Dendritic cells (DCs) are professional antigen-presenting cells, which display an extraordinary capacity to induce T-cell responses. Recent findings revealed that DCs also play a crucial role in the activation of natural killer (NK) cells representing important effectors in the innate immune defense against viruses and tumors. Here, we summarize various studies investigating the bidirectional crosstalk between human DCs and NK cells. In this context, it has been reported that DCs efficiently enhance CD69 expression, proliferation, interferon (IFN)-γ secretion and cytotoxic activity of NK cells. Cell membrane-associated molecules as well as soluble factors such as interleukin-12, tumor necrosis factor-α and type I IFNs contributed to DC-mediated NK cell activation. Reciprocally, the ability of human NK cells to enhance the immunostimulatory capacity of DCs was shown. Thus, NK cells promoted the maturation of DCs and markedly augmented their capacity to produce proinflammatory cytokines and to stimulate T-cell responses. The NK cell-mediated effects on DCs were dependent on cell membrane-associated molecules such as NKp30 and soluble factors such as tumor necrosis factor-α and IFN-γ. In conclusion, the reciprocal activating interaction between human DCs and NK cells may play a pivotal role in the immune defense against viruses and tumors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Macrophages are innate immune cells responsible for engulfing microbes and cell debris through phagocytosis and orchestrating immune responses to maintain homeostasis. While conducting immune surveillance over all types of organs and tissues, macrophages face inherently heterogeneous microenvironments with unique biophysical features. For instance, microglia residing in the brain, Kupffer cells living in the skin and bone osteoclasts are exposed to very distinct tissue stiffnesses. Despite the research done in the last decade clearly indicates that macrophages are sensitive to physical factors, how mechanical cues modulate their inflammatory response remains poorly understood. The present study aims at investigating how microenvironment stiffness influences the pro-inflammatory behaviour of macrophages. Besides characterising the regulatory effect on pro-inflammatory gene expression and cytokine production, this work examines the impact of stiffness on the inflammasome, one of the main macrophage signalling platforms. For this, an in vitro system based in 2D polyacrylamide hydrogels whose stiffness can be independently tuned was established. Using substrates with an elastic moduli between 0.2 and 33.1 kPa, bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff polyacrylamide. Upon priming with lipopolysaccharide, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels, yet there were no significant differences in the expression of other major pro-inflammatory genes. Additionally stimulating macrophages with the ionophore nigericin revealed higher secreted protein levels of IL-1β and IL-6 on compliant substrates. Interestingly, macrophages challenged on compliant polyacrylamide also displayed an enhanced formation of the NLRP3 inflammasome as well as increased levels of pyroptotic cell death. The upregulation of inflammasome assembly on compliant hydrogels was not primarily attributed to the reduced cell spreading, since spatially confining cells on micropatterns led to a decrease of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, these results show that substrate stiffness affects the pro-inflammatory response of macrophages and for the first time describe that the NLRP3 inflammasome is one of the signalling components affected by stiffness mechanosensing. The work presented here expands our understanding of how microenvironment stiffness affects macrophage behaviour and which elements of their machinery might contribute to integrate mechanical cues into the regulation of their inflammatory functions. The onset of pathological processes or the implant of foreign bodies represent immune challenges in which macrophages can face a mechanically changing environment. Therefore, a better insight on how macrophages detect and process biophysical signals could potentially provide a basis for new strategies to modulate inflammatory responses.