Medicine, Faculty of
Medicine, Department of
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UBCV
Huff, Ryan D.
Doctor of Philosophy - PhD
University of British Columbia
Air pollution is a pervasive and global problem, with 99% of the world’s population living in areas that exceed the World Health Organization exposure guidelines. Although public policies and legislation continue to lower anthropomorphic emissions, and engineering solutions such as air purifiers work to limit personal exposures, few therapeutic strategies exist to limit the cellular effects of air pollution. To address this issue, I evaluated multiple approaches to counteracting air pollution-induced oxidative stress and inflammation using several in vitro models of particulate matter (PM) exposure on human lung epithelial cells. Using immortalized and primary human epithelial cells exposed to PM, I showed that two antioxidants, tetrahydrocurcumin and quercetin, significantly attenuated cellular reactive oxygen species (ROS) and inflammatory mediator release. Next, I examined the utility of modulating lung epithelial cell responses to PM by priming them with omega-3 fatty acids, precursors of specialised pro-resolving mediators. I demonstrated that docosahexaenoic acid significantly decreased PM-induced pro-inflammatory mediator release and cellular ROS. Finally, I noted that suppressing inflammation in the lungs has been associated with increased susceptibility to viral infections, an association also observed with air pollution exposures. Thus, I investigated the effect of exposing human epithelial cells to air pollution, as well as their treatment with a corticosteroid, a common medication used to control inflammation, on rhinovirus infectivity. I demonstrated that pre-exposure of epithelial cells to the corticosteroid fluticasone propionate and PM synergistically increased rhinovirus levels, suppressed host defence responses, and increased levels of the rhinovirus entry receptor. In summary, this thesis’ investigation of air pollution interventions and possible consequences both provides novel therapeutic options and identifies a potential risk, evidence that collectively may inform comprehensive strategies to mitigate the risk associated with air pollution exposure.
INVESTIGATION OF AIR POLLUTION INTERVENTIONS IN AIRWAY EPITHELIAL CELLS by Ryan D. Huff B.M.Sc., The University of Western Ontario, 2011 M.Sc., The University of British Columbia, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2024 © Ryan D. Huff, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Investigation of Air Pollution Interventions in Airway Epithelial Cells submitted by Ryan D. Huff in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine Examining Committee: Christopher Carlsten, Professor, Respiratory Medicine, UBC Supervisor Jeremy Hirota, Assistant Professor, Department of Medicine, McMaster University Supervisory Committee Member William Lockwood, Associate Professor, Pathology and Laboratory Medicine, UBC University Examiner Andrew Sandford, Professor, Medicine, UBC University Examiner Kymberly Gowdy, Associate Professor, Division of Pulmonary, Critical Care and Sleep Medicine, Ohio State University External Examiner Additional Supervisory Committee Members: David Granville, Professor, Pathology and Laboratory Medicine, UBC Supervisory Committee Member Stephan van Eeden, Professor, Medicine, UBC Supervisory Committee Member iii Abstract Air pollution is a pervasive and global problem, with 99% of the world’s population living in areas that exceed the World Health Organization exposure guidelines. Although public policies and legislation continue to lower anthropomorphic emissions, and engineering solutions such as air purifiers work to limit personal exposures, few therapeutic strategies exist to limit the cellular effects of air pollution. To address this issue, I evaluated multiple approaches to counteracting air pollution-induced oxidative stress and inflammation using several in vitro models of particulate matter (PM) exposure on human lung epithelial cells. Using immortalized and primary human epithelial cells exposed to PM, I showed that two antioxidants, tetrahydrocurcumin and quercetin, significantly attenuated cellular reactive oxygen species (ROS) and inflammatory mediator release. Next, I examined the utility of modulating lung epithelial cell responses to PM by priming them with omega-3 fatty acids, precursors of specialised pro-resolving mediators. I demonstrated that docosahexaenoic acid significantly decreased PM-induced pro-inflammatory mediator release and cellular ROS. Finally, I noted that suppressing inflammation in the lungs has been associated with increased susceptibility to viral infections, an association also observed with air pollution exposures. Thus, I investigated the effect of exposing human epithelial cells to air pollution, as well as their treatment with a corticosteroid, a common medication used to control inflammation, on rhinovirus infectivity. I demonstrated that pre-exposure of epithelial cells to the corticosteroid fluticasone propionate and PM synergistically increased rhinovirus levels, suppressed host defence responses, and increased levels of the rhinovirus entry receptor. In summary, this thesis’ investigation of air pollution interventions and possible consequences both provides novel therapeutic options and identifies a potential risk, evidence that collectively may inform comprehensive strategies to mitigate the risk associated with air pollution exposure. iv Lay Summary The majority of people around the world breathe in polluted air due to the use of fossil fuels in everyday life. Exposure to this air pollution, particularly high levels in urban centres, is known to irritate the lungs and cause inflammation. Although small appliances like air purifiers can help reduce exposure at home, a medicine might be useful during periods of high air pollution to reduce irritation to the lungs. My thesis identified three possible medicines that reduce inflammation caused by air pollution in cells that form the lining of the lungs. Air pollution exposure also increases the risk of catching a respiratory virus, and I looked at how suppressing inflammation changes that risk. My results provide evidence of how lung cells exposed to air pollution and treated with corticosteroids, a commonly used medicine to control inflammation in those with lung disease, are more likely to be infected by rhinovirus (common cold virus). v Preface Chapter 1 was written by me and reviewed by Dr. Chris Carlsten (CC), Dr. Christopher Rider (CR), Agnes Yuen (AY), and my PhD supervisory committee members. Figure 1.1 was reproduced from the 2019 Global Burden of Disease report1 under a creative commons licence (https://creativecommons.org/licenses/by/4.0). Sections 1.6 to 1.10 and Figures 1.2 and 1.3 were adapted from a published review written by me2. Figure 1.2 was designed by Dr. Jeremy Hirota (JH) and Figure 1.3 was designed by me. As the author of this review I retained the right to reuse this material for a thesis or dissertation and permission was not required by Elsevier. Chapter 2 was written by me and reviewed by CC, CR, and my PhD advisory committee. CC, CR, and I contributed to the conception of the work. I designed the experiments. I executed the experiments and collected the data with the assistance of Hang Li (HL), Lenneth Li (LL), Agnes Yuen (AY), Tina Afshar (TA), and Vanessa Cheng (VC). HL and LL assisted with oxidative stress assays, AY and TA assisted with LDH assays, and VC assisted with primary epithelial cell culture. I performed the statistical analysis and interpretation with the guidance of CC, CR, and my PhD advisory committee. All figures and tables in this chapter were generated by me. Chapter 3 was written by me and reviewed by Dr. Emily Brigham (EB), CC, and my PhD advisory committee. EB, CC, and I conceived and designed the work. I executed all experiments and collected all the data. I performed the statistical analysis and interpretation with the guidance of EB, CC, and my PhD advisory committee. All figures and tables in this chapter were generated by me. Chapter 4 was written by me and reviewed by CC, CR, and my PhD advisory committee. CC, CR, and I contributed to the conception of the work. I designed the experiments with the assistance of CR and Dr. Emily Watts (EW). I executed the experiments and collected the data with the assistance of Nataly El-Bittar (NB). NB vi assisted with performing flow cytometry experiments and extracting RNA. I performed the statistical analysis and interpretation with the guidance of CC, CR, EW, and my PhD advisory committee. Chapter 5 was written by me and reviewed by CC, AY, and my PhD advisory committee. UBC ethics board approval was not required for the research carried out in this thesis. vii Table of Contents Abstract ......................................................................................................................... iii Lay Summary ................................................................................................................ iv Preface ............................................................................................................................ v Table of Contents ......................................................................................................... vii List of Tables ................................................................................................................ xii List of Figures ............................................................................................................. xiii List of Abbreviations ................................................................................................... xv Acknowledgements ..................................................................................................... xx Dedication ................................................................................................................... xxii Chapter 1: Introduction ................................................................................................. 1 1.1 Overview ................................................................................................................ 1 1.2 Air pollution composition ........................................................................................ 2 1.3 Impact of air pollution on morbidity and mortality ................................................... 3 1.4 Air pollution and the airways .................................................................................. 6 1.5 Air pollution, inflammation, and the lung epithelium ............................................... 9 1.6 Air pollution and oxidative stress .......................................................................... 11 1.7 Air pollution associated oxidative stress in the lung epithelium ........................... 14 1.8 Air pollution-induced oxidative stress and lung epithelial cell defence mechanisms ................................................................................................................................... 17 1.9 Consequences of air pollution-induced oxidative stress ...................................... 19 1.10 Interventions for air pollution-induced oxidative stress and inflammation .......... 20 viii 1.10.1 Inhaled corticosteroids ................................................................................. 20 1.10.2 Omega-3 polyunsaturated fatty acids .......................................................... 21 1.10.3 Antioxidants ................................................................................................. 22 1.11 Air pollution and susceptibility to viral infection .................................................. 23 1.12 Thesis objectives and hypotheses ..................................................................... 26 Chapter 2: Targeting traffic-related air pollution-induced oxidative stress in airway epithelial cells. ................................................................................................. 28 2.1 Introduction .......................................................................................................... 28 2.2 Methods ............................................................................................................... 29 2.2.1 Cell culture ..................................................................................................... 29 2.2.2 PM exposures ................................................................................................ 30 2.2.3 Air pollution interventions .............................................................................. 32 2.2.4 Oxidative stress assay ................................................................................... 32 2.2.5 Multiplex ELISA ............................................................................................. 34 2.2.6 LDH assays ................................................................................................... 35 2.2.7 Statistical analysis ......................................................................................... 35 2.3 Results ................................................................................................................. 35 2.3.1 Effect of particulate matter on epithelial cell oxidative stress and cytotoxicity35 2.3.2 Effect of small molecule interventions on particulate matter-induced oxidative stress ...................................................................................................................... 36 2.3.3 Effect of small molecule interventions on cell viability ................................... 37 ix 2.3.4 Tetrahydrocurcumin and quercetin attenuate particulate matter-induced oxidative stress in primary human bronchial epithelial cells ................................... 41 2.3.5 Effect of tetrahydrocurcumin and quercetin on particulate matter-induced immune mediator profile ......................................................................................... 44 2.4 Discussion ............................................................................................................ 48 Chapter 3: Dietary omega-3 fatty acids modify air pollution-induced inflammation in airway epithelial cells .............................................................................................. 53 3.1 Introduction .......................................................................................................... 53 3.2 Methods ............................................................................................................... 55 3.2.1 Cell culture ..................................................................................................... 55 3.2.2 PM exposure ................................................................................................. 55 3.2.3 Oxidative stress assay ................................................................................... 56 3.2.4 ELISA assays ................................................................................................ 56 3.2.5 LDH assays ................................................................................................... 57 3.2.6 Statistical analysis ......................................................................................... 57 3.3 Results ................................................................................................................. 57 3.3.1 Effect of urban particulate matter on epithelial cell oxidative stress and cytotoxicity .............................................................................................................. 57 3.3.2 DHA and EPA attenuates urban particulate matter-induced oxidative stress 58 3.3.3 DHA attenuates urban particulate matter-induced inflammatory mediator release .................................................................................................................... 61 3.4 Discussion ............................................................................................................ 63 x Chapter 4: Corticosteroid and traffic-related air pollution exposure increases human rhinovirus replication in airway epithelial cells. .......................................... 67 4.1 Introduction .......................................................................................................... 67 4.2 Methods ............................................................................................................... 69 4.2.1 Epithelial cell culture and viral infection ......................................................... 69 4.2.2 siRNA transfection ......................................................................................... 70 4.2.3 Flow cytometry ............................................................................................... 70 4.2.4 qPCR ............................................................................................................. 71 4.2.5 ELISAs ........................................................................................................... 72 4.2.6 Statistical analysis ......................................................................................... 73 4.3 Results ................................................................................................................. 73 4.3.1 RV replication increased by the corticosteroid FP ......................................... 73 4.3.2 RV replication synergistically increased by particulate matter and FP .......... 75 4.3.3 RV16 entry receptor ICAM-1 increased by particulate matter ....................... 75 4.3.4 Knockdown of ICAM-1 attenuates particulate matter-induced RV replication 78 4.3.5 NF-κB pathway inhibition suppresses particulate matter-induced ICAM-1 expression .............................................................................................................. 78 4.3.6 NF-κB pathway inhibition attenuates particulate matter and FP-induced RV16 replication ............................................................................................................... 78 4.3.7 NF-κB pathway inhibition suppresses markers of inflammatory and anti-viral gene expression ..................................................................................................... 80 4.3.8 Quercetin treatment increases RV16 replication ........................................... 80 xi 4.4 Discussion ............................................................................................................ 82 Chapter 5. Conclusion ................................................................................................. 86 5.1 Summary of findings ............................................................................................ 86 5.2 Limitations ............................................................................................................ 88 5.3 Implications .......................................................................................................... 89 5.4 Future directions .................................................................................................. 92 References ................................................................................................................... 94 xii List of Tables Table 1.1 Effects of controlled human exposures on the healthy human airways ........... 7 Table 2.1 HBEC donor characteristics ........................................................................... 30 Table 2.2 Summary of interventions selected for oxidative stress screening assays in Calu-3 and BEAS-2B cells ............................................................................................. 33 Table 2.3 Effect of interventions on air pollution-induced oxidative stress .................... 38 Table 4.1 qRT-PCR taqman assays .............................................................................. 72 xiii List of Figures Figure 1.1 Global number of deaths and percentage attributable to Level 2 risk factors, by cause and sex, 2019 ................................................................................................... 4 Figure 1.2 Respiratory mucosa in response to air pollution exposure ........................... 10 Figure 1.3 Effect of air pollution on oxidation reduction homeostasis and signaling ..... 14 Figure 2.1 SRM1650b and APEL PM dose-dependently increase ROS generation ..... 32 Figure 2.2 Overview of oxidative stress assays in Calu-3 and BEAS2-B cells .............. 37 Figure 2.3 Cytotoxicity of antioxidant interventions in calu-3 cells ................................. 41 Figure 2.4 Cytotoxicity of antioxidant interventions in BEAS-2B cells ........................... 43 Figure 2.5 Tetrahydrocurcumin and quercetin attenuate PM-induced oxidative stress in HBECs ........................................................................................................................... 44 Figure 2.6 Effect of tetrahydrocurcumin on air pollution associated immune mediators 45 Figure 2.7 Effect of quercetin on air pollution associated immune mediators ............... 46 Figure 2.8 Tetrahydrocurcumin and quercetin modulate air pollution immune mediator profile ............................................................................................................................. 47 Figure 3.1 Experimental overview ................................................................................. 56 Figure 3.2 Urban particulate matter induces oxidative stress in BEAS-2B cells ............ 58 Figure 3.3 DHA significantly attenuates urban particulate matter-induced oxidative stress ............................................................................................................................. 59 Figure 3.4 EPA significantly attenuates urban particulate matter-induced oxidative stress ............................................................................................................................. 61 Figure 3.5 DHA and EPA attenuate urban particulate matter-induced inflammatory mediator release ............................................................................................................ 62 Figure 4.1 Flow cytometric plots showing the gating strategies for measuring ICAM-1 levels .............................................................................................................................. 71 xiv Figure 4.2 FP increases RV16 replication and suppresses protein expression of RV-induced pro-inflammatory cytokines and antiviral mediators ......................................... 74 Figure 4.3 The combination of FP and PM increases RV16 replication and modulates expression of related markers ....................................................................................... 76 Figure 4.4 PM exposure increases ICAM-1 expression ................................................ 77 Figure 4.5 NF-κB pathway inhibitor suppresses RV16 replication in PM exposed and FP treated BEAS-2B cells ................................................................................................... 79 Figure 4.6 NF-κB pathway inhibitor suppresses anti-viral gene expression .................. 