Toxoplasma gondii ist ein obligat intrazellulär lebender einzelliger Parasit, der sich durch ein breites Wirtsspektrum sowie lebenslang persistierende Infektionen bei Menschen und Tieren auszeichnet. T. gondii hat für ein langfristiges Überleben unterschiedliche Mechanismen entwickelt, die ein Gleichgewicht zwischen der Pathogenität des Erregers und der intakten Immunabwehr des Wirtes gewährleisten. In diesem Kontext stellt die Modulation von Signalwegen der Wirtszelle eine wichtige Überlebensstrategie von Toxoplasmen dar. Frühere Arbeiten mit murinen Monozyten haben gezeigt, dass T. gondii die Expression von MHC-Klasse-II-Molekülen auf der Oberfläche infizierter Wirtszellen hemmt und dadurch eine effektive Antigenpräsentation an T-Helfer-Lymphozyten verhindert. Das Ziel der vorliegenden Dissertation war es herauszufinden, inwieweit Toxoplasma gondii auch die Interferon-γ-induzierte MHC-Expression von Monozyten des Menschen vermindert. Analysen mittels Immunfluoreszenzfärbung und Durchflusszytometrie zeigten, dass sowohl primäre, aus PBMC des Menschen isolierte Monozyten als auch permanente humane Monozyten (THP-1) durch eine Infektion mit T. gondii in der Expression von HLA-A, -B, -C und HLA-DR, -DP, -DQ signifikant gehemmt werden. Das Ausmaß der Inhibition ist dabei von der Infektionsdosis des Parasiten abhängig und betrifft sowohl die HLA-Expression auf der Zelloberfläche als auch den intrazellulären HLA-Pool. Interessanterweise kann dieser Effekt auch durch hohe Konzentrationen des stimulierenden Zytokins Interferon-γ nicht aufgehoben werden. Es zeigt sich außerdem eine signifikant reduzierte Expression von HLA-DR, -DP, -DQ bei Parasit-negativen Zellen einer T. gondii-infizierten Kultur, was möglicherweise durch sezernierte Proteine des Parasiten oder durch die Produktion hemmender Zytokine durch infizierte Wirtszellen begründet sein kann. Die HLA-Expression in in vitro-infizierten und nicht-infizierten primären Monozyten wurde darüberhinaus zwischen T. gondii-negativen Individuen und Spendern mit chronischer Toxoplasmose verglichen. Chronisch mit T. gondii infizierte Blutspender wurden serologisch anhand von spezifischen IgG-Antikörpern identifiziert. Durchflusszytometrische Analysen zeigten, dass Monozyten aus chronisch mit T. gondii infizierten Blut-Spendern signifikant mehr HLA-A, -B, -C und HLA-DR, -DP, -DQ exprimieren als Monozyten aus Toxoplasma-negativen Spendern. Eine Erklärung für diese gesteigerte MHC-Expression könnte eine Dominanz bestimmter Monozyten-Subpopulationen in Abhängigkeit vom Infektionsstatus ihres Spenders sein. Die Expression von HLA-A, -B, -C und HLA-DR, -DP, -DQ wird jedoch sowohl bei Monozyten von T. gondii-positiven als auch nicht-infizierten Individuen durch eine Infektion mit Toxoplasmen in vitro signifikant inhibiert. Analysen mit Hilfe von RT-qPCR zeigten deutlich, dass T. gondii mit der HLA-DR-, -DP-, -DQ-Expressions-Kaskade interferiert und die Synthese der Transkripte von IRF-1 und CIITA dosisabhängig inhibiert. Außerdem sind die Transskripte für HLA-A und HLA-DRα in infizierten Monozyten deutlich reduziert. Dies legt die Annahme nahe, dass T. gondii die Aktivierung von STAT1-abhängigen Promotoren effektiv inhibiert und so die Synthese der sich anschließenden HLA-Expressionskaskade supprimiert. Die Ergebnisse dieser Arbeit eröffnen interessante Ansätze für weitere Untersuchungen, insbesondere eine genauere Charakterisierung von Monozyten-Subpopulationen bei T. gondii-positiven Individuen sowie die Erforschung einer möglicherweise gesteigerten Immunreaktivität gegen andere Infektionserreger im Rahmen einer chronischen Toxoplasmose.