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS / Als Teil des angeborenen Immunsystems sind Makrophagen dafür verantwortlich Pathogene und Zellrückstände durch Phagozytose zu beseitigen. Sie orchestrieren Immunantworten um homöostatische Bedingungen von Organen und Geweben aufrechtzuerhalten. Dabei sind sie extrem heterogenen Mikroumgebungen ausgesetzt, welche sich jeweils durch eine einzigartige Kombination von (bio)chemischen und mechanischen Eigenschaften, vor allem Gewebesteifigkeiten, auszeichnen. Dies veranschaulichen beispielsweise im Gehirn residierende Mikroglia, Kupffer-Zellen in der Haut und Osteoklasten in Knochen. Obwohl diverse Studien aus dem letzten Jahrzehnt eindeutig zeigen, dass Makrophagen auf mechanische Signale reagieren, ist der zugrunde liegende Mechanismus, wie diese Signale eine Entzündungsreaktion modulieren, noch immer unzureichend verstanden. Die vorliegende Studie beinhaltet die systematische Untersuchung, wie die Steifigkeit der Mikroumgebung das proinflammatorische Verhalten von Makrophagen beeinflusst. Neben der Charakterisierung der regulatorischen Wirkung auf die proinflammatorische Genexpression und Zytokinproduktion untersucht diese Arbeit auch den Einfluss der Steifigkeit auf das Inflammasom; eine der wichtigsten Signalplattformen für Makrophagen. Zu diesem Zweck wurde zunächst ein Zellkultursystem mit 2D-Polyacrylamid-Hydrogelen als Zellsubstrat entwickelt, bei dem das Elastizitätsmodul der Gelsubstrate gezielt eingestellt werden kann. Unter Verwendung von Substraten mit einem Elastizitätsmodul zwischen 0,2 kPa und 33,1 kPa zeigt die mikroskopische Analyse, dass aus Knochenmark stammende Makrophagen im Vergleich zu steifem Polyacrylamid eine weniger ausgebreitete und rundere Morphologie annehmen. Nach dem Primen mit Lipopolysaccharid waren die Expressionsniveaus des Gens, das für TNF-α kodiert, auf deformierbareren Hydrogelen höher, jedoch gab es keine signifikanten Unterschiede in der Expression anderer wichtiger pro-inflammatorischer Gene. Eine zusätzliche Stimulierung von Makrophagen mit dem Ionophor Nigericin bewirkte höhere sekretierte Proteinspiegel von IL-1β und IL-6 auf deformierbaren Substraten. Makrophagen, die deformierbarem Polyacrylamid ausgesetzt waren, zeigten auch eine verstärkte Bildung des NLRP3-Inflammasoms sowie ein erhöhtes Ausmaß an pyroptotischem Zelltod. Die Hochregulierung der Inflammasom-Assemblierung auf deformierbaren Hydrogelen wurde nicht primär auf die reduzierte Zellausbreitung zurückgeführt, da räumlich begrenzte Zellen auf Mikromustern zu einer Abnahme von Inflammasom-positiven Zellen im Vergleich zu stark ausgebreiteten Zellen führten. Schließlich verringerte eine Störung der Aktomyosin-Kontraktilität die Unterschiede in der Inflammasombildung zwischen deformierbaren und steifen Substraten. Zusammenfassend zeigen diese Ergebnisse, dass die Substratsteifigkeit die proinflammatorische Reaktion von Makrophagen beeinflusst und beschreiben erstmalig, dass das NLRP3-Inflammasom eine der Signalkomponenten ist, die von der zellulären Steifheitswahrnehmung beeinflusst werden. Die hier vorgestellte Arbeit erweitert unser Verständnis davon, wie die Steifigkeit der Mikroumgebung das Verhalten von Makrophagen beeinflusst und welche Elemente ihrer Maschinerie dazu beitragen könnten mechanische Signale in die Regulierung ihrer Entzündungsfunktionen zu integrieren. Das Einsetzen pathologischer Prozesse oder die Implantation von Fremdkörpern stellen Immunherausforderungen dar, bei denen Makrophagen einer sich mechanisch verändernden Umgebung ausgesetzt sein können. Daher könnte ein besserer Einblick in die Art und Weise, wie Makrophagen biophysikalische Signale erkennen und verarbeiten, möglicherweise eine Grundlage für neue Strategien zur Modulation von Entzündungsreaktionen bieten.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS

Hematopoiesis in the adult organism is maintained by a complex, hierarchically organized cascade of differentiating cells that ultimately originate from adult hematopoietic stem cells (HSCs). HSCs have been extensively studied in perturbative settings (e.g transplantation assays), which are well suited for exploring cell potential but tell very little about cell behaviour in a physiological state. Novel \textit{in situ} techniques have made it possible to observe hematopoiesis in its natural environment, yet many questions about native HSC behaviour remain controversial. It is yet unclear whether and to what extent HSCs contribute to steady-state blood production, and whether they represent a reserve population that can be activated upon emergency. As, to