81 xv List of Abbreviations AA Arachidonic acid ACE Angiotensin-converting enzyme AhR Aryl Hydrocarbon Receptor AP-1 Activator Protein 1 AUC Area under the curve BAL Bronchoalveolar lavage BALF Bronchoalveolar lavage fluid BW Bronchial wash CCL C-C motif chemokine COPD Chronic obstructive pulmonary disease CXCL C-X-C motif chemokine ligand DALY Disability-adjusted life year DE Diesel Exhaust DHA Docosahexaenoic acid DMEM/F12 Dulbecco's modified eagle’s medium/ham’s nutrient mixture F12 DPM Diesel exhaust particulate matter EGF Epidermal growth factor ELISA Enzyme-linked immunosorbent assay EMEM Eagle's minimum essential medium EPA Eicosapentaenoic acid ERK Extracellular signal-regulated kinase FBS Fetal Bovine Serum FEV1 Forced expiratory volume in 1 second FGF Fibroblast growth factor xvi FLT-3L FMS-like tyrosine kinase 3 ligand FOXO Forkhead box O FP Fluticasone propionate FSC-A Forward scatter area FVC Forced vital capacity GBD Global burden of disease GM-CSF Granulocyte-macrophage colony-stimulating factor GPX Glutathione peroxidase GSH Glutathione GSSG Glutathione disulfide HBD2 Human b-defensin-2 HDP Host defence peptides HIF-1α Hypoxia-inducible factor 1α HO-1 Heme oxygenase HUVECs Human umbilical vein endothelial cells ICAM-1 Intercellular adhesion molecule 1 ICS Inhaled corticosteroid IFN Interferon IL Interleukin IRF1 IFN regulatory factor 1 ISG Interferon Stimulated Gene IκB Inhibitor of nuclear factor kappa B JNK c-Jun N-terminal kinase KEAP1 Kelch-like ECH-associated protein 1 LDH Lactate dehydrogenase LLOD Lower Limit Of Detection xvii LRI Lower Respiratory Infections LTB4 Leukotriene B4 M-CSF Macrophage colony-stimulating factor MAPK Mitogen-Activated Protein Kinase MCP Monocyte chemoattractant protein MDA Malondialdehyde MIG Monokine induced by interferon-gamma MPO Myeloperoxidase NAC N-acetylcysteine NADPH Nicotinamide adenine dinucleotide phosphate NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NHBE Normal Human Bronchial Epithelial NIST National institute of standards and technology NOX2 Nicotinamide adenine dinucleotide phosphate oxidase 2 NQO1 NAD(P)H quinone dehydrogenase 1 Nrf2 Nuclear factor-like 2 ODE Organic dust extract Ox-LDL Oxidized low-density lipoprotein PAH Polycyclic aromatic hydrocarbon PDGF Platelet-derived growth factor PI3K Phosphoinositide 3-Kinase PKB Protein Kinase B PM Particulate matter PM0.1 Particulate matter of ≤ 0.1 microns in aerodynamic diameter PM10 Particulate matter of ≤ 10 microns in aerodynamic diameter PM2.5 Particulate matter of ≤ 2.5 microns in aerodynamic diameter xviii PPAR Peroxisome proliferator–activated receptors PRRs Pattern Recognition Receptors PTP Protein tyrosine phosphatases RANTES Regulated on activation, normal T cell expressed and secreted RFU Relative fluorescence intensity RNS Reactive Nitrogen Species ROS Reactive Oxygen Species RSAD2 Radical S-adenosyl methionine domain containing 2 RSV Respiratory Syncytial Virus RT-qPCR Quantitative Real-Time Polymerase Chain Reaction RV Rhinovirus RvD1 Resolvin D1 SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 SFM Serum Free Media SOD Superoxide dismutase SPM Specialized pro-resolving mediator SRM Standard reference material SSC-A Side scatter area STAT3 Signal transducer and activator of transcription 3 TH2 T helper 2 TLR Toll-Like Receptor TMPRSS2 Transmembrane protease serine 2 TNF Tumour Necrosis Factor TNFR Tumor necrosis factor receptor TRAP Traffic-Related Air Pollution TSLP Thymic Stromal Lymphopoietin xix VCAM-1 Vascular Cell Adhesion Protein 1 ZO Zona Occludin xx Acknowledgements I have been very lucky to have the support of many individuals in the pursuit of this PhD. I am grateful to the University of British Columbia and the Experimental Medicine program for creating a program that allowed me to pursue this line of research. I am thankful to Vancouver General Hospital and Vancouver Coastal Health Research Institute staff for their support. Financially, the Allergy, Genes and Environment Network, Vanier Canada Graduate Scholarships, and the University of British Columbia were all instrumental in providing support, allowing me to focus on my research. I would also like to thank my advisory committee; Dr. David Granville, Dr. Stephan van Eeden, and Dr. Jeremy Hirota. They have been very helpful in advising the direction of my research and always encouraging me to push my understanding further. I am thankful for their patience and understanding as my thesis focus shifted post-COVID19 research delays. I am very grateful to friends and colleagues of the Carlsten lab who made this experience memorable and enjoyable. I would not have made it through this degree without their laughter, discussions, support, and advice. Thank you to Agnes Yuen, Dr. Min Hyung Ryu, Dr. Chris Rider, Dr. Emily Brigham, Tina Afshar, Carley Schwartz, Parteek Johal, Kevin Lau, Dr. Emily Watts, Dr. Hannah Li, Shijia (Lenneth) Li, Dr. Danay Maestre-Batlle, Juma Orach, Andrew Robinson, Wayne Tse, Vanessa Cheng, and Nataly El-Bittar. In particular, thank you to Agnes for always being supportive and ready to jump into everything from grants to experiments. The Carlsten lab would be unrecognizable without you. I would also like to thank Emily Brigham, who worked closely with me in conceiving the omega-3 experiments in chapter 3 and interpreting the data. Thank you to Min and Tina, for all our adventures and listening to all my ideas. Thank you to Chris Rider and Min for all your advice and support in planning xxi experiments, reading my work, and interpreting results. Lastly, I would like to thank the undergrad students Wayne, Vanessa, and Nataly, who contributed to my projects, helping with cell culture and collecting data. I would especially like to thank Dr. Jeremy Hirota and Dr. Chris Carlsten, my two biggest mentors and supporters of my academic career. To Jeremy, for taking a chance and starting my journey in respiratory science, encouraging me to focus and always hustle. To Chris, I am very grateful to have you as my PhD supervisor. You have been incredibly supportive through all the ups and downs of my studies and I am thankful you gave me the freedom to chase ideas. Last, I would like to recognize my family, my wife Ashley, and my daughter Charlotte. Their smiles and laughter pulled me through the tough days. Ashley has been there for me my entire academic journey, and I’m incredibly thankful for her love, support, and sacrifice through my PhD. She was the first to listen to all my ideas and my motivation to run with them. I would not have been able to take this undertaking without her. xxii Dedication To my wife Ashley, my guiding light Chapter 1: Introduction 1.1 Overview Air pollution is a pervasive and growing problem globally, and epidemiological and direct exposure clinical trials, along with toxicology, over the past three decades have highlighted the mechanisms by which exposure to air pollution is harmful to human health. These studies have been instrumental in driving informed policy, guidelines, and legislation in countries around the world in order to lower emission standards and tighten acceptable levels of ambient pollutants. However, the vast majority of the world still lives in environments that exceed the World Health Organization’s recommendations for safe levels of exposure. Alongside engineering solutions, such as air purifiers to reduce an individual’s level of exposure, priming the body’s own defences against the harmful effects of air pollution at the cellular level may have broad application as an additional harm reduction strategy. The work in this thesis is focused on evaluating the utility of small molecule interventions that target both air pollution-induced oxidative stress and inflammation within controlled air pollution exposure models using primary lung epithelial cells. However, inflammation is a natural physiological response to external insults in the lungs and although it may be beneficial to control it for the overall health of an individual, suppressing inflammation may have undesirable effects. To this end, my thesis also investigated the interaction between corticosteroids, a commonly used class of anti-inflammatory medicines used by those with chronic lung disease, and a potential intervention for air pollution-induced inflammation and particulate matter exposure on viral infectivity in vitro. This work provides mechanistic insights into why air pollution increases an individual’s susceptibility to rhinovirus infection and how vulnerability appears to be further exacerbated by corticosteroids. 2 1.2 Air pollution composition Air pollution consists of gases and particulate matter (PM), a complex mixture of black carbon and other organic and inorganic molecules suspended in the air. Although natural processes contribute to air pollution (e.g., forest fires and dust storms), this thesis will focus on urban air pollution generated as a byproduct of human activities by the deliberate combustion of fossil or biomass fuels. The emissions produced in these contexts consist of primary aerosols that include elemental and organic carbon, and metals such as aluminum, iron, zinc, copper, nickel, and manganese. However, the type, source and quality of the fuel, the engine characteristics, temperature, and humidity all impact the composition of the air pollution released into the atmosphere. For example, emissions produced by the combustion of coal are higher in aluminum, selenium, cobalt, and arsenic, whereas oil is higher in nickel, iron, and manganese3,4. In addition to these primary aerosols, secondary organic aerosols are produced by the atmospheric oxidation of emitted precursors, and depending on the fuel source, consist of sulfur, nitrogen, and organic carbon species (including volatile organic compounds and polycyclic aromatic hydrocarbons (PAHs)) at varying levels3–5. Adding to the complex and dynamic nature of air pollution, these primary and secondary aerosols also evolve and age over time in the atmosphere through photochemical reactions and agglomeration6. In terms of human health impacts, the fluctuating composition of air pollution from location to location and season to season is known to translate into differential respiratory tract responses5,7–10. Moreover, the size of air pollution PM is also an important characteristic for respiratory health. Binned into three categories based on particle diameter, course (PM10), fine (PM2.5), and ultrafine (PM0.1), PM consist of particles that have diameters less than 10, 2.5 and 0.1 μm respectively. Globally, there is a strong research and regulatory focus on PM2.5 as it penetrates deeper into the lungs 3 than the course fraction of particles included in PM10 and is thought to mediate the more harmful effects of air pollution11–13. Despite the implementation of legislation and policies that have resulted in a reduction of ambient urban PM2.5 concentrations in the Americas, Europe, and Western Pacific, from the year 2000 to 2019, the global population weighted annual average PM2.5 concentration has remained constant at 35 μg/m3 due to substantial increases in South-East Asia14. Furthermore, a large source of urban PM2.5 comes from the combustion of diesel fuel in transportation and construction industries15, which is not decreasing significantly globally. 1.3 Impact of air pollution on morbidity and mortality Air pollution is a major risk factor for human health, with 6.67 million deaths and 213 million disability-adjusted life years (DALYs) attributed to the effects of air pollution in 201916. The Global Burden of Disease (GBD) 2019 report estimated it as the 4th highest risk factor for global attributable deaths (Figure 1.1), and the leading risk factor for DALYs among environmental and occupational risks1,16. Ambient air pollution (PM2.5) in particular was estimated to cause 4.14 million deaths in 2019 and was ranked the 6th highest risk factor17. Of these 4.14 million deaths, cardiovascular diseases (2.47 million), chronic respiratory diseases (0.69 million), and respiratory infections and tuberculosis (0.33 million) were the leading three causes of ambient air pollution attributable deaths. Furthermore, ambient air pollution contributed 118 million DALYs, with the largest proportions attributed to cardiovascular diseases, chronic respiratory diseases, and respiratory infections17. With currently 99% of the world’s population living in environments that exceed the WHO guidelines for air pollution exposure13, these statistics are unlikely to decrease over the coming years. 4 Figure 1.1 Global number of deaths and percentage attributable to Level 2 risk factors, by cause and sex, 2019. The level 2 risk factors ranked by attributable deaths is shown on the Y-axis and causes of death are shown by different colours within each risk factor. Figure reproduced from GBD 20191. 5 In a recent European study, long-term exposure to PM2.5 has been associated with an all-cause increase in mortality of 13% (95% CI, 1.106 to 1.155) per 5 μg/m3 of PM2.5 (annual average). Concentration-response curves also showed stronger associations at lower exposures, an observation that has been reported in other long-term exposure studies18–21. Furthermore, at lower concentrations, a trend towards stronger associations with respiratory over cardiovascular mortality was observed22. Short term exposure to PM2.5 has also been associated with daily mortality in a pooled analysis of 110 different studies, with a 1.04% (95% CI, 0.52 to 1.56) increase per 10 μg/m3 of PM2.523. Similar to long-term exposures, deaths attributed to respiratory diseases were more strongly associated with PM2.5 levels than cardiovascular diseases in this study. Importantly, long- and short-term PM2.5 exposure concentration-response relationships have not demonstrated a safe threshold for ambient air pollution in the context of human health24. As reported in the GBD study, ambient air pollution significantly contributes to respiratory disease morbidity and mortality17,24. In particular, an abundance of studies have examined the relationship between air pollution and chronic obstructive respiratory disease (COPD) or asthma. Those living with COPD have been shown to be more susceptible to air pollution, with increased short- and long-term exposures associated with worsening symptoms of wheeze and cough, and increased exacerbations, hospitalizations, and deaths25–31. Furthermore, increased exposure to air pollution components (PM2.5, NO2, and O3) has been associated with increased incidence of COPD (PM2.5 relative risk: 1.07, 95% CI 1.06 to 1.08)32. For asthma, a meta-analysis of 19 studies has shown exposure to traffic-related air pollution (TRAP) in early childhood increased the risk of developing asthma by 14% (95% CI, 1.00 to 1.30) per 2 μg/m3 of PM2.533. This analysis also demonstrated an association between increased PM2.5 exposure and increased sensitization to aero- and food-allergens. Systematic reviews of 6 short-term air pollution exposure have also shown increased relative risk of emergency room visits and hospital emissions by 1.023 per 10 μg/m3 PM2.5 (95% CI 1.015 to 1.031)34 and exacerbations by 1.028 per 10 μg/m3 PM2.5 (95% CI 1.009 to 1.047)35. Moreover, every 1 μg/m3 PM2.5 increase in weekly exposure has been associated with a 0.82% increase in short-acting beta agonist rescue inhaler usage36, indicating a knowledge gap of effective medications that mitigate the effects of air pollution. Of ambient air pollution attributable deaths, respiratory infections (0.33 million), which were predominantly lower respiratory infections (LRIs), were the third leading cause of death reported by the GBD in 201924. Defined as Streptococcus pneumoniae, Hemophilus influenzae type b, respiratory syncytial virus (RSV) and influenza infections, LRIs were the fourth most common overall cause of death reported by the GBD37. Although overall LRI deaths and DALYs have been reported to be decreasing, the percentage attributed to ambient PM2.5 compared to household PM2.5 has dramatically increased from approximately 20% to 50% from 1990 to 2019 globally and more so in high, high-middle, and middle socio-economic areas37. 1.4 Air pollution and the airways The association of specific air pollutants such as PM2.5 with morbidity and mortality in epidemiology publications is also supported by many in vitro and in vivo studies examining the direct effects of air pollution exposures. Of these studies, controlled human exposure studies utilizing fresh diluted diesel exhaust (DE) as a model of TRAP have revealed common mechanisms by which air pollution affects lung function and tissue homeostasis over the last 30 years. As detailed in Table 1.1, healthy humans exposed to DE for 1-2 hours at levels similar to that observed in a heavily 7 Table 1.1 Effects of controlled human exposures on the healthy human airways Exposure Time* (h) N Effect of Exposure on Lung Endpoints Ref. DE (1.4 x 106 particles /cm3), 1h 18 8 ↑ BAL neutrophils, Leu3a/Leu3a+ T-lymphocytes 38 DE (1.4 x 106 particles /cm3), 1h 0 12 ↑ airway resistance, and specific airway resistance 39 DE (300 µg/m3 PM10), 1h 6 15 ↑ BW neutrophils & macrophages, ↑ BAL B-cells, methylhistamine, ↑ tissue neutrophils, T lymphocytes, mast cells, ICAM-1, & VCAM-1 40 DE (300 µg/m3 PM10), 1h 6, 24 15 ↑ 6h sputum neutrophils, macrophages, IL-6, & methylhistamine, ↑ 24h sputum neutrophils & IL-6 41 CAPs (47, 107, 206 µg/m3 PM2.5), 2h 18 38 ↑ BAL neutrophils, monocytes, & protein, ↑ blood fibrinogen, 42 DE (200 µg/m3 PM10), 2h 4, 24 10 ↑ exhaled CO, ↑ 4h sputum neutrophils & MPO, ↓4h sputum macrophages 43 DE (300 µg/m3 PM10), 1h 6 15 ↑ tissue CXCL8/IL-8 & CXCL1/GRO-alpha, ↑ BW and tissue CXCL8/IL-8 mRNA expression 44 DE (100 µg/m3 PM10), 2h 6 25 ↑ BW neutrophils (not-significant), ↑ BW GSH, L-cysteine, ↑ Airway Resistance 45 DE (100µg/m3 PM10), 2h 6 25 ↑ BW Neutrophils, IL-6, CXCL8/IL-8, ↑ BAL lymphocytes, ↑ Tissue P-selectin, & VCAM-1 46 DE (100µg/m3 PM10), 2h 18 15 ↑ BW & tissue neutrophils, ↑ tissue mast cells, ↑ BW IL-8 & MPO, ↑ BAL urate & GSH 47 DE (100µg/m3 PM10), 2h 18 23 ↑ BW & tissue neutrophils, ↑ BW IL-6 & MPO 48 DE (270 µg/m3 PM10), 1h 6 15 ↑ BAL eosinophils, ↑ tissue P-selectin, & VCAM-1 49 DE (200µg/m3 PM2.5), 2h 5, 24 20 ↑ 5h blood IL-6 ↓ 5h GSH/GSSG ratio 50 DE (20,50, & 150µg/m3 PM2.5), 4h 4, 24 17 ↑ 4h FeNO 51 Definition of abbreviations: BAL = bronchoalveolar lavage, BW = bronchial wash, ICAM-1 = intercellular adhesion molecule 1, VCAM-1 = vascular cell adhesion molecule 1, MPO = myeloperoxidase, CXCL8 = C-X-C motif chemokine ligand 8, GSH = glutathione, GSSG = glutathione disulfide *Time (hours) post-exposure endpoints were collected 8 polluted city such as Delhi, India, experienced increased air way resistance; increased lung infiltration of immune cells, including neutrophils, eosinophils, and macrophages; increased expression of receptors involved in leukocyte trafficking, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1); increased immune mediators in the bronchial fluid including interleukin (IL)-6, IL-8, myeloperoxidase (MPO); and increased anti-oxidants such as glutathione (GSH) 38–51. In summary, the findings of these studies all support the conclusion that exposure to urban air pollution reduces lung function and induces an acute inflammatory response in healthy individuals. Controlled human exposure studies have also been used to examine the effects of air pollution in the context of chronic respiratory diseases such as asthma. In a direct comparison between age matched healthy and mild asthmatics, pro-inflammatory effects such as increased infiltration of neutrophils and lymphocytes into the airways, increased BAL IL-6 and IL-8, and increased tissue expression of P-selectin and VCAM-1 were observed in heathy individuals exposed to 2h of 100 μg/m3 PM1046. However, only increases in BAL IL-8 and bronchial tissue IL-10 were observed in the mild asthmatic group. With the absence of a neutrophil response or augmentation of pre-exposure eosinophilic inflammation, the authors surmised that a 6h follow up time point might not be long enough for mild asthmatics to generate a strong response to the exposure. However, a subsequent study by the same research group examining an 18h follow up time point also observed no significant increases in markers of airway inflammation in mild asthma48. In contrast to these studies, increased airway hyperresponsiveness and resistance, and sputum levels of IL-6 were observed by Nordenhäll et al. in asthmatics exposed to 1h of 300 μg/m3 PM1052, and significant reductions in forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC), and sputum levels of MPO were observed in asthmatics after walking on a 9 polluted street53. Moreover, my research group has previously shown that reduced lung function, and increased immune cells and mediators in the airways following an inhaled challenge to allergen (notably increasing with climate change) is exacerbated by diesel exhaust exposures (2h of 300 μg/m3 PM2.5)54–57. In summary, the weight of evidence from these studies suggests that air pollution exposure impairs lung function and increases inflammation in the lungs of asthmatics. 1.5 Air pollution, inflammation, and the lung epithelium As air pollution enters the lungs, one of the first tissues it encounters is the pseudostratified bronchial epithelium, the primary defensive barrier against exogenous insults. The epithelium consists of various cell types including basal, goblet, ciliated, club, and tuft cells that function to protect the body58. To further understand the effects of air pollution on the lungs, many studies have utilized lung bronchial epithelial cells isolated from healthy individuals that are differentiated in vitro using an air-liquid interface method to model the bronchial epithelium. Leclercq at al. demonstrated that normal human bronchial epithelial (NHBE) cells exposed to urban ambient PM2.5 suspended in solution induced the expression and release of the inflammatory mediators tumour necrosis factor (TNF)α, IL-1β, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF)59,60. In addition, Leclercq demonstrated that repeated exposures induced an increased pro-inflammatory mediator response60. Similar increases in pro-inflammatory mediator responses have been observed by other groups using different sources of PM2.561–64. This inflammatory response is thought to be primarily mediated by pattern recognition receptors (PRRs) toll-like receptor (TLR) 2 and 462,65,66; and TNF, phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt), and mitogen-activated protein kinase (MAPK) signaling 10 pathways67–71 that result in activation of the transcription factors activator protein 1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)59,70,72,73. Beyond inducing a pro-inflammatory response, PM has also been shown to disrupt other functions of the epithelium (Figure 1.2). PM2.5 exposure has been shown to upregulate the expression of mucin proteins in vitro and in vivo such as MUC5AC, MUC5B, and MUC1655,74–76, possibly via an early growth response gene 1-dependent mechanism77, that can contribute to mucus hypersecretion and lung function decline. Figure 1.2 Respiratory mucosa in response to air pollution exposure. Air pollution gaseous and particle components interact with airway surface lining fluid; surfactants; host defence peptides; tight junctions; pathogen recognition receptors, including Toll-like receptors and NOD-like receptors; and redox-sensitive pathways (Fig 3) to modulate the viability and immune responses of airway epithelial cells and recruited immune cells. Figure reproduced from Huff et al. 2019 under author’s retained rights for reuse2. 11 Several studies have also demonstrated that PM2.5 disrupts the barrier integrity of epithelial cells at high doses, allowing other exogenous molecules, allergens, and pathogens deeper access into lung tissues. Specifically, PM2.5 was found to reduce transepithelial electrical resistance, a measure of barrier integrity, and downregulate tight junction proteins such as E-cadherin and zona occludin (ZO)-164,78–81. Beyond acute inflammatory responses, PM has been shown to impact T helper 2 (TH2) and immune responses in primary epithelial cells from mild asthmatics. Diesel exhaust PM has been demonstrated to increase thymic stromal lymphopoietin (TSLP) release concentration-dependently through an aryl hydrocarbon receptor (AhR) dependent mechanism82–84. Furthermore, severe asthmatics living in high urban air pollution environments, as assessed by proximity to major roadways, were found to have increased levels of TSLP in bronchial biopsy samples that were associated with AhR nuclear localization84. This study also observed similar trends for IL-33 and IL-25, further illustrating the potential of urban air pollution to enhance TH2 immune responses. In a similar study, severe asthmatics living near major roadways were observed to have increased expression of IL-17A in their airway epithelium. In addition, airway epithelial cells from these asthmatics were exposed to diesel exhaust PM, resulting in the upregulation IL-17A gene and protein expression through an NF-κB dependent mechanism85. The influence of PM on TH17 immune profiles is noteworthy as it has been associated with severe steroid-resistance in asthma86,87. 