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The Control of Inflammation in Airway Epithelial Cells

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The Control of Inflammation in Airway Epithelial Cells The control of inflammation in airway epithelial cells Claudia Correia De Paiva Academic Unit of Respiratory Medicine Department of Infection, Immunity & Cardiovascular Disease University of Sheffield Thesis submitted for the degree of Doctor of Philosophy January 2016 1 ABSTRACT COPD is a severe chronic and complex airway disease that represents a major financial burden on the healthcare and economic system. Environmental risk factors such as cigarette smoke have been associated with the predisposition to COPD. Other factors such as exposure to viral pathogens can exacerbate airway inflammation and tissue destruction generated by recruited neutrophils, culminating in altered epithelial cell responses in COPD. This thesis investigated the physiological role of Pellino-1, an E3-ubiquitin ligase, and its regulation in human primary bronchial epithelial cells (HBEpCs) in response to viral infection. The viral mimic poly(I:C) increased Pellino-1 protein and gene expression in HBEpCs. In addition, Pellino-1 gene expression was significantly increased by RV-16 and RV-1B infection in primary bronchial epithelial cells from COPD patients. Pellino-1 knockdown in HBEpCs led to a reduction in NF-κB regulated cytokines CXCL8, IL-1α and β gene expression and release of CXCL8 in response to poly(I:C) while having no measurable effect on IFNβ mRNA expression. Furthermore, the transient knockdown of Pellino-1 resulted in the decrease in IKKα/β phosphorylation. The role for Pellino-1 in the non-canonical NF-κB pathway was also investigated and while Pellino-1 knockdown did not alter the expression of the non-canonical NF-κB precursor protein NFKB1 following poly(I:C) stimulation, NFKB2 protein expression was suppressed. In contrast to Pellino-1 knockdown, the transient knockdown of NFKB2 resulted in significant increase in CXCL8 mRNA and protein expression and in turn did not regulate Pellino- 1 mRNA expression to poly(I:C). These data suggest that following viral infection in airway epithelial cells, TLR3 activation culminates in the up-regulation of Pellino-1 leading to an increase in NFKB2 expression, resulting in the suppression of NF-κB specific gene transcription. In addition to activating non-canonical NF-κB, NFKB1 regulates the activation of ERK signalling via MEK1. Treatment of HBEpCs with MEK1 inhibitors, PD98059 and U0126, resulted in a significant reduction in Pellino-1 protein and gene expression which led to the suggestion of ERK as a potential Pellino-1 regulator. Proteomics analysis of primary epithelial cells obtained from COPD patient airways further identified a potential novel mechanism of action for Pellino-1 in the NF-κB signalling pathway, wherein it Pellino-1 may inhibit A20’s negative regulatory role or its adaptor proteins TNIP1 or TAX1BP. Taken in combination these data support Pellino-1 as a potential target to down- regulate neutrophilic inflammation whilst retaining antiviral immunity by selectively mediating the TLR3 TRIF-dependent NF-κB/MAPK pathway and not TLR3-mediated IRF3 and IFNβ activation. 2 ACKNOWLEDGEMENTS Firstly I would like to extend my sincerest gratitude to my supervisors, Professor Ian Sabroe and Dr. Lisa Parker, not only for giving me the opportunity of a lifetime but for their consistent support and guidance. I would also like to thank them for the time they have invested in me and above all for believing in me even when I did not. They have not only helped me develop as a researcher, but above all they have helped me grow into the person I am today. I would also like to thank all of my past and present colleagues in the Department of Infection, Immunity and Cardiovascular Disease, who were always happy to offer their help and advice. In particular, I would like to say a special thank you to Elizabeth Prestwich as a member of ‘team Pellino’ not only for her willingness to work jointly with me through some of the harder aspects of the research, but mostly to have survived it with me. Also a massive thank you to Julie Bennett not only for her pioneering work on Pellinos which helped pave the way for my PhD, but above all for becoming one of my greatest friends and still offering her help and expertise even 4 years after having left the department. She has given me some of the best and funniest memories of my time in the department. I would also like to say a big thank you to Amanda, Louise, Laura, Vicky and Anna