Environmental Contaminants and Respiratory Innate Immunity: In Vitro Effects of Polycyclic Aromatic Hydrocarbons and Crude Oil on Tracheal Antimicrobial Peptide Gene Expression By: Laura A Bourque A Thesis Presented to The University of Guelph In partial fulfillment of requirements For the degree of Doctor of Veterinary Science In Pathobiology Guelph, Ontario, Canada © Laura Bourque, March, 2017 ABSTRACT ENVIRONMENTAL CONTAMINANTS AND RESPIRATORY INNATE IMMUNITY: IN VITRO EFFECTS OF POLYCYCLIC AROMATIC HYDROCARBONS AND CRUDE OIL ON TRACHEAL ANTIMICROBIAL PEPTIDE GENE EXPRESSION Laura Alice Bourque Advisor: University of Guelph, 2017 Dr. Jeff Caswell Respiratory disease is an important cause of morbidity and mortality in cetaceans, which are also threatened by environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs; immunotoxic constituents of crude oil). Cetaceans exposed to oil spills may inhale volatile PAHs which could potentially affect respiratory immunity. PAHs activate the aryl hydrocarbon receptor which can affect immunity through interaction with NF-κB. β-defensins are potent antimicrobial peptides that play an important role in defending the lower airway from pathogens. β-defensin expression is known to be dependent on NF-κB, and so we hypothesized that PAHs may suppress pulmonary β-defensins and to pneumonia pathogenesis. This hypothesis was modeled by measuring the effects of benzo(a)pyrene (BAP), phenanthrene, naphthalene, and crude oil on tracheal antimicrobial peptide (TAP) gene expression in primary cell cultures of bovine tracheal epithelial cells. It was found that exposure to either crude oil or PAHs was associated with suppression of TAP gene expression in cultures. However, the consistency of this result varied in significance between both PAHs and calves. BAP consistently caused a statistically significant (P = 0.002) suppression of TAP gene expression in a dose dependent manner in the majority of calves. These are the first data to show that exposure to PAHs suppresses β-defensin gene expression. Acknowledgments Successful completion of my DVSc would not have been possible without the wisdom and technical support of the following people. I would like to first thank my advisor, Dr. Jeff Caswell. Jeff indulged and encouraged my “whale project” and, throughout its duration, was a great source of encouragement and expertise for me to draw upon. I could not have wished for a better advisor. I would like to thank my advisory committee: Drs. Pierre-Yves Daoust, Stephen Raverty, and Brandon Lillie. Both Pierre-Yves and Stephen inspired me during my DVM degree to pursue my interest in pathology as a profession, and in particular to craft that interest around wildlife (one of the inspirations of this project). If not for them, I would not be here. Brandon provided invaluable expertise during my days in the lab. I knew that I could stop by his office and troubleshoot just about any PCR difficulty that came up. Thank you one and all. I would like to thank all the people who contributed to the lab work of this project, first and foremost being Mary Ellen Clark. Mary Ellen, I will not be the first graduate student to be eternally grateful for your laboratory expertise nor, I am certain, will I be the last. I would like to thank our summer students, Carmon Co and Alaina Macdonald who slaved tirelessly to extract my RNA samples before a looming deadline. I would like to thank Bruce Hollebone from Environment Canada for his donation of Alberta crude oil samples, and the National Sciences and Engineering Council of Canada (NSERC) for funding this project. As well, a huge thank you to Peel Sausage Inc. for providing the trachea for my research; without their generosity, this project would have been impossible. iii Finally, I would like to thank my family and friends (especially those from the “microscope room”) who provided the moral support during my DVSc which, ultimately, saw the completion of my DVSc. iv Declaration of Work Performed The work described in the following manuscript was completed by myself with the following exceptions: 1. Statistical analysis was completed by William Sears, statistical consultant in Population Medicine. 2. Some RNA extraction and cDNA synthesis was performed by Carmon Co and Alaina MacDonald for the BAP experiments. 3. Some RNA extraction, cDNA synthesis, and PCR analysis for the second oil and CYP450 experiments were completed by Mary Ellen Clark. v Table of Contents Acknowledgments ................................................................................................................... iii Declaration of Work Performed................................................................................................ v Table of Contents ..................................................................................................................... vi List of Tables .......................................................................................................................... viii List of Figures .......................................................................................................................... ix List of Abbreviations ................................................................................................................ x Chapter 1: Literature Review ................................................................................................... 1 1.1 Introduction ....................................................................................................................... 1 1.2 Cetacean respiratory anatomy .......................................................................................... 3 1.3 Pneumonia in cetaceans .................................................................................................. 5 1.4 Physiological stress and pneumonia ................................................................................. 9 1.5 Stress physiology and immuno-suppression ....................................................................11 1.5 Effects of glucocorticoids on NF-kB and immune defences .............................................14 1.6 Innate pulmonary immunity ..............................................................................................18 1.7 Beta defensins .................................................................................................................22 1.8 Beta defensins, stress, and air pollution ..........................................................................29 1.9 Polycyclic aromatic hydrocarbons, crude oil, and immune suppression ...........................34 1.10 Rationale and objectives ................................................................................................40 Hypothesis .........................................................................................................................42 Objectives ..........................................................................................................................42 Chapter 2: Materials and Methods .........................................................................................43 2.1 Induction of in