Mechanisms by which Staphylococcus aureus induces cytokines and cell death in human keratinocytes and mouse fibroblasts

12 Declaration . 13 Copyright Statement . 14 Acknowledgment . 15 Chapter 1 : Introduction . 16 1 1.1 Foreword . 17 1.2 Atopic dermatitis . 17 1.2.1 Epidemiology and clinical overview of the disease in humans . 17 1.2.2 Japanese NC/Nga mouse eczema model . 20 1.3 The epidermis as a physical barrier . 23 1.3.1 Epidermal structure . 23 1.3.2 Stratum corneum and its barrier function . 24 1.3.2.1 Genetic factors influencing skin barrier function . 26 1.3.2.2 Environmental factors influencing skin barrier function . 28 1.4 Keratinocytes as part of the innate immune system . 30 1.4.1 Immune sensory receptors on keratinocytes . 31 1.4.1.1 Toll-like receptors (TLRs) . 31 1.4.1.2 Protease-activated receptors (PAR) . 32 1.4.2 Keratinocyte-initiated immune responses . 36 1.4.2.1 Thymic Stromal Lymphopoietin (TSLP) . 36 1.4.2.2 Interleukin-25 . 37 1.4.2.3 Interleukin-33 . 37 1.4.3 Cell death pathways. 40 1.4.4 Downstream immune cell responses . 41 1.4.4.1 Dendritic cells (DCs) . 41 1.4.4.2 T-lymphocytes . 42 1.4.4.3 Innate lymphoid cells . 44 1.4.4. 4 Eosinophils . 45 1.4.4.5 Mast cells . 45 1.5 S. aureus, skin and AD . 46 1.5. 1 S. aureus cell wall and membrane vesicle virulence factors . 47 1.5. 2 Secreted S. aureus virulence factors . 49 1.5. 3 Cytotoxic effects of Staphylococcal Virulence factors. 51 1.6 Synopsis, hypothesis and aims. 52 Chapter 2: Materials and Methods . 54 2 2.1 Culturing, Freezing and thawing of cell lines . 55 2.1.1 Culture of Mouse Embryonic Fibroblasts (MEFs) . 55 2.1.2 Human epidermal keratinocyte (HEKa) culture . 56 2.1.3 Culture of Human skin fibroblasts . 57 2.1.4 Cryopreservation of MEFs, HEKa and human fibroblasts . 57 2.2 Preparation of staphylococcal species, their virulence factors and inhibitors . 57 2.2.1 Live Staphylococcus aureus (LiSA) and other staphylococcal species . 57 2.2.2 Establishment of Staphylococcal species growth curve . 58 2.2.3 Quantification of bacterial culture by Miles Misra method . 59 2.2.4 Collection of filtered bacterial supernatant of S. aureus and S. epidermidis culture . 59 2.2. 5 Inactivation of LiSA . 59 3 2.2. 6 Preparation of TLR2 antagonist, TLR2 ligands and S.aureus virulence factors . 60 2.2. 7 Preparation of PAR1, PAR2 agonist, NDGA, KLK5, V8 serine protease of S. aureus, different proteases inhibitors, necrostatin-1 and general caspases inhibitor . 60 2.3 Co-cultures of MEFs and HEKa . 60 2.3.1 Stimulation of HEKa and MEFs with different staphylococcal species, S. aureus filtered supernatant, killed S. aureus and TLR2 ligands . 60 2.3.2 Stimulation of mast cells by IL-33 of MEFs co-culture . 61 2.3.3 T cell polarisation . 62 2.4 Assays and Analysis . 62 2.4.1 Quantification of pro-inflammatory cytokines (TNFα, TSLP, IL-33 and ST2) by ELISA 62 2.4.2 Fluorescence activated cell sorting (FACS) assays . 64 2.4.2.1 Summary of FACS protocol . 64 2.4.2.2 Surface expression of ST2 and PAR2 expression . 64 2.4.2.3 Detection of intracellular IL-6 . 65 2.4.2.4 FACS analysis . 65 2.4.3 Cell death assays . 65 2.4.3.1 Detection of cell death by AV and PI . 65 2.4.3.2 Detection of cell death by trypan blue . 66 2.4.4 Immunohistochemistry of IL-33 . 66 2.4.5 Western blotting . 67 2.4. 6 Detection of protease genes in S. aureus by PCR . 68 2.4. 7 Protease activity assays . 70 2.4. 7.1 Skimmed milk agar . 70 2.4. 7.2 Colorimetric Protease Assay . 70 2.4. 8 Mass spectrometry (MS) for filtered supernatant of S. aureus . 71 2.4. 9 Statistical analysis . 71 Chapter 3: S. aureus-induced release of the Th2-promoting cytokines IL-33 and TSLP by 3 mouse embryonic fibroblasts and human keratinocytes . 72 3.1 Foreword . 73 3.2 Growth curves of S. aureus and other staphylococcal species in culture medium. 73 3.3 S. aureus bu