An Immunological Perspective of the Mucosal Inflammation in Chronic Rhinosinusitis – Lymphoid Neo-organogenesis and Humoral Immunity A thesis submitted in total fulfillment of the requirements for the degree of Doctor of Philosophy in The Discipline of Surgery, School of Medicine Faculty of Health Science The University of Adelaide by ADEN HO YIN LAU March 2018 Table of Contents Declaration ....................................................................................................................................... i Acknowledgements ......................................................................................................................... ii Manuscripts arising from thesis ..................................................................................................... iii Presentation arising from the thesis ............................................................................................... iv Award arising from thesis .............................................................................................................. iv List of Figures ................................................................................................................................. v List of Tables ................................................................................................................................. vi Abbreviations ................................................................................................................................ vii Abstract .......................................................................................................................................... ix Chapter 1: Introduction ................................................................................................................... 1 1.1 Background ...................................................................................................................... 1 Definition of Chronic Rhinosinusitis ....................................................................................... 1 Prevalence of CRS ................................................................................................................... 1 Classification of CRS ............................................................................................................... 2 Current Treatment Options and disease recalcitrance ............................................................. 3 Co-morbidities and risk factors of CRS ................................................................................... 4 1.2 Pathophysiology of CRS .................................................................................................. 6 Persistent infections and the microbiome in CRS ................................................................... 6 Mucosal biofilms ..................................................................................................................... 6 Innate Immunity in CRS .......................................................................................................... 9 1.3 Adaptive Immunity in CRS ............................................................................................ 18 T cells and Th type immune response .................................................................................... 18 B cells .................................................................................................................................... 19 i 1.4 Epithelial cells mediating Inflammation ........................................................................ 20 1.5 Secondary lymphoid organs and tertiary lymphoid organs............................................ 22 Overview of lymphocytes and lymphoid organs ................................................................... 22 Tertiary Lymphoid Organs .................................................................................................... 24 Identification of TLO ............................................................................................................. 26 TLOs in inflammation ........................................................................................................... 27 Potential clinical implications of TLOs ................................................................................. 28 1.6 Potential autoimmunity in CRS ..................................................................................... 29 Chapter 2: Characterization of tertiary lymphoid organs in chronic rhinosinusitis ...................... 32 2.1 Abstract .......................................................................................................................... 36 2.2 Introduction .................................................................................................................... 37 2.3 Methods .......................................................................................................................... 38 Patients and Samples ............................................................................................................. 38 Histology ................................................................................................................................ 39 Immunofluorescence .............................................................................................................. 39 Quantitative Real-time PCR .................................................................................................. 40 Statistical analysis .................................................................................................................. 40 2.4 Results ............................................................................................................................ 41 Patient demographical data .................................................................................................... 41 Relative expression of TLO-related genes in CRSwNP patients .......................................... 41 Hierarchical clustering of TLO-related gene expression ....................................................... 43 GC-like structures are identified in sinus tissue from CRSwNP patients .............................. 44 Associations of TLOs with clinical data, demographic factors and histological characteristics in CRS patients ...................................................................................................................... 45 2.5 Discussion ...................................................................................................................... 46 ii 2.6 Conclusion...................................................................................................................... 49 2.7 Supplementary data ........................................................................................................ 50 2.8 Citations ......................................................................................................................... 52 Chapter 3: The Nasal Epithelium Mediates Tertiary Lymphoid Organ Formation in Chronic Rhinosinusitis ................................................................................................................................ 56 3.1 Abstract .......................................................................................................................... 59 3.2 Introduction .................................................................................................................... 60 3.3 Methods .......................................................................................................................... 61 Patients and samples .............................................................................................................. 61 Cell culture ............................................................................................................................. 61 Quantitative PCR ................................................................................................................... 62 Histology ................................................................................................................................ 62 Immunohistochemistry / Immunofluorescence ..................................................................... 62 Statistical analysis .................................................................................................................. 63 3.4 Results ............................................................................................................................ 64 Patient demographics ............................................................................................................. 64 Nasal epithelial cells express TLO genes in response to TLR agonists ................................ 64 Expression of TLO genes by immunohistochemical analysis ............................................... 66 Clinical/demographical associations ...................................................................................... 66 3.5 Discussion ...................................................................................................................... 72 3.6 Conclusion...................................................................................................................... 73 3.7 Supplementary data ........................................................................................................ 74 Chapter 4: Reduced Serum Immunoglobulin G Levels in Chronic