1.6 Air pollution and oxidative stress A major theme of recent studies examining air pollution’s negative effects on respiratory mucosal immune responses is the disruption of oxidation-reduction (redox) homeostasis and the activity of the AhR pathway. Redox signaling and homeostasis consist of a complex network of molecules, signaling proteins, and enzymes that 12 maintain a balance between the production and catabolism of reactive oxygen species (ROS) and reactive nitrogen species (RNS) within and surrounding the cell (Figure 1.3)88,89. ROS is a term that comprises a diverse range of molecules, such as superoxide, hydroxyl radicals (OH●), peroxides (H2O2), and ozone, and similarly RNS to molecules, such as nitrate, peroxynitrite, nitrogen monoxide (NO●), and NO2. Accumulation of these species beyond the capacity of the cell’s antioxidant systems is generally referred to as oxidative stress. Indeed, mucus, host defence peptides, antiviral responses, and cytokine production all appear to be influenced by oxidative stress and the AhR pathway. I describe below recent advances in our understanding of how oxidative stress and AhR function in human airway epithelial cells in response to air pollution exposure. ROS are generated from a variety of endogenous sources, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, mitochondrial complexes (I-III), superoxide dismutases (SODs), xanthine oxidases, and the cytochrome P450 family of enzymes. To prevent oxidation and damage of cellular proteins, lipids, and other macromolecules, the amount of ROS is tightly controlled by antioxidant systems that include enzymes that catabolize the ROS species, such as catalases, thioredoxins, glutathione peroxidases, and peroxiredoxins, scavenging molecules, such as vitamin C and E, as well as transport systems, such as aquaporins and peroxiporins88,89. In the event that cellular antioxidant systems become overwhelmed by ROS, redox-sensitive transcription factors, such NF-kB, forkhead box O, hypoxia-inducible factor 1α, and nuclear factor erythroid 2-related factor 2 (Nrf2), translocate to the nucleus and orchestrate the expression of proteins involved in antioxidant systems, inflammation, survival, and repair. 13 14 Figure 1.3 Effect of air pollution on oxidation reduction homeostasis and signaling. Air pollution contains and generates ROS in the respiratory tract lining fluid, as well as in intracellular compartments through NADPH oxidase, SOD, metabolism of PAHs by using cytochrome P450, and redox cycling in the epithelial lining fluid, cytoplasm, and mitochondria. PAHs bind and activate AhR, a transcription factor that induces expression of cytochrome P450 enzymes that amplify intracellular ROS in response to air pollution. ROS can be shuttled between cellular compartments through membrane transporters and pores, such as aquaporins (AQPs). Excess ROS is scavenged by antioxidants, such as uric acid, vitamin C, and vitamin E, and catabolized by enzymes, such as glutathione peroxidases (GPX), which oxidizes GSH to glutathione disulfide. Increased ROS values are associated with increased receptor tyrosine kinase and mitogen-activated protein kinase signaling by inhibiting protein tyrosine phosphatases (PTP) through transient oxidation of active site cysteines. ROS-mediated protein oxidation contributes to dissociation of the NF-kB regulatory subunit IkB, as well as the NrF2 regulatory subunit Kelch-like ECH-associated protein 1 (KEAP1), leading to transcription of proinflammatory and antioxidant genes. ROS further amplifies inflammation through activation of the NLRP3 inflammasome, leading to increased IL-1b production. ROS directly activates forkhead box O transcription factors and indirectly increases hypoxia-inducible factor 1α (HIF-1α) activation through inhibition of prolyl hydroxylases, resulting in transcription of genes involved in metabolism and redox homeostasis. Further increases in ROS levels oxidize and damage proteins, leading to aggregates; damage cell membranes through lipid peroxidation; damage DNA; and induce apoptosis. ERK, Extracellular signal-regulated kinase; FOXO, forkhead box O; GR, glutathione reductase; GSSG, glutathione disulfide; JNK, c-Jun N-terminal kinase. Figure reproduced from Huff et al. 2019 under author’s retained rights for reuse2. 1.7 Air pollution associated oxidative stress in the lung epithelium Epithelial cells in the respiratory mucosa are also exposed to ROS and RNS from a variety of exogenous sources, including diverse compositions of air pollution. Although the composition and amount of air pollution varies depending on the time of year and the proximity to industry and transportation hubs and thoroughfares6–9, it is mainly comprised of particles, heavy transition metals, and reactive gases and organic compounds9,64,90–96. Reactive gases and organic compounds, such as ozone, hydroxyl radicals, sulfur and nitrogen species, and PAHs, participate in a number of ROS-generating reactions in the airway surface lining fluid97. A major redox reaction cycle in the airway surface lining fluid involves the donation of electrons from antioxidant compounds to transition metals, such as Cu2+ and Fe2+, as well as a class of PAHs called quinones. The resulting metal ions and semiquinones are regenerated by stripping electrons from O2, creating superoxide radicals, which are converted to H2O2 by extracellular SODs and Cu+ and Fe2+ in Fenton-like reactions97,98. Kinetic models predict the amount of ROS produced in the surface lining fluid from an exposure to 15 PM2.5 of greater than 50 mg/m3 for 2 hours would range from approximately 100 to 250 nmol/L compared with approximately 100 nmol/L found in the bronchial alveolar lavage fluid of healthy human subjects97,99. Along with the diffusion or import of ROS from airway surface lining fluid, air pollution exposure also stimulates intracellular ROS production. Many recent in vitro studies have examined the effects of various models of air pollution, which include ambient outdoor pollution around the world collected on filters9,59,85,90,91,94–96,100–103, fresh TRAP generated by gasoline or diesel engines64,92,93,104–106, standard reference materials (SRMs)60,107,108, and fresh wood smoke109–112, and have observed dose- and exposure-dependent increases in intracellular ROS or associated oxidation of proteins or DNA. Increased production of intracellular ROS on air pollution exposure is thought to be the result of increased activity of xenobiotic metabolism, disruption of mitochondrial membranes, and increased levels of nitrogen oxides. Xenobiotic metabolism and detoxification of PAHs involves the cytochrome P450 family of enzymes, such as CYP1A1 and CYP1B1, expression of which are increased by PAH-induced activation of the AhR. Indeed, air pollution and benzo(a)pyrene, a known carcinogenic PAH in air pollution, has induced expression of CYPs in BEAS-2B cells92,96,101,106, in a manner that was blocked by the AhR inhibitor α-NF96. Increased CYP expression has also been observed in the context of wood smoke exposure to A549 cells109. The metabolism of PAHs through cytochrome P450 produces ROS as a byproduct of the catabolic pathway, as well as quinones and semiquinones, which can subsequently produce more ROS through redox cycling113. Further supporting this mechanism, Leclercq et al.60,94 observed upregulation of many cytochrome genes, benzo(a)pyrene metabolites, OH-benzo(a)pyrene DNA adducts, and ROS-induced modifications of protein and DNA in differentiated primary human bronchial epithelial 16 cells that were grown at the air-liquid interface and exposed to SRM2786. Interestingly, Leclercq et al. also found that cells from donors with COPD had significantly more OH-benzo(a) pyrene DNA adducts and ROS-induced macromolecule modifications than those from healthy donors, suggesting that patients with chronic respiratory diseases might be more susceptible to the byproducts and molecular damage caused by increased ROS. Another major source of endogenous ROS are mitochondria. Oxidative phosphorylation produces superoxide molecules that are rapidly converted to H2O2 by SODs before being further catabolized by antioxidant enzymes. However, alteration of mitochondrial function can lead to increased rates of ROS formation, and disruption of the mitochondrial membrane can release stored ROS89. BEAS-2B cells exposed to a specific quinone 1,2-naphthoquinone, which is found on the surfaces of particulate matter, were found to have reduced maximal respiration through a mechanism upstream of pyruvate transport, increased spare respiratory capacity, unaffected ATP production, and increased non-mitochondrial respiration114. The increase in non-mitochondrial respiration coincided with an increase in H2O2 levels and was found to be NADPH-dependent, suggesting 1,4- naphthoquinone might be driving a redox cycle reaction. Leclercq et al.59 also examined the effects of PM2.5 on mitochondrial structure and function in a differentiated primary epithelial model. They found that PM2.5 exposure was associated with abnormalities in mitochondrial volumes, matrix brightness, and cristae number. Supporting this observation, they measured concentration-dependent increases in SOD activity and mitochondrial superoxide that corresponded with a decrease in mitochondrial membrane potential, basal oxygen consumption rate, and ATP production. However, these cells did not show signs of activated caspase cascades associated with mitochondrial-induced apoptosis. Expanding on this work, 17 Song et al.102 metabolically profiled BEAS-2B cells exposed to PM2.5 collected in Taiyuan, China. Analysis of the metabolites in PM2.5-exposed cells revealed increased purine metabolism through the xanthine oxidase pathway (produces ROS as a byproduct) and arginine metabolism. The metabolism of arginine through nitric oxide synthases produces NO, which can be converted to more reactive RNS that can inhibit or irreversibly damage mitochondrial complexes I to V. Consistent with Leclercq et al.59, metabolic profiling revealed a general shift from oxidative phosphorylation to glycolysis, an observation supported by significantly reduced basal and maximal respiration rates and decreased ATP production. Together, these studies demonstrate the negative effect of air pollution on mitochondrial function and ROS production. 1.8 Air pollution-induced oxidative stress and lung epithelial cell defence mechanisms After exposure to air pollution, the first line of defence against ROS and RNS within the respiratory mucosa is a combination of scavenging antioxidant molecules and antioxidant enzymes. GSH is one of the major antioxidant systems cells use to reduce ROS levels. GSH can be oxidized by glutathione peroxidase to glutathione disulfide, resulting in conversion of H2O2 to H2O. Subsequently, GSH can be regenerated by glutathione reductase by using NADPH88. Oxidation of GSH and subsequent increase in glutathione disulfide levels have been demonstrated in various epithelial cell models exposed to air pollution from a variety of sources9,60,95,102,103. Acute air pollution exposure has also been associated with increased GSH levels in human bronchoalveolar lavage fluid47, where ROS are scavenged by antioxidants, such as vitamin C and E115. Exposure to air pollution has been associated with increased uric acid release into the bronchoalveolar lavage fluid in human subjects47, which is likely produced and exported by lung epithelial cells116. Demonstrating the importance of 18 these mechanisms, air pollution exposure has also been correlated with decreases in lung function and circulating a-tocopherol (vitamin E) and threonate (a metabolite of vitamin C) levels117. As ROS levels continue to accumulate in airway epithelial cells exposed to air pollution, excess ROS triggers MAP Kinase signalling cascades as well as the activation of transcription factors Nrf2 and NF-κB. Oxidation of KEAP1 releases Nrf2, which then translocates to the nucleus and initiates the transcription of several antioxidant and detoxifying enzymes. Primary HBE cells exposed to ambient air pollution (PM2.5) were found to have dose-dependent increases in NRF2 binding activity and transcription of target genes including itself NFE2L2, glutathione peroxidases GPX1 and GPX2, as well as a subunit of a rate limiting enzyme in glutathione synthesis GCLM59. Interestingly, the induction of NRF2 genes was reduced in cells exposed to the highest dose of PM2.5, possibly through increased oxidation and degradation, suggesting a limit to the antioxidant response lung epithelial cells can mount. Similar results have been observed in other studies examining primary HBE cells64,71,104,105, epithelial BEAS-2B cells102,118, and alveolar A549 cells100 exposed to air pollution. In a similar mechanism to Nrf2, NF-κB can also be activated directly via the oxidation of inhibitor of nuclear factor kappa B (IκB) by ROS or indirectly through a combination of increased receptor tyrosine kinase, tumor necrosis factor receptor (TNFR), TLR, and IL-1R signalling in response to air pollution exposure. NF-κB is involved in regulating the expression of inflammatory cytokines such as IL-6, IL-8, and TNFα as well as enzymes such as glutathione peroxidase and thioredoxin-1 that are involved in antioxidant systems119. Current studies have predominately linked the activation of NF-κB by air pollution to increased inflammatory cytokine expression. For example, Liu et al. demonstrated increased NF-κB activation via AKT and signal transducer and activator of transcription 3 (STAT3) leading to increased IL-6 in A549 19 cells exposed to PM2.5107, Leclercq et al. showed a dose-dependent increase in NF-κB binding activity and TNFα and IL-6 expression in primary HBE cells exposed to PM2.559, and Weng et al. observed increased NF-κB-dependent induction of IL-17A in primary HBE cells85. However, these studies do not provide direct evidence of ROS induced NF-κB activity. 1.9 Consequences of air pollution-induced oxidative stress The consequences of increased ROS and RNS values that exceed the capacity of antioxidant defences in airway epithelial cells include DNA oxidation and damage, lipid peroxidation and compromised membrane integrity, protein oxidation and/or nitration, and cell-cycle arrest and apoptosis depending on the severity of the damage. In primary human bronchial epithelial cells, air pollution (SRM2786) has been demonstrated to induce production of malondialdehyde (a secondary product of lipid peroxidation) and increased carbonylated protein levels and DNA 8-OHdG adducts60. Consistent with the idea that loss of viability is related to air pollution–driven DNA damage by ROS, Wu et al.95 observed a dose-dependent decrease in S-phase cell counts and an increase in G2/M-phase cell counts in a BEAS-2B model, which was indicative of cell-cycle inhibition at DNA damage checkpoints. Similar results have also been observed in other recent in vitro models of air pollution exposure and studies that observed autophagy102,120, cell-cycle arrest121,122, and loss of cell viability at greater exposure concentrations59,94,95,102. Of clinical relevance, ROS and RNS have also been linked to decreased glucocorticoid receptor function123, suggesting air pollution can affect glucocorticoid receptor function. However, a more insidious consequence of air pollution–induced ROS and DNA damage is carcinogenesis124,125, which can be amplified by the known carcinogen benzo(a)pyrene126. 20 1.10 Interventions for air pollution-induced oxidative stress and inflammation 1.10.1 Inhaled corticosteroids Currently, inhaled corticosteroids (ICS) are the gold standard treatment for controlling inflammation in the lungs and are used to treat asthma symptoms and reduce the risk of exacerbations and death127. ICS function fundamentally by altering gene expression in airway cells. Specifically, ICS diffuse through the cell membrane and bind to the glucocorticoid receptor α. The ligand:receptor complex then translocates into the nucleus to both positively and negatively regulate gene expression, directly through interactions with DNA glucocorticoid response elements and/or indirectly through tethering-interactions with inflammatory transcription factors128. The results of this transcriptional-regulation include increased expression of anti-inflammatory genes, such as RGS2, that may block the bronchoconstriction induced by histamine129, and suppression of transcription factor (NF-κB and AP-1) induced pro-inflammatory cytokine expression128. At the population level, air pollution exposure is highly correlated with enhanced ICS use by asthmatics130,131. However, the effectiveness of ICS medications may be reduced by pollution exposures132, as has been seen with other environmental exposures such as cigarette smoke133. Furthermore, oxidative stress induced by H2O2 has been shown to induce ICS hypo-responsiveness in human bronchial epithelial cells123,134. Finally, ICS has been shown to increase rhinovirus (RV)16 infectivity in human airway epithelial cells by suppressing antiviral gene expression, and may enhance air pollution-associated susceptibility to viral respiratory infections135. Therefore, it is unclear how beneficial ICS medications may be for PM-induced inflammation. 21 1.10.2 Omega-3 polyunsaturated fatty acids Recently there has been increasing research into non-steroid interventions for air pollution-induced inflammation. One such class of interventions that has been explored is long-chain omega-3 polyunsaturated fatty acids. These fatty acids are essential fats that can only be acquired through diet and are known to have anti-inflammatory and pro-resolving properties. Present in high levels in fatty fish and other seafood, plant oils, and tree nuts136, omega-3 fatty acids such as eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are metabolized via enzymes in the eicosanoid pathway to give rise to a group of molecules called specialized pro-resolving mediators (SPMs), that include resolvins, protectins, and maresins136–138. These SPMs act to resolve inflammation through receptor-mediated interactions with immune cells such as neutrophils, eosinophils, macrophages, T cells, B cells, and epithelial cells 137,139,140. Moreover, omega-3 fatty acids have also been implicated in modulating oxidative stress responses as increased intake in the diet has been associated with total serum antioxidant capacity and glutathione peroxidase activity, as well as decreased levels of oxidative stress marker malondialdehyde (MDA) in a meta-analysis of 39 clinical trials141. In the context of air pollution, clinical studies have demonstrated that increasing omega-3 fatty acids in the diet mitigated associations between PM2.5 and asthma symptoms in children (trouble breathing: OR = 0.97, SE = 0.01)142 and reduced systemic pro-inflammatory fatty acid metabolites such as Leukotriene B4 (LTB4) per 10 μg/m3 PM2.5 in healthy participants143. Supporting these findings, in vitro studies have demonstrated a protective role for DHA against organic dust extract (ODE)-induced inflammation (IL-6 and IL-8) using lung epithelial BEAS-2B cells144. Furthermore, using an ODE mouse model, DHA was shown to reduce infiltration of immune cells into the lungs, reduce inflammatory mediator levels (IL-6, TNF-α, CXCL1, and CXCL2) in the 22 bronchoalveolar lavage fluid (BALF), as well as increase BALF levels of the DHA-derived SPM resolvin D1 (RvD1)144. Omega-3 fatty acids have also been shown to reduce ambient PM2.5-induced oxidative stress in vascular endothelial cells145. However, no study to date has examined the use of omega-3 fatty acids as an intervention for urban air pollution in human lung epithelial cells. 1.10.3 Antioxidants The possibility of intervening in air pollution–induced ROS signaling, and damage has been explored by several groups using compounds that target different aspects of redox signaling or homeostasis. One group found that rosiglitazone, which activates peroxisome proliferator–activated receptors (PPAR), repressed the activation of extracellular signal-regulated kinase (ERK) 1/2 and STAT signaling and apoptosis in A549 cells exposed to PM2.5146. Rosiglitazone also reduced ROS production, although it is unclear through which mechanism this occurred. Interestingly, in a more complex co-culture model of primary HBE cells, THP-1 macrophages reduced the expression of Nrf2 genes in in HBE cells in response to DEP71, possibly through enhancing phagocytosis of the particles, suggesting that modulating immune cell recruitment may be an area to further explore in this context. Additional antioxidants, such as quercetin147, tetrahydrocurcumin148, chebulic acid149 and gallic acid103, vitamin D150, and the Nrf2 activator resveratrol151, have also been implicated in attenuating air pollution–induced oxidative stress and inflammation. Of interest, the plant flavonoid quercetin has been shown to ameliorate urban PM2.5-induced oxidative stress, inflammation (IL-1β and IL-6 gene expression), and dysregulation of mitochondrial genes in the human bronchial epithelial cell line 16HBE147, likely via both the disruption of pro-inflammatory NF-κB transcription and suppression of translation152,153. Moreover, quercetin has been shown to suppress 23 neutrophil elastase-induced MUC5AC expression in 16HBE cells154. However, the antioxidant intervention that has been examined the most to-date is n-acetylcysteine (NAC), which is both an antioxidant and precursor in GSH synthesis. Several studies have demonstrated the protective effect of NAC against air pollution in vitro107,118,155,156, and NAC has been shown to be protective in vivo against increased airway hyperresponsiveness induced by acute exposure to diesel exhaust157. Although these compounds show some promise, further development of interventions targeting ROS homeostasis in the lung epithelium could have broad clinical applications. 