date, common definitions of HSCs comprise cells of heterogeneous function, incoherent results are likely linked by another fundamental question: what is the identity of the HSC population residing on the apex of the hematopoietic hierarchy? To answer these questions is essential to gain a deeper understanding of fatal human diseases like leukemia or aplastic anemia. We describe an integrative approach combining non-invasive experimental methods with mathematical inference to uncover underlying pathways and differentiation patterns of steady state hematopoiesis. By this, we identify a population of Sca-1\textsuperscript{hi} CD201(EPCR)\textsuperscript{hi} HSCs as the hithertho elusive apex population, and show that they contribute marginally, but continuously, to native steady-state hematopoiesis. Further, we clarify the architecture of a shortcut route of thrombopoiesis, which emanates directly from HSCs and links them trough a previously undefined CD48\textsuperscript{-/low} megakaryocyte progenitor population directly to platelets. Finally, we provide an extensive analysis of the dynamic label propagation and proliferation behaviour of hematopoietic stem and progenitor cells in prototypical stress situations mimicking blood loss, infection and inflammation. We find that apex stem cells do not directly contribute to emergency hematopoiesis and are only activated upon severe myeloablation and chronic inflammation. Further, innate immune training did not influence HSC contribution in response to reoccurring stimulation. In sum, we demonstrate that primitive HSCs do neither represent a major contributor to steady-state blood production, nor a reservoir for emergency supply. We thus question current dogma of hematopoiesis, and argue that the primary function of HSCs in the adult organism remains yet to be discovered.:Contents Abstract iv Zusammenfassung vii List of Figures xii List of Tables xiii List of Abbreviations xv 1 Introduction 1 1.1 Hematopoiesis and the hematopoietic system 1 1.1.1 Developmental origins 1 1.1.2 The hematopoietic hierarchy 3 1.2 Hematopoietic stem cells 5 1.2.1 Characteristics of hematopoietic stem cells 7 1.2.2 Identification of hematopoietic stem and progenitor cells 7 1.2.3 HSC heterogeneity 11 1.3 Novel genetic tools to analyse native hematopoiesis 12 1.3.1 Conditional gene targeting 12 1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15 1.4.1 Models of native Hematopoiesis 16 1.4.2 Models of HSC quiescence 23 1.4.3 Aged hematopoiesis 25 1.5 Hematopoietic stem cell behaviour in stress response 26 1.5.1 Inflammation and Infection 27 1.5.2 Myeloablation 30 1.5.3 Innate immune training 32 2 Aim of Thesis 35 3 Material and Methods 37 3.1 Materials 37 3.1.1 Antibodies 37 3.1.2 Buffer and Solutions 38 3.1.3 Chemicals and Reagents 39 3.1.4 Cultivation Media 40 3.1.5 Kit Systems 41 3.1.6 Software 41 3.2 Mice 42 3.2.1 Animal housing and husbandry 42 3.2.2 Mouse strains 42 3.3 Genotyping of mice 43 3.4 Treatments of mice 46 3.4.1 Reporter induction 46 3.4.2 Hematopoietic stress induction 46 3.5 Hemograms 48 3.6 Transplantation and in vivo repopulation assays 49 3.6.1 Preconditioning and transplantation of recipients 49 3.6.2 Competitive repopulation assay 50 3.6.3 Limiting dilution assay 50 3.6.4 Transplantation into sublethally irradiated hosts 51 3.7 Cell preparation for transplantation and flow cytometry 51 3.7.1 Bone marrow 51 3.7.2 Splenocytes 51 3.7.3 Peripheral blood 51 3.7.4 Peritoneal cells 52 3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52 3.8 Flow Cytometry 53 3.8.1 Cell staining 54 3.8.2 Cell counting 55 3.8.3 Cell identification and gating 55 3.8.4 Chimerism analysis 57 3.8.5 Cell sorting 58 3.9 Cytokine detection assay 58 3.10 in vitro single cell expansion assay 59 3.11 Sequencing 61 3.11.1 Single cell RNA sequencing 61 3.11.2 Bulk RNA sequencing 62 3.11.3 Single cell and bulk transcriptome analysis 62 3.12 Mathematical modelling 62 3.13 Data Normalization 63 3.14 Statistical Analysis 64 4 Results 67 4.1 Identification of an apex HSC population 67 4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68 4.1.2 ES HSCs reside at the top of the hierarchy 71 4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76 4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77 4.2.2 The myeloid lineage diverges within phenotypic HSCs 80 4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82 4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82 