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An Analysis of Between-Cow Variation in Innate Immunity in Relation to Mastitis Severity Filiz Korkmaz University of Vermont

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An Analysis of Between-Cow Variation in Innate Immunity in Relation to Mastitis Severity Filiz Korkmaz University of Vermont University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2018 An Analysis of Between-Cow Variation in Innate Immunity in Relation to Mastitis Severity Filiz Korkmaz University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Immunology and Infectious Disease Commons, and the Microbiology Commons Recommended Citation Korkmaz, Filiz, "An Analysis of Between-Cow Variation in Innate Immunity in Relation to Mastitis Severity" (2018). Graduate College Dissertations and Theses. 856. https://scholarworks.uvm.edu/graddis/856 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. AN ANALYSIS OF BETWEEN-COW VARIATION IN INNATE IMMUNITY IN RELATION TO MASTITIS SEVERITY. A Dissertation Presented by Filiz Korkmaz to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Cellular, Molecular and Biomedical Sciences May, 2018 Defense Date: January 9, 2018 Dissertation Examination Committee: David E. Kerr, Ph.D., Advisor Matthew E. Poynter, Ph.D., Chairperson Stephanie D. McKay, Ph.D. John Barlow, Ph.D., DVM. Jonathan Boyson, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Bovine mastitis remains one of the costliest diseases affecting the dairy industry. Individual susceptibility to mastitis and severity of infection varies between animals and can only be partially explained by genetics. As such, understanding how genetic predisposition coordinately interacts with epigenetic modifications and environmental exposures is necessary to bridge the gap in missing heritability. The role of DNA methylation in regulating the response to bacterial lipopolysaccharide (LPS) was first determined by performing reduced representation bisulfite sequencing on fibroblasts isolated from heifers at 5- and 16-months of age that exhibit an age-dependent up- regulation in LPS-responsiveness. More than 14,000 differentially methylated sites were identified between the two sets of cultures with a trend towards decreased methylation with age. Young cultures were also hyper-methylated in gene promoters regulated by NF-κB and exhibited lower expression in genes that regulate the innate immune response, suggesting that methylation contributes to gene regulation in fibroblast innate response. Previously, TLR4 expression was shown to differ in the age-dependent fibroblast model, however, it was not known if variation in TLR4 expression would affect mastitis severity. Therefore, fibroblasts were isolated from sixty lactating, adult Holstein cows and their expression of TLR4 , along with LPS-induced production of IL-8 and IL-6, was used to rank the animals from high to low. Six high responders and six low responders were then experimentally infected in one mammary gland with E. coli . Overall, severity of mastitis was quite variable, with a few notable differences between high and low responders. High responding animals had an earlier increase in somatic cell count and febrile response that coincided with more efficient bacterial clearance. However, tissue damage and milk production did not differ between the two groups, indicating that while rapid up-regulation of the innate response addresses bacterial clearance, subsequent down- regulation is required to alleviate damage within the mammary gland. Finally, one-week old bull calves were subjected to treatment with either saline or LPS to determine if neonatal exposure to endotoxin would make calves less responsive to a second LPS challenge at 32-days of age. The initial treatment showed a large effect of LPS as measured by higher plasma IL-6 and TNF-α concentrations in calves treated with LPS over saline. Subsequent treatment of all 10 calves with LPS showed a very similar response between the two treatment groups and significant inter-animal variability in clinical response. Fibroblasts and monocyte-derived-macrophages (MDMs) were also isolated following initial treatment to determine if any changes occurred at the cellular level as a result of LPS exposure. Fibroblasts isolated from calves at 20-days of age had a very low response to LPS that did not differ between the early life treatments. MDMs isolated from calves at 28-days of age were more responsive to LPS, but again no differences were detected between the early life treatments. In summary, our results suggest that DNA methylation likely plays a role in the cellular response to LPS and may partially contribute to differences between animals in severity of E. coli mastitis, however, the appropriate in vitro phenotype to detect susceptible animals still needs to be characterized before epigenetic biomarkers can be identified, and perhaps modified by environmental interventions. CITATIONS Material from this dissertation has been published in the following form: Korkmaz, F.T. & Kerr, D.E.. (2017). Genome-wide methylation analysis reveals differentially methylated loci that are associated with an age-dependent increase in bovine fibroblast response to LPS. BMC Genomics . (18), 405-423. Benjamin, A.L., Korkmaz, F.T., Elsasser, T.H. & Kerr, D.E.. (2016). Neonatal lipopolysaccharide exposure does not diminish the innate immune response to a subsequent lipopolysaccharide challenge in Holstein bull calves. Journal of Dairy Science . 99(7), 5750-5763. Material from this dissertation has been submitted for publication to Journal of Dairy Science on December 31, 2017 in the following form: Korkmaz, F.T., Elsasser, T.H. & Kerr, D.E.. Variation in fibroblast expression of TLR4 and LPS-Induced cytokine production between-animals predicts control of bacterial growth but not severity of Escherichia coli mastitis. ii ACKNOWLEDGEMENTS First and foremost, I would like to acknowledge my mentor Dr. David Kerr, for his patience and guidance throughout the four and half years we worked together to complete the research that contributed to my dissertation, and some that did not. His confidence in my abilities as a scientist were critical in my success over the years. In addition, I would like to thank Drs. Benjamin Green and Aimee Benjamin, for their assistance with laboratory techniques, large animal handling and for their good spirits that got me through many long days. Outside the lab, Aimee was also an essential part of my success through her friendship and for pushing me to do things I never thought I was capable of (two marathons!). Next, my fellow graduate students, both in the CMB and Animal Science departments, who were always there to bounce ideas off of and to make everyday life much more enjoyable; thank you. I consider myself very lucky to have met each and every one of you. In addition, it is imperative to acknowledge the UVM Miller Research Farm staff, specifically Scott Shumway, Matthew Bodette and countless undergraduate students, all of whom made sure my experiments went as smoothly as possible. Also, my wonderful family (Mom, Baba, Teoman & Maya) and friends (Andrea and Audrey), I sincerely appreciate your support throughout this journey that would have been far more arduous without you all by my side. Finally, and most importantly, I would like to thank my husband, Rob Markwith and my fur-babies, Lucy and Gonzo, for your unconditional love. Thank you. iii TABLE OF CONTENTS CITATIONS ....................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii CHAPTER 1: COMPREHENSIVE LITERATURE REVIEW ......................................... 1 1.1 Bovine Mastitis: Prevalence and Economic Impacts .................................................... 1 1.2 Causative Agents of Mastitis ........................................................................................ 2 1.3 Between-Animal Variation in Mastitis Severity ........................................................... 3 1.4 Immune Response to Mastitis ....................................................................................... 5 1.5 Inflammatory Response Regulation: What is the Appropriate Response? ................... 8 1.6 Cellular Models for in vivo Response ........................................................................ 11 1.7 Genetics of the Immune Response to Mastitis ............................................................ 13 1.8 Regulation of Immunity by Non-Genetic Mechanisms .............................................. 15 1.9 Epigenetic Regulation of Immunity in Agricultural Species ...................................... 18 1.10 Environmental Effects on Epigenetic Modifications and Immunity ........................ 20 1.11 Unanswered Questions.............................................................................................. 23 CHAPTER 2: GENOME-WIDE METHYLATION ANALYSIS REVEALS DIFFERENTIALLY METHYLATED LOCI THAT ARE ASSOCIATED WITH AN AGE-DEPENDENT INCREASE IN BOVINE
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