Characterizing innate immune cell activation by fungal pathogens

Candida albicans (C. albicans) and Candida glabrata (C. glabrata) are the two most prevalent Candida species causing bloodstream infections. Isolated NK cells get stronger activated by C. albicans than C. glabrata. In contrast, activation of blood NK cells was more pronounced during C. glabrata infection. NK cell activation in blood is mediated by humoral mediators released by other immune cells and does not depend on direct activation by fungal cells. Cross-talk between Candida-confronted monocyte-derived dendritic cells (moDC) and NK cells resulted in the same NK activation phenotype like NK cells in human blood. Blocking experiments and cytokine substitution identified interleukin 12 as a critical mediator in regulation of primary NK cells by moDC-derived cytokines. On the other hand, we focused on the identification of changes in polymorphonuclear cells (PMN) behavior induced by C. albicans and C. glabrata since these immune cells are of outstanding importance in the response against invasive Candida infections. Sorting and extraction of PMN were performed after a one-hour confrontation in human whole blood in presence of fungal cells. Then, infected isolated PMN were used for separate analysis in live cell imaging experiments to visualize their dynamic features in comparison to mock-infected PMN. PMN in the acquired microscopic images were analyzed using a migration and interaction tracking algorithm and further classified using different morphokinetics features. Compared to mock-treated PMN, PMN isolated from whole-blood infected with either C. albicans or C. glabrata presented a higher percentage of PMN with a spreading morphology. Furthermore, C. glabrata presented a significantly higher number of cells with a spreading morphology compared to C. albicans. Combination of live cell imaging with automated analysis allowed a classification and distinction of PMN isolated from mock-infected blood and Candida-infected blood based on their morphology

Quantitative automated analysis of host-pathogen interactions

This work aims to broaden knowledge about neutrophil biology in their interaction with fungi species that most frequently cause invasive fungal diseases (IFD). The questions addressed include the alteration of neutrophil morphology after interaction with Candida albicans or C. glabrata, revealing factors that modulate the production and composition of neutrophil-derived extracellular vesicles (EVs) obtained in confrontation assay with conidia of Aspergillus fumigatus and analysing EVs activity against this fungus. Alongside fundamental interests, those questions have important applied aspects in the medicine of IFD. In particular, for diagnostic purposes and infection process monitoring. The results of this work include: 1 a novel segmentation and tracking algorithm which is capable of working with low-contrast cell images, producing accurate cell contours and providing data about positions of clusters, which would improve further analysis; 2 a novel workflow algorithm for analysis of neutrophil continuous morphological spectrum without consensus-based manual annotation; 3 quantitative evidence that morphodynamics of isolated neutrophils depends on the infectious agent (C. albicans or C. glabrata) used in whole blood infection assay; 4 quantitative evidence that neutrophil-derived extracellular vesicles, obtained in confrontation assays with conidia of A. fumigatus could inhibit hyphae development and damage hyphae cell wall; 5 quantitative evidence that EVs inhibition activity is strain-specific

The specific role of neutrophil- and epithelial cell-derived extracellular vesicles in antifungal defence against Aspergillus fumigatus

oai:www.db-thueringen.de:dbt_mods_00055686In this work I contributed to the elucidation of the role and physiological meaning of extracellular vesicles (EVs) released from epithelial cells and neutrophils after interaction with the pathogenic fungus Aspergillus fumigatus. With my work I untangled more aspects of the interaction between neutrophils and this fungus and came to the finding that EVs generated from neutrophils infected with A. fumigatus are highly specific in the killing of A. fumigatus. Evaluation of the hyphal damage induced by EVs was performed with extensive imaging, based on confocal laser scanning microscopy, bioinformatic 3D reconstruction and quantification of signals, and a metabolic assay. In the course of my research on extracellular vesicles I also characterized EVs from epithelial cells. I evaluated the cell response after confrontation with different conidia morphotypes of A. fumigatus. Analysing the EVs after these different co-incubations revealed that their protein and cytokine contents are changed. This proves that the cells can sense different stimuli and modify their EV content

Agent-based modelling of the spatio-temporal interaction between immune cells and human-pathogenic fungi

Human-pathogene Pilze stellen aufgrund der zunehmenden Anzahl von immungeschwächten Patienten ein zunehmendes Problem im Gesundheitswesen dar und sind mit hohen Sterblichkeitsraten assoziiert. Das menschliche Immunsystem ein hochkomplexes System stellt ein Arsenal an Effektormechanismen bereit, die den gesunden Zustand des Wirts schützen. Vielfältige Ursachen können jedoch diese schützende Funktion des Immunsystems beeinträchtigen, was es eindringenden Mikroben erlaubt, schwere Infektionen zu verursachen. Die Forschung an Wirt-Pathogen-Interaktionen zwischen humanpathogenen Pilzen und dem Immunsystem ist essentiell für die Entwicklung neuer diagnostischer und therapeutischer Verfahren. In dieser Arbeit wurden diese Wirt-Pathogen-Interaktionen entsprechend des Konzepts der Systembiologie, untersucht. Basierend auf experimentellen Daten wurden virtuelle Infektionsmodelle entwickelt, um die treibenden Kräfte der angeborenen Immunantwort gegen die pilzlichen Erreger Candida albicans, Candida glabrata und Aspergillus fumigatus zu entschlüsseln. ..