Das Oberflächenepithel (OSE) des Ovars besteht aus einer Schicht flacher oder kubischen Zellen, die am Hilus in das flache Peritonealepithel übergehen. Das OSE zeigt zyklusabhängige Veränderungen. OSE-Zellen sollen am Prozess der Ovulation aktiv teilnehmen und die Läsion nach der Follikelruptur reparieren. Die mitotische Aktivität der OSE-Zellen um den Reparatur-Prozess könnte das Überleben von mutierten Zellen begünstigen und zum Ovarialkarzinom führen. Hierbei spielen Zell-Zell-Kontakte eine Schlüsselrolle bei der Integrität von Gewebe und der Metastasierung von Tumoren. Um das Verständnis über die Pathogenese des Ovarialkarzinoms zu verbessern, untersuchte die vorliegende Arbeit die Zell-Zell-Kontakte des OSE sowie dessen Abhängigkeit von Interferon-γ (IFN-γ), welches u.a. bei der adjuvanten Therapie des Ovarialkarzinoms verwendet wird. Abstriche von humanen und bovinen Ovarien dienten als Quelle zur Kultivierung von OSE-Zellen. Konfluente Kulturen wurden mit 200 U/ml rekombinantem, speziesspezifischem IFN-γ für 72 h behandelt oder als Kontrolle unbehandelt gelassen. Die Morphologie der OSE-Zellen vor und nach der Behandlung wurde dargestellt. Weiterhin wurden mittels immunzytologischer Färbungen sowie Western Blot Analyse E- und N-Cadherin, β-Catenin, Cytokeratin sowie Vimentin nachgewiesen. Permeabilitätsmessungen von Meerrettichperoxidase (HRP) in einem Ko-Kultursystem wurden mit und ohne IFN-γ durchgeführt. Die Arbeit zeigt eine neue Wirkung von IFN-γ. Es hat die besondere Eigenschaft, das OSE komplett über Cadherin-vermittelte „tight juctions“ abzudichten. Dies könnte die Wirksamkeit des Zytokins bei der adjuvanten Therapie des Ovarialkarzinoms erklären. Möglicherweise werden Interzellularkontakte verstärkt und die Frühinvasion maligner Zellen eingeschränkt. Das erstmals beschriebene Cadherinmuster an den Zell-zu-Zell-Ecken verweist auf die Interaktion der „tight junctions“ mit E-Cadherin.

Interferons are known cytokines that display antiviral, anti-proliferative and immuno-modulatory functions in the host. Interferon-gamma (Ifnγ) is the only type II family interferon that binds to the heterodimeric receptor consisting of two subunits, IfnγR1 and IfnγR2. This specific interaction between Ifnγ and its receptor triggers the activation of Janus Kinase (Jak) – Signal Transducer and Activator of Transcription (Stat) pathway. This triggers a cascade of events leading to modulation of a wide variety of genes resulting in a plethora of responses including antimicrobial activities, induction of Major Histocompatibility Complex encoded molecules etc. The impact of Ifnγ in regulating host defense is observed in patients who lack functional IFNγ or its receptor as they succumb to less virulent strains of intracellular bacteria such as Mycobacterium and Salmonella. Also, mice lacking important downstream signaling components such as Stat1 and Interferon Regulated Factor 1 (Irf1) are known to be highly susceptible to a variety of bacterial and viral infections. Consequently, studies on uncharacterized signaling and regulatory molecules downstream to Ifnγ are of great interest. The modulatory functions of Ifnγ have been attributed to its ability to regulate the expression of a vast number of genes in a Stat1 and Irf1 dependent manner. Also, gene regulation in response to Ifnγ in a target cell such as mouse hepatoma cell line, H6, can be categorized broadly into two subsets: Reactive Oxygen Species (ROS) - Reactive Nitrogen Intermediates (RNI) dependent (e.g. Nos2, Catalase, Id2 etc.) as well as ROS – RNI independent (e.g. Tap1, Lmp2 etc.). However, the effect of Ifnγ induced ROS and RNI in the regulation of the expression of genes at the level of transcriptome and how these could impact cellular and host responses are not well characterized. To investigate these questions, we standardized an in vitro Ifnγ responsive primary cell culture system using mouse adherent peritoneal macrophages (PMs). It needs to be highlighted that this study has, primarily, utilized unstimulated resident PMs. The adherent cells from the peritoneal cavity were positive for macrophage markers such as F4/80 and CD11b, but negative for granulocyte marker Gr1. Also, PMs were resistant to Ifnγ induced cell death, unlike cell lines such as the mouse fibroblast cell line L929, for the time points studied. There are three distinct parts to this study involving the system of PMs: I. To understand the contribution of Nitric Oxide (NO) in regulating the expression of novel Ifnγ responsive genes, PMs from C57BL/6 mice and mice lacking nitric oxide synthase 2 (Nos2-/-), the enzyme isoform responsible for