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

Mast cells are resident immune cells abundantly found in the tissue at the host-environment interface, where they play a critical role in inflammatory allergic responses. Mast cell responses may be regulated by the cytokine milieu at the site of inflammation. Recent studies have revealed microRNAs to be important in altering cytokine signaling in immune cells. Here, we demonstrate for the first time that IL-10 and IL-33 induce miR-155 and miR-146a, respectively, to alter mast cell functions. We report that IL-10 enhanced IgE induced activation of mast cells. IL-10 effects are dependent on Stat3 activation, which elicits miR-155 expression, resulting in a loss of suppressor of cytokine signaling-1 (SOCS-1). The importance of miR-155 was demonstrated by the inability of IL-10 to enhance anaphylaxis in miR-155–deficient mice. Additionally, we show that IL-33 treatment greatly enhances miR-146a expression in mast cells and in mast cell derived exosomes. miR-146a induction is dependent on MyD88 and NFκB and seems to negatively regulate ST2 signaling, which is demonstrated by the hyperresponsiveness of miR-146a knockout BMMC in response to IL-33. Our preliminary data suggest that miR-146a serves as a feedback negative regulator of IL-33 signaling by targeting IRAK proteins. miR-155 and miR-146a are key microRNAs that regulate a range of immune functions. Taken together, our results reveal two novel microRNA pathways that regulate mast cell IgE and IL-33 induced responses.

Vaccines are a highly effective means of preventing infectious disease. However, for many diseases of livestock the available vaccines are ineffective or sub-optimal. This is partly due to challenges surrounding the specific targeting of antigen presenting cells (APCs). In order to improve the delivery of protective antigens to host APCs, a novel lentiviral vector derived from visna / maedi virus (VMV) has been developed. Initial characterisation using an enhanced green fluorescent protein (eGFP) reporter transgene found that the novel VMV vector efficiently transduced a wide range of cell lines including cells of ovine, human, murine, bovine and caprine origin. In addition, the VMV vector was found to elicit sustained transgene expression for at least 4 weeks in rapidly dividing cell lines. One of the most important factors for acceptable vaccines is their safety. Therefore, in order to increase the bio-safety of the VMV vector, integration-defective and self-inactivating forms were produced. Integration-defective VMV lentiviral vectors (IDLVs) were found to produce 1-LTR circular episomes favourably over integrated provirus following the transduction of target feline and ovine cell lines. This led to a decrease in transgene expression over time in dividing cells. In contrast, in non-dividing cells transgene expression was maintained at a similar level to integration-competent VMV vectors. Self-inactivating (SIN) VMV vectors were constructed and found to have a significant decrease in LTR activity. Transgene expression was maintained by the insertion of an internal promoter derived from human cytomegalovirus (CMV) acting directly on the transgene. When self-inactivating and integration-defective modifications were incorporated into the same vector particle, a 4-fold decrease in transduction relative to the parent vector was observed. Ovine monocyte-derived dendritic cells (MDDCs) and macrophages (MDMs) were found to be efficiently transduced by the VMV vector, whereas lentiviral vectors derived from HIV-1 poorly transduced both of these primary cell populations. Following this work, the ability to deliver pathogen genes into APCs was studied using the Chlamydia abortus (C. abortus) major outer membrane protein (MOMP) as the transgene. C. abortus is the most common infectious cause of ovine abortion worldwide and MOMP has previously been shown to stimulate strong antibody responses after vaccination. Unexpectedly, the VMV vector encoding either eGFP or MOMP was found to induce apoptosis in MDDCs and MDMs using Annexin V staining. Apoptotic cells were detectable as early as 6 hours post-transduction of cells. Furthermore, release of the pro-inflammatory cytokine IL-1β was associated with the formation of late apoptotic cells. Apoptotic bodies produced post-transduction were able to be phagocytosed by immature MDDCs and the transgene efficiently cross-presented to T-cells. The ability of the novel VMV vector to induce a suitable recall immune responses was investigated using an in vitro model. Here, an autologous population of MDDCs were cultured with the apoptotic bodies produced post-transduction before the addition of autologous PBMC. Proteins from the apoptotic bodies were presented by the MDDCs to PBMC leading to a strong, antigen specific recall immune response against C. abortus MOMP. This was proven by the detection of cytokines IFNγ and IL-10 in the co-culture supernatant from PBMC activated by the MOMP transgene cross-presented by MDDCs. No release of IL-4 or IL-17A could be detected. These data presented in this thesis show the potential for improving delivery of antigens in livestock vaccines by the use of lentiviral vectors. In addition, this vector system provides a strong base for the study of other potential protective antigens in vitro.

