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Apoptotic and Inflammatory Signalling Pathways in Dendritic Cells

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Apoptotic and Inflammatory Signalling Pathways in Dendritic Cells Apoptotic and inflammatory signalling pathways in dendritic cells Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg von Diplom Biologin Beate Fraust geb. Kellert geb. am 15.06.1980 in Frankfurt / Oder Gutachter: Prof. Dr. Ingo Schmitz Prof. Dr. Henning Walczak Eingereicht am: 27.05.2019 Verteidigt am: 30.01.2020 2 Summary Summary The immune system recognizes and eliminates infiltrating microorganisms and other pathogens like viruses and parasites. Beside monocytes, macrophages and B- lymphocytes, dendritic cells (DC) belong to the antigen-presenting cells (APC) and serve as point of intersection between the innate and adaptive immune system. DC recognition of pathogen-associated molecular patterns (PAMPs) and danger- associated molecular patterns (DAMPs) by specific PAMP recognition receptors (PRRs) leads to their maturation and an upregulation of different molecules, e.g. the inflammatory cytokine IL-1β and the anti-apoptotic molecule cFLIP (cellular-FLICE inhibitory protein). The aim of this thesis was the detailed characterisation of the respective inflammatory and apoptotic signalling pathways in murine DCs also by interference via either dominant-negative protein expression or siRNA-mediated knockdown and the analysis of their impact on T cell priming. Beside DC-maturation, lipopolysaccharide, lipoteichonic acid, hyaluronic acid or a cocktail containing TNF-α, IL-4, CD40L and PGE2 together led to an activation of the inflammasome. The mRNA expression of IL-18, IL-33 and IL-1β and the protein expression of the inflammatory caspase-11 increased and IL-18 and IL-1β were secreted. The IL-1β secretion could be further increased by supplemental stimulation with the DAMP ATP, was dependent on caspase-activation as well as potassium gradient and regulated via the P2X7 receptor. DC inflammasome activation did not influence the CD4+ TC proliferation rate, but showed a complex spectrum of induced cytokines, namely TNF-α, IL-2, IL-4, IL-10, GM-CSF, IL-6, IL-5, M-CSF and IL-17 arguing for a complex immune response dependent on the infection background. Expression of a dominant-negative variant of the adapter molecule ASC (dnASC) to block the inflammasome-mediated signalling cascade led to a massive DC death, and did not allow conclusions about the role of inflammatory caspases and their intracellular network in respect to the inflammatory cytokines. DC maturation-induced upregulation of cFLIP inhibited CD95L-induced apoptosis by blocking caspase-8 at the death inducing signalling complex (DISC). A complete genetic knockout of cFLIP in DCs led to a severe spontaneous cell death rescued by expression of one cFLIP-allele. A knockdown of cFLIP also directly diminished DC lifespan, but had no specific influence on TC proliferation rate and DC survival during DC-TC interaction. These findings provide new insights in the fundamental roles of ASC and cFLIP for the lifespan of DCs and may help to further develop the DC-based immunotherapy. 3 Zusammenfassung Zusammenfassung Das Immunsystem erkennt und eliminiert eindringende Mikroorganismen und andere Pathogene wie Viren und Parasiten. Dendritische Zellen (DZ) zählen neben Monozyten, Makrophagen und B-Lymphozyten zu den professionellen Antigen- präsentierenden Zellen (APC) und fungieren als Schnittstelle zwischen dem angeborenen und dem erworbenen Immunsystem. Das Erkennen fremder molekularer Muster unterschiedlicher Pathogene (pathogen-associated molecular patterns, PAMPs) und eigener Gefahr-assoziierter Muster (danger-associated molecular patterns, DAMPs) über spezifische Rezeptoren (PAMP recognition receptors (PRRs)) führt bei DZ zur Reifung und einer Hochregulation verschiedener Moleküle, wie z.B. dem inflammatorischen Zytokin IL-1β und dem anti-apoptotischen cFLIP (cellular-FLICE inhibitory protein). Das Ziel der Dissertation ist die detaillierte Charakterisierung der zugehörigen inflammatorischen und apototischen Signalwege in murinen DZ, des Weiteren auch durch Interferenz mittels dominant-negativer Proteinexpression bzw. siRNA- vermitteltem Knockdown und deren Auswirkungen auf das T-Zell-Priming. Neben einer DZ-Reifung führen Lipopolysaccharid, Lipoteichonsäure, Hyaluronsäure oder ein Cocktail aus TNF-α, IL-4, CD40L und PGE2 zur Aktivierung des Inflammasoms. Dies führt zu einer gesteigerten Genexpression von IL-18, IL-33 und IL-1β und erhöhter Proteinexpression der inflammatorischen Caspase-11 und zur Freisetzung von IL-18 und IL-1β. Die IL-1β Freisetzung konnte über eine zusätzliche Stimulation mit dem DAMP ATP erhöht werden, war abhängig von der Caspase-Aktivität und einem Kaliumgradienten, und über den P2X7 