1.11 Air pollution and susceptibility to viral infection In terms of infection risk, increased upper and lower respiratory infections have both been associated with short-term air pollution exposures158–162. For instance, one study examining short-term increases in PM2.5 exposure found associations with increased acute upper and lower respiratory infection hospital admissions of 13.5% (95% CI, 5.6 to 22) and 22.8% (95% CI, 16.5 to 29.3) per 10 μg/m3 of PM2.5 (14 day lag period) respectively160. More recently, links between specific air pollutants, including PM2.5, NOx, and SO2, and acute viral respiratory infections in children, specifically RSV and RV, have been indentified159,163. Of note, RV is the most commonly identified virus associated with COPD and asthma exacerbation164, and concerningly, asthmatics exposed to PM2.5 and infected with rhinovirus have been shown to have synergistic decreases in lung function165. The mechanisms responsible for the population level ambient PM2.5 associations with respiratory infections are multifaceted and include altered expression of viral entry receptors and activation enzymes, and disruption of antiviral host defence responses. For example, diesel exhaust PM2.5 has been shown to induce the expression of angiotensin-converting enzyme (ACE) 2, the entry receptor for severe acute respiratory 24 syndrome coronavirus 2 (SARS-CoV-2), and transmembrane protease serine 2 (TMPRSS2), a protease required for SARS-CoV-2 entry into the cell in an alveolar epithelial cell model166,167. Another example is RV, which infects epithelial cells of the respiratory epithelium and induces cold symptoms in healthy people, with more severe consequences for those with underlying chronic respiratory diseases168. The 160 genetically distinct RVs are divided into three subclasses RV-A, RV-B, and RV-C, and the entry receptor for all of RV-B and some of RV-A (> 100 total) is cell surface receptor ICAM-1169. ICAM-1 has been demonstrated to be upregulated by air pollution in lung epithelial cells, mediated by NF-κB transcriptional events downstream of oxidative stress–induced classical PKB/AKT/STAT3 signaling107. Supporting this observation, primary nasal epithelial cells treated with diesel particulate matter (DPM) and infected with RV16 resulted in significantly increased ICAM-1 expression and RV loads170. Interactions between air pollution and the low-density lipoprotein receptor, the receptor for RV-A, or cadherin-related family member 3, the receptor for RV-C, have not been examined in the context of air pollution to date. Antiviral host defence responses limit the replication and release of new virions after a virus enters a cell. Typically, recognition of viral components by TLRs induces the expression and release of type I and type III interferons (IFNs), which results in the expression of IFN-stimulated genes (ISG) that act to either inhibit viral replication or recruit virus-specific immune effector cells 167,171,172. In the context of air pollution, basal and induced levels of the ISG C-X-C motif chemokine ligand 10 (CXCL10), a cytokine important in coordinating antiviral responses in immune cells expressing the CXCR3 receptor, were found to be attenuated in primary human airway epithelial cells via activation of the AhR by diesel exhaust particles SRM1975 from the National Institute of Standards and Technology (NIST)173. Similarly, decreased expression of the PRRs TLR3 and RIG-1, and CXCL10 was observed in primary nasal epithelial cells exposed 25 to DPM170. Although the attenuation of PRRs and CXCL10 was less pronounced in cells exposed to both DPM and infected with RV16, these studies support the hypothesis that air pollution disrupts ISG expression, thereby reducing the production of antiviral mediators in airway epithelial cells. Notably, the type and composition of air pollution exposure may have opposing effects on antiviral responses. Primary human airway epithelial cells from healthy subjects exposed to a farm dust extract prior to infection with RV1B were observed to have reduced viral load and increased expression of the ISGs radical S-adenosyl methionine domain containing 2 (RSAD2) and IFN regulatory factor 1 (IRF1)174. The protective effect of this dust is thought to be related to the presence of microbial components that prime antiviral host responses. Air pollution has also been implicated in dysregulating the expression of host defence peptides (HDPs), a class of proteins that are released into the airway surface lining fluid to disrupt the life cycle of pathogens. Urban particulate matter- (NIST SRM 1649B) exposed BEAS-2B cells were found to have suppressed human b-defensin-2 gene and protein expression and impaired Pseudomonas aeruginosa killing175. Interestingly, reconstitution with human b-defensin-2 (HBD2) or treatment with the antioxidant NAC rescued bacterial killing activity. Supporting the results of this study, diesel exhaust exposures were shown to attenuate HBD2 and S100A7 in primary human airway epithelial cells in response to nontypeable Haemophilus influenzae challenge105. However, HBD2 has been shown to be ineffective against RV16176, a result of interest in the context of this thesis. RV replication can be inhibited by the HDP LL-37177, and carbon nanoparticles (component of air pollution) have been shown to alter the structure of LL-37 and render it ineffective178. A result that implicates impairment of HDPs as a mechanism by which air pollution increases susceptibility to RV infection. 26 In summary, although ambient PM2.5 is consistently linked with increased viral infection, these studies highlight the complex gene-environment interactions that modify host defence responses to specific viral pathogens. It is clear that further studies are needed to unravel the dynamic interaction between ambient PM2.5 and respiratory infections to help protect the health and well-being of the public. 1.12 Thesis objectives and hypotheses The overall aim of my thesis is to investigate the utility and consequences of air pollution interventions in human bronchial epithelial cells in vitro. Chapter 2 describes investigation to determine whether an antioxidant applied to an in vitro bronchial epithelial cell model reduces the inflammatory potential of urban air pollution particulate matter. Hypothesis: 1. Application of an antioxidant to epithelial tissue will reduce PM-induced oxidative potential and/or epithelial barrier dysfunction, resulting in attenuated inflammatory responses. Chapter 3 investigates the utility of priming bronchial epithelial cells with omega-3 polyunsaturated fatty acids against urban air pollution particulate matter-induced oxidative stress and inflammatory mediator release. Hypothesis: 1. Treatment of bronchial epithelial cells with DHA or EPA attenuates PM-induced ROS and release of pro-inflammatory mediators. 27 Chapter 4 investigates the effects of combined air pollution exposure and corticosteroids on antiviral responses and viral replication in bronchial epithelial cells. Hypotheses: 1. PM exposure combined with corticosteroid treatment will increase human rhinovirus replication in human airway epithelial cells. 2. PM exposure will suppress antiviral ISG expression. 3. Inhibition of the NF-kB pathway will attenuate PM- and corticosteroid-induced rhinovirus replication. 28 Chapter 2: Targeting traffic-related air pollution-induced oxidative stress in airway epithelial cells. 2.1 Introduction Air pollution is a persistent and growing problem around the globe with 99% of the world’s population living in areas exceeding WHO daily air pollution exposure recommendations179. A main source of urban air pollution particulate matter is diesel fuelled machinery and vehicles, and increased PM exposure has been associated with worsening asthma symptoms, and increased hospital visits53,180,181. Although limiting exposure using particle filtering masks or air purifiers are important interventions to reduce exposure concentrations during periods of high ambient air pollution, there remains a large population of people around the world that are inevitably still exposed. In vitro and in vivo studies examining the direct effects of acute air pollution exposure on the human lungs have revealed that high levels induce oxidative stress and inflammation40,44–48. The imbalance of oxidative species in cells induced by air pollution particle exposures is thought to chemically modify and damage macromolecules of the cell, augmenting an inflammatory response driven by physical interaction of the particles with pattern recognition receptors, such as TLR2 and 459,60,62,65,182,183. The convergence of these responses results in production and release of acute pro-inflammatory mediators that include TNFα, IL-1β, IL-6, and IL-8, leading to an influx in neutrophils and macrophages40,46,48,60. Furthermore, persistent inflammation induced by air pollution exposure is associated with airway hyperresponsiveness, tissue remodelling, and the pathogenies of chronic lung disease184,185. Although public health efforts continue to push for reduced emissions, there remains a growing need for interventions to protect the airway from air pollution exposure. This remains relevant as the problem of traffic-related air pollution persists, 29 and indeed grows, as the world continues to rapidly industrialize180. To date, several interventions of air pollution-induced oxidative stress and inflammation have been explored including, NAC, vitamin D, vitamin C, resveratrol, and quercetin107,147,150,151,155. NAC has been shown to be protective against air pollution-induced airway hyperresponsiveness, though it is poorly tolerated with adverse gastrointestinal effects, such as nausea and vomiting157. No study to date has broadly examined antioxidants as an air pollution intervention in primary human airway epithelial cells. To provide insight into the utility of small molecules with antioxidant properties in this context, I tested the hypothesis that application of an antioxidant to epithelial cells will reduce TRAP-induced oxidative potential, resulting in attenuated inflammatory responses. I screened 18 molecules for their antioxidant potential in human lung epithelial cell lines, and translated two molecules, tetrahydrocurcumin and quercetin, into a differentiated primary human epithelial air-liquid interface cell culture model to measure their effect on inflammatory mediators. Overall, these molecules were observed to attenuate diesel exhaust particle-induced ROS and may suppress an associated inflammatory mediator response. 2.2 Methods 2.2.1 Cell culture Human bronchial epithelial BEAS-2B cells were cultured in Dulbecco's Modified Eagle’s Medium/Ham’s F12 (DMEM/F12) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and antibiotic-antimycotic (Thermo Fisher Scientific). Calu-3 cells were cultured in Eagle's Minimum Essential Medium (EMEM) (MilliporeSigma) supplemented with L-glutamine (Thermo Fisher Scientific), sodium bicarbonate (MilliporeSigma), HEPES (MilliporeSigma), 10% FBS, and antibiotic-antimycotic. Healthy human bronchial epithelial cells (HBECs) were 30 previously isolated from endobronchial brushes from participants (never smokers) of two previously completed clinical studies: COPA (UBC Clinical Research Ethics Board: H14-00821, clinicaltrials.gov ID: NCT02236039) and AGnES (UBC Clinical Research Ethics Board: H18-01836) (Table 2.1). HBECs were expanded using PneumaCult-EX Basal Medium (Stemcell Technologies) and seeded onto 6.5mm transwell inserts (Corning) at a concentration of 3x104 cells/insert. HBECs were then incubated in submerged cultures for 4 days in PneumaCult-EX Basal Medium, prior to air lifting using PneumaCult-ALI Maintenance Medium (Stemcell Technologies) as per the manufacturer’s instructions. Cells were then maintained for at least 21 days prior to experiments. Mucus was washed from the apical surface every 2-3 days using HBSS. Table 2.1 HBEC donor characteristics ID Study Age (years) Sex FEV1 (% Predicted) FEV1/FVC 1 AGnES 55 Male 115 0.77 2 AGnES 44 Female 86 0.7 3 COPA 58 Male 140 0.8 4 COPA 52 Male 117 0.79 5 COPA 63 Male 124 0.75 6 COPA 72 Female 162 0.79 7 COPA 50 Male 101 0.74 Summary 56 ± 9 2 F, 5 M 120 ± 25 0.76 ± 0.04 2.2.2 PM exposures Pollution exposures were produced by combining National Institute of Standards and Technology (NIST) standard reference material (SRM) 1650b (MilliporeSigma) or PM generated at the Air Pollution Exposure Laboratory (APEL PM) with EMEM and DMEM for Calu-3 and BEAS-2B exposures respectively, or HBSS for HBEC exposures. SRM1650b is a diesel exhaust particulate matter model characterized by NIST (details available in the NIST certificate of analysis). APEL PM was collected from the tailpipe of 31 an EPA Tier 3-compliant, 6.0 kW Coliseum GY6000 generator and has been previously characterized186. SRM1650b and APEL PM were sonicated prior to exposures in 20 second intervals for a total sonication time of 3 minutes in EMEM media for Calu-3 cells, DMEM media for BEAS-2B cells, and HBSS for HBECs. PM dosing was determined by an initial dose-response experiment (Figure 2.1). The dose of 200 µg/mL used for initial experiments is a high dose and is approximately equivalent to that experienced by sections of the respiratory tract after breathing in 400 µg/m3 PM2.5 for 24 hours, levels routinely experienced by those living in heavily polluted cities such as Delhi, India187. Untreated0.5mM H2O21.0mM H2O212.5 25 50 1002004000.05.0×1061.0×1071.5×1072.0×1072.5×1073.0×107H2DCFDA (R.F.U.)SRM1650bAPEL PMPM (µg/mL)********************************BEAS-2BUntreated0.5mM H2O21.0mM H2O212.5 25 50 1002004000.05.0×1061.0×1071.5×1072.0×1072.5×1073.0×107H2DCFDA (R.F.U.)SRM1650bAPEL PMPM (µg/mL)**************************Calu-3A B C D UntreatedLysis BufferH202 0.5 mMH202 1 mM400g/mL200µg/mL100µg/mL50µg/mL25µg/mL12.5µg/mL051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PM****Calu-3UntreatedLysis BufferH202 0.5 mMH202 1 mM400훍g/mL200µg/mL100µg/mL50µg/mL25µg/mL12.5µg/mL051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PM****************BEAS-2B 32 Figure 2.1 SRM1650b and APEL PM dose-dependently increase ROS generation. (A) Calu-3, and (B) BEAS-2B cells were exposed to increasing doses of APEL PM and SRM1650b following incubation with 10μM of H2DCFDA for 30 minutes. Relative fluorescence intensity (R.F.U.) of H2DCFDA was measured 24 hours post-exposure and plotted as means ± SD. Matched LDH assays (C and D) were performed using cellular supernatants collected at 24 hours. The percent cytotoxicity (percent of lysis buffer control) is plotted as mean ± SD. Data analyzed by two-way ANOVA with Dunnett’s post-hoc test (N = 4; ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05; comparisons to untreated condition). 2.2.3 Air pollution interventions Small molecules with antioxidant properties were selected as potential interventions for air-pollution induced oxidative stress in collaboration with GSK. Small molecule selections were based on a combination of literature support in the form of published studies demonstrating antioxidant effects and an internal GSK screen for molecules that induce Nrf2 activity (Table 2.2). The selection of small molecule test dose ranges was based on available literature. 2.2.4 Oxidative stress assay BEAS-2B and Calu-3 cells were seeded onto black-walled 96 well plates (Corning), at a concentration of 1.5x104 cells/well and 8x104 cells/well respectively in maintenance medium. Cells were serum starved for 24 hours prior to incubation with 10 μM of 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA, Thermo Fisher Scientific), a fluorescent reactive oxygen species sensor (ROS), for 30 minutes. Following three washes with HBSS (Thermo Fisher Scientific), cells were either preincubated with antioxidant compounds for one hour or incubated simultaneously with SRM1650b or APEL PM (200 µg/mL). Each of the 18 test molecules was tested in a five-point dose-response curve based on available literature (Table 2.2). Relative fluorescence intensity (R.F.U.) of H2DCFDA (488 nm excitation/525 nm emission) was assessed at 0 hours and at 24 hours using a florescent plate reader (Spectramax i3x, Molecular Devices). Table 2.2 Summary of interventions selected for oxidative stress screening assays in Calu-3 and BEAS-2B cells *Maximum concentration used for 5-point Log10 dose-response curve &Cells were pre-incubated with treatments for the indicated times based on the mechanism of the intervention. Free radical scavengers were applied to the cells simultaneously whereas molecules that acted on pathways were pre-loaded onto the cells. %GSK reference indicates molecules identified during an internal GSK screen for molecules with Nrf-2 activity Molecule Supplier Catalog No. Cas No. Max Conc. (µM)* Pre-Incubation (Hours)& Mechanism Ref.% NAC (N-acetylcysteine) MilliporeSigma A9165-5G 616-91-1 100 0 Glutathione precursor 63 Vitamin E (α-tocopherol) MilliporeSigma T1539-25G 59-02-09 1000 0 Peroxyl scavenger 188 Ascorbic acid (Vitamin C) MilliporeSigma A4544-25G 50-81-7 1000 0 Free radical scavenger 147 Butylated Hydroxytolune MilliporeSigma B1378-100G 128-37-0 1000 0 Free radical scavenger 189 Pterostilbene MilliporeSigma 523310-10MG 537-42-8 100 1 Antioxidant & Nrf-2 activator 190,191 Tetrahydrocurcumin MilliporeSigma SMB00370-1G 36062-04-1 100 1 Free radical scavenger & NF-κB suppression 192–195 Quercetin MilliporeSigma Q4951-10G 117-39-5 100 1 Nrf-2 activator 147,152,153,196,197 Lawsone MilliporeSigma H46805-10G 83-72-7 1000 1 Nrf-2 activator 198 Tert-Butylhydroquinone MilliporeSigma 112941-5G 1948-33-0 10 1 Nrf-2 activator 199 Dimethyl fumarate MilliporeSigma 242926-25G 624-49-7 100 1 Nrf-2 activator 200 Sulbentine MedChemExpress HY-B1133 350-12-9 100 1 Nrf-2 activator GSK Guggulsterone MilliporeSigma G4923-5MG 39025-24-6 100 1 Nrf-2 activator 201 Spermidine MilliporeSigma S0266-1G 124-20-9 100 1 Nrf-2 activator & polyamine synthesis 202 Retinyl acetate MilliporeSigma R7882-1G 127-47-9 100 1 Nrf-2 activator GSK L-Cysteine ethyl ester MilliporeSigma C121908-25G 868-59-7 100 1 Nrf-2 activator GSK MitoQ Toronto Research Chemicals Inc M372215-25 845959-50-4 10 1 Mitochondria modifier 203 Fluticasone propionate MilliporeSigma F9428-5MG 80474-14-2 1 6 Corticosteroid 204,205 Resveratrol Sigma-Aldrich R5010-100MG 501-36-0 100 1 Nrf-2 activator 191,206 For HBEC H2DCFDA assays, cells were incubated with 10μM of H2DCFDA in HBSS for 30 minutes via the basal chamber prior to the start of the experiment. Cells were then treated apically with either tetrahydrocurcumin in a half log10 7-point dose-response curve (0.0316 μM – 31.6 μM) or quercetin in a half log10 7-point dose-response curve (0.01 μM – 100 μM) for 1 hour. Treatments were then removed and SRM1650b 10 μg/cm2 was applied to the apical chamber for 4 hours simultaneously with treatments. Duration and time of exposures were based on literature59 and a limited pilot (data not shown). Exposures and treatments were then removed and the cells washed once with HBSS. Relative fluorescence intensity (R.F.U.) of H2DCFDA was assessed at 0, 2, 4, 6, 8, 22, and 24 hours after the start of the exposure. Hydrogen peroxide (MilliporeSigma) was added apically at a dose of 0.5 mM in other wells, as a positive control to induce oxidative stress. 2.2.5 Multiplex ELISA HBECs were pre-incubated apically in a half log10 7-point dose-response curve with either tetrahydrocurcumin (0.0316 μM – 31.6 μM) or quercetin (0.01 μM – 100 μM) for 1 hour. Treatments were then removed and SRM1650b 10 μg/cm2 was applied to the apical chamber for 4 hours simultaneously with treatments. The exposure and treatments were then removed and the cells were washed once with HBSS. Supernatants were collected from the basal chambers 24 hours after the start of the exposure and centrifuged at 5000g for 5 mins to remove course PM particles and cells. Analyte concentrations (pg/mL) were determined using a Human Cytokine 48-Plex Discovery Assay performed by Eve Technologies (Calgary, Alberta, Canada). Analytes found to be undetectable were eliminated from the analysis; otherwise for statistical comparisons values found to be below the lower limit of detection (LLOD) were replaced with ½ of the respective LLOD. 35 2.2.6 LDH assays Supernatants were collected and centrifuged at 5000g for 5 mins to remove course PM particles and cells. Lactate dehydrogenase (LDH) assays were performed using a CyQUANT LDH cytotoxicity assay kit (Thermo Fisher Scientific) as per the manufacturer’s instructions. Absorbance was measured using a Spectramax i3x plate reader (Molecular Devices). 2.2.7 Statistical analysis Statistical analysis was performed using Prism Graphpad 10. Data (n ≥ 4) was analyzed using a combination of z-scores, t-tests, One-way, and Two-way ANOVAs using either Tukey’s or Dunnett’s post-hoc test for multiple comparisons, as indicated in figure legends. A p-value of <0.05 was considered statistically significant. Significance (p <0.05) of pairwise comparisons is indicated by an * as indicated in figure legends. Dose-response curve analysis was performed using nonlinear regression Log10 (inhibitor) versus response (Y=Bottom + (Top-Bottom)/(1+10^(X-LogIC50))). Prior to modeling dose-response H2DCFDA R.F.U. data was transformed by subtracting baseline (0 hours) and calculating the fold change from untreated control. For HBECs, H2DCFDA R.F.U. data from each time point was transformed by subtracting baseline (0 hours) and plotted against time. The area under the curve for each condition was calculated and normalized as fold change from the untreated control. 2.3 Results 2.3.1 Effect of particulate matter on epithelial cell oxidative stress and cytotoxicity Initial experiments were performed to assess the oxidative stress potential of the two test pollution models SRM1650b and APEL PM over a range of doses (12.5-400 μg) in both Calu-3 and BEAS-2B cells at 24 hours. Both PM models significantly and 36 dose-dependently increased the fluorescence of the ROS sensor H2DCFDA compared to untreated cells (Figure 2.1A-B). A dose of 200 μg/mL was chosen for use in intervention experiments as ROS levels were significantly increased by 2.1 and 2.2-fold by SRM1650b and APEL PM in Calu-3 cells, and 1.6 and 1.2-fold BEAS-2B cells respectively. Cytotoxicity, as measured by LDH release assays, was also assessed in the same PM dose-response experiments (Figure 2.1C-D). In Calu-3 cells, no significant increases in LDH release were observed over the range of SRM1650b and APEL PM doses tested apart from the highest dose of APEL PM (400 μg/mL). In BEAS-2B cells, no significant increases in LDH release were observed over the range of SRM1650b doses tested. However, a trend for increased LDH release was observed for APEL PM, with minimal, but significant, increases at 200 and 400 μg/mL. 2.3.2 Effect of small molecule interventions on particulate matter-induced oxidative stress To assess the utility of the test molecules at attenuating air pollution-induced oxidative stress Calu-3 and BEAS-2B cells were exposed to a fixed dose of 200 μg/mL of SRM1650b and APEL PM in combination with each of the 18 small molecules in a five-point dose-response experiment (Figure 2.2). The efficacy of each molecule at attenuating air-pollution induced ROS was determined using nonlinear regression of the Log10(inhibitor) versus response and is summarized in Table 2.3. In total, 9 of 18 molecules significantly attenuated SRM1650b-induced ROS levels in Calu-3 cells as determined by the inhibitory maximum (SRM1650b condition vs Imax). Of these nine molecules, the Imax of seven were calculated to be below that of untreated controls. In BEAS-2B cells, six of these seven molecules were also determined to have Imax values that were significantly decreased compared to 37 Figure 2.2 Overview of oxidative stress assays in Calu-3 and BEAS2-B cells. Cells were exposed to 200µg/mL of either SRM1650b or APEL PM for 24 hours. Cells were pre-incubated with compounds predicted to modulate pathways for 1 hour prior to exposure. Compounds were tested in 5-point dose curve in both cell lines. Calu-3 and BEAS-2B submerged monolayer cultures were used to examine oxidative stress using an H2DCFDA fluorescent dye-based method, and cytotoxicity by measuring LDH release over the 24-hour exposure. SRM1650b exposed cells. Finally, three molecules, were also found to have significant Imax values in APEL PM exposed cells. Ranked by the average Imax value across all conditions and cell models, these three molecules were vitamin C, NAC, and tetrahydrocurcumin. 