DOCTOR OF PHILOSOPHY Elucidation of the roles of PTN and ISG15 in RSV cytopathogenesis: possible biomarkers of severe disease? Groves, Helen Award date: 2018 Awarding institution: Queen's University Belfast Link to publication Terms of use All those accessing thesis content in Queen’s University Belfast Research Portal are subject to the following terms and conditions of use • Copyright is subject to the Copyright, Designs and Patent Act 1988, or as modified by any successor legislation • Copyright and moral rights for thesis content are retained by the author and/or other copyright owners • A copy of a thesis may be downloaded for personal non-commercial research/study without the need for permission or charge • Distribution or reproduction of thesis content in any format is not permitted without the permission of the copyright holder • When citing this work, full bibliographic details should be supplied, including the author, title, awarding institution and date of thesis Take down policy A thesis can be removed from the Research Portal if there has been a breach of copyright, or a similarly robust reason. 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Helen Elizabeth Groves MB BCh BAO (Hon) School of Medicine, Dentistry and Biomedical Sciences, The Queen’s University of Belfast Thesis offered to The Queen’s University of Belfast for the degree of Doctor of Philosophy June 2018 Acknowledgements I would like to thank everyone who has supported and assisted me over the past few years in the preparation of this thesis work. I would particularly like to thank both of my supervisors, Dr Ultan Power and Prof. Mike Shields for their patience and encouragement throughout. I thank Dr Ultan Power for teaching me the scientific method, from the basics of how to pipette, the intricacies of scientific experimental design, through to critical thinking, problem solving and the drive for innovative science. I thank Prof. Shields for his encouragement, pragmatism and enthusiasm for the clinical application of scientific research. I commend him for his passion in teaching me the joy of robust statistical methodology and I hope one day to understand this as well as he does. I would like to thank Dr Lindsay Broadbent, Dr Hong Guo‐Parke, Dr Lyndsey Ferguson and Ms Andrena Millar for their training, guidance and friendship throughout my PhD work, as well as all of the other colleagues and students who supported me in their journey through the fourth floor of the Medical Biology Centre. I am grateful to all of the parents and the infants who took part in this research. Without them this work could not have been possible. I would also like to thank the staff of the Royal Jubilee Maternity Hospital, Belfast and the Royal Belfast Hospital for Sick Children for their assistance and support in recruiting infants. I thank the Wellcome Trust for their backing and financial support in completing this PhD. I am deeply grateful to my fiancé Andrew who has supported me unwaveringly during the business and challenges of this PhD work. I love him all the more for the faithfulness, kindness, patience and love he has shown me. 2 I thank my parents for their encouragement and unshakable backing throughout a lifetime of education, especially to my mother who taught me how to learn. Finally, I praise God, who enabled me to undertake this work and has given me innumerable blessings. For from Him and through Him and for Him are all things. 3 Statement of work contribution All work included in this thesis was conducted by myself (Dr Helen Groves). This included consenting and recruiting all participants, nasal sampling and processing of samples, as well as all laboratory work and data analysis detailed in this thesis. Acknowledgements of other contributions Prior to commencing this thesis work, an extensive microarray study was undertaken comparing pan‐genomic transcriptome responses in WD‐PNECs derived from cohorts of infants with histories of mild and severe RSV disease. This work was completed by Dr Hong Guo‐Parke who kindly contributed pleiotrophin microarray data. In addition, Dr Guillermo Lopez Campos performed analysis of the microarray data for pleiotrophin. This pleiotrophin data is presented in figure 25a. Dr Olivier Touzelet kindly contributed pleiotrophin ELISA data shown in figure 25b. qRT‐PCR analysis of cDNA from WD‐PNECs derived from cohorts of infants with histories of mild and severe RSV disease is presented in figure 39. I performed this work while being trained by Dr Lindsay Broadbent and she therefore contributed the data for analysis. Dr Lindsay Broadbent kindly contributed data for PTN pretreatment in WD‐PBECs shown in figure 31c. 4 Abstract Respiratory syncytial virus (RSV) infection is the most common cause of severe lower respiratory tract infection in infants under two years old. Sparking seasonal epidemics, RSV contributes to around 20,000 admissions to hospital in the UK annually and many require intensive care support. Peak incidences of severe RSV disease occur between 6 weeks and 6 months of age. Despite over 60 years of research since its discovery, no RSV vaccine or specific therapy exists. The only preventative strategy against RSV is the monoclonal antibody palivizumab, which is very expensive and, accordingly, only available to those infants at known high risk of severe RSV disease, including preterm infants and those with congenital heart conditions or bronchpulmonary dysplasia. As the majority of infants hospitalised with RSV have no known predisposing risk factor for severe illness, the benefits of palivizumab for managing the impact of RSV is very restricted. Considerable challenges in performing research in vulnerable young infants has slowed progress in RSV research. Animal models only partially reflect RSV‐human host interactions and thus extrapolation of results from these models to human responses is of limited value. Study of autopsy specimens from infants who succumbed to RSV infection identified that the infection is restricted primarily to the airway epithelium. As such, research aimed at detecting innate immune responses of human airway epithelium to RSV is likely to yield crucial insights into the cytopathogenesis of this infection. It has previously been demonstrated that an ex‐vivo/in‐vitro model of human airway epithelium/RSV interactions, termed well‐differentiated primary paediatric nasal epithelial cell cultures (WD‐PNECs), provides a reasonable surrogate for in vivo responses. Because of the strong association of severe RSV disease with prematurity and young age, the first part of this thesis describes the utilisation of the WD‐PNEC model to establish and 5 characterise unique nasal samples from preterm and term infants at birth and repeated at one‐year old. This work is the first description of morphologically and physiologically authentic WD‐PNEC cultures generated from term and preterm newborn infants and as such represents an exceptional opportunity to study RSV‐human host interactions in early life. We found that newborn term‐ and preterm‐derived WD‐PNECs were morphologically indistinguishable under light or fluorescent microscopy analysis. However, interestingly, newborn WD‐PNECs demonstrated significantly higher proportions of goblet cells compared to one‐year repeat WD‐PNECs. This finding indicates the possibility of increased mucous production in newborn infants, which may, in part, explain their susceptibility to more severe RSV disease. Importantly, we demonstrated nasal sampling to be a safe, minimally invasive method performed consistently with high rates of success. Furthermore, we were also able to successfully freeze, thaw, and subsequently differentiate the nasal epithelial cells. This confirmed the exciting possibility of storing newborn ‘‘naive’’ airway epithelial cells (AECs) indefinitely for use in subsequent experimentation, e.g., once clinical phenotypes, like severe RSV or asthma, have been established. We next sought to establish if differential RSV‐induced innate immune responses of airway epithelial cells could account, at least in part, for the increased susceptibility of preterm and very young infants to severe RSV disease. To investigate this, we infected (or mock‐infected) WD‐PNEC cultures established from term and preterm infants at birth and repeated at one‐ year‐old. No significant differences in cytopathology or viral growth kinetics were evident in WD‐PNECs derived from any cohort following RSV infection. However, crucially, we observed significantly higher secretion of interferon 1 (IL‐29) (P<0.01), IP‐10 (CXCL‐10) (P<0.05) and RANTES (CCL‐5) (P<0.05) following RSV infection of one‐year‐derived WD‐PNEC cultures compared to newborn‐derived cultures. These novel findings suggest airway 6 epithelium innate immune responses