4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88 4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92 4.4 Ageing modulates the dynamics of fate mapping 95 4.5 HSCs in times of crisis: Contribution to stress recovery 99 4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100 4.5.2 Severe myeloablation provokes HSC activation 105 4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107 4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113 4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118 4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120 5 Discussion 123 5.1 Labels matter: What is a true HSC? 123 5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124 5.1.2 ES HSCs reside at the top of the hierarchy 126 5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127 5.1.4 The myeloid lineage diverges within phenotypic HSCs 130 5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132 5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132 5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135 5.3 Fundamental dynamics of label propagation are preserved in aged mice 136 5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138 5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139 5.4.2 Severe myeloablation provokes HSC activation 143 5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145 5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149 5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150 6 Conclusion 153 References 155 Acknowledgements 185 Appendices 187 Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189 Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190 / Die Blutbildung in erwachsenen Organismen wird durch eine komplexe, hierarchisch organisierte Kaskade von sich fortwährend differenzierenden Zellen aufrechterhalten, und hat ihren Ursprung in adulten h\'amatopoietischen Stammzellen (HSZs). HSZs wurden ausgiebig unter unphysiologischen Extrembedingungen (z.B. Transplantation) untersucht, in welchen sich viel über das maximale potential einer Zelle, aber wenig über ihr Verhalten in ungestörtem Zustand sagen lässt. Obwohl durch moderne \textit-Techniken mittlerweile h\'amatopoietische Differenzierung in ihrer ursprünglichen Umgebung beobachtet werden kann, bleiben etliche Fragen zum nativen Verhalten von HSZs offen. So ist es noch unklar, ob und in welchem Ausmaß HSZs überhaupt zur stationären Blutbildung beitragen, oder ob sie eine Reservepopulation darstellen, die im Notfall aktiviert werden kann. Da bis dato gebr\'auchliche Definitionen von HSZs verschiedene Sub-Populationen mit heterogener Funktionalität umfassen, sind die inkohärenten Ergebnisse wahrscheinlich mit einer anderen grundlegenden Frage verknüpft: Was ist die wahre Identit\'at der HSZ-Population, die an der Spitze der h\'amatopoetischen Hierarchie steht? Die Beantwortung dieser Fragen ist essenziell für ein besseres Verst\'andnis von fatalen humanen Krankheiten, wie beispielsweise Leuk\'amie oder aplastische An\'amie. Wir beschreiben einen integrativen Ansatz, der nicht-invasive experimentelle Methoden mit mathematischer Modellierung vereint, um die zugrundeliegenden Pfade und Differenzierungsmuster der station\'arer H\'amatopoese zu ergründen. Auf diese Weise können wir eine Population von Sca-1\textsuperscript{hi} und CD201(EPCR)\textsuperscript{hi} HSZs als die bisher unbekannte Apex-Population identifizieren, und zeigen, dass diese Zellen geringf\'ugig, aber kontinuierlich, zur nativen H\'amatopoese beitragen. Darüber hinaus klären wir die Architektur eines direkten thrombozytischen Differenzierungspfades, welcher unmittelbar von HSZs abzweigt und diese über eine neu definierte CD48\textsuperscript{-/lo} Megakaryozyten-Vorläuferpopulation direkt mit Thrombozyten ver- bindet. Schließlich liefern wir eine umfassende Analyse des dynamischen Differenzierungs- und Proliferationsverhaltens von hämatopoetischen Stamm- und Vorläuferzellen in prototypischen Stresssituationen, die Blutverlust, Infektion und Entzündung nachahmen. Wir zeigen, dass primitive Stammzellen nicht direkt zur Notfall-Hämatopoese beitragen und nur bei schwerer Myeloablation und chronischer Entzündung aktiviert werden. Ebenfalls hat das Training des angeborenen Immunsystems keinen Einfluss auf die Aktivität von HSZs bei wiederholter Stimulation. Unsere Ergebnisse belegen, dass primitive HSZs weder einen wesentlichen Beitrag zur stationären Blutbildung leisten, noch ein Reservoir für die Notfallversorgung