vitro TAP gene expression in bovine tracheal epithelial cells ....................43 2.1.1 Acquisition of bovine trachea.....................................................................................43 2.1.2 Culture of bovine tracheal epithelial cells ..................................................................43 2.1.3 Lipopolysaccharide stimulation of tracheal epithelial cells .........................................45 2.2 Polycyclic aromatic hydrocarbon exposure of tracheal epithelial cells ..............................45 2.4 Crude oil exposure of tracheal epithelial cells grown at an air-liquid interface ..................46 2.5 RNA extraction and cDNA synthesis from tracheal epithelial cell cultures ........................47 2.6 Real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) for tracheal antimicrobial peptide gene expression .....................................................................48 2.7 Statistical analysis ...........................................................................................................50 vi Chapter 3: Results ..................................................................................................................53 3.1 Phenotypic characterization of tracheal epithelial cell cultures .........................................53 3.2 Effects of lipopolysaccharide (LPS) and dimethyl sulfoxide (DMSO) on tracheal antimicrobial peptide gene expression ...................................................................................53 3.3 Effects of benzo(a)pyrene on tracheal antimicrobial peptide gene expression .................53 3.4 Statistical analysis of effects of 5 μM BAP for 8 hours .....................................................54 3.5 Effects of naphthalene on tracheal antimicrobial peptide gene expression ......................55 3.6 Effects of phenanthrene on tracheal antimicrobial peptide gene expression ....................56 3.7 Effects of benzo(a)pyrene on cytochrome P450 1A1 (CYP450-1A1) gene expression ....57 3.8 Effects of crude oil on tracheal antimicrobial peptide gene expression ............................57 Chapter 4: Discussion ............................................................................................................70 Conclusion ..............................................................................................................................89 References ..............................................................................................................................91 Appendices ........................................................................................................................... 118 Appendix 1: The use of tracheas from different calves in the various in vitro experiments ... 118 Appendix 2: Representative experimental layout for well cell culture plates ......................... 119 Appendix 3: Experimental protocol for cryopreservation of tracheal mucosa for epithelial cell culture. ................................................................................................................................ 120 Appendix 4: Sequence of bovine CYP450-1A1 amplicon .................................................... 123 Appendix 5: Standard curve and efficiency for TAP primers ................................................ 124 Appendix 6: Standard curve and efficiency for GAPDH primers .......................................... 125 Appendix 7: Raw RT-RTqPCR data for BAP analyses ........................................................ 126 Appendix 8: Raw statistical data calculated using Proc MIXED (SAS 9.2). .......................... 138 Appendix 9: Preliminary sequence analyses of putative cetacean β-defensins .................... 149 vii List of Tables Page Table 1. Primer sequences and product sizes for RT-qPCR analysis........................................51 Table 2. The use of tracheas from different calves in the various in vitro experiments ……….118 Table 3. A typical 24-well plate layout for a dose-response experiment………………………...118 Table 4. A typical 24-well plate layout for a time-course experiment……………………………119 Table 5. A typical 12-well layout for air liquid interface for oil exposure experiment…………..119 viii List of Figures Page Figure 1. β-Defensin peptide sequence…………………………………………………………..…23 Figure 2. Typical polycyclic aromatic hydrocarbon structure……………………………………...35 Figure 3. Chemical structures representative of experimental PAHs…………………………….51 Figure 4. Representative image of tracheal epithelial cell microcolonies………………………..52 Figure 5. Representative image of 100% tracheal epithelial cell confluency……………………52 Figure 6. Immunohistochemical analysis of cultured bovine tracheal epithelial cells…………..59 Figure 7. Effects of different times of exposure to BAP on TAP gene expression……………...60 Figure 8. Effects of varying concentrations of BAP on TAP gene expression…………………..61 Figure 9. Effects of varying concentrations of BAP on TAP gene expression (5 calves)………62 Figure 10. TAP expression in 9 calves exposed to 5 μM of BAP for 8 hours …………………..63 Figure 11. Effects of different times of exposure to naphthalene on TAP gene expression…...64 Figure 12. Effects of varying concentrations of naphthalene on TAP gene expression………..65 Figure 13. Effects of different times of exposure to phenanthrene on TAP gene expression…66 Figure 14. Effects of varying concentrations of phenanthrene on TAP gene expression……...67 Figure 15. Effects of BAP on cytochrome (CY) P450-1A1 gene expression……………………68 Figure 16. Effects of crude oil on TAP gene expression…………………………………………..69 Figure 17. A hypothesized relationship of how the interaction between AHR and NF-κB affects TAP gene expression……………………………………………………………..88 ix List of Abbreviations ACTH adrenocorticotropic hormone ANOVA analysis of variance AHR aryl hydrocarbon receptor BAP benzo(a)pyrene BRDC bovine respiratory disease complex bTEC bovine tracheal epithelial cell CCR chemokine receptor CD cluster of differentiation cDNA complementary DNA CMV cetacean morbillivirus CRH corticotropin releasing hormone DEP diesel exhaust particles DNA deoxyribonucleic acid DMSO dimethyl sulfoxide FBS fetal bovine serum GAPDH glyceraldehyde-3-phosphate dehydrogenase GM-CSF granulocyte macrophage-colony stimulating factor GR glucocorticoid receptor GRE glucocorticoid response element HEK human embryonic kidney cell HPA Hypothalamus pituitary adrenal hBD human beta-defensin iDC immature dendritic cells IFN-γ interferon gamma IκB inhibitor of NF-κB IL interleukin LAP lingual antimicrobial peptide LC-DC Langerhan cell like dendritic cell LPS lipopolysaccharide MAPK mitogen-associated protein kinase mRNA messenger ribonucleic acid NAPH naphthalene NF-κB nuclear factor-κB PAMP pathogen associated molecular pattern PAH polycyclic aromatic hydrocarbons PBS phosphate buffered saline PCR polymerase chain reaction PHEN phenanthrene PRR pattern recognition receptor PTG peptidoglycan rhLZ human recombinant lysozyme RNA ribonucleic acid RNase H ribonuclease H ROFA residual oil fly ash RT-PCR reverse transcription PCR RT-qPCR reverse transcription quantitative PCR RSV Respiratory syncytial virus x