2.3.3 Effect of small molecule interventions on cell viability To assess the effect of the test molecules on cytotoxicity, LDH release assays were performed in parallel with oxidative stress assays (Figure 2.3 and 2.4). In Calu-3 cells, SRM1650b and APEL PM induced significant, but minimal, cytotoxicity in several iterations of the dose-response experiments (Figure 2.3D, E, G, Q, and R). However, when the data from all the iterations were combined there was no significant difference in cytotoxicity between untreated (M = 6.78, SD = 2.95), SRM1650b (M = 5.24, SD = 2.88), and APEL PM (M = 6.57, SD = 3.20) exposed cells. The test molecules minimally H2DCFDA 30 mins. Antioxidants / free radical scavengers PM 200µg/mL Pathway modulators (e.g. NRF2 activators) PM 200µg/mL H2DCFDA fluorescence: 0 and 24 hours 1 hour 30 mins. LDH Assays: Supernatants collected at 24 hours 24 hours 24 hours Table 2.3 Effect of interventions on air pollution-induced oxidative stress Rank% Molecule PM Model Calu-3 (n=4) BEAS-2B (n=4) IC50 ± SE (μM) Imax ± SE (%Untreated) § IC50 ± SE (μM) Imax ± SE (% of Untreated) § 1 Vitamin C SRM1650b 40.22 ± 1.54 6.65 ± 15.36* 141.90 ± 1.17 40.72 ± 4.47* APEL PM 1.05 ± 14.93 5.45 ± 9.93* 328.60 ± 1.50 69.33 ± 4.16* 2 N-acetyl cysteine SRM1650b 31.27 ± 1.52 0.01 ± 15.88* 2.35 ± 1.50 68.72 ± 5.53* APEL PM 19.11 ± 1.60 34.28 ± 17.07* 1.51 ± 1.85 58.85 ± 3.72* 3 Lawsone SRM1650b 7.96 ± 1.56 10.17 ± 10.17* ~ ~ APEL PM 172.50 ± 1.58 13.29 ± 13.29* 431.30 ± 1.64 20.72 ± 12.99* 4 Tetrahydrocurcumin SRM1650b 0.16 ± 1.43 62.78 ± 3.08* 30.70 ± 1.28 35.72 ± 7.68* APEL PM 0.07 ± 1.47 93.38 ± 2.84* 21.59 ± 4.82 82.17 ± 7.69* 5 L-Cysteine Ethyl Ester SRM1650b 2.14 ± 1.88 61.29 ± 13.67* 48.35 ± 2.33 43.56 ± 32.17* APEL PM 5.70 ± 11.51 97.88 ± 17.15 ~ ~ 6 Quercetin SRM1650b 1.96 ± 1.43 67.78 ± 10.01* 5.73 ± 1.30 31.6 ± 8.78* APEL PM 1.28 ± 12.25 164.70 ± 13.61 7.45 ± 4.45 118.30 ± 4.60 7 Sulbentine SRM1650b 16.76 ± 1.38 20.38 ± 12.83* 17.70 ± 1.32 54.06 ± 6.56* APEL PM ~ ~ ~ ~ 8 Resveratrol SRM1650b 0.29 ± 4.05 156.60 ± 7.97 1.02 ± 1.68 121.3 ± 5.29 APEL PM ~ ~ 21.52 ± 2.37 82.41 ± 11.84* 9 Fluticasone propionate SRM1650b ~ 164.90 ± 4.41 0.16 ± 1.35 57.87 ± 6.87* APEL PM 0.01 ± 4.91 137.70 ± 4.12 ~ ~ 10 Vitamin E SRM1650b ~ ~ ~ ~ APEL PM 0.27 ± 127 112.00 ± 13.72* 0.81 ± 14.79 114.00 ± 2.23 11 Spermidine SRM1650b 0.28 ± 1.13 107.40 ± 5.35* 8.4 ± 3.96 192.6 ± 6.03 APEL PM 0.25 ± 1.15 96.11 ± 6.25* 8.79 ± 17.42 149.90 ± 11.41 39 Rank% Molecule PM Model Calu-3 (n=4) BEAS-2B (n=4) IC50 ± SE (μM) Imax ± SE (%Untreated) § IC50 ± SE (μM) Imax ± SE (% of Untreated) § 12 Pterostilbene SRM1650b ~ ~ 19.62 ± 4.99 148.7 ± 17.24 APEL PM ~ ~ 50.86 ± 16.52 99.39 ± 26.41 13 Tert-Butylhydroquinone SRM1650b 0.01 ± 2.12 110.90 ± 5.14* 1.45 ± 3.49 230.90 ± 12.86 APEL PM 0.01 ± 3.69 103.90 ± 6.10* 0.02 ± 32.96 126.50 ± 11.41 14 MitoQ SRM1650b 0.08 ± 3.37 134.90 ± 7.51 0.16 ± 3.81 202.30 ± 6.48 APEL PM 0.22 ± 20.89 140.30 ± 7.66 0.01 ± 9.32 130.10 ± 3.75 15 Dimethyl fumarate SRM1650b ~ 164.80 ± 8.52 1.74 ± 3.34 181.7 ± 5.85 APEL PM ~ ~ 0.65 ± 5.17 119.3 ± 2.84 16 Guggulsterone SRM1650b ~ ~ 0.44 ± 7.65 182.4 ± 5.75 APEL PM ~ ~ 2.194 ± 2.71 129.2 ± 3.59 17 Butylated Hydroxytolune SRM1650b ~ ~ 643.00 ± 47.75 168.1 ± 26.21 APEL PM ~ 204.00 ± 10.97 4.02 ± 16.52 124.2 ± 3.18 18 Retinyl acetate SRM1650b ~ ~ ~ ~ APEL PM ~ ~ ~ ~ 5-point dose-response curves were analyzed using nonlinear regression of Log10(inhibitor) versus response. ~ Ambiguous fit of nonlinear regression of log(inhibitor) versus response. §Inhibitory maximum (Imax) was determined using nonlinear regression of the log(inhibitor) versus response. Bolded numbers represent cases where the maximum inhibition of oxidative stress as measured by H2DCFDA was predicted to be below that of untreated (100%). *Indicates significantly decreased oxidative stress levels compared to SRM1650b or APEL PM (P < 0.05, t-test) %Interventions were ranked based on the average maximum inhibition predicted by dose-response model. If a curve was ambiguous with no predicted value then the precent change of the exposure compared to untreated (SRM1650b or APEL PM) was used as it represented no inhibition. 40 A B C D E F G H I J K L M N O P Q R UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMNAC(µM)UntreatedLysis BufferSRM1650bAPEL PM 0.1 1 10 1001000051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMButylated Hydroxytolune (BHT)(µM)**** ********UntreatedLysis BufferSRM1650bAPEL PM 0.001 0.01 0.1 1 10051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMQuercetin(µM)*******UntreatedLysis BufferSRM1650bAPEL PM 0.001 0.01 0.1 1 10051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMDimethyl Fumarate(µM)UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMSpermidine(µM)UntreatedLysis BufferSRM1650bAPEL PM 0.001 0.01 0.1 1 10051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMMitoQ(µM)UntreatedLysis BufferSRM1650bAPEL PM 0.1 1 10 1001000051015202595100105Percent Cytotoxicity(LDH Release) Vitamin E (α-tocopherol)(µM)SRM1650bAPEL PM*UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMPterostilbene(µM)**************************UntreatedLysis BufferSRM1650bAPEL PM 0.1 1 10 1001000051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMLawsone(µM)****UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMSulbentine(µM)UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMRetinyl Acetate(µM)UntreatedLysis BufferSRM1650bAPEL PM0.00010.001 0.01 0.1 1051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMFlucticasone Propionate(µM)*UntreatedLysis BufferSRM1650bAPEL PM 0.1 1 10 1001000051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMAscorbic acid (Vitamin C)(µM)**********UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMTetrahydrocurcumin(µM)UntreatedLysis BufferSRM1650bAPEL PM 0.001 0.01 0.1 1 10051015202595100105Percent Cytotoxicity(LDH Release) Tert-Butylhydroquinone(µM)SRM1650bAPEL PMUntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMGuggulsterone(µM)UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PML-Cysteine ethyl ester(µM)UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMResveratrol(µM)**** 41 Figure 2.3 Cytotoxicity of antioxidant interventions in calu-3 cells. Cells were exposed to 200 µg/mL of either SRM1650b or APEL PM in combination with increasing doses of an antioxidant as indicated in Table 2.2 and Figure 2.2. The percent cytotoxicity (percent of lysis buffer control) is plotted as mean ± SD. Data analyzed by two-way ANOVA with Dunnett’s post-hoc test (N = 4; ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05; comparisons to untreated condition). impacted cell viability, with significant cytotoxicity observed at the top end of the dose-response curve for pterostilbene, lawsone, and resveratrol. No protective dose-response was observed for any of the molecules for the given test PM exposures. In BEAS-2B cells, no significant differences in cytotoxicity were observed in SRM1650b exposed cells (M = 4.33, SD = 1.63) compared to untreated cells (M = 4.49, SD = 1.97) when all iterations were combined, and a small significant difference (P < 0.05) was observed for APEL PM exposure (M = 13.77, SD = 5.30) (Figure 2.4). Similar to observations made in Calu-3 cells, the test molecules minimally impacted cell viability, with significant cytotoxicity observed at the top end of the dose-response curve for NAC, Lawsone, and dimethyl-fumarate. Again, no protective dose-response was observed for any of the molecules for the given test exposures. 2.3.4 Tetrahydrocurcumin and quercetin attenuate particulate matter-induced oxidative stress in primary human bronchial epithelial cells Based on oxidative stress and LDH assays in Calu-3 and BEAS-2B cells, two molecules were selected to further examine in primary human epithelial cells using an air-liquid interface culture model. These two compounds, tetrahydrocurcumin and quercetin, were the first two compounds with the lowest average Imax in which dose-response curves could be modeled, narrow IC50 ranges, and no dose-dependent increases in cytotoxicity in both cell line and particulate matter exposure models (Table 2.3, Figure 2.3 and 2.4). Using H2DCFDA assays and a seven-point dose-response curve, both tetrahydrocurcumin and quercetin significantly and dose-dependently 42 A B C D E F G H I J K L M N O P Q R UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 1000102030405095100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMNAC(µM)*******************UntreatedLysis BufferSRM1650bAPEL PM 0.1 1 10 1001000010203095100105Percent Cytotoxicity(LDH Release) Butylated Hydroxytolune (BHT)(µM)SRM1650bAPEL PM*******************UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 10001020304095100105Percent Cytotoxicity(LDH Release) Quercetin(µM)SRM1650bAPEL PM****** ****** *** **UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 10001020304095100105Percent Cytotoxicity(LDH Release) Dimethyl Fumarate(µM)SRM1650bAPEL PM*******************UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) Spermidine(µM)SRM1650bAPEL PM******************UntreatedLysis BufferSRM1650bAPEL PM 0.001 0.01 0.1 1 10051015202595100105Percent Cytotoxicity(LDH Release) MitoQ(µM)SRM1650bAPEL PM**UntreatedLysis BufferSRM1650bAPEL PM 0.1 1 10 100100001020304095100105Percent Cytotoxicity(LDH Release) Vitamin E (α-tocopherol)(µM)SRM1650bAPEL PM************************UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) Pterostilbene(µM)SRM1650bAPEL PM*****UntreatedLysis BufferSRM1650bAPEL PM 0.1 1 10 1001000010203095100105Percent Cytotoxicity(LDH Release) Lawsone(µM)SRM1650bAPEL PM*************************UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMSulbentine(µM)***********************UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) Retinyl Acetate(µM)SRM1650bAPEL PM***********UntreatedLysis BufferSRM1650bAPEL PM0.00010.001 0.01 0.1 101020304095100105Percent Cytotoxicity(LDH Release) Flucticasone Propionate(µM)SRM1650bAPEL PM***********************UntreatedLysis BufferSRM1650bAPEL PM 0.1 1 10 10010000102030405095100105Percent Cytotoxicity(LDH Release) SRM1650bAPEL PMAscorbic acid (Vitamin C)(µM)************UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 10001020304095100105Percent Cytotoxicity(LDH Release) Tetrahydrocurcumin(µM)SRM1650bAPEL PM********************UntreatedLysis BufferSRM1650bAPEL PM 0.001 0.01 0.1 1 10051015202595100105Percent Cytotoxicity(LDH Release) Tert-Butylhydroquinone(µM)SRM1650bAPEL PMUntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) Guggulsterone(µM)SRM1650bAPEL PM************UntreatedLysis BufferSRM1650bAPEL PM 0.01 0.1 1 10 100051015202595100105Percent Cytotoxicity(LDH Release) L-Cysteine ethyl ester(µM)SRM1650bAPEL PM************************UntreatedLysis BufferSRM1650bAPEL PM 0.001 0.01 0.1 1 10051015202595100105Percent Cytotoxicity(LDH Release) Resveratrol(µM)SRM1650bAPEL PM************** 43 Figure 2.4 Cytotoxicity of antioxidant interventions in BEAS-2B cells. Cells were exposed to 200 µg/mL of either SRM1650b or APEL PM in combination with increasing doses of an antioxidant, as indicated in Table 2.2 and Figure 2.2. The percent cytotoxicity (percent of lysis buffer control) is plotted as mean ± SD. Data analyzed by two-way ANOVA with Dunnett’s post-hoc test (N = 4; ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05; comparisons to untreated condition). decreased ROS levels (Figure 2.5A and B). Log(inhibitor) versus response modeling of the curves estimated IC50 and Imax mean and standard error values of 0.23 ± 3.46 μM and 107 ± 8.5 % of untreated respectively for tetrahydrocurcumin. Estimates for IC50 and Imax values for quercetin were 18.63 ± 2.23 μM and 91.7 ± 17 % of untreated respectively. No effect on cell viability was detected for either molecule across the doses tested (Figure 2.5C and D). B A D C Untreated PMH 2O 2Vehicle0.01 0.1 1 10 1000102030% Cytotoxicity(LDH Release)[Tetrahydrocurcumin ], Log µMUntreated PMH 2O 2Vehicle0.01 0.1 1 10 1000.51.01.52.0H2DCFDA (Fold Change AUC)[Tetrahydrocurcumin], Log µM✱✱IC50 = 0.23 ± 3.46 µM✱✱✱Untreated PMH 2O 2Vehicle0.1 1 10 1000.51.01.52.0H2DCFDA (Fold Change AUC)[Quercetin], Log µM✱✱✱IC50 = 18.44 ± 2.23 µMUntreated PMH 2O 2Vehicle0.1 1 10 1000102030% Cytotoxicity(LDH Release)[Quercetin ], Log µM 44 Figure 2.5 Tetrahydrocurcumin and quercetin attenuate PM-induced oxidative stress in HBECs. Primary cells grown under ALI conditions were loaded with 10 µM of H2DCFDA for 30 minutes prior to pretreatment with increasing doses of either tetrahydrocurcumin or quercetin for 1 hour. Cells were then exposed to either 10 µg/cm2 of SRM1650b (PM) in combination with treatments, vehicle (DMSO 0.1%), or H2O2 (0.5mM) for 4 hours. (A and B) Fluorescence was measured 0, 2, 4, 6, 8, 22, and 24 hours after the start of the exposure. Data represent fold change area under the curve (AUC) from untreated cells and plotted as mean ± SE. Nonlinear regression of the log(inhibitor) versus response was used to model dose-response curves. IC50 values represent mean ± SEM. Data was analyzed using one-way ANOVAs with Dunnett’s post-hoc test (N = 5-7, *P < 0.05, comparisons either to untreated condition for controls or PM for dose-response data). (C and D) LDH assays were performed using supernatants collected 24 hours after the start of the exposure. The percent cytotoxicity (percent of lysis buffer control) is plotted as mean ± SEM. Data analyzed by one-way ANOVA with Dunnett’s post-hoc test (N = 5-7; *P < 0.05; comparisons to untreated condition). 2.3.5 Effect of tetrahydrocurcumin and quercetin on particulate matter-induced immune mediator profile After confirming that tetrahydrocurcumin attenuated ROS levels in HBECs, multiplex assays measuring immune mediator concentrations in the collected cell supernatants were performed (Figure 2.6 and 2.7). As seen by z-score transformation normalization, SRM1650b altered the levels of many mediators compared to untreated cells (Figure 2.6A). Ranking by the magnitude of the z-score difference between SRM1650b exposed and untreated cells revealed pro-inflammatory IL-1β as the top mediator. Other pro-inflammatory mediators known to be induced by PM such as CXCL1, IL-6, and TNF were also highly ranked. Similar results were also observed for quercetin experiments (Figure 2.7A). When examining known PM associated pro-inflammatory mediators, SRM1650b-induced upregulation of IL-1β, IL-6, and TNFα was suppressed by tetrahydrocurcumin (Figure 2.6C, D, and F). Whereas quercetin mediated SRM1650b-induced upregulation of TNFα and CCL2 (Figure 2.7F and H). Although there was a dose-dependent trend for tetrahydrocurcumin and quercetin for these mediators, no significant differences was observed between doses. To better understand the overall effect of tetrahydrocurcumin and quercetin on SRM1650b exposed cells, the log fold change from the untreated conditions of both 45 Figure 2.6 Effect of tetrahydrocurcumin on air pollution associated immune mediators. Primary cells grown under ALI conditions were pretreated with increasing doses of tetrahydrocurcumin (THC) for 1 hour. Cells were then exposed to either 10 µg/cm2 of SRM1650b (PM) in combination with increasing doses of THC, vehicle (DMSO 0.1%), or H2O2 (0.5mM) for 4 hours. Supernatants were collected 24 hours after the start of the exposure. (A) Heat map representing the normalized (z-score) mean concentration (pg/mL) of 39 immune mediators (columns) in untreated (media), PM, or PM + THC (rows). Rows are sorted (left to right) in order of the magnitude of the difference between media and PM conditions. (B-H) Concentration of immune mediators is plotted as mean ± SEM. Data analyzed by one-way ANOVA with Dunnett’s post-hoc test (N = 7; *P < 0.05; comparisons to media condition). B C D E F G H A Media PMPM + THC 0.1 µMPM + THC 1 µMPM + THC 10 µMVehicle05101520IL-1αpg/mLMedia PMPM + THC 0.1 µMPM + THC 1 µMPM + THC 10 µMVehicle02000400060008000CXCL1pg/mLMedia PMPM + THC 0.1 µMPM + THC 1 µMPM + THC 10 µMVehicle0123pg/mLIL-1β✱✱Media PMPM + THC 0.1 µMPM + THC 1 µMPM + THC 10 µMVehicle0200400600CCL2pg/mLMedia PMPM + THC 0.1 µMPM + THC 1 µMPM + THC 10 µMVehicle020406080100IL-6pg/mL✱Media PMPM + THC 0.1 µMPM + THC 1 µMPM + THC 10 µMVehicle02000400060008000IL-8pg/mLMedia PMPM + THC 0.1 µMPM + THC 1 µMPM + THC 10 µMVehicle0246810pg/mLTNFα✱✱IL-1βCXCL1IL-1αCCL3 IL-6TNFαCCL11CCL7 IL-9CCL2sCD40LTGFα IL-8CXCL10PDGF-AB/BBIL-12p40 IL-2PDGF-AAIL-27CCL5IFNγM-CSFIL-22IL-15IL-4IL-13CCL22IL-7FGF-2TNFβFLT-3LEGFIL-1RACXCL9VEGF-ACX3CL1IL-12p70G-CSFGM-CSFMediaPMPM + THC 0.1 µMPM + THC 1 µMPM + THC 10 µM-0.5 0 0.5 46 Figure 2.7 Effect of quercetin on air pollution associated immune mediators. Primary cells grown under ALI conditions were pretreated with increasing doses of quercetin (Qu) for 1 hour. Cells were then exposed to either 10 µg/cm2 of SRM1650b (PM) in combination with increasing doses of Qu, vehicle (DMSO 0.1%), or H2O2 (0.5mM) for 4 hours. Supernatants were collected 24 hours after the start of the exposure. (A) Heat map representing the normalized (z-score) mean concentration (pg/mL) of 39 immune mediators (columns) in untreated (media), PM, or PM + Qu (rows). Rows are sorted (left to right) in order of the magnitude of the difference between media and PM conditions. (B-H) Concentration of immune mediators is plotted as mean ± SEM. Data analyzed by one-way ANOVA with Dunnett’s post-hoc test (N = 5; *P < 0.05; comparisons to media condition). B C D E F G H A Media PMPM + Qu 1 µMPM + Qu 10 µMPM + Qu 100 µMVehicle051015IL-1αpg/mLMedia PMPM + Qu 1 µMPM + Qu 10 µMPM + Qu 100 µMVehicle0200040006000CXCL1pg/mLMedia PMPM + Qu 1 µMPM + Qu 10 µMPM + Qu 100 µMVehicle0123IL-1βpg/mLMedia PMPM + Qu 1 µMPM + Qu 10 µMPM + Qu 100 µMVehicle0100200300400500CCL2pg/mL✱Media PMPM + Qu 1 µMPM + Qu 10 µMPM + Qu 100 µMVehicle020406080100IL-6pg/mLMedia PMPM + Qu 1 µMPM + Qu 10 µMPM + Qu 100 µMVehicle010002000300040005000IL-8pg/mLMedia PMPM + Qu 1 µMPM + Qu 10 µMPM + Qu 100 µMVehicle02468pg/mLTNFα✱CCL2IL-1βCXCL10TNFαTGFαFGF-2IL-8EGFG-CSFIL-6CCL7IL-1αIL-2IL-12p40CCL3CCL5CXCL9PDGF-AAIL-1RAM-CSFCX3CL1CCL22IL-7FLT-3LIFNγVEGF-AIL-4TNFβ IL-9IL-13CXCL1CCL11GM-CSFIL-22IL-27sCD40LIL-15PDGF-AB/BBIL-12p70MediaPMPM + Qu 1 µMPM + Qu 10 µMPM + Qu 100 µM-0.5 0 0.5 47 SRM1650b and SRM1650b, and tetrahydrocurcumin (1 μM) or quercetin (10 μM) was plotted for each mediator (Figure 2.8). Providing support for the modulating effects of tetrahydrocurcumin, linear regression of these data revealed a slope of 0.58 (r2 = 0.37), indicative of an overall suppressive effect from the treatment. Although individual mediators differ, a similar overall effect was also observed for quercetin (slope = 0.60, r2 = 0.57). No significant differences between SRM1650b with and without treatments were observed for any individual mediator (multiple paired t-test, FDR = 5%). Figure 2.8 Tetrahydrocurcumin and quercetin modulate air pollution immune mediator profile. The concentration of immune mediators was plotted as the Log10 fold change of PM from media control versus the Log10 fold change of (A) PM + THC 1μM or (B) PM + Qu 10 μM from media control. Data was analyzed using linear regressions and the relationship (solid blue lines) and 95% confidence interval (dashed grey lines) is indicated. A -0.2 0 0.2-0.200.2Log Fold Change PMLog Fold Change PM + THC - 1µMsCD40LEGFFGF-2FLT-3LG-CSFGM-CSFIFNγIL-1αIL-1βIL-1RAIL-2IL-4IL-6IL-7IL-8IL-9IL-12p40IL-12p70IL-13IL-15IL-22IL-27M-CSFPDGF-AAPDGF-AB/BBTGFαTNFαTNFβVEGF-ACCL11CX3CL1CXCL1CXCL10CCL2CCL7CCL22CXCL9CCL3CCL5Slope = 0.58r2 = 0.37B -0.2 0 0.2 0.4-0.200.20.4Log Fold Change PMLog Fold Change PM + Qu - 10µM sCD40LEGFFGF-2FLT-3LG-CSFGM-CSFIFNγIL-1αIL-1βIL-1RAIL-2IL-4IL-6IL-7IL-8IL-9IL-12p40IL-12p70IL-13IL-15IL-22IL-27 M-CSFPDGF-AAPDGF-AB/BBTGFαTNFαTNFβVEGF-ACCL11CX3CL1CXCL1CXCL10CCL2CCL7CCL22CXCL9CCL3CCL5 Slope = 0.60r2 = 0.57 48 2.4 Discussion Exposure to elevated levels of ambient fine air pollution is associated with increased exacerbations and hospitalizations of chronic lung diseases, such as asthma and COPD53,180,181. For those exposed, PM is thought to elevate levels of oxidative stress and inflammation in respiratory tract tissue, leading to dysfunctional immune responses40,41,43,47,50. Although reducing air pollution emissions and exposure to PM remains the best strategy for maintaining the long-term health of the respiratory system, there are still those exposed that might benefit from a therapeutic to mitigate the harmful effects of air pollution. To this end, I examined the utility of small molecules with antioxidant properties, to determine if they could attenuate the negative effects of PM on lung epithelial cells. Using a combination of cell lines and primary HBECs, I demonstrated a potential role for tetrahydrocurcumin and quercetin in disrupting PM-induced oxidative stress and inflammation. Oxidative stress in human cells can be interpreted as an imbalance between extracellular sources, and cellular processes that contribute to, or generate, molecules with oxidative potential, and the capacity of cellular mechanisms to reduce these species. As levels of ROS, a common class of molecules that contribute to oxidative stress, increase beyond the capacity of ROS scavenging mechanisms, such as the GSH/GSSG cycle, the transcription factor Nrf-2 upregulates the expression of cellular detoxifying enzymes, such as NAD(P)H quinone dehydrogenase 1 (NQO1), superoxide dismutase 1 (SOD1), and glutathione-disulfide reductase (GR)88,207. Simultaneously, increased levels of ROS leads to the upregulation of inflammatory cytokines and chemokines through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)2,207. PM is known to both contribute to and induce ROS through its production as a by-product of xenobiotic catabolism, and excessive ROS is thought to be one of the main mechanisms that PM damages or kills cells in the 49 lungs2,208. Indeed, a previous study exposing primary HBECs to similar levels of PM has demonstrated increases in damage markers associated with oxidative stress such as carbonylated protein and 8-hydroxy-2’-deoxyguanosine60. Therefore, selection of antioxidant air pollution interventions was focused on small molecules with Nrf-2 pathway activating, free radical scavenging or similar activity (Table 2.2). In this study, I demonstrated a role for tetrahydrocurcumin in attenuating oxidative stress and inflammation induced by SRM1650b, a model of traffic related air pollution. Tetrahydrocurcumin is the primary metabolite of curcumin, which is readily found in the dietary spice turmeric. Derived from the rhizome of the Curcuma longa plant, curcumin has been demonstrated to have broad applications as an antioxidant, anti-inflammatory, and anti-cancer cancer treatment209–211. Although curcumin has been shown to be an effective, the systemic bioavailability is low, and a number of studies have instead focused on characterizing the effects of its metabolite tetrahydrocurcumin212. In line with the results of our study, tetrahydrocurcumin has been previously shown to reduce IL-1β, IL-6, and TNF-α in a carrageenan-induced inflammation model via suppression of IκBα phosphorylation, preventing the activation of NF-κB in vivo210. Furthermore, tetrahydrocurcumin has been demonstrated to reduce T helper 2 (Th2) allergic airway inflammation in murine models of asthma213,214. We did not observe any significant differences in IL-4, IL-5, or IL-13; however, we did not observe pollution-induced release of these immune mediators and our model was confined to lung epithelial cells. Finally, we demonstrated that tetrahydrocurcumin modulates oxidative stress, likely via its capacity as a free radical scavenger195. However, another potential mechanism is the induction of antioxidant enzymes, such as SOD2, NQO1, and Nrf-2, which has been demonstrated in a lipopolysaccharide mouse model examining cardiomyocytes. Although, it is unclear if this occurs via a direct or indirect activation of 50 Nrf-2 transcriptional networks215. Future studies are needed to clarify this mechanism of action in the context of air pollution exposures. Quercetin is a polyphenolic flavonoid that is commonly found in nuts, and fruits and vegetables, such as berries, apples, cabbage, cauliflower, dill, and onions. As with many members of the flavonoid family, it has a broad pharmacological utility for metabolic and allergic diseases and has been demonstrated to have antiviral, anticancer, and anti-inflammatory activity216–218. Previous studies have demonstrated that quercetin suppresses NF-κB-induced expression of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, CCL2, and TNF-α, via nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), and PI3K pathways in LPS and TNF-α stimulated lung epithelial cells152,153. Furthermore, in the context of air pollution, quercetin has been shown to reduce IL-1β and IL-6 release from the lung epithelial cell lines 16HBE and HBEC stimulated with Shanxi urban air pollution, or benzo[a]pyrene, a common component of pollution, via NF-κB suppression147,197. In line with these studies, we observed that quercetin suppresses the pro-inflammatory mediators TNF-α and CCL2, and this is the first time it has been demonstrated in a primary HBEC model cultured under ALI conditions. In vitro and in vivo studies utilizing quercetin have previously demonstrated broad immunomodulatory effects on allergic diseases, such as asthma, rhinitis, and atopic dermatitis, that include suppression of Th-2 mediators, serum IgE, histamine release, and airway hyperresponsiveness218. We observed no differences in Th-2 immune mediators assessed in our model, although we did not include an allergen challenge, and the effectiveness of quercetin in a combined allergen and pollution model is unknown. Previous studies using quercetin have also shown it to supress mucin5AC (MUC5AC) expression in human lung epithelial cells, the accumulation of which is associated with decreased lung function in asthma and COPD154. As MUC5AC has 51 been shown to be increased in the lungs following an acute air pollution exposure55, this may be an area of interest to further explore. Quercetin has also been shown to be effective at reducing oxidative stress in response to air pollution particulate matter in 16HBE cells147. Although the mechanism(s) is not clear, it may include inducing heme oxygenase (HO-1). This is a key enzyme in the conversion of heme into the antioxidant biliverdin and has been shown to be up-regulated by quercetin in murine lung epithelial cells stimulated with H2O2 196. Another possible mechanism of action is the activation of the Nrf-2 pathway, however this has only been demonstrated in other contexts, such as liver toxicity models219. Interestingly, in a mouse carbon tetrachloride hepatic injury model quercetin has been shown to inhibit the activation of TLR2 and TLR4, important pattern recognition receptors that mediate cellular responses to pollution particles220. Our results further support a role for quercetin in suppressing oxidative stress induced by air pollution particulate matter in human lung epithelial cells, but more studies are needed to identify the mechanism of action. The results of this study indicate that tetrahydrocurcumin and quercetin may have utility in attenuating air pollution-induced oxidative stress and inflammation and potentially be helpful in managing environmental exposures for at-risk individuals. Both molecules have been shown to be safe in clinical trials investigating a range of conditions221–228. For quercetin, although ingestion of up to 2000 mg/day was found to have no effect on lung function of mild to severe COPD patients in a small pilot study 223, decreases in serum IL-6 and TNF-α have been observed in women with polycystic ovary syndrome taking 500 mg/day for 40 days orally. No clinical studies have been performed investigating the effects of tetrahydrocurcumin on chronic lung disease, however a study examining the effects of oral curcumin supplementation in asthmatics is ongoing229. Although these studies examined oral supplementation of these 52 molecules, in the context of air pollution exposures an inhaled delivery system may be more effective at protecting cells of the lungs and should be explored. While the fight to reduce global air pollution emissions through conservation and alternative technologies continues, 4.14 million deaths were attributed to air pollution ambient particulates in 2019230. Reducing pollution-induced oxidative stress and lung inflammation by direct intervention may help ameliorate the detrimental health effects for those exposed, potentially reducing the morbidity and mortality of at risk populations, In this study we demonstrate the potential of antioxidants as interventions for air pollution-induced inflammation and advocate for further research examining the utility of these molecules and the possibility of combination therapies in respiratory focused clinical trials. 