THE ROLE OF BPIFA1 IN OTITIS MEDIA APOORVA MULAY A thesis submitted for the degree of Doctor of Philosophy (Ph.D.) September 2016 University of Sheffield Faculty of Medicine, Dentistry and Health Department of Infection, Immunity and Cardiovascular Disease Supervisors: Professor Colin Bingle Dr Lynne Bingle Professor Michael Cheeseman I Dedicated to my grandfather, Mr Vasant Mulay You are an inspiration and I miss you everyday! I ABSTRACT Otitis Media (OM) is the most common paediatric disease and a leading cause of conductive hearing impairment. This multifactorial disease shows significant involvement of innate immunity genes and epithelial abnormalities are also commonly implicated. BPIFA1, a member of BPI fold containing family of putative innate defence proteins, is one of the most abundant secretory proteins in the upper airways and SNPs in BPIFA1 have been associated with OM susceptibility. Recent studies suggest that BPIFA1 plays a pleiotropic host defense role. This thesis describes experiments aimed at investigating the role of BPIFA1 in protection of the middle ear and in the development of OM. Bpifa1-/- mice do not spontaneously develop OM and do not demonstrate increased nasopharyngeal carriage of the human otopathogen, NTHi. However, deletion of Bpifa1 in Junbo (Evi1Jbo/+) mice, an established model of chronic OM, leads to significant exacerbation of OM severity and ME mucosal thickness. This thesis also describes the development of a novel in vitro model of the murine middle ear epithelium. Using a combination of transcriptional and proteomic approaches, I demonstrate that the model closely mimics the native middle ear epithelium and differentiates into ciliated cells, goblet cells and secretory cells and also supports infection by NTHi. Attempts were made to recapitulate the OM phenotype in vitro using this culture system. Overall, the data from this thesis indicate that BPIFA1 is involved in maintaining homeostasis within the middle ear under steady state conditions through nonspecific defence of the middle ear mucosa. Loss of BPIFA1 in presence of infection or inflammation increases the sensitivity of the epithelium and leads to an exacerbated host defence response and excessive epithelial remodelling. Furthermore, the novel in vitro culture I system can be applied as an effective tool to study the interaction between the middle ear epithelium and various otopathogens. II ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor, Professor Colin Bingle. I came to the UK 6 years ago as a Master’s student in Colin’s lab and he has always been extremely supportive and approachable. This thesis would not have been possible without his invaluable help, guidance and encouragement. Also a heartfelt thank you to Dr Lynne Bingle for her very helpful advice with planning experiments during this study and invaluable suggestions while drafting the paper. A special thank you to Dr Khondoker Akram for being an amazing teacher and guide throughout my PhD. I have learnt a lot about being a researcher from you! I would like to thank Professor Steve Brown for providing me with the opportunity to spend the first year of my PhD at MRC, Harwell and always making the taking the time to regularly meet and discuss my project. Thank you to Professor Michael Cheeseman for the support and scientific discussions throughout. I am grateful to Dr Derek Hood for always patiently tending to my microbiology doubts. I would like to thank the past and present members of the deafness group and especially Tom Purnell, Hayley Tyrer and Lauren Chessum for helping me settle into the PhD life initially and always making me feel welcome when I went back to Harwell for experiments. Special thanks to Dr Nanda Rodrigues and Dr Sneha Anand for all their advice in helping me figure out the logistics of working between two places. I would like to thank our collaborating researchers: Professor Ralph Shohet at University of Hawaii for generating the Bpifa1-/- mice and Professor James Stewart and Stuart Armstrong at the University of Liverpool for the MS secretome analysis. My thanks also go to the staff of the Mary Lyon Centre: Sara Wells, Lucie Vizor and Lisa Ireson, in particular, for looking after my mouse lines and co-ordinating complicated breeding for multiple simultaneous experiments. I am grateful to the core facilities at MRC Harwell: Deen Quwailid, Adele Traynor and Rumana Zaman from the GEMS core for genotyping my mice, and Adele Austin, Caroline Barker and Naomi Busk from histology and necropsy for processing the large number of histological sections used in my project. A huge thank you to Debbie Williams for coming to my rescue with her RT-qPCR expertise. You are a star! Thank you to Roland Quinney for organising mouse exports