the generation of NO in PMs, were stimulated with Ifnγ for 8 h and microarray analysis was performed. Detailed analysis led to identification of several annotated genes that were uniquely regulated in C57BL/6 and Nos2-/- PMs. Further analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified several differentially regulated pathways. Interestingly, a large number of metabolism related pathways, Butirosin and neomycin, Galactose, Phenylalanine and glyoxylate and dicarboxylate, were specifically up-regulated in the C57BL/6 PMs treated with Ifnγ. Similarly, other metabolism related pathways were differentially regulated by Ifnγ in PMs from C57BL/6 and Nos2-/- mice. One of the pathways that was up regulated in a Nos2 dependent manner in the C57BL/6 PMs upon Ifnγ treatment was that of circadian rhythm, which consisted of genes Per1, Bhlhb2 and Bhlhb3. All three are known circadian rhythm regulators, with Bhlhb2 and Bhlhb3 being transcriptional repressors that bind to E-box consensus sequence (CANNTG) as heterodimers, using the basic helix-loop-helix (bHLH) domains, along with other transcriptional regulators. Both Bhlhb2 and Bhlhb3 were up regulated at RNA and protein levels in a kinetic manner upon Ifnγ treatment in L929 cells. Studies with inhibitors to ROS and RNI revealed that up regulation of Bhlhb2 and Bhlhb3 was RNI, but not ROS, dependent in response to Ifnγ. Interestingly, the RNI inhibitor, NG-Methyl-L-Arginine (LNMA) rescued Ifnγ induced ROS. On the other hand, ROS inhibitors, e.g. Apocyanin and polyethylene glycol Catalase (PEG-Catalase), did not affect the nitrite amounts in the supernatant. These experiments suggested that RNI was upstream to ROS in L929 cells and both contributed to Ifnγ induced cell death. The knockdown of Bhlhb3 using specific siRNAs in the untreated L929 cells increased Bhlhb2 amounts, but not vice versa. This observation is consistent with the fact that Bhlhb3 is a known repressor of Bhlhb2. However, this repression of Bhlhb2 by Bhlhb3 was not detected upon Ifnγ treatment in L929 cells possibly because of heterodimerization of Bhlhb3 with other Ifnγ induced transcriptional modulators. Finally, knockdown of either of the proteins did not affect induced nitrite but decreased ROS amounts resulting in significant rescue of Ifnγ induced cell death of L929 cells. Thus, Bhlhb2 and Bhlhb3 are novel Ifnγ induced proteins that are NO dependent and contribute to Ifnγ induced cell death. Ifnγ and Nos2 are known to elicit antibacterial defense in the host. Interestingly, recent studies have implicated circadian rhythm to regulate bacterial infection in mice. Therefore, regulation of both Bhlhb2 and Bhlhb3 upon Ifnγ treatment and during Salmonella enterica Serovar Typhimurium (S. typhimurium) infection in the bone marrow derived macrophages (BMDMs) was performed. Both Bhlhb2 and Bhlhb3 were induced in a Nos2 dependent manner upon Ifnγ addition in BMDMs. Similar to L929 cells, Bhlhb3 repressed Bhlhb2 in the untreated BMDMs. Also, infection of BMDMs with S. typhimurium increased the protein amounts of Bhlhb2, while repressing Bhlhb3. Importantly, knockdown of Bhlhb2 resulted in higher colony forming units (CFU), whereas knockdown of Bhlhb3 reduced CFU in BMDMs 18 h post infection with S. typhimurium. Thus, Bhlhb2 induced whereas Bhlhb3 repressed antibacterial defense in BMDMs. The exact mechanism downstream to these two proteins and their inter-relationship in regulating S. typhimurium infection is of considerable interest and will be studied in future. II. Macrophages are known to produce a large number of different cytokines and chemokines upon activation. To identify novel cytokines and chemokines that may be differentially regulated in response to Ifnγ, a protein array was performed using the supernatants of C57BL/6 PMs treated with and without Ifnγ. Chemokine Ccl3 was found to be repressed by Ifnγ in the supernatant of PMs. Further analysis using Enzyme Linked Immuno-Sorbent Assay (ELISA) revealed that both Ccl3 and Ccl4, highly homologous proteins that chemo-attract almost all types of leukocytes, were repressed upon Ifnγ treatment. This response was ligand and cell specific as Lip polysaccharide (LPS) stimulation of PMs and Ifnγ stimulation of thioglycollate elicited PMs did not result in repression of Ccl3 and Ccl4. Also, studies with fludarabine, an inhibitor to Stat1, revealed that the repression of Ccl3 and Ccl4 as well as induction of Cxcl10 in response to Ifnγ was Stat1 dependent. Importantly, the use of LNMA as well as PMs from Nos2-/- mice established the role of Nos2 in the repression of Ccl3 and Ccl4, but not Cxcl10 induction, in