53 Chapter 3: Dietary omega-3 fatty acids modify air pollution-induced inflammation in airway epithelial cells 3.1 Introduction Every day we breathe over 10,000 L of air that contains a variety of exogenous insults including pollutants and infectious agents the body disposes of or defends against. An important part of responding to these insults is restoring tissue homeostasis by resolving inflammation generated by the body’s defence mechanisms. Over the past few decades omega-3 polyunsaturated fatty acids have been identified as important regulators of inflammation and post-inflammatory resolution and have been associated with better respiratory health137. Acquired only through the diet in humans, higher body levels of essential omega-3 fatty acids have been associated with a lower incidence of respiratory illness231 as well as reduced lung function decline as measured by FVC and FEV1232. Moreover, higher omega-3 fatty acids levels have been associated with better symptom control and lower medication use in asthmatics233. Therefore, as air pollution continues to negatively impact respiratory health around the world, omega-3 fatty acid treatment or dietary supplementation may represent a strategy to mitigate the harmful effects of air pollutants in the lungs. EPA and DHA are two common omega-3 fatty acids that are found in high levels in fatty fish and have been shown to regulate immune responses by several different mechanisms137,234. First, EPA and DHA have been shown to be metabolized into SPMs such as resolvins, protectins, and maresins via enzymes in the eicosanoid pathway. These SPMs have receptor-mediated tissue- and cell-specific pro-resolving and anti-inflammatory effects, and have been demonstrated to regulate neutrophil, eosinophil, macrophage, T cell, B cell, and epithelial cell function137,139,140. Aside from being metabolic precursors, DHA and EPA may also directly suppress inflammation induced 54 by arachidonic acid (AA) -derived eicosanoids by competing with AA for cell membrane incorporation and COX and LOX enzymes140,235,236. Finally, EPA and DHA have also been implicated in suppressing pro-inflammatory NF-κB transcriptional activity through direct interaction with the G protein coupled receptors and disruption of TLR4 signaling cascades237–239. Overall, these mechanisms act to both reduce inflammatory signaling and resolve inflammation. In the context of air pollution-induced inflammation, increased omega-3 fatty acids levels have been associated with a delayed systemic inflammatory response per interquartile range increase in pollution as measured by plasma oxidized low-density lipoprotein (Ox-LDL) and IL-6 levels240. However, no PM protective effect was observed on lung function measures FEV1 and FVC. Higher omega-3 intake has also been associated with a reduced effect of indoor PM2.5 on symptoms in asthmatic children142. Furthermore, in vitro studies have demonstrated that treating umbilical vein endothelial cells with omega-3 fatty acids reduces the release of pro-inflammatory mediators IL-8 and C-C motif chemokine 2 (CCL2)241. Omega-3 fatty acids have also been shown to reduce particulate matter-induced pro-inflammatory mediator release and oxidative stress in vascular endothelial cells145,242. Similarly, in human lung epithelial cells and mouse models DHA has been shown to be protective against organic dust-induced inflammation (IL-6 and IL-8) and immune cell infiltration144. However, the effect of EPA and DHA on human lung epithelial cells exposed to an urban air pollution model is unknown. Therefore, I hypothesized that DHA and EPA would suppress immune mediator release and oxidative stress induced by ambient urban air pollution particulate matter in human lung epithelial cells. 55 3.2 Methods 3.2.1 Cell culture Human bronchial epithelial BEAS-2B cells were cultured in Dulbecco's Modified Eagle Medium F12 (DMEM/F12) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and antibiotic-antimycotic (Thermo Fisher Scientific). 3.2.2 PM exposure Pollution exposures were produced by combining NIST SRM1648a (MilliporeSigma) with DMEM media for BEAS-2B exposures. SRM1648a is an atmospheric particulate matter model collected in an industrialized urban environment. The composition of SRM1648a was characterized by NIST. SRM1648a was sonicated prior to exposures in 20 second intervals for a total sonication time of 3 minutes in DMEM media. BEAS-2B cells were seeded onto black-walled 96 well plates (Corning) at a concentration of 1.5x104 cells/well in maintenance medium. Cells were serum starved 24 hours prior to preincubation with either 100 μM of eicosapentaenoic acid (EPA, Cayman Chemical, CAS No. 10417-94-4), 100 μM of docosahexaenoic acid (DHA, MilliporeSigma, CAS No. 6217-54-5), vehicle control (1% EtOH), or media for four hours (Figure 3.1). Timing and dosing were based on previous experiments performed using BEAS-2B cells243. The EPA and DHA dose used was relatively high as a previous in vitro study using 16-HBE cells demonstrated that a dose of 30 μM of EPA or DHA increased the percentage of total cell fatty acids by 10.33 and 12.88 %244, and a DHA and EPA supplementation study (1.4 g EPA, 200 mg DHA per day for 12 weeks) has demonstrated increases of 1.9 and 0.8% in plasma samples245. Cells were simultaneously exposed to SRM1648a (25, 50, or 100 μg/mL) or H2O2 (0.5 mM, MilliporeSigma) alone for 24 hours. 56 3.2.3 Oxidative stress assay Cells were incubated with 10 μM of 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA, Thermo Fisher Scientific), a fluorescent reactive oxygen species (ROS) sensor, for 1 hour and washed three washes with HBSS (Thermo Fisher Scientific) prior to pre-treatments with EPA or DHA. Relative fluorescence intensity (R.F.U.) of H2DCFDA (488 nm excitation/525 nm emission) was assessed at -4, -2, 0, 2, 20 and 24 hours after the addition of SRM1648a using a florescent plate reader (Spectramax i3x, Molecular Devices). Figure 3.1 Experimental overview. Experimental set-up and outcomes measured for BEAS-2B cells treated with Vehicle (1% EtOH), EPA (100µM), or DHA (100µM) prior to SRM1648a exposure (n=4). 3.2.4 ELISA assays Supernatants were collected and centrifuged at 5000g for 5 mins to remove course PM particles and cells. IL-6 and C-C motif chemokine 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1) duoset ELISAs (R&D Systems) were performed according to manufacturer’s instructions. Absorbance was measured using a Spectramax i3x plate reader (Molecular Devices). H2DCFDA (10 μM) 1 hour 4 hour Treatments: 1) Media 2) Vehicle (1% EtOH) 3) EPA (100 μM) 4) DHA (100 μM) SRM1648a Exposure: 1) Untreated 2) 25 μg/mL 3) 50 μg/mL 4) 100 μg/mL 24 hours Collection Supernatants H2DCFDA fluorescence: 0, 2, 4, 6, 20, and 24 hours ELISAs LDH Assays 57 3.2.5 LDH assays Supernatants were collected and centrifuged at 5000g for 5 mins to remove course PM particles and cells. LDH assays were performed using a CyQUANT LDH cytotoxicity assay kit (Thermo Fisher Scientific) as per manufacturer’s instructions. Absorbance was measured using a Spectramax i3x plate reader (Molecular Devices). 3.2.6 Statistical analysis Statistical analysis was performed using Prism Graphpad 10. H2DCFDA R.F.U. data from each time point was transformed by subtracting baseline and plotted against time. The area under the curve for each condition was calculated and normalized as fold change from the untreated control. Data (n = 4) was analyzed using One-way ANOVAs with with a Bonferroni’s post-hoc test as indicated in figure legends. A P value of < 0.05 was considered statistically significant. Significance (P <0.05) of pairwise comparisons is indicated by an * as indicated in figure legends. 3.3 Results 3.3.1 Effect of urban particulate matter on epithelial cell oxidative stress and cytotoxicity To establish a dose of PM for intervention experiments, the oxidative stress potential of SRM1648a on human lung epithelial cells (BEAS-2B) was assessed using the florescent ROS sensor H2DCFDA. SRM1648a significantly and dose-dependently (25, 50, and 100 μg/mL) increased relative fluorescence over 24 hours (AUC) compared to untreated controls (Figure 3.2A). There was no effect on cytotoxicity as measured by LDH release assays observed. 58 Figure 3.2 Urban particulate matter induces oxidative stress in BEAS-2B cells. BEAS-2B cells were incubated with H2DCFDA for one hour prior to being exposed to SRM1648a particles. (A) H2DCFDA fluorescence was measured at 0, 2, 20, and 24 hours. Baseline (0 hour) was subtracted from subsequent timepoints and area under the curve (AUC) was calculated. Fold change AUC from untreated is plotted as mean ± SD. (B) Cell supernatants were collected 24 hours post exposure and LDH cytotoxicity assays were performed. The percent cytotoxicity (percent of lysis buffer control) is plotted as mean ± SD. Results were analyzed by ANOVA with a Bonferroni’s post-hoc test (N = 4; ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05). 3.3.2 DHA and EPA attenuates urban particulate matter-induced oxidative stress The effect of DHA and EPA on SRM1648a-induced oxidative stress was assessed using H2DCFDA assays in BEAS-2B cells (Figure 3.3A). AUC analyses of the pre-treatment phase revealed a small but significant reduction of ROS in cells treated with DHA compared to untreated controls. However, this effect was reversed over the subsequent 24 hours, with a significant 1.8-fold increase in ROS levels observed in DHA exposed cells (Figure 3.3B and C). In combination with SRM1648a, DHA treatment significantly attenuated ROS levels by 31% and 44% in cells exposed to 50 and 100 μg/mL respectively. A B UntreatedH 2O 2 25 50 1000510152025Percent Cytotoxicity(LDH Release) SRM1648a (μg/mL)UntreatedH 2O 2 25 50 10002468H2DCFDA (Fold Change AUC)SRM1648a (µg/mL)✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱ 59 Figure 3.3 DHA significantly attenuates urban particulate matter-induced oxidative stress. BEAS-2B cells were incubated with H2DCFDA for one hour prior to being treated with DHA (100µM) or vehicle (1% EtOH) for four hours. Cells were then exposed to increasing doses of SRM1648a particles. (A) H2DCFDA fluorescence was measured at -4, -2, 0, 2, 20, and 24 hours relative to the start of SRM1648a exposure. (B) Baseline (-4 hour) was subtracted from -2 and 0 hour timepoints and area under the curve (AUC) was calculated. Fold change AUC from untreated is plotted as mean ± SD. (C) Baseline (0 hour) was subtracted from 2, 20, and 24 hour timepoints and area under the curve (AUC) was calculated. Fold change AUC from untreated is plotted as mean ± SD. Results were analyzed by ANOVA with a Bonferroni’s post-hoc test (N = 4; ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05). Not all comparisons shown. A B C -4 -2 0 0 2 20 240.05.0×1071.0×1081.5×1082.0×108HoursH2DCFDA (R.F.U.)PM AddedPost-Exposure AUCPre-Treat AUCUntreatedVehicleDHASRM1648a 25 µg/mLSRM1648a 25 µg/mL + DHASRM1648a 50 µg/mLSRM1648a 50 µg/mL + DHASRM1648a 100 µg/mLSRM1648a 100 µg/mL + DHAUntreatedVehicleDHA0.00.51.01.5H2DCFDA (Fold Change AUC) ns✱✱✱UntreatedDHA 25DHA + 25 50DHA + 50 100DHA + 10002468H2DCFDA (Fold Change AUC)✱✱✱✱✱✱✱✱✱✱✱ ns ✱✱✱✱SRM1648a (µg/mL)✱✱✱✱✱✱✱✱ 60 For EPA, a significant decrease in ROS levels was observed in the pre-treatment phase (27% compared to untreated) (Figure 3.4A). Although, no significant differences between EPA treated and untreated cells were observed over the following 24 hours. EPA did significantly attenuate ROS levels by 18% in cells exposed to 100 μg/mL of SRM1648a, however the effect was substantially less than that observed in DHA treated cells. No effect was observed at lower doses of SRM1648a. -4 -2 0 0 2 20 240.05.0×1071.0×1081.5×1082.0×108HoursH2DCFDA (R.F.U.)UntreatedVehicleEPASRM1648a 25 µg/mLSRM1648a 25 µg/mL + EPASRM1648a 50 µg/mLSRM1648a 50 µg/mL + EPASRM1648a 100 µg/mLSRM1648a 100 µg/mL + EPAPM AddedPost-Exposure AUCPre-Treat AUCUntreatedVehicleEPA0.00.51.01.5H2DCFDA (Fold Change AUC) ns✱✱✱UntreatedEPA 25EPA + 25 50EPA + 50 100EPA + 10002468H2DCFDA (Fold Change AUC)✱✱✱✱✱✱✱✱ns ns nsSRM1648a (µg/mL)✱✱✱✱✱✱✱✱A B C 61 Figure 3.4 EPA significantly attenuates urban particulate matter-induced oxidative stress. BEAS-2B cells were incubated with H2DCFDA for one hour prior to being treated with EPA (100µM) or vehicle (1% EtOH) for four hours. Cells were then exposed to increasing doses of SRM1648a particles. (A) H2DCFDA fluorescence was measured at -4, -2, 0, 2, 20, and 24 hours relative to the start of SRM1648a exposure. (B) Baseline (-4 hour) was subtracted from -2 and 0 hour timepoints and area under the curve (AUC) was calculated. Fold change AUC from untreated is plotted as mean ± SD. (C) Baseline (0 hour) was subtracted from 2, 20, and 24 hour timepoints and area under the curve (AUC) was calculated. Fold change AUC from untreated is plotted as mean ± SD. Results were analyzed by ANOVA with a Bonferroni’s post-hoc test (N = 4; ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05). Not all comparisons shown. 3.3.3 DHA attenuates urban particulate matter-induced inflammatory mediator release To investigate whether DHA and EPA modulate particulate matter-induced inflammatory responses, the levels of pro-inflammatory markers IL-6 and CCL2 were measured 24 hours post SRM1648a exposure (Figure 3.5). SRM1648a was observed to significantly increase IL-6 levels at 50 μg/mL (Figure 3.5A), an effect that was nullified by DHA treatment. Furthermore, DHA treatment in combination with all SRM1648a doses significantly and substantially reduced IL-6 levels. Although not significant, a trend for reduced IL-6 was also observed in the DHA alone condition compared to untreated. No differences in IL-6 levels were observed in response to EPA treatment (Figure 3.5B). SRM1648a also significantly increased CCL2 release (Figure 3.5C and D). Treatment with either DHA or EPA significantly and substantially decreased SRM1648a-induced CCL2 levels in all three exposure conditions. Compared to untreated, a trend for attenuated CCL2 levels was also observed for EPA and DHA treated cells. 62 Figure 3.5 DHA and EPA attenuate urban particulate matter-induced inflammatory mediator release. (A and B) IL-6 and (C and D) MCP-1/CCL2 levels were measured in the supernatants collected at 24 hours post SRM1648a exposure using ELISAs. Concentration (pg/mL) is plotted as mean ± SD. Results were analyzed by ANOVA with a Bonferroni’s post-hoc test (N = 4; ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05). Not all comparisons shown. A B C D UntreatedDHA 25DHA + 25 50DHA + 50 100DHA + 100050100150IL-6 (pg/mL)ns✱✱nsns✱✱✱✱✱✱✱✱SRM1648a (µg/mL)UntreatedDHA 25DHA + 25 50DHA + 50 100DHA + 1000200040006000800010000CCL2 (pg/mL)nsns✱✱✱✱✱✱✱✱✱✱✱✱✱✱SRM1648a (µg/mL)UntreatedEPA 25EPA + 25 50EPA + 50 100EPA + 100050100150IL-6 (pg/mL)SRM1648a (µg/mL)nsns✱nsnsnsnsUntreatedEPA 25EPA + 25 50EPA + 50 100EPA + 1000200040006000800010000CCL2 (pg/mL)ns✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱SRM1648a (µg/mL) 63 3.4 Discussion As air pollution continues to impact populations across the globe, finding solutions to reduce the long-term morbidity and mortality associated with exposure is becoming a pressing concern. This study supports the hypothesis that the lung epithelium, the primary barrier against external insults, can be primed to limit inflammation and oxidative stress caused by air pollution particulate matter. Furthermore, this is the first study that has examined the effect of directly supplying omega-3 fatty acids to epithelial cells exposed to an urban air pollution model. The observations from this study further support a role for DHA in decreasing particulate matter-induced oxidative stress and inflammatory mediator release acutely in human lung epithelial cells. Oxidative stress and inflammation are intrinsically linked in the lung epithelium’s response to external stimuli. The induction of an inflammatory response is often associated with increased ROS as a result of both xenobiotic catabolism and immune cell defence mechanisms, and increased ROS results in increased production of pro-inflammatory mediators2. In several different tissue types, omega-3 fatty acids have been linked with increased cellular antioxidant capacity and decreased ROS both in vitro and in vivo141,145,246–250. Moreover, DHA and EPA have been demonstrated to up-regulate the transcriptional activity of Nrf-2 leading to transcription of cytoprotective antioxidant genes NQO1, HO-1, and GSTM2, increasing the capacity of the cell to respond to increased ROS246,247,249,251 Independent of this pathway, DHA and EPA have also been shown to have free radical scavenging activity248. However, these mechanisms have yet to be demonstrated in a PM exposure model of lung epithelial cells. In the context of ambient PM exposures, two studies to date have demonstrated an antioxidant effect of omega-3 fatty acids in pulmonary and human umbilical vein 64 endothelial cells (HUVECs)145,251. Our study provides additional evidence supporting DHA and EPA as effective treatments against PM-induced oxidative stress, and directly show this effect in human lung epithelial cells. These findings imply that increased availability of omega-3 fatty acids, possibly through diet, may help prime the lung epithelium, the body’s barrier to the outside world, to be more resilient to air pollution-associated oxidative insults. IL-6 is an important mediator in both innate and adaptative immunity and is expressed by the lung epithelium in response to exposures in vitro and 41,46,48,60–62,65. Supporting the findings of this study, omega-3 fatty acids have previously been shown to suppress the production of IL-6 in lung epithelial and HUVECs cells in response to organic dust and particulate matter144,145. Although the mechanism is unclear, this effect may be due to the convergence of several different pathway effects. First, omega-3 fatty acids have been shown to disrupt TLR4 signaling, which is known to induce NF-κB transcription of proinflammatory genes such as IL-6 in response to air pollution particulates237,238. Second, increased DHA-derived SPMs resolvin D1 and maresin-1 have been shown to inhibit IL-6 production through PPARγ and PKC-α dependent mechanisms respectively that converge on modulating NF-κB activity144,252,253. Finally, whether by competitive inhibition of eicosanoid enzymes or displacement of AA from cell membranes, DHA suppresses the production of PGE2, which is known to induce the transcription of IL-6144. Future studies are needed to clarify which of these mechanisms are responsible for attenuating PM-induced IL-6. As with IL-6, CCL2 production has been associated with air pollution exposure254, and in line with our results omega-3 fatty acids have been previously demonstrated to regulate its expression241,255,256. Specifically, EPA has been shown to suppress CCL2 expression by NF-κB transcriptional activity in endothelial cells241. Taken together, our 65 results provide further support for the immunomodulatory role of omega-3 fatty acids in the context of air pollution exposures. IL-6 is known to have several immunomodulatory roles such as recruitment of leukocytes and regulation of CD4 T-helper cells and B-cells257,258 and CCL2 is known to be a potent monocyte chemoattractant259. Beyond their production in normal acute inflammatory responses, both have been implicated in asthma. IL-6 is thought to contribute to the pathogenesis of asthma through regulation of T-cell differentiation and subsequent production of adaptive effector cytokines IL-4, IL-5, and IL-13 that promote eosinophilia, mucus hypersecretion, B-cell class switching, and activation of mast cells258. Furthermore, elevated levels of IL-6 in the airways of asthmatic patients has been associated with decreased lung function and asthma severity260. Similarly, marked increases in CCL2 and macrophages have been observed in the lungs of mild allergic asthmatics upon allergen challenge259. The regulation of IL-6 and CCL2 by omega-3 fatty acid derived SPMs may partially explain why omega-3 fatty acid levels have been associated with better lung function and reduced asthma incidence and morbidity136 as well as proven protective against the negative effects of air pollutants on lung function in healthy adults exposed to NO2, O3, and PM, and asthmatic children exposed to PM142,240,261. This study has several limitations. As a proof of concept study, it was focused on exploring the role omega-3 fatty acids as an intervention against air pollution in lung cells. Future studies are needed to identify the mechanisms of action by which DHA and EPA exert their anti-inflammatory and antioxidant effects in this context. Furthermore, more complex lung models such as differentiated primary epithelial cell cultures with mucociliary properties are necessary to confirm the findings reported here. Finally, as DHA supplementation and specific SPMs have been shown to limit inflammatory mediator release and migration of neutrophils262,263, the focus on epithelial cells in this 66 study may not recapitulate the complex cross-talk and signaling between the epithelium and infiltrating immune cells in vivo. Controlling and resolving inflammation is crucial to maintaining tissue homeostasis in the lung in the short term by minimizing macromolecular damage and cell death, and the long term by preventing tissue remodeling and disease development137,264,265. In this study we provide further support for omega-3 fatty acids as an effective anti-inflammatory intervention for air pollution particulate matter and advocate future supplementation clinical trials using controlled human air pollution exposures to confirm these findings. 