Abstract Introduction: Current evidence suggests that the lungs of infants with the debilitating disorder, bronchopulmonary dysplasia (BPD), react to the challenges of extra-uterine adaptation with inappropriately aggressive inflammation. The reasons for this are not entirely clear and this study hypothesizes that a deficiency of interleukin (IL)-10, a potent anti-inflammatory mediator, leads to the functional and architectural changes characteristic of BPD. Aim: To characterize the behaviour of IL-10 and neutrophil apoptosis in the tracheal fluids (TF) of infants at risk of developing BPD. Method: TF from intubated infants of varying gestations at the Royal Hospital for Women, Randwick was spun and ILs 8, 10 and 16 were measured in the supernatant. The residual pellets of white cells were used to determine differential white cell counts and neutrophil apoptosis. Results: None of the 20 TF specimens from the extremely premature infants with BPD (n=11) had detectable IL-10, compared to 14/20(70%) of the specimens from preterm infants without BPD (n=20) and to 5/19 (26%) of the specimens from term infants (n=19). BPD infants also had a significantly lower number of apoptotic neutrophils during the 1st week of life. Premature infants with TF IL-10 &gt5pg/ml did not develop BPD. Levels of IL-8, a neutrophil chemotaxin, and white cell counts, while not differing significantly between the groups, increased considerably towards the end of the first week of life in the BPD group. IL-16, a chemotaxin for inflammatory CD4+ cells, was also detected in more BPD than non-BPD specimens (BPD: 16/46 (35%) v 1/30 (0.3%) non-BPD preterm and 2/7 (28%) term TF specimens). Conclusions: Extremely premature infants prone to BPD have decreased pulmonary anti-inflammatory activity as demonstrated by decreased IL-10 and apoptotic neutrophils in tracheal fluids. The lack of a counter-regulatory response to the inflammatory processes that are an inevitable consequence of extra-uterine adaptation may therefore place the extremely premature newborn infant at a considerable risk of developing BPD.

Gene therapy has emerged as a field to modulate cell functions by introducing genes of interest to target cells. An emerging focus in this field is to employ non-viral vectors to deliver immunosuppressive cytokines to dendritic cells (DCs) to attenuate damaging immune responses. DCs serve as potential targets for suppression of T cell responses. In this work, we investigated the ability of polycation-modified polymeric particles complexed with interleukin-10 (IL-10) gene to modulate DCs. The delivery systems (designated as PSO10H6 and PLGAO10H6) were formed by coating cationic peptide O10H6 (O: ornithine; H: histidine) on the polystyrene (PS) and poly (lactic-co-glycolic acid) (PLGA) particulates. A mouse IL-10 encoding plasmid (pIL-10) was loaded on the surface of PSO10H6 and PLGAO10H6 via ionic interactions. Physical characterization of these particles revealed stable colloidal dispersions (diameters: 297.2±14nm in PLGAO10H6-pIL-10 and 126.0±8nm in PSO10H6-pIL-10). DNA molecules carried by PSO10H6 and PLGAO10H6 were protected from serum digestion. Results from in vitro gene transfection studies showed two-fold enhancement of IL-10 expression in bone marrow-derived DCs transfected with PSO10H6-pIL-10 and PLGAO10H6-pIL-10 compared to untransfected DCs. Their suppressive functions were evaluated in an in vitro mixed lymphocyte model. Results indicated that PSO10H6-pIL-10 and PLGAO10H6-pIL-10 modified DCs elicited weakest proliferation of allogeneic bulk T cells as well as CD4 and CD8 T cells among all the delivery modes. Using cell-embedded Matrigel as a surrogate graft, we showed that IL-10 gene-modified DCs suppressed host cell infiltration in vivo. These data suggested PSO10H6-pIL-10 and PLGAO10H6-pIL-10 deliver an overriding suppressive signal to T cells. Further studies revealed T cells stimulated by the IL-10 gene-modified DCs exhibited characteristics of regulatory T (Treg) cells, as evident by up-regulation of a Treg cell marker forkhead-type transcription factor 3 (Foxp3). This result was concomitant with an increase in of transforming growth factor beta (TGF-beta) production. <br>Taken together, this work demonstrated that PSO10H6 and PLGAO10H6 are effective in delivering pIL-10 to modulate DCs to suppress T cell responses. Collectively, the results raise the prospects of using PSO10H6 and PLGAO10H6 as vectors to deliver immunosuppressive genes to modulate T cell responses in vivo. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmaceutics / PhD / Dissertation

