ABSTRACT WALTER, KATHLEEN ROSE. Enhancement of Porcine
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ABSTRACT WALTER, KATHLEEN ROSE. Enhancement of Porcine Alveolar Macrophage Response to Respiratory Pathogens by Dietary Supplementation of Long-chain PUFA. (Under the direction of Dr. Jack Odle and Dr. Debora Esposito). Despite common medicinal treatments, respiratory infections are one of the most reported health problems afflicting the swine industry. Leading causes of respiratory-related diseases and deaths in the swine industry are attributed to gram-negative bacteria and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Novel approaches involving the use of dietary nutrients as alternative methods or used in conjunction with vaccines to alleviate the severity of respiratory infections in swine are sparse. Dietary long-chain polyunsaturated fatty acids (LC PUFA), such as arachidonic acid (ARA; n-6) and eicosapentaenoic acid (EPA; n-3), demonstrate immunomodulatory effects on host immune response to infection. A key link between immune cell function and long-chain PUFA is the production of lipid mediators, specifically eicosanoids. Arachidonic acid synthesizes potent eicosanoids, such as prostaglandin E2 (PGE2), which promotes pro-inflammatory and antiviral signaling at the onset of an acute challenge, and subsequently hinders its initial response to promote anti-inflammatory and pro-resolving cascades (termed lipid-mediator class switching). Previous studies geared toward assessing benefits and potential negative impacts of long-chain PUFA supplementation during infection are reviewed in Chapter I. This research assessed the utilization of dietary pre-formed ARA or EPA to enrich the long-chain PUFA content of piglet alveolar macrophages (PAM), and their ability to modify eicosanoid and cytokine production following LPS or PRRSV challenge. In Chapter II, we evaluated if feeding milk-replacer supplemented with LC n-6 PUFA to neonatal pigs could provide a heighted but balanced innate immune response following in situ LPS stimulation. Day-old pigs were allotted to one of three dietary groups for 21 days (n = 20/diet), and received either a control diet (CON, arachidonate = 0.5% of total fatty acids), an arachidonate (ARA)-enriched diet (LC n-6, ARA = 2.2%), or an eicosapentaenoic (EPA)- enriched diet (LC n-3, EPA = 3.0%). Results demonstrated a 2-fold increase of ARA enrichment in PAM from LC n-6 fed pigs compared to CON and LC n-3. A similar enrichment was observed in EPA enrichment from LC n-3 fed pigs, while the concentration of ARA decreased 2-fold. Abundance of COX-2 and TNF-α mRNA (P < 0.0001), and PGE2 secretion (P <0. 01) were higher in LC n-6 PAM vs CON after 24 hours of LPS stimulation, although abundance of ALOX5 was 1.6-fold lower than CON. Abundance of ALOX12/15 was 4-fold higher in abundance (P < 0.0001) in CON and LC n-6 PAM compared to LC n-3. A dietary interaction for oxidative burst was not observed. These findings suggest LC n-6 supplementation in neonatal pig diets can enrich the ARA content in PAM, which results in increased mRNA abundance of pro- inflammatory cytokines and eicosanoid production, while initiating early signaling for lipid- mediator class switching within a 24-hour period. In Chapter III, PUFA-enriched PAM were utilized to determine if ARA-enrichment could improve the innate immune response of PAM and dampen some of the immunosuppressive effects elicited when challenged with PRRSV in situ. By 12 hours post infection (hpi), LC n-6 PAM displayed a 73% increase in mRNA abundance of antiviral cytokines, while having 67% less transcription of viral nucleocapsid compared to LC n-3. Additionally, by 12hpi pro- inflammatory cytokine mRNA abundance was upregulated while anti-inflammatory (IL-10) mRNA was downregulated. These results suggest ARA-enriched PAM can dampen immunosuppressive effects of PRRSV by upregulating antiviral and pro-inflammatory cytokine mRNA abundance and downregulating viral replication. In conclusion, dietary LC n-6 PUFA demonstrates an effective means to enhance a stronger, well-balanced innate immune response to respiratory challenges in neonatal pigs, and could provide an alternative therapeutic agent to promote a quicker return to a stable herd-health status, and lessen the overall economic impact of respiratory diseases in the swine industry. © Copyright 2019 by Kathleen Rose Walter All Rights Reserved Enhancement of Porcine Alveolar Macrophage Response to Respiratory Pathogens by Dietary Supplementation of Long-chain PUFA by Kathleen Rose Walter A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Animal Science & Poultry Science Raleigh, North Carolina 2019 APPROVED BY: _______________________________ _______________________________ Dr. Jack Odle Dr. Debora Esposito Committee Co-Chair Committee Co-Chair _______________________________ _______________________________ Dr. Lin Xi Dr. Matthew Koci DEDICATION My work is dedicated to my dad Michael A. Walter and in loving memory of my mom Clara A. Walter. Thank you for your endless and unconditional love, support and encouragement to pursue my dreams. My accomplishments would have never been possible without the both of you. Everything I am is because of the amazing parents you are. I could not be more blessed. Thank you. I love you! ii BIOGRAPHY Kathleen Rose Walter was born in Falls Church, Virginia. Her and her family relocated to Charlotte, NC when she was six years old. After high school, Kathleen moved to Raleigh, NC for her undergraduate degree at North Carolina State University. She received a Bachelor’s of Science in Animal Science and minor in Genetics. In 2015, she did a PhD curriculum transfer to the Animal Science department at North Carolina State University where she joined Dr. Jack Odle’s lab. From her previous experiences in Dr. Sunny Liu and Dr. Chad Stahl’s labs, Kathleen was able to merge her previous training and knowledge with the work accomplished in Dr. Odle’s lab. In 2016, Kathleen was introduced to Dr. Debora Esposito who is stationed at NC State’s satellite location in Kannapolis, NC at the North Carolina Research Campus. At the end of 2016, Dr. Esposito became Kathleen’s co-PI and invited her to finish her research at the Plants for Human Health Institute on the NC Research Campus. Kathleen graciously accepted and has been building a strong network with other research groups in Kannapolis since. From Kathleen’s unconventional path through the PhD program, she was able to design and develop her project which merges her knowledge and experience of all labs. Her work focuses on the use of dietary lipids to improve the response of respiratory immune cells to common pathogens that afflict the swine industry. Kathleen’s passion centers around the desire to improve the quality of life in humans and animals. As such, she is dedicated to continuing her career in the field of nutritional immunology. iii ACKNOWLEDGMENTS I would like to give a special thanks to my advisor, Dr. Jack Odle, for his continuous guidance, support and encouragement. I am extremely honored to have been given the opportunity to work with him. I would also like to give a special thanks to my co-advisor, Dr. Debora Esposito, for graciously welcoming me into her lab, as well as her guidance and support throughout the remainder of my degree. Additionally, I would like to extend my most sincere gratitude to the rest of my committee, Dr. Lin Xi and Dr. Matthew Koci for all of their assistance, invaluable feedback and contributions throughout my project. Thank you to all of you for recognizing my potential and motivating me to overcome all the obstacles I have faced throughout my graduate school career. Special appreciation is extended to Dr. Tobias Käser and his lab at NC State’s Veterinary School for their time, resources, training and collaboration on my research project. This project would not have been made possible without the mentoring and training I received in previous labs before joining Dr. Odle and Dr. Esposito. A huge thank you to Dr. Sunny Liu for her advising throughout my undergraduate degree and giving me my first opportunity in graduate school. Dr. Liu’s teaching and training helped me to realize that my true passion lies in research. I am forever grateful. I would also like to thank Dr. Julie Hicks from Dr. Liu’s lab for all of her training, assistance and friendship over the years. A big thank you to Dr. Chad Stahl, the training received in his lab gave me my appreciation and interest of nutrition’s role in regulating gene expression. The brief time in his lab, also gave me to opportunity to be introduced and welcomed into Dr. Odle’s lab. My education, training and experience in Dr. Liu’s and Dr. Stahl’s labs made the conceptualization and execution of my research project possible. iv Special thanks are given to Dr. Jia Xiong and Sierra Boney in the Esposito lab for their help and support since coming to the NC Research Campus. A huge thank you to Dr. Slavko Komarnytsky and Mickey Wilson for their assistance, training and allowing use of equipment that was vital for my research. Appreciation is extended to the faculty and staff in the Animal Science department, thank you for the wonderful opportunities and support I have received over the years. My academic journey would not have been made possible without the love and support of family and friends. To my parents Michael and Clara Walter, thank you for your unconditional love, encouragement and support. Thank you for giving me an amazing childhood filled with love and laughter. Thank you for always guiding me to aim for my dreams and be the best person I can be. For teaching me to put everything in God’s hands, and that “what is meant to be, will be”.