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The Role of Mast Cells in the Immune Response Against Bacterial Infections

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The Role of Mast Cells in the Immune Response Against Bacterial Infections The Role of Mast Cells in the Immune Response against Bacterial Infections vorgelegt von Dipl. Mol. Med., Carolin Zimmermann, geb. in Stuttgart von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Naturwissenschaften -Dr.rer.nat.- genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Roland Lauster Gutachter: Prof. Jens Kurreck Gutachter: Prof. Marcus Maurer Tag der wissenschaftlichen Aussprache: 19.05.2016 Berlin, 2016 Table of contents Table of contents I. List of abbreviations ............................................................................................................. VII II. Summary .................................................................................................................................. X III. Zusammenfassung ................................................................................................................... XI 1. Introduction ............................................................................................................................... 1 1.1. Mast cell morphology and distribution pattern ...................................................................... 1 1.2. Mast cell origin and differentiation........................................................................................ 2 1.3. Mast cell heterogeneity .......................................................................................................... 3 1.4. Mast cell-deficient mouse models ......................................................................................... 4 1.5. Classical mast cell activation ................................................................................................. 5 1.6. Mast cell activation within the innate immune system .......................................................... 5 1.7. Mast cell functions in response to pathogens ........................................................................ 8 1.7.1. Mast cell involvement in innate responses to bacterial infections 9 1.7.2. Role of mast cells in skin innate immunity against bacteria 14 1.7.3. Role of mast cells in skin wound healing 15 1.8. Skin wound infection with Pseudomonas aeruginosa ......................................................... 18 1.8.1. Skin wound infection 18 1.8.2. Pseudomonas aeruginosa 18 1.9. Aim of the study .................................................................................................................. 19 2. Materials ................................................................................................................................. 21 2.1. Cell sources .......................................................................................................................... 21 II Table of contents 2.2. Mast cell-deficient mouse strains......................................................................................... 21 2.3. Genetically modified mice ................................................................................................... 21 2.4. Cell culture media and supplements .................................................................................... 22 2.5. Bacterial culture media and supplements ............................................................................ 22 2.6. Buffers, reagents and chemicals .......................................................................................... 22 2.7. Antibodies ............................................................................................................................ 23 2.8. Cytokines ............................................................................................................................. 24 2.9. Primers ................................................................................................................................. 24 2.10. Commercial kits ................................................................................................................... 24 2.11. Consumables ........................................................................................................................ 25 2.12. Devices and technical support ............................................................................................. 25 2.13. Analysis software ................................................................................................................. 26 2.14. Manufacturers and distributers ............................................................................................ 26 3. Methods ................................................................................................................................... 29 3.1. Isolation and culture of bone marrow-derived cultured MCs (BMCMCs) ......................... 29 3.2. Murine keratinocyte cell culture .......................................................................................... 29 3.3. Isolation and culture of primary human keratinocytes ........................................................ 30 3.4. Bacterial culture ................................................................................................................... 30 3.5. Mouse model: Skin wound infection ................................................................................... 32 3.6. Histology .............................................................................................................................. 34 3.7. In vivo live imaging ............................................................................................................. 35 3.8. Skin explant infection .......................................................................................................... 35 3.9. Cell culture infection experiments ....................................................................................... 35 3.10. Enzyme linked immunosorbent assay (ELISA) ................................................................... 37 3.11. Quantitative real time polymerase chain reaction (qRT-PCR) ............................................ 37 III Table of contents 3.12. Flow cytometry .................................................................................................................... 38 3.13. Myeloperoxidase assay ........................................................................................................ 39 3.14. Data presentation and statistical analysis ............................................................................. 39 4. Results ..................................................................................................................................... 41 4.1. Genetically mast cell-deficient KitW/KitW-v mice show impaired wound closure upon skin wound infection with P. aeruginosa .................................................................................... 41 4.2. Local mast cell reconstitution of genetically mast cell-deficient KitW/KitW-v mice normalises wound closure upon skin wound infection with P. aeruginosa ........................ 43 4.3. Mast cells control bacterial numbers in P. aeruginosa-infected skin wounds .................... 45 4.4. P. aeruginosa reduction requires mast cell-keratinocyte interaction................................... 47 4.5. Mast cell-derived IL-6 induces antimicrobial defence in keratinocytes upon P. aeruginosa infection ........................................................................................................ 51 4.6. IL-6 production in mast cells is dependent on stimulation by IL-1 family members .......... 51 4.7. Mast cell-derived IL-6 protects from skin wound infections with P. aeruginosa ............... 55 4.8. Topical treatment with recombinant IL-6 enhances defence against P. aeruginosa skin wound infection ................................................................................................................... 57 4.9. Recombinant IL-6 treatment stimulates the antibacterial response of human keratinocytes ........................................................................................................................ 57 5. Discussion ............................................................................................................................... 61 5.1. Mast cells are required for closure of infected skin wounds ............................................... 61 5.2. Mast cells reduce the bacterial load within P. aeruginosa infected skin wounds ............... 64 5.3. Mast cell-mediated bacterial clearance is independent of immune cell recruitment ........... 68 5.4. Mast cells promote the release of antimicrobial peptides by keratinocytes......................... 69 5.5. Skin antibacterial capacity requires mast cell-derived IL-6 ................................................ 71 5.6. Keratinocytes induce IL-6 production and release in mast cells ......................................... 72 5.7. Mast cell-derived IL-6 is required for normal healing of infected skin wounds in mice .... 74 5.8. Strengths and limitations ..................................................................................................... 75 IV Table of contents 5.9. Conclusion and outlook ....................................................................................................... 75 6. List of figures .......................................................................................................................... 76 7. List of tables ...........................................................................................................................
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