response to Ifnγ. Furthermore, activation of p38 Mapk, but not Jnk, was downstream to Nos2 activation and contributed functionally to the repression of Ccl3 and Ccl4 in response to Ifnγ. Finally, the transcriptional repressor, Activating Transcription Factor 3 (Atf3), was induced in a Stat1-Nos2-p38 Mapk dependent manner and knockdown of Atf3 using siRNAs established the functional role of the same in the repression of Ccl3 and Ccl4 in response to Ifnγ. Further, to understand the regulation of Ccl3 and Ccl4, their modulation upon S. typhimuirum infection of BMDMs was performed. Apart from regulating the CFU in BMDMs, Ifnγ and Nos2 functionally repressed Ccl3 and Ccl4 upon S. typhimurium infection. Oral infection of mice with S. typhimurium was performed and mice lacking Ifnγ and Nos2 were found to have greater CFU in their organs as well as more leukocytes in the infected liver sections in comparison to the infected C57BL/6 mice. Importanly, absence of Ifnγ as well as Nos2 increased the amounts of Ccl3 and Ccl4 in the sera upon S. typhimurium infection in comparison to the C57BL/6 infected mice. Overall, this part of the study identified Ifnγ and Nos2 to repress chemokines Ccl3 and Ccl4 in macrophages and in mice upon S. typhimurium infection. III. While working on the above mentioned studies, it was noticed that addition of Ifnγ to PMs induced in a dose and time dependent manner aggregation of cells. Experiments with LPS, TG PECs and BMDMs established that Ifnγ induced aggregation of PMs was ligand and cell type specific. A panel of cell surface integrins and selectins were screened for regulation upon Ifnγ addition, namely Icam1, Lfa1, CD11b, P-selectin and E-selectin. Most of the cell surface integrins were repressed by Ifnγ in a kinetic manner. Interestingly, CD11b as well as E-Selectin co-localized to the sites of interactions between the PMs upon Ifnγ treatment. Studies with Reopro, a purified F(ab’)2 to glycoprotein GPIIb that is also known to functionally block CD11b, revealed the functional contribution of CD11b during Ifn induced aggregation of PMs. Further, studies with specific inhibitors identified RNI, but not ROS, to contribute to Ifnγ induced PM aggregation. Also, lack of Nos2 in PMs upon Ifnγ treatment resulted in minimal aggregation together with morphological changes, e.g. flattening of cells. Since differences in the morphology of PMs was observed in the absence of Nos2 upon Ifnγ treatment, the regulation and roles of cytoskeleton proteins, Actin and tubulin, during Ifnγ induced aggregation of PMs was studied. Upon Ifnγ stimuli, actin and tubulin get stabilized. On the other hand, the absence of Nos2 leads to depolymerization of actin, while tubulin failed to stabilize to the membrane, in response to Ifnγ. Further, addition of actin and tubulin depolymerizing agents, Cytochalasin D and Colcemid respectively, decreased Ifnγ induced aggregation of PMs. Live cell imaging studies revealed that PMs needed actin, but not tubulin or CD11b, for mobility. Upon Ifnγ treatment, PMs from C57BL/6 mice exhibited reduced mobility and aggregated with each other. The Nos2-/- PMs exhibited lower mobility compared to C57BL/6 PMs and, upon Ifnγ treatment, underwent morphological changes with time, e.g. flattening. On the other hand, Nos2 is important for endogenous mobility and maintaining the cellular morphology in response to Ifnγ. To understand the physiological relevance of our observations, oral infection of C57BL/6 and Nos2-/- mice with S. typhimurium was performed. Four days post infection, no significant differences in the number of peritoneal cells were found. Importantly, PMs from infected Nos2-/- mice had higher CFU in comparison to C57BL/6 mice. However, the amounts of cytokines such as Ifnγ, Tnfα, Il6 and Il1β in the peritoneal lavage were not significantly different between the two infected strains. Interestingly, PMs isolated from infected Nos2-/- mice displayed distinct morphology, e.g. flattening. In comparison, infected C57BL/6 PMs aggregated when cultured for 24 h in vitro. In the future, it will be interesting to address the functional roles of aggregates of macrophages during physiologically relevant responses such as combating intracellular bacterial infection. This part of the study adds a new dimension to the ability of Ifnγ in the regulation of macrophage-macrophage interactions and their roles during intracellular bacterial infections. Overall, the present study has elucidated hitherto uncharacterized roles of Nos2 and mechanisms involved in regulation of novel functional responses of PMs to Ifnγ and during S. typhimurium infection.

