Cytosolic DNA sensing plays a key role in autoimmune and autoinflammatory diseases. STING is a cytosolic adaptor protein which upon activation leads to induction of type I interferons and inflammatory cytokines. Recently, gain-of-function mutations in STING have been identified in patients with an autoinflammatory disease called STING-associated vasculopathy with onset in infancy (SAVI). We compared two independent SAVI mutant mouse models and revealed a hierarchy of immune abnormalities which were dependent on SAVI mutation in lymphocytes. We also showed that bone marrow from the V154M mutant mice transfers disease to the wild-type host, whereas the N153S does not, indicating mutation-specific disease outcomes. Collectively, these mutant mice recapitulate disease features seen in SAVI patients and highlight mutation-specific functions of STING. Other autoimmune mouse models such as DNAseII and DNAseIII-deficient mice, that fail to degrade DNA result in activation of the cGAS STING pathway. Deficiency of this pathway in these mouse models ameliorates lethality. By contrast, we previously reported that STING potently suppresses inflammation in a pristane-induced model of autoimmunity. In this model, we show that both cGAS- and STING-deficient mice exhibit exacerbated disease phenotypes compared to controls. We report that STING constrained TLR activation, and thereby limited autoimmune manifestations. Consistent with this premise, cGAS or STING deficient mice that lack a common TLR chaperone UNC93b develop less severe systemic autoimmunity than cGAS or STING deficient mice that are UNC93b sufficient. Overall, this study demonstrates that STING activation constrains systemic autoimmune disease and has important implications for cGAS STING-directed therapies.

Background: Type I interferonopathies comprise a heterogenous and phenotypically diverse range of diseases, characterized by an elevated level of type I interferon (IFN) exhibited in patients accompanied by high interferon stimulated gene (ISG) scores. Type I interferonopathies are difficult to treat, especially in the acute phases of the disease, and typically chronic, requiring lifelong treatment and care. A patient, exhibiting symptoms of a type I interferonopathy was identified by whole exome sequencing to have a compound heterozygous mutation in the type III IFN receptor, IFNLR1. The compound mutation is comprised of two discrete truncating mutations, c.532_535dupCATG (p.G179AfsX37) and c.904dupG (p.V302GfsX30), termed mutant 1 or mutant 2 (M1, M2), respectively. The M1 and M2 IFNLR1 proteins were shown to be expressed, but were demonstrated to be non-functional. Hypothesis: Mutant isoforms of IFNLR1 interfere with the normal function of the closely related IL-10 family of cytokines and receptors through a shared β subunit receptor chain IL10RB. It is hypothesized that M1 and M2 IFNLR1 are able to impair correct IL-10 or IL-22 receptor formation by preventing coupling with IL10RB resulting in immune dysregulation. Materials and Methods: Multiple CRISPR-Cas9 tools were implemented to create several cell lines with genome edits up to a single base pair resolution to model deficiencies in each of the IFN receptors: IFNLR1, IFNGR1, and IFNAR1. The knock-out cell lines were