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Toll-like receptors (TLRs) expressed by cells of the immune system play a central role in the generation of immune responses against pathogens. Following TLR ligation, both pro-inflammatory and anti-inflammatory mediators are produced in order to elicit an immune response that controls the microbial infection while limiting tissue damage. Among these mediators, the proinflammatory cytokine IL-12 and the anti-inflammatory cytokine IL-10 are known to play major roles. Here, we show that in vitro or in vivo stimulation with flagellin, the TLR5 ligand, does not result in IL-10 production. Furthermore flagellin inhibits IL-10 production by other specific TLR ligands at the protein and mRNA levels while increasing IL-12p70 production. Several studies have linked the activation of extracellular signal regulated kinases (ERKs) with IL-10 induction by TLRs. Our findings that LPS-induced ERK activation is significantly decreased in flagellin-treated macrophages suggest that this pathway might play a role in the inhibition of IL-10 production by flagellin. Flagellin-mediated IL-10 inhibition was not observed in cells that do not express TLR5 supporting that this effect is TLR5-dependent.Flagellin used as an adjuvant is capable of priming antigen specific T cell responses in an in vivo model of tolerance using high dose peptide. Furthermore, DCs differentiated in tolerogenic conditions (tolerogenic-DCs) express higher levels of TLR5 mRNA than standard BM-DCs and respond more vigorously to flagellin stimulation. Antigen presentation by LPS-matured tolerogenic-DCs results in the differentiation of IL-10 producing T cells with a Tr1-like phenotype. On the contrary, antigen presentation by tolerogenic-DCs that have been stimulated with flagellin results in the differentiation of a typical Th1 responseThis study provides a new insight of the role of flagellin recognition by TLR5 in shaping the immune response elicited by flagellated microorganisms.

Η cAMP αποτελεί ένα σημαντικό δεύτερο μήνυμα που ρυθμίζει την ανοσολογική απόκριση. Η αύξηση της ενδοκυττάριας cAMP αυξάνει την παραγωγή της IL-10 από μονοκύτταρα. Σκοπός της μελέτης είναι η αποσαφήνιση της συμμετοχής της cAMP στην παραγωγή της IL-10 από Τ-λεμφοκύτταρα όπου τα δεδομένα είναι ακόμα ασαφή. Ανθρώπινα Τ-λεμφοκύτταρα περιφερικού αίματος διεγέρθηκαν με anti-CD3/anti-CD28, anti-CD3 ή Ionomycin/PMA παρουσία ή απουσία παραγόντων που αυξάνουν την ενδοκυττάρια cAMP (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram και 10-6 Μ 8-Br-cAMP). Το πρωτεϊνικό προϊόν της IL-10 μετρήθηκε με ELISA ενώ η παραγωγή mRNA της IL-10 με Real Time PCR. Η ενεργότητα του υποκινητή της IL-10 ελέγχθηκε με διαμόλυνση των κυττάρων με πλασμίδια που φέρουν τον υποκινητή του γονιδίου (1327 bp) ή τμήματα αυτού (-1010, -500, -310, -235, -135 bp). Η δέσμευση των μεταγραφικών παραγόντων MEF-2 και CREB ελέγχτηκε σε πυρηνικά πρωτεϊνικά εκχυλίσματα με πειράματα EMSA, ενώ η ενεργότητα τους ελέγχθηκε με πειράματα διαμολύνσεως με πλασμίδια που ελέγχουν την ενεργότητα της λουσιφεράσης υπό τον έλεγχο των MEF-2 και CREB. Αύξηση της cAMP ελαττώνει την παραγωγή της IL-10 σε πρωτεϊνικό επίπεδο κατά 50-60% μετά από διέγερση με Ion/PMA, και κατά 80-90% με anti-CD3 ή με anti-CD3/anti-CD28. Η IL-10 παράγεται ακόμα και μετά από διέγερση μόνο με anti-CD3, εύρημα ειδικό για την IL-10, καθώς δεν παρατηρήθηκε αύξηση της παραγωγής άλλων κυτταροκινων (IL-2 & IL-4). Η ελάττωση της παραγωγής της IL-10 αντανακλάται και σε επίπεδο mRNA όπου οι αντίστοιχες μειώσεις είναι κατά 50% με όλους τους τρόπους διέγερσης. Η ενεργότητα του υποκινητή της IL-10 δεν επηρεάζεται από αλλαγές στα επίπεδα της cAMP όταν η διεγερση παρακάμπτει τον Τ κυτταρικό υποδοχέα. Ωστοσο, μειώνεται παρουσία αυξημένων συγκεντρώσεων cAMP όταν τα κύτταρα διεγείρονται μέσω του Τ κυτταρικού υποδοχέα. Το τμήμα του υποκινητή της IL-10 που επηρεάζεται από την ανασταλτική δραση της cAMP (50 % αναστολή) βρίσκεται στις πρώτες 500 bp πριν το TATA box, και περιέχει σημεία πρόσδεσης των μεταγραφικών παραγόντων MEF-2 και CREB, όπως ελέγχθηκε με το πρόγραμμα Consite. Η δέσμευση του MEF-2 σε πυρηνικά εκχυλίσματα διεγερμένων Τ-λεμφοκυττάρων