Deciphering the function of DNGR-1 in cross-presentation through the characterisation of phagosomal compartments in cDC1

Induction of antigen specific cytotoxic CD8+ T lymphocyte (CTL) responses by dendritic cells (DCs) is essential for clearance of infected or malignantly transformed cells. Antigens derived from such cells are presented to naïve CD8+ T cells in the form of short antigenic peptides associated with major histocompatibility complex I (MHC I) on the DC surface. This process, called cross-presentation, often involves transfer of antigens from dying infected or malignantly transformed cells to DCs and is facilitated by innate receptors that sense dead cell-derived damage-associated molecular patterns (DAMPs). These receptors include the C-type lectin receptor DNGR-1, which allows DCs to detect the presence of dead or dying cells by binding to filamentous actin (F-actin) exposed by dead cell corpses. DNGR-1 promotes cross-presentation of dead cell-associated antigens but the mechanism involved is still poorly understood. The aim of my PhD project was to dissect the mechanism by which DNGR-1 facilitates cross-presentation of dead cell-associated antigens. I found it involved proteasomal degradation and was enhanced by inhibition of lysosomal proteases. Further, the cytoplasmic tail of DNGR-1 and, therefore likely DNGR-1 signalling, was essential to promote cross-presentation post cargo uptake. Since DNGR-1 was recruited to antigen-bearing phagosomes, I studied the characteristics of those DNGR-1+ phagosomes. Combined analysis of antigen degradation and staining for DNGR-1 and LAMP-2 revealed two distinct phagosome populations with varying degradative potential and MHC I recruitment: a DNGR-1+LAMP-2-MHC I+ that showed strikingly lower degradative potential, in contrast to DNGR-1-LAMP-2+MHC I- phagosomes. However, DNGR-1+ phagosomes eventually acquired LAMP-2+ resulting in an increase in antigen degradation. To test whether DNGR-1 ligand engagement was shaping the phagosomal proteome in cDC1s, I analysed FACS-purified DNGR-1+ and LAMP-2+ phagosome populations by mass spectrometry. A strong enrichment of the calcium pump SERCA1 and the autophagy initiator beclin-1 was observed in DNGR-1+ phagosomes containing DNGR-1 ligand. Preliminary experiments further revealed that the phagosomal lumen became accessible for cytosolic galectins in a DNGR-1-dependent manner suggesting that DNGR-1 might be involved in antigen to cytosol transfer. In summary, this thesis offers novel insights into the mechanisms by which dead cell antigens are cross-presented by cDC1 through the engagement of DNGR-1, which potentially regulates the stability of antigen-containing phagosomes and thus, might mediate the transfer of antigen from the phagosome into the cytosol

Chloroquine enhances human CD8+ T cell responses against soluble antigens in vivo

The presentation of exogenous protein antigens in a major histocompatibility complex class I–restricted fashion to CD8+ T cells is called cross-presentation. We demonstrate that cross-presentation of soluble viral antigens (derived from hepatitis C virus [HCV], hepatitis B virus [HBV], or human immunodeficiency virus) to specific CD8+ T cell clones is dramatically improved when antigen-presenting dendritic cells (DCs) are pulsed with the antigen in the presence of chloroquine or ammonium chloride, which reduce acidification of the endocytic system. The export of soluble antigen into the cytosol is considerably higher in chloroquine-treated than in untreated DCs, as detected by confocal microscopy of cultured cells and Western blot analysis comparing endocytic and cytosolic fractions. To pursue our findings in an in vivo setting, we boosted groups of HBV vaccine responder individuals with a further dose of hepatitis B envelope protein vaccine with or without a single dose of chloroquine. Although all individuals showed a boost in antibody titers to HBV, six of nine individuals who were administered chloroquine showed a substantial CD8+ T cell response to HBV antigen, whereas zero of eight without chloroquine lacked a CD8 response. Our results suggest that chloroquine treatment improves CD8 immunity during vaccination

Crosspresentation by dendritic cells,”

MHC class I-loading complex A series of endoplasmic reticulum chaperone proteins that stabilize empty MHC class I molecules and control the loading of high-affinity peptides onto MHC class I molecules. Cross-presentation by dendritic cells Abstract | The presentation of exogenous antigens on MHC class I molecules, known as cross-presentation, is essential for the initiation of CD8 + T cell responses. In vivo, cross-presentation is mainly carried out by specific dendritic cell (DC) subsets through an adaptation of their endocytic and phagocytic pathways. Here, we summarize recent advances in our understanding of the intracellular mechanisms of cross-presentation and discuss its role in immunity and tolerance in the context of specialization between DC subsets. Finally, we review current strategies to use cross-presentation for immunotherapy