darstellen. Wir stellen somit das derzeitige Dogma der Hämatopoese in Frage und argumentieren, dass die primäre Funktion der HSZ im erwachsenen Organismus noch zu entdecken ist.:Contents Abstract iv Zusammenfassung vii List of Figures xii List of Tables xiii List of Abbreviations xv 1 Introduction 1 1.1 Hematopoiesis and the hematopoietic system 1 1.1.1 Developmental origins 1 1.1.2 The hematopoietic hierarchy 3 1.2 Hematopoietic stem cells 5 1.2.1 Characteristics of hematopoietic stem cells 7 1.2.2 Identification of hematopoietic stem and progenitor cells 7 1.2.3 HSC heterogeneity 11 1.3 Novel genetic tools to analyse native hematopoiesis 12 1.3.1 Conditional gene targeting 12 1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15 1.4.1 Models of native Hematopoiesis 16 1.4.2 Models of HSC quiescence 23 1.4.3 Aged hematopoiesis 25 1.5 Hematopoietic stem cell behaviour in stress response 26 1.5.1 Inflammation and Infection 27 1.5.2 Myeloablation 30 1.5.3 Innate immune training 32 2 Aim of Thesis 35 3 Material and Methods 37 3.1 Materials 37 3.1.1 Antibodies 37 3.1.2 Buffer and Solutions 38 3.1.3 Chemicals and Reagents 39 3.1.4 Cultivation Media 40 3.1.5 Kit Systems 41 3.1.6 Software 41 3.2 Mice 42 3.2.1 Animal housing and husbandry 42 3.2.2 Mouse strains 42 3.3 Genotyping of mice 43 3.4 Treatments of mice 46 3.4.1 Reporter induction 46 3.4.2 Hematopoietic stress induction 46 3.5 Hemograms 48 3.6 Transplantation and in vivo repopulation assays 49 3.6.1 Preconditioning and transplantation of recipients 49 3.6.2 Competitive repopulation assay 50 3.6.3 Limiting dilution assay 50 3.6.4 Transplantation into sublethally irradiated hosts 51 3.7 Cell preparation for transplantation and flow cytometry 51 3.7.1 Bone marrow 51 3.7.2 Splenocytes 51 3.7.3 Peripheral blood 51 3.7.4 Peritoneal cells 52 3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52 3.8 Flow Cytometry 53 3.8.1 Cell staining 54 3.8.2 Cell counting 55 3.8.3 Cell identification and gating 55 3.8.4 Chimerism analysis 57 3.8.5 Cell sorting 58 3.9 Cytokine detection assay 58 3.10 in vitro single cell expansion assay 59 3.11 Sequencing 61 3.11.1 Single cell RNA sequencing 61 3.11.2 Bulk RNA sequencing 62 3.11.3 Single cell and bulk transcriptome analysis 62 3.12 Mathematical modelling 62 3.13 Data Normalization 63 3.14 Statistical Analysis 64 4 Results 67 4.1 Identification of an apex HSC population 67 4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68 4.1.2 ES HSCs reside at the top of the hierarchy 71 4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76 4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77 4.2.2 The myeloid lineage diverges within phenotypic HSCs 80 4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82 4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82 4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88 4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92 4.4 Ageing modulates the dynamics of fate mapping 95 4.5 HSCs in times of crisis: Contribution to stress recovery 99 4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100 4.5.2 Severe myeloablation provokes HSC activation 105 4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107 4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113 4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118 4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120 5 Discussion 123 5.1 Labels matter: What is a true HSC? 123 5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124 5.1.2 ES HSCs reside at the top of the hierarchy 126 5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127 5.1.4 The myeloid lineage diverges within phenotypic HSCs 130 5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132 5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132 5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135 5.3 Fundamental dynamics of label propagation are preserved in aged mice 136 5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138 5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139 5.4.2 Severe myeloablation provokes HSC activation 143 5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145 5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149 5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150 6 Conclusion 153 References 155 Acknowledgements 185 Appendices 187 Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189 Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190

No description available.

