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Recognition of Microbial Viability Via TLR8 Promotes Innate and Adaptive Immunity

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Recognition of Microbial Viability Via TLR8 Promotes Innate and Adaptive Immunity Recognition of microbial viability via TLR8 promotes innate and adaptive immunity Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von Matteo Ugolini, M.Sc. Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-ing Dr. Sabine Kunst Dekan der Lebenswissenschaftliche Fakultät der Humboldt-Universität zu Berlin Prof. Dr. Bernhard Grimm Gutacher/innen 1. Prof. Dr. Leif Erik Sander 2. Prof. Dr. Kai Matuschewski 3. Dr. Benedikt Beckmann Tag der mündlichen Prüfung: 12. April 2019 All life is an experiment. The more experiments you make the better. Ralph Waldo Emerson, 1841 Acknowledgements There are many people I would like to thank and without whom I would have never be able to achieve what you are about to read in the next one-hundred or so pages. Unfortunately, or fortunately, I am not proficient in these moments, so I will be brief. First and foremost, my gratitude goes to Prog. Leif Erik Sander for being the best supervisor one student can dream of, a good friend and, last but not least, an impressive traditional Greek dancer (video evidence will be kindly provided upon request). I will never forget the time spent discussing experiments, writing papers or even arguing on the right font choice for a figure. Your drive and passion for science is something I will always cherish. Next, a well-deserved thanks goes to all the members of the Sander group, for their endless support and also for being able to cope with having disorganized me working around for so many years. Elisa, Jenny, Philipp, Ling, Moritz, Daniel, Sarah (strictly in the order they joined the lab), thanks for creating a wonderful atmosphere even in the darkest of times. Same goes for all the other member of the department at the Charité. In particular my gratitude goes to Prof. Dr. Norbert Suttorp for welcoming me and allowing my do join his department. A special mention goes to Elena. If it was our common obsession with South Park or our questionable interest in shady dictators, I am not able to say but we got along from the very start. Our friendship is something that goes way beyond the wall of the lab! Mariagrazia, who would have thought that after meeting in the remotes of South Africa we would have ended up in the next lab and to IKEA every other evening? Thanks for being such a good friend and an inspiration for what a scientist should strive for. Moreover, Daniela, who would have thought that after many years our path would cross again in this new home far from home. Your questionable sense of style has never and will never been an obstacle to our friendship or to my admiration for the passion you put in what you do. How not to thanks all my other friends who were not physically in the lab but supported me and, most importantly, endured my many complaints. Nikoleta, you have been always there, and I hope to be such a splendid friend for you as you are for me. Mirna, the only person that talks more than I do, and Carla, whose musical tastes are beyond my comprehension, a big thanks goes to you both. Finally, to my family back in Italy…mia madre, mio padre, mia sorella e mia zia. Chi avrebbe pensato quel giorno del giugno di dieci anni fa che sarei rimasto via dall’Italia così tanti anni? Il tempo è passato ma il vostro affetto e il vostro supporto non sono diminuiti. Ogni traguardo che ho, o spero di superare in futuro è merito vostro tanto quanto mio! I II Table of contents List of figures .................................................................................................................. VI List of tables ................................................................................................................... VII Summary ........................................................................................................................ IX Zusammenfassung ............................................................................................................ X 1 INTRODUCTION .................................................................................................. 1 1.1 Innate immunity: receptors and responses ............................................................. 1 1.2 Nucleic acid sensing receptors .............................................................................. 3 1.3 Toll-like receptors ............................................................................................... 4 MyD88 and TRIF-dependent pathways .................................................................... 6 TLR8........................................................................................................................ 7 1.4 RNA sensing in the cytosol ............................................................................... 10 1.5 Bacterial ‘viability sensing’ and vita-PAMPs ....................................................... 12 1.6 The adaptive immune response and its control by innate immunity .................... 14 T Lymphocytes ....................................................................................................... 14 T-helper cell differentiation ..................................................................................... 16 Characteristics and development of T-follicular helper (TFH) cells ............................ 18 Roles of TFH cells in health and diseases ................................................................... 22 1.7 Aims of this study .............................................................................................. 24 2 MATERIALS & METHODS .................................................................................. 25 2.1 Reagents, materials and instruments ................................................................... 25 Reagents ................................................................................................................. 25 Materials ................................................................................................................. 27 Kits ......................................................................................................................... 27 Buffers and cell culture media .................................................................................. 28 Antibodies ............................................................................................................... 29 Instruments ............................................................................................................. 30 III 2.2 Methods ............................................................................................................ 30 Cell isolation and culture ......................................................................................... 30 Bacteria ................................................................................................................... 32 Infection experiments .............................................................................................. 32 Cells stimulation experiments .................................................................................. 33 Enzyme-linked immunosorbent assay (ELISA) ......................................................... 34 RNA Isolation ........................................................................................................ 34 Quantitative RT-polymerase chain reaction (RT-qPCR) ........................................ 35 Gene Array ............................................................................................................. 36 RNA interference ................................................................................................... 37 T-cells differentiation studies ................................................................................... 38 Flow cytometry and cell sorting ............................................................................... 39 Animal experiments ................................................................................................. 41 Anti-S. enterica IgG ELISA ..................................................................................... 45 Immunohistochemistry ............................................................................................ 45 Phylogenetic analyses............................................................................................... 45 Statistical analyses .................................................................................................... 46 3 RESULTS ............................................................................................................... 49 3.1 Sensing of bacterial viability modifies cytokine responses of APC ....................... 49 3.2 Recognition of live bacteria induces a specific cytokine secretion pattern ........... 55 3.3 Presence of vita-PAMPs in different bacterial species .......................................... 57 3.4 APC maturation occurs independently of bacterial viability ................................ 58 3.5 Evolutionary conservation of ‘viability recognition’ ............................................ 58 Sus scrofa domestica ................................................................................................ 59 Salmo salar .............................................................................................................. 60 3.6 Involvement of TLRs in the recognition of bacterial viability ............................. 62 3.7
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