Rezeptor reguliert. Die Aktivierung des Inflammasoms in DZ hatte keinen Einfluss auf die CD4+ T-Zell-Proliferationsrate, zeigte aber ein komplexes Spektrum induzierter Zytokine, wie TNF-α, IL-2, IL-4, IL-10, GM-CSF, IL-6, IL-5, M-CSF und IL-17, welches für eine komplexe Immunantwort in Abhängigkeit vom Infektionshintergrund spricht. Die Expression einer dominant-negativen Form des Adaptermoleküls ASC (dnASC), welche den inflammatorischen Signalweg blockieren sollte, führte zu einem massiven DZ-Sterben und ließ keine Rückschlüsse über die Rolle der inflammatorischen Caspasen und ihr intrazelluläres Netzwerk im Hinblick auf die inflammatorischen Zytokine zu. Die durch DZ-Reifung induzierte Hochregulation des Apoptose-Inhibitors cFLIP führt zu einer Resistenz gegenüber der CD95L-induzierten Apoptose durch die Inhibition der Caspase-8 im CD95 Rezeptorkomplex (DISC). Ein kompletter genetischer Verlust von cFLIP führte zu einem spontanen DZ-Zelltod, welcher bereits durch das Vorhandensein eines cFLIP-Allels verhindert werden konnte. Eine Verringerung der cFLIP Expression in DZ führte ebenso zu einer Verkürzung ihrer Lebensdauer, zeigte aber keinen spezifischen Einfluss auf die TZ-Proliferationsrate und das Überleben der DZ im Interaktionsmodell. 4 Zusammenfassung Diese Befunde erlauben einen tieferen Einblick in die fundamentalen Rollen von ASC und cFLIP in dendritischen Zellen und helfen möglicherweise bei der Weiterentwicklung der DZ-basierten Immuntherapie. 5 Content Content Summary 3 Zusammenfassung 4 Content 6 1. Introduction 11 1.1. The immune system 11 1.1.1. Physiological function of the immune system .............................................. 11 1.1.2. Function of dendritic cells ............................................................................ 12 1.1.3. The inflammasome ...................................................................................... 18 1.1.4. Processing and function of IL-1β, IL-18 and IL-33 ....................................... 21 1.1.5. Inhibition of the inflammasome by pathogens ............................................. 25 1.1.6. The role of the inflammasome in DCs ......................................................... 27 1.2. Role of cell death for regulation of DC immune response 28 1.2.1. The different faces of cell death................................................................... 28 1.2.2. The intrinsic cell death pathway................................................................... 29 1.2.3. CD95L-mediated apoptosis as an extrinsic cell death pathway ................... 30 1.2.4. Inhibition of CD95-mediated apoptosis by cFLIP ......................................... 32 1.2.5. Apoptosis in dendritic cells .......................................................................... 35 1.3. Aims 38 2. Material and Methods 40 2.1. Material 40 2.1.1. Chemicals .................................................................................................... 40 2.1.2. Enzymes and molecular biology reagents ...................................................... 41 2.1.3. Ready-made reaction systems (Kits) .............................................................. 42 2.1.4. Phosphatase and protease inhibitors.............................................................. 42 6 Content 2.1.5. Pharmacological stimulating substances ........................................................ 43 2.1.6. Stimulating Cytokines ..................................................................................... 43 2.1.7. Molecular weight markers ............................................................................... 44 2.1.8. Protein molecular weight markers................................................................... 44 2.1.9. Table 2: Primary antibodies for Western blot analysis .................................... 44 2.1.10. Table 3: Antibodies for FACS analysis ......................................................... 45 2.1.11. Vector backbones ......................................................................................... 46 2.1.12. Prokaryotic cells............................................................................................ 47 2.1.13. Eukaryotic cells ............................................................................................. 47 2.1.14. Animals ......................................................................................................... 48 2.1.15. Culture media for bacterial cells ................................................................... 48 2.1.16. Cell culture media and reagents for cells ...................................................... 49 2.1.17. Generally used buffers .................................................................................. 49 2.1.18. Buffer and reagents for protein
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