67 Chapter 4: Corticosteroid and traffic-related air pollution exposure increases human rhinovirus replication in airway epithelial cells. 4.1 Introduction The indirect and direct health consequences of air pollution exposures on the respiratory system range from increased respiratory tract symptoms and infections, to exacerbations of chronic lung disease and excess mortality. Beyond the direct effects of air pollution exposure, increased susceptibility to infection is a pressing concern in the management of chronic respiratory diseases, as it is an independent trigger of exacerbations and cause of increased mortality164,165. Respiratory tract infections are often associated with periods of high air pollution, in particular correlating with increased levels of PM2.5161. One of the earliest associations stems from the Great Smog of London incident of 1952 during which pneumonia deaths tripled266. More recent studies have identified links between specific air pollutants, including PM2.5, NOx, and SO2, and acute viral respiratory infections in children, specifically RSV and human RV159,163. Furthermore, the association between air pollution and RV is of particular interest as it is the most commonly identified virus associated with COPD and asthma exacerbation164. Impairment of antiviral host defence responses by pollutant-induced oxidative stress and inflammation have been theorized to be the main cause driving increased RV infection rates159. Several studies have sought to elucidate these mechanisms using in vitro models of the epithelium, the respiratory tract’s barrier against external insults. For example, primary human nasal epithelial cells exposed to diesel particulates were shown to have significant increases in viral load 20 hours post-infection with either RV1b or RV16170. These viral increases were associated with significantly higher concentrations of pro-inflammatory cytokines (IL-6, IL-1b), a response that was also 68 shown in a study using NO2 and O3 as models of environmental pollutants with RV16 infected bronchial epithelial cells267. Furthermore, PM has been shown to increase the expression of RV16 cell-surface entry receptor ICAM-1, through a NF-kB signal transduction pathway107,268. At the population level, air pollution exposure is highly correlated with increased use of ICS130, the front-line therapy used to control inflammation and prevent exacerbations of chronic respiratory diseases, such as asthma and COPD. ICS suppresses inflammation by modulating the expression of networks of pro- and anti-inflammatory genes via the glucocorticoid receptor by directly binding gene promotor elements, and through direct or indirect interactions with transcription factors, such as NF-kB and AP-1128,269. ICS also suppress the expression of some genes involved in the body’s antiviral host defence responses270. However, they may increase others such as the PRR TLR2271. Nevertheless, using cell line models of the lung epithelium, the ICS fluticasone propionate (FP) has been shown to increase the infectivity of RV1B and decrease the expression of type-1 IFN genes135. Accordingly, those on high doses of ICS have a significantly higher risk of respiratory tract infections272,273. Among recent studies focusing on the development of anti-viral medications, the flavonoid quercetin has been identified as a potential therapeutic agent. Quercetin, which was shown to attenuate air pollution-induced oxidative stress and inflammation in Chapter 2, has also been shown to inhibit the replication of RV in vitro and in vivo274. Moreover, quercetin has been demonstrated to suppress the expression of ICAM-1 and the pro-inflammatory cytokines IL-6 and IL-1β147,275. The utility of quercetin in the context of RV infection and PM exposure has not been explored to date. ICS therapies are foundational in altering the trajectory of chronic respiratory diseases and are prescribed for approximately one third of the Centers for Disease Control and Prevention’s estimate of 25 million (7.8%) asthmatic Americans276. 69 However, there remains an elevated risk of respiratory infections for these patients, and this risk may be intensified by air pollution exposure. Given the widespread use of ICS and inescapability of ambient air pollution exposures, we investigated the interaction of FP and air pollution exposure on viral replication in RV16 exposed human bronchial epithelial cells. We also examined inflammatory and antiviral host defence responses in this context. Finally, we assessed the role of a NF-kB pathway in upregulating the expression of the RV16 entry receptor ICAM-1 in co-exposed cells. 4.2 Methods 4.2.1 Epithelial cell culture and viral infection BEAS-2B cells were cultured in Dulbecco's modified Eagle’s medium/Ham’s nutrient mixture F12 (DMEM/F12; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and antibiotic-antimycotic (Thermo Fisher Scientific). Human RV16 (104.5 TCID50/mL) was supplied by Dr. David Proud (University of Calgary). BEAS-2B cells were seeded at a concentration of 5.0×104 cells per well in 24-well plates and incubated at 37°C with 5% CO2. After 48 hours cells were serum starved overnight prior to replacement of serum free media (SFM; 500µL) alone as a control or pre-treatment with Fluticasone Propionate 250nM (FP; Sigma-Aldrich), National Institute of Standards and Technology (NIST) standard reference material (SRM) 2975 200µg/mL (PM; Sigma-Aldrich), or FP + PM in SFM for 1 hour. SFM was then aspirated and replaced with either SFM alone or containing FP, PM, or FP+PM in combination, or not, with RV16 which was incubated with cells for 1 hour at room temperature. Cells were then washed three times using 250µL of HBSS (Thermo Fisher Scientific). Finally, SFM either alone or supplemented with FP, PM, or FP+PM was added to the wells and cells incubated for 24 hours at 37°C with 5% CO2. For NF-kB pathway inhibition experiments, cells were prepared as described above and pretreated 70 with 10µM of Bay 11-7082 (Cedarlane Labs) for 1 hour. Cells were harvested and supernatants collected and centrifuged at 5000g for 5 mins to remove course PM particles and cytotoxicity was assessed using CyQUANT LDH Cytotoxicity Assays (Thermo Fisher Scientific). 4.2.2 siRNA transfection BEAS-2B cells were seeded in 24-well plates at a density of 1.0x104 cells per well. The cells were grown for 24 hours in growth medium containing serum. Cells were then transfected with either 10pmol of siRNAs targeting ICAM-1 (s7086) or Silencer Select Negative Control No. 1 siRNA (Thermo Fisher Scientific) in Opti-MEM (Thermo Fisher Scientific) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to manufacturer’s instructions. After 6 hours, the transfection media was exchanged for growth medium with serum for 18 hours. Cells were then serum starved for 24 hours prior to exposures. 4.2.3 Flow cytometry Surface expression of ICAM-1 was measured by flow cytometry (BD FACS Canto II, BD Biosciences). BEAS-2B cells were trypsinized (0.5%) (Thermo Fisher Scientific) and reconstituted in flow cytometry staining buffer containing 2mM EDTA (Sigma-Aldrich) and 0.5% BSA (Thermo Fisher Scientific) in PBS (Thermo Fisher Scientific). Cells were then blocked using TruStain (BioLegend) and stained with either FITC anti-CD54 (ICAM-1) or FITC Mouse IgG1, κ Isotype control Antibody (BioLegend) according to manufacturer’s instructions. Cells were then washed three times and stained with 7-AAD viability dye (BioLegend) for live cell discrimination. Cells were gated by side scatter area (SSC-A) versus forward scatter area (FSC-A) to eliminate debris and then by forward scatter height (FSC-H) versus FSC-A to select single cells. 71 Dead cells were then eliminated from the analysis based on PerCP-Cy5.5-area (7-AAD). Collected data was analyzed using FlowJo version 10.9 (BD Biosciences). Isotype and untreated controls were used to examine nonspecific binding and background signal (Figure 4.1). Figure 4.1 Flow cytometric plots showing the gating strategies for measuring ICAM-1 levels. Epithelial cells were selected using FSC-A vs SSC-A and single cells were selected based on FSC-A vs FSC-H. Live cells were discriminated using 7-AAD based on FSC-A vs PerCP-Cy5-5-A. ICAM-1 expression was determined by FITC fluorescence in unstained (A), isotype control (B), and ICAM-1 stained cells (C). 4.2.4 qPCR Total RNA was extracted from cellular lysates post exposure using a RNeasy mini kit (Qiagen). cDNA was generated using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time PCR (RT-qPCR) was ICAM1 Isotype Control Unstained Control A B C 72 performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) on an StepOnePlus RT-PCR machine (Applied Biosystems). TaqMan primers were ordered from Thermo Fisher Scientific and their sequences are indicated in Table 4.1. Gene expression (DDCt) was calculated relative to GAPDH and untreated controls. Table 4.1 qRT-PCR taqman assays Gene Details Sequence / Taqman Assay ID* RV-16 Forward 5′-TCCTCCGGCCCCTGAA-3′ Reverse 5′-ACTGGATTGTGTGCACTGGCT-3′ Probe 5′-FAM-TGGCTAACCTTAAACCT-MGB-3′ RSAD2/Viperin Forward 5′-GCGCATATATTCATCCAGAATAAGG-3′ Reverse 5′-GCTGGGACCTGACGGTGA-3′ Probe 5′-FAM-ACCAGAAGATGAAAGACT-MGB-3′ ISG15 Forward 5′-TGGAGCTGCTCAGGGACAC-3′ Reverse 5′-GAAATTATTCCTGCAAGCCAATTT-3′ Probe 5′-FAM-ATGCTGGCGGGCAACGAATTCC-MGB-3′ CXCL10/IP10 Forward 5′-GAAATTATTCCTGCAAGCCAATTT-3′ Reverse 5′-TCACCCTTCTTTTTCATTGTAGCA-3′ Probe 5′-FAM-TCCACGTGTTGAGATCA-MGB-3′ CXCL8/IL-8 Assay # Hs00174103_m1 ICAM-1 Assay # Hs00164932_m1 GAPDH Catalog # 4333764T *All Taqman assays purchased from Thermo Fisher Scientific 4.2.5 ELISAs Supernatants were collected and centrifuged at 5000g for 5 mins to remove course PM particles and cells. Analyte concentrations (pg/mL) were determined using a Human Cytokine 48-Plex Discovery Assay performed by Eve Technologies (Calgary, Alberta, Canada). Analytes found to be not detectable were eliminated from the analysis, otherwise for statistical comparisons values found to be below the LLOD were replaced with ½ of the respective LLOD. 73 4.2.6 Statistical analysis Statistical analysis was performed using Prism Graphpad 9. Data (n ≥ 3) was analyzed using a combination of t-tests, One-way, and Two-way ANOVAs using either Tukey’s or Dunnett’s post-hoc test for multiple comparisons as indicated in figure legends. A P-value of <0.05 was considered statistically significant. Significance (P <0.05) of pairwise comparisons is indicated either by different letters or a * (as indicated in figure legends). 4.3 Results 4.3.1 RV replication increased by the corticosteroid FP Prior to investigating the interaction between corticosteroid and particulate matter, we assessed the effect of FP on inflammatory and anti-viral mediators, and RV16 replication in BEAS-2B cells. As previously shown, treatment of cells with FP (250nM) induced a significant 2.15-fold increase in RV16 viral RNA (Figure 4.2A). However, we were also interested in assessing the broader effect of RV16 and FP on inflammatory mediator levels and performed a multiplex assay measuring analyte concentrations in the collected cell supernatants. Infection with RV16 induced an inflammatory response that was suppressed by pre-treatment with FP (Figure 4.2B). More specifically, significant increases were observed in pro-inflammatory mediators (TNFα, IL-1β, IL-6, IL-8, CCL2, CXCL1) and anti-viral mediators (CCL5, and CXCL10) (Figure 4.2C-J). Treatment with FP prior to infection with RV16 significantly attenuated protein expression of 7/8 of these mediators by greater than 90%. 74 Figure 4.2 FP increases RV16 replication and suppresses protein expression of RV-induced pro-inflammatory cytokines and antiviral mediators. BEAS-2B cells were either not treated (NT) or were treated with fluticasone propionate (FP; 250 nM) for 1h prior to being infected with 103.5 TCID50/mL of RV16 for 1 h. Treatments (NT/FP) were then added back for a further 24h. Cell supernatants were collected, and total RNA was extracted from cells. (A) RV16 expression levels relative to GAPDH and NT were assessed by qRT-PCR. (B) Heat map representing the normalized (z-score) mean concentration (pg/mL) of the 28 measured immune mediators (columns) in NT, RV16, or FP+RV16 treated cells (rows). Rows are sorted (left to right) in order of the magnitude of the difference between NT and RV16 conditions. (C-J) Concentrations of immune mediators TNFα, IL-1b, IL-6, IL-8, MCP-1/CCL2, GROα, RANTES/CCL5, and CXCL10 were measured by ELISA. RV16 results (A) were analyzed by one-tailed T-test (N = 6, P < 0.05). ELISA results (C-J) were analyzed by two-way ANOVA with Tukey’s post-hoc test (N = 3, P < 0.05). Data represent either mean z-score or mean ± SD and significantly different comparisons are denoted by different letters, whereas bars that are not significantly different share the same letter. A B C D E F G H I J NTRV16FP + RV160.00.51.01.5TNFαpg/mLABANTRV16FP + RV160500100015002000MCP-1/CCL2pg/mLABANTRV16FP + RV160.00.51.01.5IL-1βpg/mL ABANTRV16FP + RV160100200300IL-6pg/mLABANTRV16FP + RV160246810RANTES/CCL5pg/mLABANTRV16FP + RV160246IL-8pg/mLABANTRV16FP + RV1601020304050CXCL10pg/mLABANTRV16FP + RV160.00.51.01.5GROα/CXCL1pg/mLABBM-CSFCXCL10TNFαMCP-1/CCL2 IL-6FGF-2IL-8GROα/CXCL1TGFαIL-18IL-1βRANTES/CCL5MCP-3/CCL7IL-15TNFβVEGF-APDGF-AAIL-12p40MIG/CXCL9FLT-3LIL-10IFNγEotaxinIL-12p70IL-17F IL-2EGFIL-27NTRV16FP + RV16-0.5 0 0.5Z-scores1.0NT FP0123RV16/GAPDH (Fold Change)AB 75 4.3.2 RV replication synergistically increased by particulate matter and FP To evaluate the effects of particulate matter and corticosteroids on viral replication, we assessed RV16 viral RNA levels in BEAS-2Bs treated with FP (250nM), PM (SRM2975 200µg/mL), or FP and PM prior to RV16 infection (Figure 4.3A). We observed that treatment with PM alone significantly increased viral RNA by approximately 12-fold over untreated cells, compared to the 2-fold rise induced by FP. When combined, FP and PM induced a significant increase of viral RNA by approximately 17-fold relative to NT. This increase in RV16 RNA was synergistic (FP alone added to PM alone fold change vs FP + PM fold change, unpaired t-test, P = 0.03). To better understand this synergistic interaction, we assessed the effects of PM and FP on several inflammatory and anti-viral markers (Figure 4.3C-F). Unlike with FP, we observed that PM exposure induced significant increases in IL-8 and CXCL10 and a trend for RSAD2, and ISG15. These PM-induced expression increases were reduced by the addition of FP (PM + FP treatment). No significant differences in cytotoxicity as measured by LDH assays were observed for any of the treatment conditions (Figure 4.3B). 4.3.3 RV16 entry receptor ICAM-1 increased by particulate matter Given the observed increases in select antiviral markers following PM exposure, we examined the expression of another key component of the viral life-cycle, the RV16 entry receptor ICAM-1. Exposing BEAS-2B cells to PM significantly increased expression of ICAM-1 both at the RNA and protein level (Figure 4.4A-B). ICAM-1 expression remained unaffected by FP treatment. 76 Figure 4.3 The combination of FP and PM increases RV16 replication and modulates expression of related markers. BEAS-2B cells were either not treated (NT) or treated with fluticasone propionate (FP, 250 nM), SRM2975 diesel exhaust particles (PM, 50 μg/cm2), or PM and FP and subsequently infected with RV16 at a dose of 103.5 TCID50/mL for 1h. After washes to remove virus, treatments were added back on for a further 24h. Cell supernatants were collected, and total RNA was extracted from cells. (A, C-F) Relative gene expression levels were assessed by qRT-PCR and calculated using GAPDH and the NT condition. (D) Percent cytotoxicity for control and RV16 infected BEAS-2B cells was measured in no NT, FP, PM, or FP and PM exposure groups by an LDH assay. Data represent mean ± SD. Statistical analysis performed by two-way ANOVA with Tukey post-hoc test (N = 6-8, P < 0.05). Significantly different comparisons are denoted by different letters, whereas bars that are not significantly different share the same letter. NT FP PM FP + PM-4-20246RSAD2/GAPDH(Log2 Fold Change)ABCABC CDAB ABCControl RV16NT FP PM FP + PM02468CXCL10/GAPDH(Log2 Fold Change)Control RV16AABAACBCABC ABNT FP PM FP + PM0246810IL-8/GAPDH(Log2 Fold Change)Control RV16AABB BCDEDENT FP PM FP + PM-3-2-1012ISG15/GAPDH(Log2 Fold Change)ABCABDBCDACBDBCDDControl RV16NT FP PMFP + PM0510152025RV16/GAPDH (Fold Change)ABCDNT FP PM FP + PM0246810% Cytotoxicity (LDH Release)Control RV-16A A A A A AAAA B C D E F 77 Figure 4.4 PM exposure increases ICAM-1 expression. BEAS-2B cells were either not treated (NT) or were treated with FP (250 nM), PM (50 μg/cm2), or both FP and PM for 1h, prior to being infected with RV16 at a dose of 103.5 TCID50/mL for 1h. After washes, treatments were added back on for a further 24h. For siRNA experiments cells were transfected with either control (Silencer Select Negative Control No. 1) or ICAM-1 siRNAs 48 h prior to treatments. (A, C, and E )Total RNA was extracted from cells and gene expression relative to GAPDH and the NT condition was assessed by qRT-PCR. Data represent either log2 mean or mean ± SD. (B and D) Cell surface ICAM-1 expression was measured using flow cytometry. Data represent fold change in mean fluorescence intensity (MFI). (F) Percent cytotoxicity was measured by LDH assay and data represent mean ± SD. Statistical analysis was performed either by (B) unpaired one-tailed T-test or two-way ANOVA with a (A, D, and F) Tukey post-hoc test or (C and E) Dunnett’s post-hoc test (N = 4-8). For T-tests and Tukey post-hoc tests significantly different comparisons (P < 0.05) are denoted by different letters, whereas comparisons that are not significantly different share the same letter. For Dunnett post-hoc tests comparisons are to control condition within each exposure grouping (*P < 0.05). A B C D E F NT FP PM FP + PM01234ICAM-1/GAPDH(Log2 Fold Change)Control RV16A ABCCCAANT PM0.51.01.5ICAM-1 (Fold Change MFI)ABNTsham siRNAICAM-1 siRNA PMPM + ICAM-1 siRNA0.00.51.01.5ICAM-1 (Fold Change MFI)ABC CANT FP PM FP + PM0246810RV16/GAPDH(Fold Change)Control⋇⋇sham siRNA ICAM-1 siRNANT FP PM FP + PM-2024ICAM-1/GAPDH(Log2 Fold Change)RV16RV16 sham siRNARV16 ICAM-1 siRNA⋇ ⋇⋇ ⋇Control⋇⋇⋇⋇NT FP PM FP + PM051015% Cytotoxicity (LDH Release)ControlRV16RV16 + sham siRNARV16 + ICAM-1 siRNAAABCDBCDABCD ABCBCDABCDABCD BCD BCDABCDCD BCD DABCDCD 78 4.3.4 Knockdown of ICAM-1 attenuates particulate matter-induced RV replication To further investigate the role of ICAM-1 in PM-induced RV16 replication I knocked down ICAM-1 using siRNAs. Knockdown efficiency was assessed by measuring the transcription and expression of ICAM-1 using RT-qPCR and flow cytometry (Figure 4.4C-D). A significant reduction in ICAM-1 transcript levels that corresponded to a 41% reduction in cell surface ICAM-1 was observed in control conditions, and in PM treated cells the increase in ICAM-1 expression was significantly reversed. Knockdown of ICAM-1 also significantly attenuated RV16 viral loads in PM and FP and PM exposed cells by 38% and 51% respectively (Figure 4.4E), with little effect on cytotoxicity (Figure 4.4F). 4.3.5 NF-κB pathway inhibition suppresses particulate matter-induced ICAM-1 expression Given the observed effect of particulate matter on ICAM-1 expression and a previously published link between a NF-kB pathway and ICAM-1 expression, we assessed whether blocking this pathway would prevent PM-induced ICAM-1 expression in our model (Figure 4.5). Using an irreversible inhibitor of IκBα phosphorylation (Bay 11-7082) to suppress NF-κB activity, we observed a significant reduction in ICAM-1 expression by 41% and 39% in RV16 infected cells exposed to PM with and without FP respectively (Figure 4.5A). This result translated to a significant reduction in cell surface ICAM-1 expression in PM exposed cells (1.14 to 0.91 fold change, Figure 4.5B). 4.3.6 NF-κB pathway inhibition attenuates particulate matter and FP-induced RV16 replication After establishing that Bay 11-7082 attenuated PM-induced ICAM-1 expression, we then examined the effect of Bay 11-7082 on RV16 replication in cell exposed to PM 79 and FP plus PM (Figure 4.5C). We observed that Bay 11-7082 significantly reduced RV16 viral RNA by 26% in cells pre-treated with FP and exposed to PM. A similar trend was observed in cells exposed to PM alone (8% reduction). Figure 4.5 NF-κB pathway inhibitor suppresses RV16 replication in PM exposed and FP treated BEAS-2B cells. Cells were not treated (NT) or were incubated with 10µM of Bay 11-7082 or quercetin for 1h prior to treatment with FP (250 nM), PM (50 μg/cm2), or FP plus PM for 1h. Cells were then infected with 103.5 TCID50/mL of RV16 for 1h. FP, PM, and quercetin treatments (but not Bay 11-7082) were added back to cells for a further 24h. (A and C) Total RNA was extracted from cells and gene expression relative to GAPDH and the NT condition was assessed by qRT-PCR. Data represent either log2 mean ± SD or mean ± SD, and were analyzed by two-way ANOVA with Dunnett’s post-hoc test (N = 4, *P < 0.05, comparisons to RV-16 condition within each exposure grouping). (B) Relative cell surface ICAM-1 expression levels were measured using flow cytometry. Statistical analysis was performed by one-way ANOVA with Tukey post-hoc test (N = 3, P < 0.05, significantly different comparisons are denoted by different letters, whereas comparisons that are not significantly different share the same letter). NT PMPM + Bay-11-7082PM + Quercetin0.51.01.5ICAM-1 (Fold Change MFI)AC CADBNT PM FP + PM-101234ICAM1/GAPDH(Log2 Fold Change)ControlRV16 RV16 + QuercetinRV16 + Bay 11-7082⋇⋇ ⋇⋇⋇NT PM FP + PM010203040HRV16/GAPDH (Fold Change)RV-16 RV16 + Bay 11-7082RV16 + Quercetin⋇⋇⋇A B C 80 4.3.7 NF-κB pathway inhibition suppresses markers of inflammatory and anti-viral gene expression As the NF-kB pathway is important in mediating cell inflammatory responses to exogenous stimuli, we also assessed the effects of inhibition of a NF-kB pathway on the expression of inflammatory and anti-viral genes. Similar to the effect of FP demonstrated in Figure 4.3, Bay 11-7082 treatment significantly impaired RV16 induced expression of CXCL10, RSAD2, ISG15, and IL-8 in untreated cells (Figure 4.6A-D). A similar result was observed for RV16 infected cells treated with FP and exposed to PM, with significant decreases of CXCL10 and IL-8 expression observed. 4.3.8 Quercetin treatment increases RV16 replication Given recent publications demonstrating the use of quercetin as an anti-inflammatory intervention for PM exposure in addition to having anti-viral effects in vitro, we explored the utility of quercetin in preventing PM induced increases in RV16 replication (Figure 4.5C). Interestingly, we observed no significant differences in innate anti-viral responses (Figure 4.6A-D), and a significant increase in RV16 viral RNA in PM exposed cells (Figure 4.5C). However, a small but significant reduction in PM-induced surface ICAM-1 expression was observed in quercetin treated cells (Figure 4.5B). No decreases in cell viability were observed in cells treated with Quercetin (Figure 4.6E). 