μειώνεται κατά 70% παρουσία αυξημένης cAMP ενώ η ενεργότητα του δεν επηρεάζεται σημαντικά. Αντίθετα, η αύξηση της cAMP αυξάνει τόσο τη δέσμευση (x 2,5) όσο και την ενεργότητα του CREB (x 2). Η δράση της cAMP στην παραγωγή της IL-10 είναι ειδική για τα Τ-λεμφοκύτταρα και εξαρτάται από τον τρόπο διέγερσής τους. Η ρύθμισή της γίνεται τόσο σε μεταγραφικό όσο και σε μετά-μεταγραφικό επίπεδο. Η αύξηση της cAMP μπορεί να επηρεάσει την παραγωγή IL-10 από τα Τ-λεμφοκύτταρα παρεμβαίνοντας στην δέσμευση και την ενεργότητα των παραγόντων μεταγραφής MEF-2 και CREB. Ο τρόπος της αλληλεπίδρασης/συνεργασίας των MEF-2 και CREB παραμένει υπό διερεύνηση. / cAMP is a second messenger playing a crucial role in the signal transduction which controls the immune response, while IL-10 is considered to be an important regulator of this response. Elevation of intracellular concentration of cAMP has been shown to increase IL-10 production by monocytes. The aim of this study was the elucidation of the role of cAMP in IL-10 production by normal T lymphocytes, a mechanism that remains unclear. Fresh Human Τ-lymphocytes derived from PBMC of healthy donors where stimulated with anti-CD3/anti-CD28 or Ionomycin/PMA, in the presence or absence of cAMP elevating agents (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram and 10-6 Μ 8-Br-cAMP). The protein product of IL-10 was measured by ELISA, the production of IL-10 mRNA by Real Time PCR and IL-10 mRNA stability was determined by the use of Actinomycin D (10 μM). The activity of IL-10 promoter was measured by luciferase reporter assay, after transfection of cells with plasmids carrying the wild type promoter (1037bp) or promoter fragments (constructs of -1010, -500, -310, -235, -135bp). PKA role was examined either by cotransfection experiments with a plasmid carrying a constitutively active mutant of the catalytic subunit of PKA-α isoform, or by the use of a specific PKA inhibitor Rp-8- Br-cAMP (10-50 μM). The presence of binding sites of transcription factors in the first 500bp of the IL-10 promoter, was validated using the web-based program CONSITE. Binding of the transcription factors MEF2 and CREB was investigated in nuclear extracts of stimulated human T cells with EMSA experiments. The activity of MEF2 and CREB was investigated independently with transfection experiments using plasmids containing the lusiferase reporter under the control of the transcription factors. Intracellular cAMP elevation, inhibits IL-10 protein production by 50-60%, when T cells are stimulated with Ionomycin/PMA, and by 80-90% after stimulation with anti-CD3 or anti-CD3/anti-CD28, while PKA blocking by Rp-8- Br-cAMP reversed cAMP mediated inhibition.. IL-10 steady state mRNA levels follow the same pattern of inhibition only after anti-CD3/anti-CD28 stimulation. cAMP elevation decreases IL-10 mRNA stability after I/PMA stimulation, whereas in the anti-CD3/anti-CD28 stimulated cells, the mechanism of inhibition is mainly transcriptional. IL-10 promoter activity is reduced up to 60% when cells are stimulated with anti CD3/anti CD28 in the presence of cAMP elevating agents, but is not affected after stimulation with Ionomycin/PMA or cotransfection of the cells with constitutively active PKA mutant. Transfection assays with the different IL-10 promoter fragments revealed that the most responsible part of IL-10 promoter to cAMP mediated inhibition, is the first 500 bp after the TATA box. This part contains binding sites for the transcription factors MEF-2 and CREB, as validated by the web-based program Consite. Increased intracellular cAMP reduces the binding of MEF2 to nuclear extracts of stimulated T cells by 70 %, however its activity is not affected significantly. On the contrary, both the binding and the activity of CREB are increased in the presence of elevated cAMP. cAMP mediated inhibition of IL-10 production is PKA mediated and specific for T lymphocytes, depending on the nature/strength of stimulation. cAMP-dependent regulation of IL-10 production is controlled by transcriptional and/or post-transcriptional mechanisms depending on the nature of stimulus. Transcriptional mechanisms involve the transcription factors MEF2 and CREB, however the exact mechanisms of action of these factors deserves further elucidation. Cell and stimulus specific mechanism of regulation of IL-10 production is necessary for its immunoregulatory function.