Aus der Klinik und Poliklinik für Dermatologie und Allergologie der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Dr. h.c. Thomas Ruzicka Psoriasin (S100A7) and koebnerisin (S100A15) in the model of inflammation: functional characterization in the inflammation cascade. Dissertation zum Erwerb des Doktorgrades der Humanbiologie an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Stephanie Zwicker, M.Sc. aus Berlin 2014 - 2 - Mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Berichterstatter: PD Dr. med. Ronald Wolf Mitberichterstatter: Prof. Dr. med. Dr. phil. Johannes Ring Prof. Dr. med. Hendrick Schulze-Koops Prof. Dr. med. Marc Heckmann Dekan: Prof. Dr. med. Dr. h.c. M. Reiser, FACR, FRCR Tag der mündlichen Prüfung: 30. Juli 2014 - 3 - Für meine Liebsten - 4 - - 5 - Life is what happens while you are busy making other plans. (Leben ist das, was passiert, während du dabei bist, andere Pläne zu schmieden.) John Lennon, 1940-1980 (British musician) - 6 - - 7 - Table of contents List of abbreviations ............................................................................................................... - 11 - 1. Introduction ....................................................................................................................... - 13 - 1.1 Structure and barrier function of human skin.................................................................... - 13 - 1.2 Innate and adaptive immunity in the skin ......................................................................... - 15 - 1.2.1 The inflammasome ..................................................................................................... - 17 - 1.2.2 Antimicrobial peptides................................................................................................ - 21 - 1.2.3 Psoriasin (S100A7) and koebnerisin (S100A15) – highly homologous but distinct .... - 22 - 1.3 Plaque psoriasis ................................................................................................................. - 25 - 1.3.1 Therapy of psoriasis .................................................................................................... - 27 - 1.4 Aims ................................................................................................................................... - 28 - 2. Materials and Methods ....................................................................................................... - 31 - 2.1 Materials ............................................................................................................................ - 31 - 2.1.1 Technical devices ........................................................................................................ - 31 - 2.1.2 Plastic consumables ................................................................................................... - 31 - 2.1.3 Cells, medium, buffer & solutions ............................................................................... - 31 - 2.1.4 Antibodies, primers & siRNAs ..................................................................................... - 32 - 2.1.5 Chemicals, reagents & stimulants .............................................................................. - 34 - 2.1.6 Kits .............................................................................................................................. - 35 - 2.1.7 Western blot consumables ......................................................................................... - 35 - 2.1.8 Software ..................................................................................................................... - 35 - 2.2 Methods ............................................................................................................................. - 35 - 2.2.1 Patients and skin samples .......................................................................................... - 35 - 2.2.2 Cell culture and stimulation ........................................................................................ - 36 - 2.2.3 RNA-isolation and cDNA synthesis ............................................................................. - 36 - 2.2.4 Real-time-PCR (qPCR) ................................................................................................. - 38 - 2.2.5 siRNA design and transfection ................................................................................... - 38 - 2.2.6 Protein isolation and Western blot ............................................................................. - 39 - 2.2.7 ELISA ........................................................................................................................... - 40 - 2.2.8 Immunofluorescent staining ....................................................................................... - 40 - 2.2.9 Statistical analysis ...................................................................................................... - 41 - 3. Results ............................................................................................................................... - 43 - 3.1 Psoriasin (S100A7) and koebnerisin (S100A15) in skin homeostasis ................................. - 43 - - 8 - 3.1.1 Psoriasin (S100A7) and koebnerisin (S100A15) have different expression patterns in healthy skin.......................................................................................................................... - 43 - 3.2 Regulation of psoriasin (S100A7) and koebnerisin (S100A15) in skin inflammation ......... - 46 - 3.2.1 Psoriasin (S100A7) and koebnerisin (S100A15) are up-regulated in non-lesional and lesional psoriatic skin .......................................................................................................... - 46 - 3.2.2 Psoriasin (S100A7) and koebnerisin (S100A15) are differently regulated through Th17 and Th22 and TNFα cytokines ............................................................................................. - 48 - 3.2.3 The Th1/Th17 cytokines IFNγ and IL-17A synergize to induce S100 expression in human epidermal keratinocytes ...................................................................................................... - 50 - 3.2.4 UVB regulates the S100 gene expression in human epidermal keratinocytes ........... - 51 - 3.3 Function of psoriasin (S100A7) and koebnerisin (S100A15) during inflammation ............ - 54 - 3.3.1 IL-1β, pro-inflammatory caspases and inflammasome complexes are up-regulated in lesional psoriatic skin .......................................................................................................... - 54 - 3.3.2 Th1/Th17 cytokines are crucial for the regulation of IL-1β and pro-inflammatory caspases in human epidermal keratinocytes ...................................................................... - 56 - 3.3.3 Psoriasin (S100A7) is a key regulator of IL-1β and pro-inflammatory caspases under the Th1/Th17 cytokine-milieu .................................................................................................... - 59 - 3.3.4 Psoriasin (S100A7) and koebnerisin (S100A15) synergize as ‘alarmins’ to regulate pro- inflammatory cytokines and other antimicrobial peptides in epidermal keratinocytes...... - 61 - 3.3.5 IFNγ and double stranded DNA are required to activate inflammasomes in human epidermal keratinocytes ...................................................................................................... - 62 - 3.3.6 Th17 cytokine IL-17A suppresses IFNγ induced release of IL-1β by human epidermal keratinocytes ....................................................................................................................... - 64 - 3.3.7 S100-proteins induce IL-1β release ............................................................................. - 65 - 3.3.8 Cytokine-milieu regulates the inflammasome dependent IL-1β release .................... - 66 - 3.3.9 Role of psoriasin (S100A7) and koebnerisin (S100A15) during UVB-induced acute inflammation ....................................................................................................................... - 67 - 3.4 Function of vitamin D in chronic inflamed psoriatic skin ................................................... - 70 - 3.4.1 T cell supernatants from patients with psoriasis increase S100 expression in keratinocytes ....................................................................................................................... - 70 - 3.4.2 IL-17A but not TNFα is the main inducer of S100-‘alarmins’ in psoriasis ................... - 71 - 3.4.3 1.25-Dihydroxyvitamin D3 (1.25D3) inhibits the Th17-induced expression of psoriasin (S100A7) and koebnerisin (S100A15) and suppresses the S100-mediated pro-inflammatory loop in psoriasis ................................................................................................................... - 72 - - 9 - 3.4.4 Vitamin D3 analog calcipotriol inhibits the expression of psoriasin (S100A7) and koebnerisin (S100A15) in situ .............................................................................................. - 74 - 4. Discussion .......................................................................................................................... - 77 - 4.1 Psoriasin (S100A7) and koebnerisin (S100A15) in skin homeostasis ................................. - 77 - 4.2 Regulation of psoriasin (S100A7) and koebnerisin (S100A15) ..........................................