81 Figure 4.6 NF-κB pathway inhibitor suppresses anti-viral gene expression. Cells were not treated (NT) or were incubated with 10µM of Bay 11-7082 or quercetin for 1h prior to treatment with FP (250 nM), PM (50 μg/cm2), or FP plus PM for 1h. Cells were then infected with 103.5 TCID50/mL of RV16 for 1h. FP, PM, and quercetin treatments (but not Bay 11-7082) were added back to cells for a further 24h. (A-D) Total RNA was extracted from cells and relative gene expression levels were assessed by qRT-PCR and calculated relative to GAPDH and the NT condition. Data represent either log2 mean ± SD, and were analyzed by two-way ANOVA with Dunnett’s post-hoc test (N = 4, *P < 0.05, comparisons to RV-16 condition within each exposure grouping). (E) Percent cytotoxicity was measured by LDH assay and data represent mean ± SD. Statistical analysis performed by two-way ANOVA with Tukey post hoc test (N=4, P<0.05, significantly different comparisons are denoted by different letters, whereas comparisons that are not significantly different share the same letter) NT PM FP + PM-4-202468CXCL10/GAPDH(Log2 Fold Change)ControlRV16 RV16 + QuercetinRV16 + Bay 11-7082⋇⋇⋇⋇⋇NT PM FP + PM-4-2024ISG15/GAPDH(Log2 Fold Change)ControlRV16 RV16 + QuercetinRV16 + Bay 11-7082⋇⋇⋇⋇⋇NT PM FP + PM-6-4-20246RSAD2/GAPDH(Log2 Fold Change)ControlRV16 RV16 + QuercetinRV16 + Bay 11-7082⋇⋇⋇⋇⋇⋇NT PM FP + PM-20246810IL-8/GAPDH(Log2 Fold Change)ControlRV16 RV16 + QuercetinRV16 + Bay 11-7082⋇⋇⋇⋇⋇ ⋇NT PM FP + PM051015% Cytotoxicity (LDH Release)ControlRV16 RV16 + QuercetinRV16 + Bay 11-7082A AABB B B B B B B BA B C D E 82 4.4 Discussion Air pollution exposure and inhaled corticosteroids have separately been linked to viral susceptibility and demonstrated to increase viral load in vitro, yet the effects of the combination of the two were previously unknown. Here we investigated the effects of combining this common exposure and treatment on rhinovirus replication, using a model of traffic-related air pollution NIST SRM2975 and the asthma medication fluticasone propionate in the human BEAS-2B lung epithelial cell line. Complementing previous studies, we provide further evidence in vitro that antiviral and inflammatory immune responses are impaired by the corticosteroid FP, inducing an increase in RV16 levels. Similarly, we demonstrate that PM exposure increases RV16 levels. However, when we combined PM and FP we observed a synergistically increase in RV16 levels. This result was concurrent with a significant and marked PM-dependent increase in the RV16 entry receptor ICAM-1. To further investigate this observation, I knocked down ICAM-1 and inhibited the NF-kB pathway that had previously been reported to play a role in PM induced ICAM-1 expression. Notably, inhibition of a NF-kB pathway significantly reduced PM-induced ICAM-1 expression, which was associated with a reduction of RV16 viral RNA in PM and FP treated cells. Collectively, these results demonstrate that the combination of air pollution exposure and corticosteroid treatment facilitate a cellular state that is primed for rhinovirus infection through distinct mechanisms of increasing the expression of the ICAM-1 entry receptor and impairing specific innate antiviral immune responses respectively. The inhaled corticosteroid FP is a common therapy for the management of chronic respiratory diseases277. The effect of FP on innate anti-viral immune responses and the type I IFN signalling pathway in human bronchial epithelial cells has previously been detailed in vitro135. Leveraging the findings of this paper, we confirmed that FP at a dose of 250 nM led to a significant increase in RV16 replication and a significant 83 reduction in the expression of the interferon stimulated gene (ISG) RSAD2/Viperin. We further explored how RV16 infection and FP treatment effected the inflammatory mediator profile of bronchial epithelial cells. In line with previous reports, we observed significant RV-induced expression of innate pro-inflammatory mediators TNFα, IL-1β, IL-6 and IL-8, CCL2, and CXCL1 and anti-viral mediators CCL5 and CXCL10 that was suppressed by FP135,278,279. Our results add further support the hypothesis that ICS, such as FP, impair specific immune responses in the bronchial epithelium and may increase the susceptibility of the human airways to viral infection. Previous in vitro studies have also investigated the potential mechanisms by which air pollution increases viral infections. In primary nasal epithelial cells, significantly increased RV16 expression was observed following exposure to diesel exhaust particulate matter NIST SRM 1650b. It was postulated in this study that increased viral loads were caused by DPM-induced downregulation of PRRs MDA-5, TLR3, and RIG-1 and the antiviral mediator CXCL10, impairing the recognition and antiviral response of the cells170. However, this effect on PRRs and CXCL10 largely went away when the epithelial cells were infected with RV16 and exposed to DPM. In contrast we observed that PM exposure induced a significant increase of the PRR RIG-1 and ISGs RSAD2/Viperin and CXCL10, and upon infection with RV16, RSAD2/Viperin and CXCL10 remained elevated. Our findings do not support PM impairment of classical antiviral immune responses. Host defence peptides produced by the epithelium are another layer of defence against viral and bacterial infection, and the expression of these peptides has previously been shown to be decreased by air pollution exposure55,175,280. More specifically, RV replication can be inhibited by LL-37177, and its effectiveness is reduced by exposure to carbon nanoparticles 178. Therefore, we also measured the expression of the gene 84 CAMP which encodes LL-37. However, the transcript levels of CAMP were below the limit of detection (data not shown). Rhinoviruses fall under the Enterovirus genus of the Picornaviridae family of viruses, and there are over 160 genetically distinct RVs that are classified into either A, B, or C species. Over 100 of these RV serotypes use the cell surface receptor ICAM-1 as their entry receptor, including RV16169. Physiologically, ICAM-1 plays a role in stabilizing cell-cell interactions and the adhesion of neutrophils in inflamed tissues281. Previous studies have reported that particulate matter-induced inflammation also leads to increased ICAM-1 expression107,268 We therefore decided to assess the expression of ICAM-1 at the RNA and protein level in response to PM and RV16 infection. Notably, we demonstrated a significant and marked increase in transcription levels, as well as cell surface levels of ICAM-1, suggesting that PM may be facilitating RV16 replication by increasing the expression of RV16’s entry receptor. The synergistic increase in RV16 viral RNA we observed in cells concurrently exposed to PM and treated with FP may also be explained by the induction of ICAM-1 by PM exposure. Indeed, when we knocked down ICAM-1 we saw a marked decrease in RV16 viral RNA. Furthermore, when targeting the NF-kB pathway, which has previously been reported to induce ICAM-1 expression upon PM exposure107,268, we saw a significant reduction in both ICAM-1 and RV16 levels in FP treated and PM exposed cells. It is possible that PM induced ICAM-1 expression may also be regulated by other pathways such as the PI3K-AKT or p38 MAPK pathways, and ICAM-1 has also shown to be induced by oxidative stress, a common effect of PM exposure107,268. This may explain why we only saw a partial reduction in RV16 RNA levels following NF-kB pathway inhibition. However, the NF-κB pathway is also activated by TLR3 following RV infection and involved in the induction of inflammatory mediators and ISGs171,282,283. Therefore, targeting the NF-κB pathway to interrupt the induction of ICAM-1 by PM may 85 also be impairing the induction of ISGs, an effect we observed with RSAD2, CXCL10, and ISG15 expression that may also explain the partial reduction in RV16 RNA levels following NF-κB pathway inhibition. Finally, we explored the use of the flavonoid quercetin as potential antiviral in the context of PM exposures. Previously quercetin been shown to inhibit RV replication in vitro and in vivo and suppress RV-induced inflammatory responses274. Moreover, quercetin has also been shown to suppress PM-induced inflammation, as well as the induction of ICAM-1 by IL-1β147,275. In contrast to these studies, we observed that treatment with quercetin significantly increased RV16 replication in PM exposed cells. This increase in RV16 RNA was not associated with significant differences in ISG expression or IL-8. Further studies are needed to identify the mechanisms by which quercetin increases RV infection following PM exposures. In conclusion, we provide the first evidence that air pollution exposure and ICS treatment increase the infectivity of RV16 in human bronchial epithelial cells. Furthermore, we demonstrated that PM exposure and FP treatment may synergistically increase RV16 susceptibility through mechanisms affecting different aspects of host defence response and the viral infection cycle. As air pollution exposure continues to be a pressing concern around the world, this study identifies a potential risk for those with chronic respiratory diseases taking lung disease controller medications. Further research is needed on the effects of ICS and air pollution on a broader range of viral pathogens to understand the scope of this risk factor. 86 Chapter 5. Conclusion 5.1 Summary of findings The work in this thesis explores the use of different interventions to mediate the harmful effects of air pollution at the cellular level in lung epithelial cells. These effects largely stem from increased pro-inflammatory signaling, and oxidative stress-induced macromolecular damage and cell death. Furthermore, this work investigates the association between air pollution and rhinovirus, and highlights a potential compounding effect when a commonly used anti-inflammatory medicine is added to a polluted exposure. The results of this thesis suggest that targeting different pathways that mediate different aspects of air pollution toxicity may have broad application to reduce air pollution associated morbidity and mortality. In chapter 2, I tested the hypothesis that application of an antioxidant to epithelial tissue will reduce PM-induced oxidative potential and/or epithelial barrier dysfunction, resulting in attenuated inflammatory responses. Utilizing a focused screening method, I demonstrated that 6/18 small molecules were significant and effective antioxidants against diesel exhaust particulate matter model (NIST SRM1650b) in human bronchial epithelial cell lines BEAS-2B and Calu-3. I then demonstrated that two of these candidates, tetrahydrocurcumin and quercetin, significantly reduced ROS levels induced by SRM1650b in a more complex model using differentiated primary bronchial epithelial cells from healthy individuals grown at air-liquid interface. These molecules were also observed to dose-dependently mediate SRM1650b-induced upregulation of pro-inflammatory mediator TNFα. These findings support the use of tetrahydrocurcumin and quercetin as interventions for traffic-related air pollution-induced oxidative stress and inflammation. 87 In chapter 3, I examined the use of an air pollution intervention that promoted pro-resolving immune pathways by supplying precursor omega-3 fatty acids to bronchial epithelial cells. I tested the hypothesis that treatment of bronchial epithelial cells with DHA or EPA attenuates PM-induced ROS and release of pro-inflammatory mediators. In this chapter, I demonstrated that DHA was effective at lessening urban air pollution particulate matter- (SRM1648a) induced release of IL-6 and CCL2, as well as reduced ROS levels in BEAS-2B cells. I also demonstrated that EPA was effective at reducing levels of CCL2 and ROS, although to a lesser degree than DHA. These results are in line with a growing body of evidence supporting the notion that cells can be primed to attenuate the responses of canonical signaling pathways to external insults such as air pollution. In chapter 4, I examined the relationship between an anti-inflammatory corticosteroid and air pollution particulate matter exposure on susceptibility to viral infection. I first tested the hypothesis that PM exposure combined with corticosteroid treatment will increase human rhinovirus replication in human airway epithelial cells. I demonstrated that pre-exposure of BEAS-2B cells to the corticosteroid fluticasone propionate and diesel exhaust particulate matter (SRM 2975) synergistically increased RV16 levels. Next, to understand the mechanisms by which PM was increasing RV16 levels in my model, I tested the hypothesis that PM exposure will suppress antiviral ISG expression. Contrary to a previous study that examined the effect of PM on the ISG CXCL10170, I found that PM exposure induced significant increases in the ISGs RSAD2 and CXCL10. I also showed that corticosteroid treatment significantly suppressed ISG expression as previously reported135. Finally, previous studies have implicated PM exposure via the NF-κB pathway in the upregulation of the RV16 entry receptor ICAM-1107,268. Therefore, I tested the hypothesis that inhibition of the NF-κB pathway will attenuate PM- and corticosteroid-induced rhinovirus replication. I demonstrated that 88 blocking the NF-κB pathway using a small molecule inhibitor decreased the RNA and protein expression of ICAM-1, and this was associated with a significant reduction in RV16 levels in PM and corticosteroid treated cells. These findings support the notion that the combination of air pollution exposure and corticosteroid treatment may prime lung epithelial cells for infection by ICAM-1 specific rhinoviruses by increasing the expression of the ICAM-1 entry receptor and impairing specific innate antiviral immune responses respectively. The scale and growing impact of air pollution exposures on human health necessitates the development of a comprehensive approach to both reduce and mitigate the associated risks of exposure. The in vitro exploration of potential air pollution interventions, including consequences, in this thesis is an important step in informing treatment guidelines and moving alternative therapeutic approaches to the clinic where they may help those inevitably exposed. 5.2 Limitations There are several limitations associated with the work presented in this thesis. Using a pseudostratified primary epithelial model differentiated at air-liquid interface was a strength of the work in chapter 2. However, the majority of the work performed in chapters 3 and 4 relied on the use of the immortalized epithelial cell line BEAS-2B in submerged monocultures. Although this model is effective at investigating toxicology, basic epithelial cell responses, and mechanisms of action, it may not be representative of the complex pseudostratified mucosal epithelium. Moreover, the epithelium functions in concert with cells of the immune system to respond and resolve insults, and without these cells, the epithelium may not function as in situ. Although the work in this thesis has possible benefits and implications for those living with chronic respiratory diseases, the primary and cell line models used in this 89 thesis are representative of a healthy individual. Previous studies have highlighted differences in cell responses between primary cells isolated from healthy, asthmatic, and COPD patients60,94,284, typically indicating a more pronounced inflammatory response from diseased cells. When considering the applicability of the interventions tested in this thesis in this context, they may have differential effects. The pollution models used in this thesis were sourced from the National Institute of Standards and Technology and were a mix of urban and traffic-related air pollution standard reference materials. The advantages of using SRMs is knowing the detailed characteristics and molecular make-up of the particulate matter as well as the ability of my work to be tested and replicated in other settings. However, exposures performed with SRMs in solution do not replicate the exact particle size and secondary organic aerosol characteristics or contain the gaseous component of freshly generated air pollution. Even though SRMs induce acute inflammatory responses that share features with in vivo responses, this difference may induce differential effects in cells exposed. Finally, although sub-lethal doses of SRMs were used in this thesis as measured by LDH assays, there remains the possibility that the SRM doses and treatments used may be inducing apoptosis, which would not be reflected in the LDH assay results. 5.3 Implications In the search for strategies to reduce the risk of morbidity and mortality associated with air pollution, the concept of an air pollution therapeutic intervention may need to be expanded. The majority of guidelines and recommendations to health care professionals give details on avoiding or reducing air pollution exposure at the personal level through the use of engineering solutions such as air cleaners and face masks, or strategies such as changing routes of travel or minimizing outdoor activity285. However, current recommendations for therapeutic interventions that may control an acute 90 inflammatory response to air pollution only pertain to individuals with chronic respiratory conditions such as asthma127,285. These recommendations advise to maximize control partially through use of inhaled corticosteroids, yet the effectiveness of these medications in this setting has been recently questioned132,285 and current Global Initiative for Asthma (GINA) guidelines do not consider any adverse effects127. Thus, there is a growing need for therapeutic options as an additional tool to mitigate the harmful effects of air pollution. The concept of using an antioxidant to mitigate the effects of air pollution has been illustrated in vivo using NAC157, and it is also used in the treatment of severe smoke inhalation286. However, NAC has also been associated with negative side effects such as nausea and vomiting157. The work presented in this thesis is the first to broadly examine the utility of antioxidant interventions, demonstrating a potential role for quercetin and tetrahydrocurcumin in mitigating air pollution-induced inflammation. Importantly, the effect of these interventions was demonstrated in cells from healthy individuals, meaning there may be a role for therapeutics outside at-risk population groups given the association between air pollution and development of asthma in childhood33. Although, strategies for best use (timing, dose, and delivery) would need to be studied and defined. For example, it may be plausible that use of a therapeutic might be warranted when acute PM2.5 levels reach a certain threshold to minimize cumulative damage and inflammation, similar to the use of antihistamines during pollen seasons. I believe the findings in this thesis provide a basis for further mechanistic and clinical studies on the use of therapeutics for air pollution-induced signaling and damage in the lungs. In addition to investigating the use of antioxidants, I demonstrated that the polyunsaturated omega-3 fatty acid DHA, and to a lesser extent EPA, mitigated particulate matter-induced increases in ROS and release inflammatory mediators in an 91 urban air pollution model. A key difference in the mechanism of action of these fatty acids compared to an intervention such as a glucocorticoid with a more defined target is that they can prime a cell to be less reactive to pro-inflammatory insults by shifting the balance of fatty acid precursors towards pro-resolving. This distinction is worth noting, as the underlying biological signalling pathways are not being temporally inhibited or dysregulated by synthetic means. Moreover, increasing the supply of these omega-3 fatty acids can be accomplished through changes in the diet, which may become an important tool at the population level in mitigating air pollution effects across large regions. However, the delivery of these fatty acids directly to frontline tissues, such as the lung epithelium, through the use of an inhaler might be an area of interest for future research. Finally, the findings presented in chapter 4 of this thesis have potential implications for those currently taking corticosteroids. Although current GINA treatment guidelines identify air pollution as a factor that increases the risk of exacerbation, there is no consideration of adverse ICS effects in this context127. The epidemiological associations between air pollution exposure and increased risk of hospitalization, exacerbation, and death of asthmatic individuals may in part be explained by a synergistic increase in rhinovirus susceptibility by those taking ICS medications. I believe these findings are important as rhinovirus is a major cause of asthma exacerbations and the most common virus detected during exacerbations164. Although this suggests corticosteroid use should be reconsidered during periods of increased air pollution, ICS provide an essential function in supressing long-term inflammation and progression of chronic respiratory disease. Instead, the use of contact precautions and masks may need to be strengthened in line with air quality warnings, and health care providers should be aware of this increased risk to patients through updated guidelines. Although further work in more complex cell and exposure models is needed to confirm 92 my findings, my results further support the overarching conclusion of epidemiological, controlled exposure, and in vitro studies that it is important to limit exposure to air pollution in general and especially for at-risk populations such as asthmatics. 5.4 Future directions There are several exciting avenues of investigations that stem from the results in this thesis. First, is the concept that air pollution, as a complex mix of molecules and metals, exerts its harmful effects on lung cells by multiple receptors and pathways in a combination of both direct and indirect mechanisms. As such, targeting one pathway or one aspect of inflammatory signaling may only partially mitigate the effects of an air pollution exposure. Using a combination therapy approach targeting multiple facets of air pollution effects such as a free radical scavenger, Nrf2 agonist, and an anti-inflammatory may be much more effective than any one target in particular. Second, although the research on air pollution antioxidant interventions presented in this thesis was focused on two specific endpoints, general ROS levels and inflammatory mediator responses, there are other endpoints of interest that would provide a more detailed picture of how antioxidant interventions impact cellular defences and air pollution-induced damage. These include examining oxidative stress damage markers such as lipid peroxidation, carbonylated protein, and DNA adducts (8-hydroxy-20-deoxyguanosine) and the status of detoxifying enzymes and molecules such as levels of super oxide dismutase, Nrf2 activation, glutathione disulfide/reduced glutathione ratio, and total antioxidant status60. As omega-3 fatty acids were also found to impact ROS levels induced by air pollution, these endpoints would also add additional mechanistic information on the potential limits of DHA and EPA as an intervention. Finally, the use of DHA and EPA, which are metabolized into SPMs, brings up the question of which SPMs are mediating the effects of air pollution. Assessing levels of 93 known SPMs in the context of omega-3 fatty acid as well as antioxidant interventions may reveal common mechanisms of action. Indeed marsin-1, a metabolite of DHA, has been demonstrated to attenuate inflammatory responses in epithelial cells exposed to organic dust252. Beyond investigating the potential effects of these interventions, identifying the specific mechanisms of action are also necessary to move these therapies towards clinical trials. Third, moving toward a live aerosol exposure model to test interventions will generate information that is likely more relevant to real world exposures. This would entail both acute and chronic exposure modeling where cells would be exposed for short intervals over multiple days. Successful interventions tested in this type of model will likely also be more effective in vivo. Last, there are several avenues of interest to test potential air pollution interventions in more complex biological models. These include using primary cells isolated from not just healthy, but asthmatic and COPD patients. As those living with chronic respiratory diseases are more at risk to the effects of air pollution, these interventions may have direct impact on the morbidity and mortality of these diseases. However, in vitro studies are needed to demonstrate the proof of concept. Another example of a complex model that has potential applications is the rhinovirus pollution model. 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Carlsten, Christopher Russell
Investigation of air pollution interventions in airway epithelial cells
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