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 ...........................................................................................................................