used as models for the expression of M1 and M2 IFNLR1, IL10RA, and IL10RB, to study the relationship between the IFN-λ, IL-10 and IL-22 pathways. Stimulation of these model pathways and expression systems with IL-10, IL-22, and IFN-λ helped further our understanding between these proteins. The relationship between the IL-10 and IFN-λ pathways was further explored by stimulation of whole blood derived from the patient, parents, and controls which was conducted to further quantify the role IL-10 signalling played in the pathogenesis of the disease. Results: M1 was demonstrated to promote the spontaneous phosphorylation of STAT1, STAT2, and STAT3 independent of stimulation. This phosphorylation was independent of type I, type II, or type III IFN signalling, with phosphorylation persisting in knock-out lines of all IFN receptors singly or in combination. Consequently, the closely related IL-10 family of cytokines was examined for its role in the pathogenesis of the disease. M1 and M2 were demonstrated to interact with IL10RA and IL10RB on the protein level and were demonstrated to influence the phosphorylation of STAT3 by IL-10 or IL-22 stimulation. Further analysis of whole blood derived from the patient, parents and controls demonstrated a lack of IL-10-mediated regulation of IL-12 solely in the patient. Elevated basal and stimulatory levels of IL-18, CXCL10, and IFN-γ were also detected in the patient. Conclusions: The patient maintained IL-10 regulatory capacity in all but IL-12 signalling, which is a pathway known to be directly controlled by IL-10. IL-12 is mainly produced in cells like dendritic cells, which are one of the only cell types to naturally express IFNLR1, IL10RA and IL10RB. The loss of IL-12 regulation by IL-10 likely stems from interference by the M1 and M2 IFNLR1 present in patient dendritic cells, inhibiting proper formation of the IL-10 receptor and preventing its regulatory function. The elevated levels of IL-12 in conjunction with elevated IL-18 levels, which functions synergistically with IL-12 result in secretion of high levels of IFN-γ. IFN-γ likely participates in a positive feedback loop with CXCL10, resulting in prolonged and heightened immune response after immune challenge in the patient resulting in autoinflammation.:List of Tables v List of Figures vi List of Abbreviations ix 1 Introduction 1 1.1 CRISPR-Cas9-Mediated Editing 1 1.1.1 Guide Efficiency and Off-Target Prediction 11 1.2 Type I Interferonopathies 13 1.3 Pathogen Detection by the Innate Immune System 14 1.3.1 TLR Dependent Nucleic Acid Sensing 14 1.3.2 Non-TLR-Mediated Detection of Nucleic Acids 17 1.4 Interferon-Mediated Innate Immunity 19 2 Hypotheses and Goals 24 2.1 Hypotheses 24 2.2 Goals 24 3 Methods and Materials 26 3.1 Table of Materials and Software Used 26 3.2 Cell Culture 30 3.2.1 Adherent Cell Culture 31 3.2.2 Suspension Cell Culture 31 3.2.3 Cell Counting and Seeding 32 3.2.4 Cell Defrosting and Freezing 33 3.2.5 Cytokine Stimulation 34 3.2.6 Transfection 35 3.3 Target Prediction 37 3.4 CRISPR-Cas9 Cloning 39 3.4.1 Insert Generation, Plasmid Digestion and Ligation 40 3.4.2 Transformation and Clonal Selection 43 3.4.3 In-Fusion Cloning 44 3.4.4 DNA-Miniprep 44 3.4.5 DNA-Maxiprep 45 3.5 PCR 46 3.5.1 DNA Extraction 47 3.5.2 