An Investigation of the Role of Bacillus oleronius in the Pathogenesis of Rosacea Niamh O’Reilly B.Sc. Thesis submitted to the National University of Ireland for the degree of Doctor of Philosophy May 2012 Supervisor Head of Department Dr. Kevin Kavanagh, Prof. Kay Ohlendieck Department of Biology, Department of Biology, National University of Ireland, National University of Ireland, Maynooth, Maynooth, Co. Kildare Co. Kildare Table of Contents Declaration of Authorship ................................................................................................... viii Acknowledgments .................................................................................................................. ix Publications ............................................................................................................................. x Abbreviations ........................................................................................................................ xii List of Figures ....................................................................................................................... xiv List of Tables ......................................................................................................................... xx Abstract ................................................................................................................................. xxi CHAPTER 1 Introduction 1.1 Rosacea .......................................................................................................................... 1 1.2 Diagnostic Features ....................................................................................................... 4 1.3 Rosacea Subtypes .......................................................................................................... 5 1.3.1 Erythematotelangiectatic Rosacea ......................................................................... 5 1.3.2 Papulopustular Rosacea ......................................................................................... 5 1.3.3 Phymatous Rosacea ............................................................................................... 6 1.3.4 Ocular Rosacea ...................................................................................................... 6 1.3.5 Rosacea Variants.................................................................................................... 9 1.4 Factors that trigger rosacea symptoms ........................................................................ 11 1.5 Demodex Mites (Demodex folliculorum and Demodex brevis) ................................... 13 1.5.1 The pathogenic properties of Demodex mites ..................................................... 15 1.6 Endosymbiotic bacteria and bacterial antigens in human disease ............................... 18 1.6.1 Role of Bacillus oleronius in the induction of rosacea ........................................ 19 1.7 Treatment of Rosacea .................................................................................................. 20 1.8 Neutrophils and the innate immune response .............................................................. 24 1.9 Innate immunity of the ocular surface ......................................................................... 28 1.10 Results of previous studies .......................................................................................... 31 1.11 Aims of this study ........................................................................................................ 32 CHAPTER 2 Materials and Methods 2.1 Study Population ......................................................................................................... 33 2.1.1 Ocular Rosacea Study Population ........................................................................ 33 2.1.2 Erythematotelangiectic Rosacea Study Population ............................................. 33 2.1.3 Neutrophils .......................................................................................................... 34 2.2 General Chemicals and Reagents ................................................................................ 34 2.3 Sterilisation Procedure ................................................................................................. 34 2.4 Culture Media .............................................................................................................. 34 2.4.1 Nutrient Agar ....................................................................................................... 34 2.4.2 Nutrient Broth ...................................................................................................... 34 2.4.3 2XYT Broth ......................................................................................................... 35 2.5 Culture Conditions ....................................................................................................... 35 2.6 Glycerol Stocks ........................................................................................................... 35 2.7 Phosphate Buffered Saline (PBS) ................................................................................ 36 2.8 Bacillus Lysis Buffer (pH 7.2) .................................................................................... 36 2.9 FPLC Binding Buffer (pH 4.8) .................................................................................... 36 2.10 FPLC Elution Buffer (pH 4.8) ..................................................................................... 36 2.11 Bradford Protein Assay ............................................................................................... 36 2.12 Protein extraction from Bacillus oleronius .................................................................. 39 2.13 Preparation of bacteria for confocal microscopy ......................................................... 40 2.14 Fractionation by Q-Sepharose charge separation ........................................................ 40 2.15 Development of ELISA for testing reactivity of Human serum to B. oleronius antigens ..................................................................................................................................... 41 2.16 Acetone Precipitation .................................................................................................. 42 2.17 Sodium Dodecyl Sulphate-Poly acrylamide (SDS-PAGE) Gel Electrophoresis ......... 42 2.18 10X running buffer, pH8.9 .......................................................................................... 44 2.19 10X Sample Buffer ...................................................................................................... 44 2.20 Staining of SDS-PAGE Gels ....................................................................................... 45 2.21 Two-Dimensional Gel Elextrophoresis ....................................................................... 46 2.22 Liquid Chromatography Mass Spectrometry .............................................................. 48 2.23 Western Blotting .......................................................................................................... 50 2.24 Trypan blue exclusion assay ........................................................................................ 53 2.25 Assessing the effect of Bacillus antigens on Neutrophils ............................................ 53 2.26 Isolation of human neutrophils .................................................................................... 53 2.27 Neutrophil Migration assay ......................................................................................... 54 2.28 Quantification of release of MPO MMP-9 and hCAP-18 (LL-37) as markers of degranuation. ............................................................................................................... 55 2.29 Production of neutrophil culture supernatants for cytokine assays ............................. 55 2.30 General Cell Culture Techniques ................................................................................ 56 2.30.1 Cell Preservation .................................................................................................. 56 2.30.2 Cell Recovery ...................................................................................................... 56 2.30.3 Cell counting and viability testing ....................................................................... 56 2.30.4 Harvesting and subculture of cells ....................................................................... 57 2.31 Assays using Human corneal Epithelial Cells (hTCEpi) ............................................. 57 2.31.1 Toxicity Assay ..................................................................................................... 58 2.31.2 Wound healing assay ........................................................................................... 58 2.31.3 Corneal cell Migration assay ............................................................................... 58 2.31.4 Invasion Assay ..................................................................................................... 59 2.31.5 Adhesion Assay ................................................................................................... 59 2.32 Isolation of protein from corneal cells ......................................................................... 59 2.33 Electrophoresis and Western Blotting ........................................................................