Individual-based modeling and predictive simulation of fungal infection dynamics

The human-pathogenic fungus Aspergillus fumigatus causes life-threatening infections in immunocompromised patients and poses increasing challenges for the modern medicine. A. fumigatus is ubiquitously present and disseminates via small conidia over the air of the athmosphere. Each human inhales several hundreds to thousands of conidia every day. The small size of conidia allows them to pass into the alveoli of the lung, where primary infections with A. fumigatus are typically observed. In alveoli, the interaction between fungi and the innate immune system of the host takes place. This interaction is the core topic of this thesis and covered by mathematical modeling and computer simulations. Since in vivo laboratory studies of A. fumigatus infections under physiological conditions is hard to realize a modular software framework was developed and implemented, which allows for spatio-temporal agent-based modeling and simulation. A to-scale A. fumigatus infection model in a typical human alveolus was developed in order to simulate and analyze the infection scenario under physiological conditions. The process of conidial discovery by alveolar macrophages was modeled and simulated with different migration modes and different parameter configurations. It could be shown that chemotactic migration was required to find the pathogen before the onset of germination. A second model took advantage of evolutionary game theory on graphs. Here, the course of infection was modeled as a consecutive sequence of evolutionary games related to the complement system, alveolar macrophages and polymorphonuclear neutrophilic granulocytes. The results revealed a central immunoregulatory role of alveolar macrophages. In the case of high infectious doses it was found that the host required fully active phagocytes, but in particular a qualitative response of quantitatively sufficient polymorphonuclear neutrophilic granulocytes.Der human-pathogene Schimmelpilz Aspergillus fumigatus verursacht tödliche Infektionen und Erkrankungen vorrangig bei immunsupprimierten Patienten und stellt die moderne Medizin vor zunehmende Herausforderungen. A. fumigatus ist ubiquitär präsent und verbreitet sich über sehr kleine Konidien durch Luftströmungen in der Athmosphäre. Mehrere Hundert bis Tausende dieser Konidien werden täglich durch jeden Menschen eingeatmet. Die geringe Größe der infektiösen Konidien erlauben es dem Pilz bis in die Alveolen der Lunge des Wirtes vorzudringen,in denen eine Primärinfektionen mit A. fumigatus am häufigsten stattfindet. Die Alveolen sind der zentrale Schauplatz der Interaktion zwischen dem Pilz und dem angeborenen Immunsystem, welche Gegenstand dieser Arbeit ist. Diese Interaktion wird mit Hilfe von mathematischen Modellen und Computersimulationen nachgestellt und untersucht, da eine A. fumigatus Infektion im Nasslabor in vivo unter physiologischen Bedingungen nur sehr schwer realisiert werden kann. Als Grundlage für dieses Vorhaben wurde ein modulares Software-Paket entwickelt, welches agentenbasierte Modellierung und entsprechende Simulationen in Raum und Zeit ermöglicht. Ein maßstabsgetreues mathematisches Infektionsmodell in einer typischen menschlichen Alveole wurde entwickelt und die Suchstrategien von Alveolarmakrophagen unter der Berücksichtigung verschiedener Parameter wie Migrationsgeschwindigkeit, dem Vorhandensein von Chemokinen, dessen Diffusion und Chemotaxis untersucht. Es zeigte sich, dass Chemotaxis, notwendig ist, um die Konidie rechtzeitig finden zu können. In einem weiteren Modell, welches auf das Konzept evolutionärer Spieltheorie auf Graphen zurückgegriff, wurde der Infektionsverlauf als aufeinanderfolgende Serie evolutionärer Spiele mit dem Komplementsystem, Alveolarmakrophagen und Neutrophilen nachgestellt. Aus den Simulationsergebnissen konnte eine zentrale immunregulatorische Rolle von Alveolarmakrophagen entnommen werden

Accessible software frameworks for reproducible image analysis of host-pathogen interactions

