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Role of IRF6 in Epithelial Cell-Mediated Host Defence and Inflammation

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Role of IRF6 in Epithelial Cell-Mediated Host Defence and Inflammation Role of IRF6 in epithelial cell-mediated host defence and inflammation Divya Ramnath M. Molecular Biology A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Institute for Molecular Bioscience Abstract Abstract Members of the Interferon Regulatory Factor (IRF) family of transcription factors have essential roles in developmental pathways, as well as immune function and host defence. IRF6 has a well- characterized role in epithelial cell differentiation, but has not been studied in the context of immune function. In this thesis, I investigated IRF6 involvement in Toll-like Receptor (TLR) responses in epithelial cells. In contrast to the ubiquitous and myeloid-restricted expression patterns of IRF3 and IRF8, respectively, IRF6 was selectively expressed in a range of human epithelial cell types, particularly keratinocytes. Keratinocyte differentiation also increased IRF6 mRNA and protein expression. Primary human keratinocytes, as well as the keratinocyte cell line HaCaT, responded weakly to most TLR ligands, with the exception of the TLR3 agonist poly(IC). Poly(IC) robustly upregulated mRNA expression of IFN-β, as well as the chemokines IL-8, CCL5 and CCL20 in both of these cell types. Surprisingly, poly(IC) also selectively upregulated mRNAs encoding IL-23p19 and EBI3, two members of the IL-12 family that have not been shown to heterodimerize. Although IRF6 enhanced poly(IC)-inducible IFN-β promoter activity in HEK- TLR3 cells, silencing IRF6 expression did not decrease poly(IC)-inducible IFN-β mRNA expression in primary keratinocytes. Several other inducible genes were also unaffected, however knock-down of IRF6 did impair poly(IC)-inducible IL-23p19 mRNA expression in primary keratinocytes. Conversely, ectopic expression of IRF6 in the bladder epithelial cell line T24 conferred poly(IC)-inducible IL-23p19 mRNA expression. To investigate the mechanisms responsible, the IL-23p19 promoter was cloned and transient transfection analyses were performed. These experiments indicated that IRF6 enhanced IL-23p19 promoter activity upon poly(IC) stimulation. Poly(IC) also trigged nuclear translocation of IRF6 in keratinocytes. However, IL- 23p19 protein was not detected in supernatants or cell lysates from poly(IC)-activated keratinocytes. Therefore, the effect of inflammatory stimuli, like IFN-γ, on keratinocyte responses to poly(IC) was examined. IFN-γ priming enhanced poly(IC)-inducible gene expression but still did not license IL-23p19 secretion. Interestingly, keratinocyte differentiation increased poly(IC)- inducible IL-23p19 mRNA expression, but decreased poly(IC)-inducible IFN-β mRNA expression upon IFN-γ priming. Collectively, these data suggest that IRF6 promotes a sub-set of TLR3 responses in human keratinocytes. Studies were also undertaken to determine whether IRF6 is involved in MyD88-dependent signalling responses in keratinocytes. Most MyD88-dependent TLR agonists were generally ineffective at triggering inflammatory responses in these cells. Investigation of other MyD88- ii dependent ligands revealed that the TLR7 agonist imiquimod and the TLR2/6 agonist FSL-1 did upregulate inflammatory chemokine expression in keratinocytes. Knock-down studies suggested that IRF6 contributed to TLR7-inducible CCL5 and IFN-β mRNA expression in these cells. Given the modest effects of most MyD88-dependent TLR agonists on inflammatory responses in keratinocytes, factors that may enhance these responses were also investigated. Keratinocyte differentiation increased imiquimod-inducible CCL5 mRNA expression in keratinocytes, whereas there was no clear effect of IFN-γ priming. The role of IRF6 in promoting TLR2/6 responses in keratinocytes remains unresolved. In total, my findings posit IRF6 as a transducer of a sub-set of MyD88-dependent and -independent TLR responses in keratinocytes, implicating it as a sentinel transcription factor in pathogen sensing by the skin. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature Conference Abstracts Ramnath, D., Bergot, A.S., Scholz, G.M., Sweet, M.J. (2011) IRF6: An epithelial cell-specific component of TLR signalling? Brisbane Immunology Group 12th Annual Meeting, Sea-world Resort, Gold Coast, QLD, Australia. Ramnath, D., Bergot, A.S., Scholz, G.M., Sweet, M.J. (2012) IRF6: An epithelial cell-specific component of TLR signalling. TLROZ 2012 conference, Melbourne, VIC, Australia. Ramnath, D., Bergot, A.S., Sturm, R.A., Scholz, G.M., Sweet, M.J. (2013) IRF6: An epithelial cell-specific component of TLR signalling. Australasian society of Immunology 43rd Annual Scientific Meeting, Wellington, New Zealand. (I was unable to attend this conference since I did not receive a visa for travel. This talk was kindly presented by Dr. Katryn Stacey, University of Queensland, on my behalf). Publications included in this thesis No publications included. v Contributions by others to the thesis A/Prof. Matthew Sweet made a significant and substantial contribution to the conception and design of this project, as well as interpretation of data and critical revision of the thesis. A/Prof. Glen Scholz also provided significant input in project design and provided plasmids used in these studies. A/Prof. Richard Sturm provided support in technical work related to culturing of human keratinocytes and critically revised this thesis. Dr. Anne-Sophie Bergot, Dr. Makrina Totsika, Dr. Timothy Barnett, Dr. Nilesh Bokil and A/Prof. Kiarash Khosrotehrani provided cells or tissue samples used in these studies, as indicated in materials and methods. Dr. Kolja Schaale and Dr. Ronan Kapetanovic also contributed to critical revision of this thesis. Mr. Daniel Hohenhaus and Mr. John Griffin provided technical support with microscopy studies and Russell Medical Centre, Queensland provided skin samples used in this thesis. Statement of parts of the thesis submitted to qualify for the award of another degree None. vi Acknowledgements I have been privileged to work with a number of people during my PhD, who have supported and inspired me during this challenging duration of my study. I would first like to give my appreciation and thanks to my PhD advisor, A/Prof. Matt Sweet for his invaluable support, encouragement and guidance throughout my PhD, without which I would never have made it to the end. I would also like to thank my co-supervisors A/Prof. Glen Scholz and A/Prof. Rick Sturm for their help and guidance in my project. I would also like to extend my sincere thanks to my thesis committee members, Dr. Kate Schroder, A/Prof. Stuart Kellie and Prof. Mark Walker, for their comments and criticism that were very useful for improving my project. I would also like to thank UQ and IMB for providing me this wonderful opportunity to pursue my PhD here. My experience working in the Sweet group has been both comfortable and stimulating, for which I would like to thank all the members of Group Sweet, past and present. Thanks Dan, Greg, Juliana, Kolja, Katja, Mel, Nabilah, Nilesh, Ronan, Selene, Sian and Tam; you have been a fantastic group to work with and made it such an enjoyable and memorable experience to work in the lab. I would also like to thank the people who shared the PhD “Fishbowl” office with me for your cheerful chats and being such great friends. Also thanks to all the members of Groups Schroder and Stacey for the useful discussions and comments during lab meetings. Thank you to all the fellow scientists on Level 5, IMB for just being around and the many conversations that made working late at night or on weekends more bearable. Many thanks to everyone who provided technical support and/or reagents that were essential for this work. Thanks to all the support staff, in particular Dr. Amanda Carozzi and Mrs. Anne Tobin, and the scientists in the IMB for being such a pleasure to work with. A special thanks to Dr. Terry Russell and his staff at Russell Medical Centre for their support throughout my PhD. I would also like to give my appreciation to Dr. David Pennisi, Dr. Richa Dave and Dr. Steve Broomfield for their cordial support and guidance,
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