Effect of Estrogen on Host-Pathogen Interactions in ex vivo and in vitro Models of the Inflammatory Phase of Age-Related Impaired Healing A thesis submitted in partial fulfilment of the requirements of Manchester Metropolitan University for the degree of Doctor of Philosophy Mohamed El Mohtadi Department of Life Sciences Manchester Metropolitan University 2019 Table of Contents List of Abbreviations………………………………………………………………………………………VI List of Figures………………………………………………………………………….……………………..X List of Tables………………………………………………………………………………………………….XIII Abstract…………………………………………………………………………………………………………XIV Declaration and Copyright Statements…………………………………………………………..XVI The Author…………………………………………………………………………………………………….XVII Acknowledgments………………………………………………………………………………………...XVIII Dedication……………………………………………………………………………………………………..XX : Introduction .................................................................................... 1 1.1 Acute Wound Healing ..................................................................................... 2 1.1.1 Haemostasis ................................................................................................................... 4 1.1.2 Inflammatory Phase ....................................................................................................... 5 1.1.3 Proliferative Phase ......................................................................................................... 8 ECM Formation ..................................................................................................... 8 Re-epithelialisation ............................................................................................... 9 Angiogenesis ......................................................................................................... 9 1.1.4 Remodelling Phase ....................................................................................................... 10 1.2 Abnormal Wound Healing ............................................................................ 11 1.2.1 Age-Related Impaired Healing ...................................................................................... 12 1.2.2 Chronic Wounds ........................................................................................................... 13 Venous Ulcers ..................................................................................................... 15 Diabetic Ulcers .................................................................................................... 16 Pressure Ulcers ................................................................................................... 16 1.3 Wounds and Bacteria ................................................................................... 17 1.3.1 Wound Infection ........................................................................................................... 17 1.3.2 Bacterial Species in Chronic Wounds ........................................................................... 17 Staphylococcus aureus ........................................................................................ 18 Pseudomonas aeruginosa ................................................................................... 19 1.3.3 Chronic Wounds and Biofilms ...................................................................................... 20 1.3.4 Bacterial Invasion of Host Tissues ................................................................................ 21 Host-Pathogen Interactions ................................................................................ 22 The Phagocytosis Process ................................................................................... 25 Monocytes/Macrophages ................................................................................... 27 1.4 Estrogen ...................................................................................................... 31 1.4.1 Estrogen Biosynthesis ................................................................................................... 32 1.4.2 Estrogen Receptors ...................................................................................................... 34 I 1.4.3 Effect of Estrogen on Skin ............................................................................................ 35 1.4.4 Estrogen and Wound Healing ....................................................................................... 36 Effect of Estrogen on the Inflammatory Phase of Wound healing ..................... 37 Effect of Estrogen on the Proliferative Phase of Wound Healing ....................... 38 Effect of Estrogen on the Remodelling Phase of Acute Wound Healing ............ 39 1.4.5 Estrogen and Chronic Wounds ..................................................................................... 39 1.5 Aim and Objectives ...................................................................................... 40 : Materials and Methods ................................................................. 42 2.1 Materials ..................................................................................................... 43 2.2 Methods ...................................................................................................... 47 2.2.1 Preparation of RPMI-1640 Complete Medium ............................................................. 47 2.2.2 Estrogen Treatment Preparation.................................................................................. 47 2.2.3 Cell Culture ................................................................................................................... 47 2.2.4 Differentiation of Monocytes into M0 Macrophages .................................................. 48 2.2.5 Generation of M1 and M2 Macrophages ..................................................................... 48 2.2.6 Flow Cytometry ............................................................................................................ 49 Differentiation of Monocytes into M0 Macrophages ......................................... 49 Polarisation of M0 Macrophages into M1/M2 Macrophages ............................ 50 2.2.7 Bacterial Culture Preparation ....................................................................................... 50 2.2.8 In vitro and Ex vivo Host-Pathogen Interaction Assays ................................................ 52 Interaction of M0 Macrophages with MRSA and PAO1 ...................................... 52 Interaction of M1/M2 Macrophages with MRSA and PAO1 ............................... 53 2.2.9 Interaction of M0 Macrophages with GFP- S. aureus ................................................... 53 2.2.10 Gentamicin Protection Assay ................................................................................... 54 2.2.11 Visualising Host-Pathogen Interactions ................................................................... 55 Fluorescence Microscopy ................................................................................... 55 Scanning Electron Microscopy ............................................................................ 55 2.2.12 Estrogen Receptor Stimulation/Blockade ................................................................ 56 2.2.13 Investigating Mediators of Host-Pathogen Interactions ......................................... 57 Preparation of Buffers ........................................................................................ 57 Protein Extraction and Quantification ................................................................ 57 Immunoblotting .................................................................................................. 58 : Effect of 17β-estradiol on the Phagocytosis of MRSA and P. aeruginosa by Human M0 Macrophages ............................................................ 60 3.1 Introduction ................................................................................................. 61 3.1.1 Estrogen Promotes Wound Healing ............................................................................. 61 II 3.1.2 Macrophages and Wound Healing ............................................................................... 63 3.1.3 Effect of Estrogen on Macrophages ............................................................................. 64 3.2 Aim and Objectives ...................................................................................... 66 3.2.1 Aim ............................................................................................................................... 66 3.2.2 Objectives ..................................................................................................................... 66 3.3 Methods ...................................................................................................... 68 3.3.1 Generation of U937- and HPBM-derived M0 Macrophages ........................................ 68 3.3.2 Effect of Estrogen on Bacterial Growth ........................................................................ 68 3.3.3 Effect of Estrogen on the Clearance of MRSA/PAO1 by M0 Macrophages .................. 68 Phenol Red-Free Medium Controls..................................................................... 68 Multiplicity of Infection (MOI) ............................................................................ 69 In vitro and Ex Vivo Host-Pathogen Interaction Assays ...................................... 69 3.3.4 Interaction of U937 Estrogen-Treated M0 Macrophages with GFP- S.aureus ............