Amplification Reaction (Standard PCR) 48 3.5.3 PCR Clean 49 3.5.4 In Vitro sgRNA Synthesis 50 3.5.5 pegRNA Templates 52 3.6 Measurement of DNA and RNA Concentration 54 3.7 Agarose Gel Electrophoresis 54 3.7.1 Gel Preparation 54 3.7.2 Electrophoresis Parameters 55 3.7.3 Gel Extraction 55 3.8 Gene Editing 57 3.8.1 CRISPR-Cas9-Mediated Gene Editing 57 3.8.2 Cutting Assays 60 3.8.3 Isolation of Single Clones 62 3.9 Sanger Sequencing 64 3.10 Immunostaining 64 3.10.1 Protein Extraction and BCA Assay 64 3.10.2 Western Blotting 66 3.10.3 Co-Immunoprecipitation 69 3.11 Whole Blood Assays 71 3.11.1 Whole Blood Stimulation 71 3.11.2 Flow Cytometry 72 3.11.3 Statistical Analysis 73 4 Results 74 4.1 Implementation of CRISPR-Cas9 Techniques 74 4.2 In Vitro sgRNA Synthesis for CRISPR-Cas9 Editing 75 4.3 Validation of CRISPR-Cas9 Editing 75 4.4 Cas9 Editing 78 4.4.1 IFNLR1 Editing 78 4.4.2 IFNLR1 Rescue 80 4.4.3 IFNGR1 Knock-Outs 81 4.4.4 IFNAR1 Knock-Outs 82 4.4.5 Targeted Installation of Mutations 84 4.5 Investigation of a Type I Interferonopathy 88 4.5.1 Characterization of a Patient with Complete IFNLR1 Deficiency 88 4.5.2 Basal Level of pSTAT1 and pSTAT3 in Patient Cells 89 4.5.3 IFNLR1 Expression in Patient 91 4.5.4 Overexpression of IFNLR1 Isoforms 93 4.5.5 Spontaneous Induction of STAT1 Phosphorylation 94 4.5.6 Independence of Immune Activation from IFN Signalling Pathways 96 4.6 IL-10 and IL-22 Stimulations 98 4.6.1 IL-10 Stimulation 98 4.6.2 Interaction of IL10RA, IL10RB, and IFNLR1 Isoforms 102 4.6.3 IL-22 Stimulation 106 4.7 Whole Blood Assays 107 4.7.1 TNF-α 107 4.7.2 IFN-γ 108 4.7.3 IL-12 and IL-18 109 4.7.4 CXCL10 111 4.7.5 IL-10 112 5 Discussion 114 5.1 Cell Model Creation by CRISPR-Cas9 Techniques 114 5.1.1 Cutting Assays and CRISPR-Cas9 Validation 114 5.1.2 IFN Receptor Knock-outs 116 5.1.3 Base Editing and Prime Editing 117 5.2 Characterization of a Complete IFN-λ Receptor Deficiency 121 5.2.1 Establishing an IFNLR1 Overexpression System 121 5.2.2 IL-10 Family of Cytokines 124 5.3 Whole Blood Assays 129 6 Conclusions 133 7 Summary 135 8 Zusammenfassung 137 9 Scientific Output 139 10 Literature 140 11 Acknowledgements 155 12 Appendix 156 12.1.1 Supplementary Tables 156 12.1.2 Supplementary Figures 160 12.1.3 Declarations 161 / Hintergrund: Typ-I-Interferonopathien umfassen ein heterogenes und phänotypisch vielfältiges Spektrum von Krankheiten, die sich durch einen erhöhten Typ-I-Interferon (IFN)-Spiegel bei Patienten auszeichnen, der mit einer hohen Expression IFN-stimulierter Gene (ISG) einhergeht. Typ-I-Interferonopathien sind vor allem in den akuten Phasen der Krankheit oft schwer zu behandeln und verlaufen in der Regel chronisch, so dass eine lebenslange Behandlung erforderlich ist. Bei einer Patientin mit Symptomen einer Typ-I-Interferonopathie wurde durch eine Exom-Sequenzierung eine compound heterozygote Mutation im IFNLR1-Gen, das den Typ-III-IFN-Rezeptor bzw. INF-λ-Rezeptor kodiert, festgestellt. Dabei handelt es sich um zwei trunkierende Mutationen, c.532_535dupCATG (p.G179AfsX37) und c.904dupG (p.V302GfsX30), die als Mutante 1, beziehungsweise Mutante 2 (M1, M2) bezeichnet wurden. Es konnte gezeigt werden, dass die M1- und M2-IFNLR1-Proteine zwar exprimiert wurden, allerdings nicht funktionell waren. Hypothese: Mutierte