The effects of atopy and asthma on in vivo human nasal responses to Toll-like receptor agonists Dr Akhilesh Jha A thesis submitted for the degree of Doctor of Philosophy (PhD) Centre for Respiratory Infection National Heart and Lung Institute (NHLI), St Mary’s Campus Imperial College London, Norfolk Place, London W2 1PG July 2018 Abstract Acute respiratory viral infections cause significant morbidity and mortality, especially in vulnerable individuals, and it is important to study viral pathogenesis and the host immune response in humans. Toll-like receptors (TLRs) play a critical role in the detection of viral nucleic acids, and airway TLR receptors respond to nucleic acid patterns in the RNA viruses that cause respiratory infections. However, a reliable method of measuring mucosal innate immune responses to viral infections is lacking. TLR3 agonists (poly(I:C) and poly-ICLC) and the combined TLR7/8 agonist (resiquimod, R848) are synthetic analogues of double stranded RNA (dsRNA) and single stranded RNA (ssRNA) respectively. Nasal challenge with these TLR agonists was carried out, and serial sampling using nasosorption and nasal curettage was performed. Mucosal immune responses were measured and the effect of different host factors (e.g. asthma) on these responses was studied. Poly(I:C) and poly-ICLC were well tolerated but failed to induce significant and reliable nasal mucosal innate immune responses. R848 at a higher dose (10 µg/100 µL per nostril) induced significant mucosal interferon and cytokine responses but caused mild to moderate flu-like symptoms in three out of nine volunteers. A lower dose of R848 (0.02 µg/kg/100 µL, mean dose 1.5 µg/100 µL) was subsequently utilised in three groups of volunteers: healthy non-atopic (n=12), allergic rhinitis (n=12) and allergic asthma (n=11). This was well tolerated and induced significant release of nasal mucosal IFNs (IFN-α, IFN-γ), proinflammatory cytokines (TNF-α, IL-2, IL-12p70) and chemokines (CXCL10, CCL2, CCL3, CCL4 and CCL13) when compared to saline. Participants with allergic rhinitis and allergic asthma had similar IFN-α, CCL3 and CCL13 levels that were increased compared to healthy volunteers. In volunteers with atopy, baseline nasal mucosal gene expression of the anti-inflammatory secretoglobin SCGB1A1 had a strong negative correlation with subsequent innate immune activation by R848. Eight hours after R848 challenge, several mucosal IFN stimulated genes (ISGs) were upregulated (IFIT3, OAS2, IRF7, MX1, MYD88, DDX58 and STAT1) as well as SOCS1, TLR3, TLR7, KRT5 and CLEC4C, whilst IFNAR1 and Dr Akhilesh Jha, Thesis for PhD submission, Imperial College London, March 2018 1 ADGRG1 were downregulated. Volunteers with asthma had increased DDX58, MX1 and IFIT3 when compared to those with allergic rhinitis and healthy volunteers. This research has led to the successful development of a non-invasive and well tolerated method to induce and precisely measure nasal mucosal innate immune responses to the ssRNA analogue and TLR7/8 agonist R848, and this methodology has been used to demonstrate heightened innate immune activation in volunteers with allergic rhinitis and asthma. This technique can be extended to examine responses in a range of host conditions and diseases as well as to assess the adjuvant potential of R848 in conjunction with mucosal vaccines targeted against infection and cancer. Dr Akhilesh Jha, Thesis for PhD submission, Imperial College London, March 2018 2 Declaration I hereby declare that this thesis is my own work and has been written by myself. Any work not done by me has been acknowledged in the text. Part of the work described in this thesis has been submitted for publication as a book chapter (Jha et al., Precision Mucosal Sampling and Biomarkers in Allergic Rhinitis and Asthma, Handbook of Biomarkers and Personalized Medicine, CRC Press). Copyright Statement The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Dr Akhilesh Jha, Thesis for PhD submission, Imperial College London, March 2018 3 Acknowledgements I wish to express my sincere gratitude to Professor Peter Openshaw for teaching me the importance of relentless scientific enquiry and assisting in the transition from clinical medicine to experimental research. He gave me sage advice and opened many doors for future collaborations. Professor Trevor Hansel led the study and provided great support, having conceived the original concept as a transition from established allergen challenge models to those based on a panel of Toll-like receptor agonists. His persistence in developing the appropriate techniques has enabled mucosal sampling with great precision. Professor Robin Shattock provided expert advice on TLR agonists and Dr Saranya Sridhar gave me valuable encouragement. Dr Ryan Thwaites has been an incredible asset to the study, keeping a cool head and offered practical lab assistance. Jennifer Brimley and Joan Nanan (research nurses) were indispensable and put in the early (and late) hours for recruitment and sampling. I am indebted to Tanushree Tunstall for her administrative, statistical and all round brilliant support. I am grateful to Professor Onn Min Kon for his supervision and have great respect for his clinical acumen. Dr Jie Zhu, Marta Jamroziak and Professor Rob Goldin were wonderful at trialling immunohistochemistry on tiny curettage specimens. I experienced superb collaborations with Professor Maggie Dallman’s group working on zebrafish (with Dr Franze Progatzky and Dr Laurence Bugeon) and at the Royal Brompton with Professors Miriam Moffatt and William Cookson, and Dr Leah Cuthbertson. Thanks to all members of the Openshaw lab (especially Dr Cecilia Johansson, Dr Chris Chiu and Dr Spyros Makris). I would like to acknowledge the UK National Institute for Health Research (NIHR) Comprehensive Local Research Networks (CLRNs), the Biomedical Research Centre (NIHR Imperial BRC), the Health Protection Research Unit in Respiratory Infections in partnership with Public Health England (PHE) at Imperial College London (NIHR HPRU RI) and the volunteers on the study. I greatly appreciate my parents in laying the foundations for my pursuit of a career in medicine. Finally, I wish to thank my wife Neena who listened to endless stories about noses and interferons, and my sons Aryan and Kiaan who were born whilst I was a clinical research fellow at Imperial College and kept me smiling and sane. Dr Akhilesh Jha, Thesis for PhD submission, Imperial College London, March 2018 4 Table of Contents Abstract ................................................................................................................................................... 1 Declaration .............................................................................................................................................. 3 Copyright Statement ............................................................................................................................... 3 Acknowledgements ................................................................................................................................. 4 List of Tables ......................................................................................................................................... 12 List of Abbreviations ............................................................................................................................. 13 1 Introduction ................................................................................................................................... 15 1.1 Airway mucosal immunity and infection ................................................................................ 15 1.1.1 Immune response to infection ......................................................................................... 15 1.1.2 Airway mucosal innate immunity .................................................................................... 17 1.1.2.1 Overview ................................................................................................................... 17 1.1.2.2 Physical and chemical barriers .................................................................................. 19 1.1.2.3 Soluble mediators ..................................................................................................... 20 1.1.2.4 Interferons ................................................................................................................ 21 1.1.2.5 Dendritic cells ............................................................................................................ 23 1.1.2.6 Macrophages ............................................................................................................ 23 1.1.2.7 Natural Killer Cells ..................................................................................................... 24 1.1.2.8 Neutrophils ............................................................................................................... 25 1.1.2.9 Eosinophils ................................................................................................................ 25