to Sheffield and to Dr Helen Marriott and Lynne Williams for their assistance with dissections at Sheffield. I would also like to thank III Hannah and Catherine for helping me with RT-PCRs and Immunohistochemistry experiments. A big thank you to the lovely members of the Bingle group and my PhD buddies who have made this process an extremely enjoyable experience. I do not know how I would have survived in a quiet office! Thank you Chloe, Emily, Lucy, Renata, Priyanka, Andreea, and Jess for the numerous gossip sessions and cakes! A big thank you to my amazing roomie, Sayali and to Furaha for always comforting me when I was struggling with experiments. A massive thank you to Hrishi for going through my drafts and making sure I hang in there in my most stressful moments. Most importantly, a huge thank you to my family: Mom, for being a sponge that absorbs all my worries; Dad, for your perpetual enthusiasm and encouragement to follow my dreams and Renu, for your unconditional love. I could never have reached this stage without you three. Finally, I would like to extend my gratitude to the University of Sheffield and the Medical Research Council for funding my PhD and the Biochemical Society, Genetics society and Action on Hearing Loss for enabling me to attend a number of international meetings during the course of my PhD. IV PUBLICATIONS ñ Apoorva Mulay, Khondoker Akram, Debbie Williams, Hannah Armes, Catherine Russell, Derek Hood, Stuart Armstrong, James P. Stewart, Steve D. M. Brown, Lynne Bingle, Colin D. Bingle “An in vitro model of murine middle ear epithelium” Disease Models & Mechanisms Sept. 2016 [Epub ahead of print]. ñ Apoorva Mulay, Debbie Williams, Michael Cheeseman, Derek Hood, Thomas Purnell, Catherine Russell, Steve D. M. Brown, Lynne Bingle, Colin D. Bingle “Loss of the homeostatic protein, BPIFA1 leads to exacerbation of Otitis media”, Manuscript in preparation. CONFERENCE PRESENTATIONS ñ Poster presentation: The role of BPIFA1 in Otitis media. British Association of Lung Research Summer Meeting, Sheffield, UK July 2016. ñ Poster presentation: The role of BPIFA1 in Otitis media. 10th International Molecular Biology of Hearing and Deafness Meeting, Hinxton, UK, May 2016. ñ Podium presentation: The role of BPIFA1 in Otitis media. 2nd International Annual Florey Symposium, Sheffield, UK September 2015. ñ Podium presentation: Isolation and characterisation of middle ear and nasal th epithelial cells for development of an in vitro ototpathogenic infection model. 18 International Symposium on Recent Advances in Otitis Media, Maryland, USA, June 2015. ñ Seminar: An in vitro otopathogenic model of the middle ear epithelium. The Children’s National Medical Centre, Washington D.C, USA, June 2015 ñ Podium presentation: An in vitro otopathogenic model of the middle ear epithelium 51st International Inner Ear Biology Workshop, Sheffield, UK, April 2014. ñ Podium presentation: Mucociliary abnormalities lead to altered expression th BPIF/PLUNC proteins in murine models of otitis media of 17 Extraordinary International Symposium on Otitis Media, Stockholm, Sweden, June 2013. V VI ABBREVIATIONS ABR Auditory Brainstem Response ALI Air liquid interface ANOVA analysis of variance AOM Acute Otitis media AP-1 Activator protein 1 ASL Airway surface liquid ASOM Acute suppurative Otitis media B.cepacia Burkholderia cepacia BAL Broncheoalvealoar lavage BHI Brain heart infusion BPI Bacterial permeability increasing protein BPIF protein Bacterial permeability increasing fold-containing protein BPIFA1 Bacterial permeability increasing fold-containing protein A1 BPIFB1 Bacterial permeability increasing fold-containing protein B1 CD Cluster of differentiation CETP Cholesterol ester transfer protein CF Cystic fibrosis COME Chronic Otitis media with effusion COPD Chronic obstructive pulmonary disease CSOM Chronic supparative Otitis media CT Cycle threshold CtBP Carboxy terminal binding protein dB decibels DPPC Dipalmitoylphosphatidylcholine e embryonic day ECM extracellular matrix ENaC Epithelial sodium channel ENU N-Ethyl Nitrosourea ERP Ethical Review Process ET Eustachian tube EVI1 Ecotropic viral integration site-1 FACs Fluorescence associated cell sorting FBS Foetal bovine serum FBXO11 F-box 11 Foxj1 Forkhead box protein J1 G Generation GWAS Genome-wide association study GWLS Genome-wide linkage study H&E haematoxylin and eosin HBE cells Human bronchial epithelial cells HIF Hypoxia inducible factor Hm Hemin hpi Hours post infection VII HSC Haematopeitic stem cell IFC Fluorescence Immunocytochemistry IHC Immunohistochemistry IL Interleukin IN Intranasal JNK c-JUN kinase K.pneumoniae Klebsiella pneumoniae kDa Kilo dalton LBP Lipopolysaccharide binding protein LEV Levinthals LPLUNC1 Long palate lung and nasal epithelium