Isoformen von IFNLR1 beeinträchtigen die normale Funktion der eng verwandten Interleukin-10-Familie von Zytokinen und Rezeptoren durch die gemeinsame β Untereinheit, der Rezeptorkette IL10RB. Es wird angenommen, dass M1 und M2 die korrekte IL-10 oder IL-22-Rezeptorbildung beeinträchtigen, indem sie die Kopplung mit IL10RB verhindern, was zu einer Dysregulation des Immunsystems führt. Material und Methode: Mittels verschiedener CRISPR-Cas9-Methoden, teilweise mit einer Genauigkeit bis zu einem Basenpaar, wurden mehrere Zelllinien mit editierten Gensequenzen erzeugt, um Funktionsverluste der verschiedenen IFN-Rezeptoren, IFNLR1, IFNGR1 und IFNAR1, zu modellieren. Die Knock-out-Zelllinien wurden dann als Modelle für die Expression von M1 und M2 IFNLR1 sowie IL10RA und IL10RB verwendet, um die Beziehung zwischen den IFN-λ, IL-10 und IL 22-Signalwegen zu untersuchen. Die Untersuchung dieser modellierten Expressionssysteme mit IL-10, IL 22 und IFN-λ trug zu einem besseren Verständnis der Beziehungen zwischen diesen Proteinen bei. Die Beziehung zwischen den IL-10 und IFN λ Signalwegen wurde durch die Stimulierung von Blutproben der Patientin, deren Eltern, sowie von Kontrollpersonen weiter untersucht, um die Rolle der IL-10-Signalübertragung bei der Pathogenese der Autoinflammation weiter zu chrakterisieren. Ergebnisse: Es wurde gezeigt, dass M1 zu einer spontanen, stimulationsunabhängigen Phosphorylierung von STAT1, STAT2 und STAT3 führt. Diese Phosphorylierung zeigte sich unabhängig vom Typ-I-, Typ-II- oder Typ III IFN Signalweg, wobei die Phosphorylierung in Knock-out-Zelllinien aller IFN Rezeptoren, einzeln oder in Kombination, bestehen blieb. Daraufhin wurde die eng verwandte IL-10-Familie von Zytokinen auf ihre Rolle bei der Pathogenese der Krankheit untersucht. Es wurde nachgewiesen, dass M1 und M2 mit IL10RA und IL10RB auf Proteinebene interagieren und die Phosphorylierung von STAT3 durch IL-10 oder IL-22 Stimulation beeinflussen. Weitere Analysen des Vollbluts der Patientin, der Eltern und von Kontrollpersonen zeigten, dass IL-10 die Regulation von IL-12 einzig bei der Patientin nicht beeinflusste. Ebenfalls wurden bei der Patientin erhöhte Basal- und Stimulationswerte von IL 18, CXCL10 und IFN-γ festgestellt. Schlussfolgerungen: Die Patientin behielt die regulierende Funktion von IL-10 in allen Bereichen bei, mit Ausnahme des IL-12-Signalwegs, von dem bekannt ist, dass er direkt durch IL-10 kontrolliert wird. IL-12 wird hauptsächlich in dendritischen Zellen produziert, die zu den einzigen Zelltypen gehören, die natürlicherweise IFNLR1, IL10RA und IL10RB exprimieren. Der Verlust der IL 12 Regulierung durch IL-10 ist wahrscheinlich auf eine Störung durch die in den dendritischen Zellen der Patientin vorhandenen M1- und M2-IFNLR1-Mutationen zurückzuführen, die die ordnungsgemäße Bildung des IL-10-Rezeptors hemmen und seine Regulierungsfunktion verhindern. Die erhöhten IL-12-Spiegel in Verbindung mit erhöhten IL-18-Spiegeln, die synergistisch mit IL-12 wirken, führen zu einer hohen Sekretion von IFN-γ. ermutlich ist IFN γ an einer positiven Rückkopplungsschleife mit CXCL10 beteiligt, die nach einer Stimulierung des Immunsystems zu einer verlängerten und verstärkten Immunantwort bei der Patientin führt, die wiederum in einer