No description available.

Background Growing evidence emphasizes the relevance of sphingolipids for metabolism and immunity of antigen-presenting cells (APC). APCs are key players in balancing tolerogenic and encephalitogenic responses in immunology. In contrast to the well-known prominent effects of sphingosine-1-phosphate (S1P) on lymphocyte trafficking, modulatory effects on APCs have not been fully characterized. Methods Frequencies and activation profiles of dendritic cell (DC) subtypes, monocytes, and T cell subsets in 35 multiple sclerosis (MS) patients were evaluated prior and after undergoing fingolimod treatment for up to 24 months. Impact of fingolimod and S1P on maturation and activation profile, pro-inflammatory cytokine release, and phagocytotic capacity was assessed in vitro and ex vivo. Modulation of DC-dependent programming of naïve CD4+ T cells, as well as CD4+ and CD8+ T cell proliferation, was also investigated in vitro and ex vivo. Results Fingolimod increased peripheral slanDC count—CD1+ DC, and monocyte frequencies remained stable. While CD4+ T cell count decreased, ratio of Treg/Th17 significantly increased in fingolimod-treated patients over time. CD83, CD150, and HLADR were all inhibited, but CD86 was upregulated in DCs after incubation in the presence of fingolimod. Fingolimod but not S1P was associated with reduced release of pro-inflammatory cytokines from DCs and monocytes in vitro and ex vivo. Fingolimod also inhibited phagocytic capacity of slanDCs and monocytes. After fingolimod, slanDCs demonstrated reduced potential to induce interferon–gamma-expressing Th1 or IL-17-expressing Th17 cells and DC-dependent T cell proliferation in vitro and in fingolimod-treated patients. Conclusions We present the first evidence that S1P-directed therapies can act additionally as immunomodulators that decrease the pro-inflammatory capabilities of APCs, which is a crucial element in DC-dependent T cell activation and programming.

Background Growing evidence emphasizes the relevance of sphingolipids for metabolism and immunity of antigen-presenting cells (APC). APCs are key players in balancing tolerogenic and encephalitogenic responses in immunology. In contrast to the well-known prominent effects of sphingosine-1-phosphate (S1P) on lymphocyte trafficking, modulatory effects on APCs have not been fully characterized. Methods Frequencies and activation profiles of dendritic cell (DC) subtypes, monocytes, and T cell subsets in 35 multiple sclerosis (MS) patients were evaluated prior and after undergoing fingolimod treatment for up to 24 months. Impact of fingolimod and S1P on maturation and activation profile, pro-inflammatory cytokine release, and phagocytotic capacity was assessed in vitro and ex vivo. Modulation of DC-dependent programming of naïve CD4+ T cells, as well as CD4+ and CD8+ T cell proliferation, was also investigated in vitro and ex vivo. Results Fingolimod increased peripheral slanDC count—CD1+ DC, and monocyte frequencies remained stable. While CD4+ T cell count decreased, ratio of Treg/Th17 significantly increased in fingolimod-treated patients over time. CD83, CD150, and HLADR were all inhibited, but CD86 was upregulated in DCs after incubation in the presence of fingolimod. Fingolimod but not S1P was associated with reduced release of pro-inflammatory cytokines from DCs and monocytes in vitro and ex vivo. Fingolimod also inhibited phagocytic capacity of slanDCs and monocytes. After fingolimod, slanDCs demonstrated reduced potential to induce interferon–gamma-expressing Th1 or IL-17-expressing Th17 cells and DC-dependent T cell proliferation in vitro and in fingolimod-treated patients. Conclusions We present the first evidence that S1P-directed therapies can act additionally as immunomodulators that decrease the pro-inflammatory capabilities of APCs, which is a crucial element in DC-dependent T cell activation and programming.

