In Vitro Effects of Polycyclic Aromatic Hydrocarbons and Crude Oil o

164 Pages·2017·5.07 MB·English

by  Laura Bourque

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.
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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.
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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.
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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
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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

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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

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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
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