who I am also very privileged to call my friends, for their support and encouragement in the form of laughs, coffees and happy memories. To my husband Richard, who has lived through every single day of this PhD with me, through some of the best and worst moments of my life, I owe you everything. I am thankful for your strength, your pride in me, for always believing in me (despite not possibly having any foundations for such) and above all for never ever failing me. Thank you to the Paiva clan for their unconditional love. However first and foremost I would like to thank my mom and brother for their permanent support and always reminding me of what I am capable of. Both have shown me what great strength looks like and have taught me to never give up in the face of adversity. And finally I would like to dedicate this thesis, this part of my life, to my dad. He may no longer be with me, but without him, his spirit and his unwavering confidence in me I would never have accomplished this chapter of my life. 3 TABLE OF CONTENTS Acknowledgements 3 List of Figures 8 List of Tables 11 Abbreviations 12 Chapter 1 - Introduction 16 1.1 Chronic Obstructive Pulmonary Disease (COPD) 16 1.2 Cells involved in COPD 16 1.2.1 Cytokines 19 1.2.1.1 Interleukin-1 (IL-1) 20 1.2.1.2 Interleukin-8 (IL-8) or CXCL8 21 1.2.1.3 Interferon (IFN) 22 1.2.1.4 Regulated on activation, normal T cell expressed and secreted (RANTES) or CCL5 24 1.3 Exacerbations of COPD 24 1.3.1 Cigarette smoke 25 1.3.2 Rhinovirus 26 1.4 Pattern recognition receptors (PRRs) and Innate Immunity 28 1.4.1 Toll-like Receptors (TLRs) 29 1.4.2 RIG-I-like receptors (RLRs) 33 1.5 TLR signalling 34 1.5.1 MyD88-dependent signalling 34 1.5.2 MyD88-independent signalling 35 1.6 PRR-induced transcription factors 40 1.6.1 NF-κB 40 1.6.1.1 Canonical NF-κB signalling 40 1.6.1.2 Non-canonical NF-κB signalling 41 1.6.2 Interferon Regulatory Factors (IRFs) 44 1.6.3 MAPK Signalling 45 1.7 Pellino 48 1.7.1 Pellino-1 48 1.7.2 Ubiquitination 49 4 1.7.3 Pellino-1 phosphorylation 52 1.7.4 Pellino-1 sumoylation 52 1.7.5 Pellino-1 Physiological function 52 1.8 Hypothesis and Aims 54 Chapter 2 - Materials and methods 55 2.1 Materials 55 2.2 Mammalian Cell Culture 55 2.2.1 BEAS-2B cell line maintenance 55 2.2.2 THP-1 cell line maintenance 56 2.2.3 Human bronchial epithelial primary cell (HBEpC) maintenance 56 2.3 Peripheral Blood Mononuclear Cell (PBMC) isolation 57 2.3.1 Cell separation by OptiPrepTM gradient 57 2.4 Negative magnetic selection of monocytes 58 2.4.1 Flow cytometry for monocyte purity 58 2.5 Enzyme-linked immunosorbent assay (ELISA) 59 2.6 Reverse transcription PCR (RT-PCR) 59 2.6.1 RNA isolation from cells 59 2.6.2 DNase treatment of purified RNA 60 2.6.3 cDNA synthesis 60 2.6.4 Reverse transcription polymerase chain reaction (RT-PCR) 61 2.6.5 Primers for PCR 62 2.6.6 Gel electrophoresis 63 2.7 Quantitative PCR (qPCR) 63 2.7.1 qPCR standards and plasmid calculations 64 2.8 Western blot 64 2.9 Transient siRNA knockdown 66 2.10 Preparation of cigarette smoke extract (CSE) 67 2.11 Rhinoviral infection in vitro 67 2.12 Caspase-8 activity 68 2.13 Statistical Analysis 68 Chapter 3 - Results. Establishing an in vitro chronic model of inflammation 70 3.1 Comparison of LPS-induced CXCL8 release in monocytes and VitD3-differentiated THP-1 cells 71 5 3.2 Comparison of direct interaction between monocytic cells and BEAS-2B in an acute model of inflammation 75 3.3 The effect of cell culture inserts on the interaction between monocytic cells and BEAS-2B in an acute model of inflammation 77 3.4 Establishing a chronic model of inflammation 80 3.4.1 Utilising a model system with cell separation via cell culture inserts 80 3.4.2 Utilising a model system with direct cell-to-cell contact 85 3.5 IRFs 1,2,3,6,7 and 9 are expressed in BEAS-2B cells 89 3.6 Establishing a model of inflammation using cigarette smoke 91 3.7 CSE potentiates proinflammatory responses to TLR agonists in a chronic model of inflammation 95 3.8 MyD88 and IRF3 stable knockdown differentially regulate CXCL8 secretion to multiple proinflammatory stimuli in BEAS-2B cells 97 3.9 Altered acute model results in increased CXCL8 production to poly(I:C) stimulation in the presence of CSE 99 3.10 Pretreatment with CSE decreased BEAS-2B cells antiviral response to RV-16 101 3.11 CSE enhances BEAS-2B proinflammatory responses to RV-16 103 3.12 CSE differentially regulates proinflammatory responses to TLR agonists in a chronic model of inflammation in human bronchial epithelial primary cells 105 3.13 Summary 107 Chapter 4 - Results.
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