Um die Mechanismen hinter lebensgefährlichen Krankheiten zu verstehen, müssen die zugrundeliegenden Interaktionen zwischen den Wirtszellen und krankheitserregenden Mikroorganismen bekannt sein. Die kontinuierlichen Verbesserungen in bildgebenden Verfahren und Computertechnologien ermöglichen die Anwendung von Methoden aus der bildbasierten Systembiologie, welche moderne Computeralgorithmen benutzt um das Verhalten von Zellen, Geweben oder ganzen Organen präzise zu messen. Um den Standards des digitalen Managements von Forschungsdaten zu genügen, müssen Algorithmen den FAIR-Prinzipien (Findability, Accessibility, Interoperability, and Reusability) entsprechen und zur Verbreitung ebenjener in der wissenschaftlichen Gemeinschaft beitragen. Dies ist insbesondere wichtig für interdisziplinäre Teams bestehend aus Experimentatoren und Informatikern, in denen Computerprogramme zur Verbesserung der Kommunikation und schnellerer Adaption von neuen Technologien beitragen können. In dieser Arbeit wurden daher Software-Frameworks entwickelt, welche dazu beitragen die FAIR-Prinzipien durch die Entwicklung von standardisierten, reproduzierbaren, hochperformanten, und leicht zugänglichen Softwarepaketen zur Quantifizierung von Interaktionen in biologischen System zu verbreiten. Zusammenfassend zeigt diese Arbeit wie Software-Frameworks zu der Charakterisierung von Interaktionen zwischen Wirtszellen und Pathogenen beitragen können, indem der Entwurf und die Anwendung von quantitativen und FAIR-kompatiblen Bildanalyseprogrammen vereinfacht werden. Diese Verbesserungen erleichtern zukünftige Kollaborationen mit Lebenswissenschaftlern und Medizinern, was nach dem Prinzip der bildbasierten Systembiologie zur Entwicklung von neuen Experimenten, Bildgebungsverfahren, Algorithmen, und Computermodellen führen wird

Aspf2 From Aspergillus fumigatus Recruits Human Immune Regulators for Immune Evasion and Cell Damage

The opportunistic fungal pathogen Aspergillus fumigatus can cause life-threatening infections, particularly in immunocompromised patients. Most pathogenic microbes control host innate immune responses at the earliest time, already before infiltrating host immune cells arrive at the site of infection. Here, we identify Aspf2 as the first A. fumigatus Factor H-binding protein. Aspf2 recruits several human plasma regulators, Factor H, factor-H-like protein 1 (FHL-1), FHR1, and plasminogen. Factor H contacts Aspf2 via two regions located in SCRs6–7 and SCR20. FHL-1 binds via SCRs6–7, and FHR1 via SCRs3–5. Factor H and FHL-1 attached to Aspf2-maintained cofactor activity and assisted in C3b inactivation. A Δaspf2 knockout strain was generated which bound Factor H with 28% and FHL-1 with 42% lower intensity. In agreement with less immune regulator acquisition, when challenged with complement-active normal human serum, Δaspf2 conidia had substantially more C3b (>57%) deposited on their surface. Consequently, Δaspf2 conidia were more efficiently phagocytosed (>20%) and killed (44%) by human neutrophils as wild-type conidia. Furthermore, Aspf2 recruited human plasminogen and, when activated by tissue-type plasminogen activator, newly generated plasmin cleaved the chromogenic substrate S2251 and degraded fibrinogen. Furthermore, plasmin attached to conidia damaged human lung epithelial cells, induced cell retraction, and caused matrix exposure. Thus, Aspf2 is a central immune evasion protein and plasminogen ligand of A. fumigatus. By blocking host innate immune attack and by disrupting human lung epithelial cell layers, Aspf2 assists in early steps of fungal infection and likely allows tissue penetration