Autoinflammation resultiert.:List of Tables v List of Figures vi List of Abbreviations ix 1 Introduction 1 1.1 CRISPR-Cas9-Mediated Editing 1 1.1.1 Guide Efficiency and Off-Target Prediction 11 1.2 Type I Interferonopathies 13 1.3 Pathogen Detection by the Innate Immune System 14 1.3.1 TLR Dependent Nucleic Acid Sensing 14 1.3.2 Non-TLR-Mediated Detection of Nucleic Acids 17 1.4 Interferon-Mediated Innate Immunity 19 2 Hypotheses and Goals 24 2.1 Hypotheses 24 2.2 Goals 24 3 Methods and Materials 26 3.1 Table of Materials and Software Used 26 3.2 Cell Culture 30 3.2.1 Adherent Cell Culture 31 3.2.2 Suspension Cell Culture 31 3.2.3 Cell Counting and Seeding 32 3.2.4 Cell Defrosting and Freezing 33 3.2.5 Cytokine Stimulation 34 3.2.6 Transfection 35 3.3 Target Prediction 37 3.4 CRISPR-Cas9 Cloning 39 3.4.1 Insert Generation, Plasmid Digestion and Ligation 40 3.4.2 Transformation and Clonal Selection 43 3.4.3 In-Fusion Cloning 44 3.4.4 DNA-Miniprep 44 3.4.5 DNA-Maxiprep 45 3.5 PCR 46 3.5.1 DNA Extraction 47 3.5.2 Amplification Reaction (Standard PCR) 48 3.5.3 PCR Clean 49 3.5.4 In Vitro sgRNA Synthesis 50 3.5.5 pegRNA Templates 52 3.6 Measurement of DNA and RNA Concentration 54 3.7 Agarose Gel Electrophoresis 54 3.7.1 Gel Preparation 54 3.7.2 Electrophoresis Parameters 55 3.7.3 Gel Extraction 55 3.8 Gene Editing 57 3.8.1 CRISPR-Cas9-Mediated Gene Editing 57 3.8.2 Cutting Assays 60 3.8.3 Isolation of Single Clones 62 3.9 Sanger Sequencing 64 3.10 Immunostaining 64 3.10.1 Protein Extraction and BCA Assay 64 3.10.2 Western Blotting 66 3.10.3 Co-Immunoprecipitation 69 3.11 Whole Blood Assays 71 3.11.1 Whole Blood Stimulation 71 3.11.2 Flow Cytometry 72 3.11.3 Statistical Analysis 73 4 Results 74 4.1 Implementation of CRISPR-Cas9 Techniques 74 4.2 In Vitro sgRNA Synthesis for CRISPR-Cas9 Editing 75 4.3 Validation of CRISPR-Cas9 Editing 75 4.4 Cas9 Editing 78 4.4.1 IFNLR1 Editing 78 4.4.2 IFNLR1 Rescue 80 4.4.3 IFNGR1 Knock-Outs 81 4.4.4 IFNAR1 Knock-Outs 82 4.4.5 Targeted Installation of Mutations 84 4.5 Investigation of a Type I Interferonopathy 88 4.5.1 Characterization of a Patient with Complete IFNLR1 Deficiency 88 4.5.2 Basal Level of pSTAT1 and pSTAT3 in Patient Cells 89 4.5.3 IFNLR1 Expression in Patient 91 4.5.4 Overexpression of IFNLR1 Isoforms 93 4.5.5 Spontaneous Induction of STAT1 Phosphorylation 94 4.5.6 Independence of Immune Activation from IFN Signalling Pathways 96 4.6 IL-10 and IL-22 Stimulations 98 4.6.1 IL-10 Stimulation 98 4.6.2 Interaction of IL10RA, IL10RB, and IFNLR1 Isoforms 102 4.6.3 IL-22 Stimulation 106 4.7 Whole Blood Assays 107 4.7.1 TNF-α 107 4.7.2 IFN-γ 108 4.7.3 IL-12 and IL-18 109 4.7.4 CXCL10 111 4.7.5 IL-10 112 5 Discussion 114 5.1 Cell Model Creation by CRISPR-Cas9 Techniques 114 5.1.1 Cutting Assays and CRISPR-Cas9 Validation 114 5.1.2 IFN Receptor Knock-outs 116 5.1.3 Base Editing and Prime Editing 117 5.2 Characterization of a Complete IFN-λ Receptor Deficiency 121 5.2.1 Establishing an IFNLR1 Overexpression System 121 5.2.2 IL-10 Family of Cytokines 124 5.3 Whole Blood Assays 129 6 Conclusions 133 7 Summary 135 8 Zusammenfassung 137 9 Scientific Output 139 10 Literature 140 11 Acknowledgements 155 12 Appendix 156 12.1.1 Supplementary Tables 156 12.1.2 Supplementary Figures 160 12.1.3 Declarations 161