the liver of healthy market weight pigs. Using expression microarrays, innate immune genes were identified that exhibited widely variable hepatic gene expression. The promoter regions of these variably expressed genes were characterized and novel promoter SNPs, insertions and deletions were identified in genes coding for proteins such as secretoglobin, family 1A, member 1; porcine surfactant protein D; retinoic acid-inducible gene 1 and porcine hepcidin antimicrobial peptide. Some of these SNPs were found to be more prevalent in animals with either impaired or enhanced hepatic gene expression. Genotyping healthy and diseased pigs using MassArray MALDI-TOF mass spectrometry revealed that genetic defects were widely variable in their frequency in different breeds of pigs and some were more or less frequent in pigs with certain common infectious diseases and/or pathogens. Information from these studies will be used to develop a comprehensive panel of genetic markers that can be used to breed for pigs with increased disease resistance. Such a panel could help improve pig production, promote animal health and welfare, and decrease the need for antimicrobial drugs. ACKNOWLEDGEMENTS I would like to extend my most sincere gratitude to my advisor and mentor, Dr. Brandon Lillie, whose support, guidance and continued encouragement was an integral component to the success of my research project and graduate training as a veterinary pathologist. I am also extremely grateful to the pathologists within the Department of Pathobiology for their mentorship and training throughout my journey towards becoming a successful veterinary pathologist and to my thesis advisory committee, Dr. Jeff Caswell, Dr.Tony Hayes and Dr. Jim Squires for their invaluable insights, advice and support throughout my DVSc studies. I would also like to sincerely thank Dr. Jutta Hammermueller for sharing her knowledge and experimental expertise and to Dr. Jim Squires (Department of Animal & Poultry Science), Dr. Alex Martinez (CFIA), Dr. Balu Odedra (CFIA) and the pathologists at the Animal Health Laboratory, University of Guelph for assistance in obtaining liver samples. I am very appreciative of the assistance provided by research assistants and summer research students involved in this project; especially Kelsie Jagt, Sarah Mavin, Eric Nham and David Balogh. I am also extremely grateful to Dr. Edana Cassol for sharing her expertise and guidance in generating microarray expression heat maps and Dr. Sharon Cassol for always being available to assist me in whichever way possible. I express my gratitude to all funding agencies (Ontario Veterinary College; NSERC; OMAFRA, Ontario Pork and the Canadian Centre for Swine Improvement) for their financial assistance. Without their support this research would not have been possible. To my parents and family: “Ek wil ook baie graag my ouers en familie bedank vir hul onvoorwaardelike liefde en ondersteuning vir alles wat ek aanpak in my lewe”. Finally, I am eternally grateful for the endless love and support of my wife, Roberta, and children, Zander and Myra, whom have made my Canadian journey especially rewarding. iv DECLARATION OF WORK PERFORMED With few exceptions (as listed below), all of the work in this thesis was performed by Heindrich Nicolaas Snyman under the supervision of Dr. Brandon Lillie and an advisory committee composed of Dr. Jeff Caswell, Dr. Tony Hayes and Dr. Jim Squires. Dr. Brandon Lillie assisted with the collection of liver samples. Dr. Jutta Hammermueller (Department of Pathobiology, University of Guelph) assisted with some of the nucleic acid extractions and real-time PCR assays. Kelsie Jagt, a summer research student (Department of Molecular and Cellular Biology, University of Guelph) under my supervision performed some of the real-time PCR, promoter region sequencing and restriction fragment length polymorphism analyses for surfactant protein D. David Balogh, an in-course master’s degree student (Department of Biomedical Science, University of Guelph) under my supervision performed some of the initial primer design and promoter amplification for peptidoglycan recognition proteins 1 and 2. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................................... iv DECLARATION OF WORK PERFORMED ........................................................................................ v LIST OF TABLES ............................................................................................................................... viii LIST OF FIGURES ............................................................................................................................... xi LIST OF ABBREVIATIONS .............................................................................................................. xiii GENERAL INTRODUCTION, HYPOTHESES AND OBJECTIVES ................................................. 1 GENERAL INTRODUCTION .......................................................................................................... 1 HYPOTHESES ................................................................................................................................... 3 OBJECTIVES ..................................................................................................................................... 3 CHAPTER 1: STRUCTURE, FUNCTION AND GENETICS OF PORCINE INNATE IMMUNE PROTEINS…… ..................................................................................................................................... 4 INTRODUCTION .............................................................................................................................. 4 TOLL-LIKE RECEPTORS ................................................................................................................ 6 COLLECTINS AND FICOLINS (COLLAGENOUS LECTINS) ................................................... 13 C-TYPE LECTIN RECEPTORS AND SCAVENGER RECEPTORS ........................................... 21 GALECTINS .................................................................................................................................... 22 RETINOIC ACID-INDUCIBLE GENE-1-LIKE RECEPTORS ..................................................... 26 NUCLEOTIDE OLIGOMERIZATION DOMAIN-LIKE RECEPTORS ....................................... 30 ANTIMICROBIAL PEPTIDES ....................................................................................................... 34 PEPTIDOGLYCAN RECOGNITION PROTEINS ......................................................................... 38 COMPLEMENT ............................................................................................................................... 42 GENETIC POLYMORPHISM AND THE INNATE IMMUNE GENOME ................................... 46 CHAPTER 2: CONSTITUTIVE VARIATION IN THE HEPATIC INNATE IMMUNE TRANSCRIPTOME OF HEALTHY PIGS .......................................................................................... 53 ABSTRACT ..................................................................................................................................... 53 INTRODUCTION ............................................................................................................................ 54 MATERIALS AND METHODS ..................................................................................................... 56 RESULTS ......................................................................................................................................... 62 DISCUSSION ..................................................................................................................................

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