Early peripheral immunological events dictate chronic wasting disease

2013 Spring.Includes bibliographical references.Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, like scrapie, has been shown to involve the immune system, which most likely contributes to their relatively proficient horizontal and environmental transmission. While CWD prions probably interact with the innate immune system immediately following peripheral exposure, little is known about this initial encounter. In the first chapter of this dissertation we examined initial events in lymphotropic and intranodal prion trafficking by tracking highly enriched, fluorescent CWD prions from infection sites to draining lymph nodes. We observed biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. CWD prions rapidly reached draining lymph nodes in a cell autonomous manner within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) showed a strong dependence on Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells comprised the majority of prion-bearing cells in the mediastinal lymph node by six hours. As most B cells are mainly located in the follicles, acquisition of prions by these cells most likely occurred through interaction with resident DCs, subcapsulary sinus macrophages, or directly from the follicular conduit system. These data highlight a novel mechanism of cell autonomous prion transport, and a vital role for B cells in intranodal prion trafficking. Upon entry into the draining lymph nodes, prion accumulation and replication on follicular dendrtic cells (FDCs) is greatly facilitated by the complement system. Complete elimination of CD21/35 significantly delays splenic prion accumulation and terminal prion disease in mice inoculated intraperitoneally with mouse-adapted scrapie prions. In the second chapter of this thesis we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. Ablation of complement receptors CD21/35 greatly diminished splenic prion accumulation and replication throughout the course of disease, similar to CD21/35 deficient murine PrP mice infected with mouse scrapie. Mice with deficiencies in CD21/35 showed a reduction in severity of neuropathology and deposition of misfolded, protease-resistant PrP associated with CWD. Prion infection resulted in translocation of CD21/35 to lipid rafts in B cells, and FDC expression of CD21/35 mediated a strong germinal center response that may be conducive to prion amplification. Complement component C3 is a central protein in the complement system whose activation is essential for the elimination of infectious pathogens. C3 is the most abundant complement protein, being found in the blood at physiological concentrations of 1 mg/ml. Among the complement proteins, C3 is perhaps the most adaptable and multifunctional protein identified to date, having evolved structural characteristics that allow it to associate with over 25 different proteins. Previous experiments suggest a vital role of C3 in scrapie prion pathogenesis. In the last chapter of my thesis we showed that lack of C3 expression by 5037 mice either transiently or genetically leads to delays in prion pathogenesis. C3 impacts disease progression in the early stages of disease by slowing the kinetic rate of accumulation and/or replication of PrPRES. This slower kinetic increase in PrPRES correlates with an increase in survival time in mice deficient in C3. This delay in disease is in sharp contrast to the complete rescue we saw in CWD infected Tg 5037;CD21-/- mice. This suggests a role for CD21/35 in peripheral prion pathogenesis independent of their endogenous ligands. Taken together we show that the innate immune system dictates the course of CWD. We have discovered novel immune cells, trafficking pathways, and complement components important in CWD pathogenesis. These data not only highlight the key role of the innate immune system in CWD, but also provide a strong foundation for future immunological studies of prion diseases

Modular Multimodal Iron Oxide-Based Nanocarriers for Image-Guided dsRNA Immunostimulation and Platinum Anticancer Drug Design

237 p.El objetivo de este proyecto se centra en la síntesis de nanopartículas inorgánicas de óxido de hierro con una composición, superficie y tamaño especialmente diseñados para activar el sistema inmune y tener capacidad anticancerígena y/o para su utilización como agente de imagen multimodal.Para la activación del sistema inmune se ha biofuncionalizado el sistema con un patrón molecular asociado a patógeno (PAMPs) de interés clínico, llamado poly (I:C) (Polyinosinic:polycytidylic acid). Este PAMP es capaz de activar un receptor tipo Toll (TLR3) presente en las células del sistema inmune.La capacidad anticancerígena, se consigue mediante a la incorporación en el sistema de un complejo de Pt (IV) inerte que actúa como profármaco de cisplatino y que junto con el poly (I:C) como agente inmunoestimulador se usan para combinar los efectos de dos terapias complementarias para matar células tumorales in vitro e in vivo.La incorporación de fluorescencia en el sistema mediante un fosfolípido modificado con rodamina B disponible en el mercado, así como el uso del agente radioactivo fac-[99mTc(OH2)3(CO)3]+ y las propiedades magnéticas intrínsecas del sistema, permitieron la visualización del mismo in vitro e in vivo mediante diferentes técnicas de imagen molecular

The role of oxygen on the interaction of Candida albicans with intestinal epithelial cells

The opportunistic fungal pathogen Candida albicans frequently occurs as commensal in the gastrointestinal tract of humans. While it is known that intestinal epithelial cells are highly sensitive to ischemia-reperfusion (IR) injury, the exact role of oxygen for IR-mediated fungal translocation has however not been addressed so far. Thus, the aim of this study was to investigate C. albicans-enterocyte interactions in dependence of oxygen availability. Enterocytes were able to maintain barrier function across a range of oxygen concentrations. Their susceptibility to C. albicans infection was however significantly influenced by oxygen: The shift from low to high oxygen enhanced susceptibility to infection, likely mediated by increased intracellular oxidative stress and alterations of tight junctions facilitating increased invasion of the fungus. In contrast, low oxygen concentrations and especially hypoxic shock were associated with less damage and partially retained barrier function. Additionally, fungal translocation was reduced after hypoxic shock. HIF-1α contributed to the protective effect, independent of the antimicrobial cathelicidin LL-37. Furthermore, peracute hypoxic preconditioning, compared to enterocytes constantly cultured at low oxygen, led to enhanced adhesion but reduced invasion and reduced hyphal growth of C. albicans, suggesting that oxygen-mediated changes of enterocytes also directly influence the fungus. Fungal adaptation to oxygen availability contributes to these interactions, as a C. albicans TYE7 deletion mutant showed oxygen-dependent alterations in virulence. Finally, this study provides evidence that the early stages of the infection process determine the fate of enterocytes during their interaction with C. albicans in an oxygen-dependent manner. In summary, this study demonstrates that oxygen availability significantly influences the interaction between C. albicans and the intestinal barrier by affecting both host cells and pathogen