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Automated Template B: Created by James Nail 2011V2.1 Validating pasture heaves as an equine model of neutrophilic asthma: a systems biology approach By Lauren A Bright A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Veterinary Medical Research in the College of Veterinary Medicine Mississippi State, Mississippi May 2015 Copyright by Lauren A Bright 2015 Validating pasture heaves as an equine model of neutrophilic asthma: a systems biology approach By Lauren A Bright Approved: ____________________________________ Cyprianna E. Swiderski (Major Professor) ____________________________________ Shane C. Burgess (Co-Major Professor) ____________________________________ Fiona M. McCarthy (Committee Member) ____________________________________ Bindu Nanduri (Committee Member) ____________________________________ Andy D. Perkins (Committee Member) ____________________________________ Andrew J. Mackin (Graduate Coordinator) ____________________________________ Kent H. Hoblet Dean College of Veterinary Medicine Name: Lauren A Bright Date of Degree: May 8, 2015 Institution: Mississippi State University Major Field: Veterinary Medical Research Major Professor: Dr. Cyprianna E. Swiderski Title of Study: Validating pasture heaves as an equine model of neutrophilic asthma: a systems biology approach Pages in Study: 197 Candidate for Degree of Doctor of Philosophy Asthma is a chronic respiratory disease characterized by reversible airway obstruction, persistent airway hyperresponsiveness, chronic airway inflammation, and chronic airway remodeling. Most adult asthmatics have neutrophilic airway inflammation that correlates to increasing disease severity, and fail to respond to corticosteroid therapies that mitigate other asthma endotypes. Accordingly, there is a need to investigate the molecular mechanisms responsible for neutrophilic asthma. Pasture heaves, a respiratory disease affecting horses housed on pasture in conditions of high heat and humidity, shares the aforementioned characteristics of human asthma, including neutrophilic inflammation. The cause is undetermined, but genetic propensities for reactivity to seasonally inhaled, pasture-associated, aeroallergens are presumed. Complexities of diseases like asthma and pasture heaves, that include temporal interactions between environmental and genetic factors, lend themselves to exploration using -omics technologies. An emergent paradigm in disease pathogenesis views disease as the result of imbalances in a biological system of thousands of proteins that maintain eukaryotic homeostasis. Consistent with this paradigm, this dissertation describes systematic efforts to identify groups of proteins in the bronchoalveolar lavage fluids of horses with pasture heaves that are altered in a manner that influences neutrophilic airway inflammation, and are similarly changed in human asthma. This is the first use of -omics technologies to investigate pasture heaves. This was accomplished first by improving functional annotation of the equine genome by providing functional annotation for an equine oligoarray, thereby facilitating future functional modeling of equine gene products. Next, through comparative modeling of protein functions in normal bronchoalveolar lavage fluid (BALF) proteomes from horse, human, and mouse, we demonstrated conservation of protein functions in lung fluids across these species. Finally, comparative modeling of pasture heaves-affected and non-diseased BALF proteomes demonstrated that proteins in diseased BALF favor airway neutrophilic inflammation by increasing neutrophil migration, chemotaxis, adhesion, detachment, transmigration, and degranulation, while reducing activation, cell spreading, infiltration, phagocytosis, respiratory burst, apoptosis, and clearance. Collectively, these molecular events contribute to airway neutrophilic inflammation in pasture heaves, and are conserved in human asthma. This method further validates pasture heaves as a robust model for human neutrophilic asthma, and highlights proteins of potential clinical and therapeutic relevance. DEDICATION To Mom and Dad, for believing in your little miss "selfie-do." I love you, always. ii ACKNOWLEDGEMENTS I would like to sincerely thank my advisor, Dr. Cyprianna Swiderski, for her guidance, patience, and most importantly, for her friendship during my graduate studies at Mississippi State University. Her mentorship was paramount in my success in the dual- degree program. She has always been very supportive and encouraging, and leads by example through dedication and ability, as a clinician, a researcher, and in life. For everything you have done for me, Dr. Swiderski, I thank you. I am truly fortunate to have worked with you, and I look forward to our continued friendship. I would also like to thank my committee members Drs. Shane Burgess, Fiona McCarthy, Bindu Nanduri, and Andy Perkins. Throughout the years I have known you, you have always supported me and looked out for my best interests. Thank you for the guidance, thought-provoking questions, and collegiality you have shown me. A special thanks to Dr. Lais Costa, for providing the samples I used in my research. Without your efforts, this project would never have taken off. Thank you to Courtney Hunter, who shared the dual degree journey with me. Your sense of humor helped keep things light. You are a rock star and will continue to impress those around you for many years. Thanks to Nisma Mujahid and Santosh Kumar TK for your research support. I enjoyed our conversations in the laboratory, and your help was invaluable. iii I would also like to thank Drs. Kent Hoblet, Mark Lawrence, and Andrew Mackin for support of my research efforts and of the DVM/PhD program throughout the years. Thanks also to the Mississippi State University College of Veterinary Medicine faculty who contributed to aspects of this research: Drs. Andrew Claude, Alison Eddy, Michael Brashier, and Jackie Bowser. Finally, I would like to thank my family. My parents, Steve and Laura Bright, did everything right. You guys held my hand when I was little, and let me fly when I was ready. Because of your faith in me, I was able to be just as ambitious as I needed to be. My siblings, Beau and Kristen Bright, are two unfortunate souls who had to grow up with me. You two may be younger than me, but you teach me more than you realize, thank you. My fiancé, Chris Bradley, is truly an amazing man. You have helped me weather the storm over the past few years, and your presence in my life has made even the bad times bearable. Thank you for always having faith in me, and of course for taking care of sweet Zoe, Petunia, and Agatha Faye. I love you and I am looking forward to many more years together. iv TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .............................................................................................................x CHAPTER I. INTRODUCTION .............................................................................................1 References ..........................................................................................................6 II. LITERATURE REVIEW ................................................................................10 Asthma overview .............................................................................................10 History and prevalence of asthma ..............................................................10 Causes of asthma........................................................................................11 Signs, symptoms, and diagnosis of asthma ................................................13 Salient features of asthma ..........................................................................14 Reversible airway obstruction ..............................................................14 Airway hyperresponsiveness ...............................................................16 Airway inflammation ...........................................................................18 Histopathology of asthma: airway remodeling ....................................20 Airway smooth muscle thickening.................................................21 Airway epithelial thickening ..........................................................21 Epithelial barrier function ..............................................................22 Goblet cell hyperplasia/metaplasia, mucus gland hypersecretion ....................................................................23 Airway fibrosis...............................................................................25 Treatment of asthma ..................................................................................26 Therapeutic controversies in asthma ....................................................29 Asthma endotypes ......................................................................................32 Neutrophilic airway inflammation in asthma ......................................33 Animal models of asthma ..........................................................................36 Murine