Untersuchung intrazellulärer Überlebensstrategien von Staphylococcus aureus unter Verwendung eines photokonvertierbaren Reporter-Systems als Biosensor für den metabolischen Status der Bakterien

Staphylococcus aureus is a human pathogen with an extra-/intracellular dual lifestyle enabling to invade, survive and proliferate in host cells, protected from the host immune system and antibiotic treatment. A better understanding of S. aureus intracellular lifestyle may facilitate the development of new therapeutic approaches. Therefore, the overall aim of this study was to investigate the mechanisms used by S. aureus to survive and replicate intracellularly within different host cells. For this purpose, an S. aureus strain encoding the mKikumeGR proliferation reporter system was used in a dual RNA-seq approach to determine the correlation between gene expression changes and the metabolic state of internalized bacteria. The host transcriptional profiles, dominated by the expression of pro-inflammatory genes, did not differ between macrophages harboring metabolically active/proliferating S. aureus and those harboring metabolically inactive S. aureus, suggesting that the metabolic state of the internalized bacteria was not dictated by heterogeneity within the host cell population. On the pathogen side, metabolically active/proliferating and metabolically inactive S. aureus exhibited a common response comprising a core set of genes representing a general stress response to the intracellular host milieu. Beside this, a specific transcriptional signature was identified. While metabolic active intracellular S. aureus showed higher expression of genes involved in proliferation, the red metabolically inactive bacteria had higher expression of oxidative stress-related genes and ribosome hibernation. The influence of the internalization route on the fate of intracellular S. aureus was also investigated. The internalization pathway used by S. aureus to access an intracellular compartment permissive for bacterial replication was found to be cell-type specific. While in macrophages, S. aureus used an α5ß1-integrin-mediated internalization pathway likely via macropinocytosis, the invasion mechanism in epithelial cells seems to be caveolae-mediated and induced by secreted Hla. Furthermore, the polarization state of macrophages influenced the fate of S. aureus since, in contrast to M1 macrophages, only M2 macrophages were permissive for intracellular proliferation. In summary, this study provides evidence for an internalization pathway of S. aureus where clathrin-independent endocytic vesicles might display a compartment for intracellular replication of S. aureus.Staphylococcus aureus ist ein Humanpathogen mit einem intra-/extrazellulären Lebenszyklus der die Invasion von Wirtszellen ermöglicht um dort, geschützt vor dem Immunsystem und Antibiotika, zu überleben und sich zu teilen. Da ein besseres Verständnis über den intrazellulären Lebenszyklus von S. aureus die Entwicklung neuer therapeutischer Ansätze fördern könnte, war es das Ziel, die von S. aureus verwendeten Mechanismen zum Überleben in verschiedenen Wirtszellen zu untersuchen. Der hierfür verwendete Stamm exprimiert das mKikumeGR Reporter-System, das erlaubt die intrazelluläre metabolische Aktivität zu verfolgen und wurde genutzt, um in einem dualen RNA-Seq Ansatz Genexpressionsänderungen und mit dem metabolischen Status der Bakterien zu korrelieren. Die Transkriptionsprofile des Wirts, die hauptsächlich die Expression von pro-inflammatorischen Genen zeigen, unterschieden sich nicht zwischen den Makrophagen, die metabolisch aktive S. aureus und denen die metabolisch inaktive Bakterien enthielten. Daraus lässt sich schließen, dass der metabolische Status der Bakterien nicht zurückzuführen ist auf eine Heterogenität innerhalb der Wirtszellpopulation. Die metabolisch aktiven und inaktiven S. aureus exprimierten ein gemeinsames, grundlegendes Gen-Set, das die allgemeine Stressantwort auf das intrazelluläre Milieu darstellen. Außerdem konnten spezielle transkriptionelle Signaturen einzelnen bakteriellen Stoffwechselzuständen zugeordnet werden. Während metabolisch aktive S. aureus wichtige Gene für die Proliferation stärker exprimierten, zeigen metabolisch inaktive S. aureus ein Expressionsprofil, das auf oxidativen Stress und den Ruhezustand von Ribosomen hinweisen. Zudem wurde der Einfluss des Aufnahmeweges in die Wirtszellen auf das Überleben von S. aureus untersucht. Es konnte gezeigt werden, dass das Bakterium einen zelltypabhängigen Weg nutzt um ein Kompartiment zu erreichen, in dem es sich teilen kann. Während eine α5ß1-Integrin vermittelte Aufnahme vermutlich über Macropinocytose genutzt wird um in Makrophagen zu gelangen, scheint die Invasion von Epithelzellen Hla-induziert über Caveolae abzulaufen. Außerdem beeinflusst die Polarisation von Makrophagen das Überleben von S. aureus, da im Gegensatz zu M1- nur M2- Makrophagen die intrazelluläre Replikation zuließen. Zusammengefasst liefert diese Studie Beweise für einen Aufnahme von S. aureus bei dem Clathrin-unabhängige endozytische Vesikel Kompartimente darstellen könnten, die eine Replikation zulassen