Transaldolase-Mangel (TALDO) ist ein extrem seltener, angeborener Stoffwechseldefekt, von dem weltweit nur 34 Fälle bekannt sind. Der Defekt geht auf den Verlust des Enzyms Transaldolase 1 aus dem nicht-oxidativen Pentosephosphat-Weg (nicht-oxPPW) zurück und äußert sich in einem weiten Spektrum klinischer Symptome. Die schwerwiegendsten Folgen sind Leber- und Nierenmangelfunktionen, die zum sehr frühen Tod führen können. Desweiteren leiden 15 % der Patienten an wiederkehrenden Infektionen. Neutrophile Granulozyten (Neutrophile) sind die häufigsten weißen Blutkörperchen im Menschen und essentiell für die angeborene Immunantwort gegen Infektionserreger. Ich habe hier funktionale Aspekte von TALDO-Neutrophilen untersucht. Der oxidative Pentosephosphat-Weg (oxPPW) stellt das Reduktionsäquivalent NADPH bereit, welches indirekt für die Entstehung von reactive oxygen species (ROS)-abhängigen Neutrophil Extracellular Traps (NETs) verantwortlich ist. Der Beitrag des nicht-oxPPW zur ROS-abhängigen NET-Bildung ist bislang nicht bekannt. In dieser Arbeit konnte ich für Neutrophile aus drei TALDO-Patienten eine jeweils komplett abwesende Entstehung ROS-abhängiger NETs und einen deutlich verringerten oxidativen Burst nach PMA-Stimulation zeigen. Um diese Beobachtungen in einem unabhängigen Modelsystem zu bestätigen, habe ich mit Hilfe des CRISPR-Cas9-Systems, ‚knock-out‘ Mutanten von Transaldolase 1 und dessen Partnerenzym Transketolase in der Neutrophil-ähnlichen Zelllinie PLB-985 hergestellt. Die dergestalt genetisch manipulierten Zellen waren nicht mehr zu PMA-induziertem Zelltod in der Lage. Dies ist somit der erste genetische Beweis für die Abhängigkeit des oxidativen Burst und der Bildung von NETs vom nicht-oxPPW. Diese Erkenntnis trägt zum einen zum mechanistischen Verständnis der NET-Entstehung bei und liefert zum anderen eine potentielle Erklärung für einige der bei TALDO beobachteten Symptome. Desweiteren wurden einige der metabolischen Erfordernisse für die Bildung von NETs mit Hilfe von Inhibitoren untersucht. Die erhaltenen Erkenntnisse zeigen, dass das initiale Maximum des oxidativen Bursts für NET-Bildung unerheblich ist und vielmehr die ROS-Generierung nach ca. 50 Minuten entscheidende Bedeutung für diese hat. / Transdaldolase 1-deficiency (TALDO) is a rare genetic disease with only 34 described cases globally. Transaldolase 1 is part of the non-oxidative pentose phosphate pathway (PPP) and its deficiency results in many clinical symptoms including kidney and liver failure, which can lead to early child-mortality. Some of these patients suffer from recurrent infections, for example in the respiratory tract. Neutrophils are the most abundant white blood cells and essential for the innate immune defence against bacterial and fungal pathogens. The PPP generates reduced NADPH that is crucial for the generation of superoxide by the NADPH oxidase NOX2. In turn, NOX2 is essential for neutrophil extracellular trap (NET) formation. NETs occur through the neutrophil-specific cell death netosis and consist of chromatin decorated with granular proteins. Here I report that neutrophils of three TALDO patients did not make NETs. Deletion of transaldolase 1, and its partner enzyme transketolase, in the neutrophil-like PLB-985 cell line reduced ROS generation and cell death. This confirms that transaldolase 1 is required for NET formation. We present, to the best of our knowledge, the first genetic evidence that the non-oxidative PPP is required for ROS generation and NET formation. Furthermore, some of the metabolic requirements for NET formation were assessed. The obtained data indicate that the initial peak of the oxidative burst is irrelevant for NET formation but the ROS generation after 50 minutes on the contrary has crucial significance.