CD14 Signaling Restrains Chronic Inflammation through Induction of p38-MAPK/SOCS-Dependent Tolerance

Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes

Non-Lytic Egress of Mycobacterium Tuberculosis from Human Macrophages

Macrophages are the target cell for Mycobacterium tuberculosis (Mtb) infection; a major reservoir for productive infection, as well as crucial effector cells for the control of Mtb. However, the majority of bacteria in the caseous necrotic centre of the TB granuloma are extracellular. Extracellular bacteria may arise from host cell necrosis and release of infecting bacteria, but mycobacteria have also been shown to exit from amoeba using a non-lytic strategy and other pathogens have also developed versatile strategies to exit from their host cell, providing an alternative mechanism to escape intracellular restriction. In this thesis I have developed a high throughput flow cytometric assay to quantify intracellular and extracellular bacteria in an in vitro model of human monocyte derived macrophages infected with fluorescent Mtb. I show that extracellular Mtb accumulate in parallel to intracellular bacteria, and make a significant contribution to the overall bacillary burden. These extracellular bacteria do not arise as a result of cellular necrosis in this low multiplicity of infection model. I use computational modelling to predict that a non-lytic process is necessary to explain the observed data. I demonstrate experimentally, for the first time that extracellular Mtb arise by non-lytic egress from human macrophages. I show that this process is related to virulence, since the attenuated Mycobacterium Bovis Bacillus Calmette-Guérin does not display this behaviour

Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of Cryptococcus neoformans

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication

Visualising the subcellular distribution of antibiotics against tuberculosis

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), remains the world’s deadliest infectious disease. Although treatable, effective chemotherapy requires at least six months of treatment with a minimum of four antibiotics. Novel antibiotics are needed to quell the pandemic. However, we do not fully understand why current treatments take so long to work in patients. Mtb has a dynamic intracellular lifestyle, and this thesis tests the hypothesis that not all antibiotics penetrate into, or are effective within, all compartments containing Mtb during infection. Our understanding of the intracellular pharmacokinetics of drugs against TB has been limited by a lack of technologies for studying the subcellular distribution of antibiotics. This work developed a correlative imaging workflow incorporating fluorescence, electron and nanoscale ion microscopy (CLEIM) to map the subcellular distribution of two antibiotics, bedaquiline (BDQ) and pyrazinamide (PZA), at sub-micrometre resolution in Mtb-infected human macrophages. This workflow was complemented with orthogonal methods, including high-content live-cell imaging, to study the dynamic processes that contribute to antibiotic activity. BDQ accumulated primarily in host cell lipid droplets (LD), but heterogeneously in Mtb within a variety of intracellular compartments. Surprisingly, LD did not sequester the antibiotic but constituted a transferable reservoir that enhanced antibacterial efficacy. Lipid binding therefore facilitated drug trafficking by host organelles to an intracellular target. PZA is a pro-drug, and the accumulation of its active metabolite pyrazinoic acid has been hypothesised to depend on the bacteria being in an acidic environment. Direct analysis of antibiotic accumulation by ion microscopy, combined with live-cell imaging at the single cell level, revealed that, whilst acidic intracellular environments support PZA activity, they are not necessary for antibiotic efficacy. Many intracellular pathogens interact with LD or reside in partially acidified vacuoles, and these results therefore have broad implications for our understanding of antibiotic activity