Infektionen mit H. pylori führen zu Veränderungen im Aufbau und der zellulären Zusammensetzung des Magenepithels. Aktuelle Studien haben gezeigt, dass Stromazellen, die sich in unmittelbarer Nähe der epithelialen Stammzelle befinden, die funktionelle Nische für dieses Kompartiment bilden und wichtige Faktoren für die Regulierung des Stammzellumsatzes und Differenzierung darstellen. Daher konzentriere ich mich bei dieser Arbeit auf die Rolle der Myh11+ Myofibroblasten, die sich sowohl unter als auch zwischen den Epitheldrüsenzellen befinden. Ich fand heraus, dass die Myofibroblasten in der Homöostase einen BMP-Gradienten entlang der Drüsenachse mit erhöhter Expression von Bmp2 im oberen Teil erzeugen, während die Drüsenbasis von Zellen umgeben ist, die Bmp-Inhibitoren produzieren. Basierend auf der Funktionsanalyse mit 3D-Organoidmodellen ist der Bmp-Signalweg ein zentraler Faktor für die rasche Differenzierung von Stammzellen in faveoläre mukusproduzierende Zellen. Darüber hinaus wurde ein auto-parakriner Signalweg gefunden, der zur Bildung von Bmp2 in Epithelzellen führt. Meine Untersuchungen Daten zeigten eine Verringerung des BMP-Signalwegs in H. pylori-infizierten Myofibroblasten. Darüber hinaus führte eine Infektion mit H. pylori nicht nur zum Verlust der stromalen, sondern auch der epithelialen Bmp2-Expression. Diese Beobachtung ging mit einer Zunahme von IFNγ einher, was auf eine Verbindung zwischen beiden Signalwegen hinwies. Tatsächlich zeigten anschließende Experimente mit Organoiden, dass IFNγ den Bmp-Signalweg hemmt und dadurch die terminale Differenzierung blockiert. Zusammenfassend zeigen meine Untersuchungen, dass das Schicksal einer Zelle, die zur Oberfläche der Magendrüse wandert, eine Induktion des BMP-Signalwegs erfährt. Die Reduktion dieses Signals durch IFNγ deutet auf einen Mechanismus hin, der Veränderungen in der zellulären Differenzierung und die Entwicklung von prämalignen epithelialen Läsionen im Verlauf einer H. pylori-Infektion bewirkt. / Infection with Helicobacter pylori (H. pylori) leads to alterations of the topology and cellular composition of the gastric epithelium. Recent studies have shown that stromal cells located in close proximity to the epithelial stem cell are creating the niche for this compartment and provide crucial factors that regulate stem cell turnover and fate decisions. Therefore, this thesis focuses on of Myh11+ myofibroblasts that are located beneath and between the epithelial gland cells. I found that during homeostasis, the myofibroblasts generate a BMP gradient along the gland axis with strong expression of Bmp2 in the upper part, whereas the base is surrounded by cells producing BMP inhibitors. Based on functional analysis with 3D organoid models, the BMP gradient occurs to be a main factor responsible for rapid differentiation of stem cells into pit surface mucous cells. Moreover, experiments led me to identify an auto-paracrine feed-forward BMP2 loop in epithelial cells, which further stabilizes BMP signaling once it is activated and induces terminal differentiation. Data presented in this study demonstrated a reduction of BMP signaling in H. pylori-infected myofibroblasts. Furthermore, infection with H. pylori resulted in loss of not only stromal, but also epithelial Bmp2 expression. This observation was accompanied with increase of Interferon γ (IFNγ), which indicated a link between both pathways. Consistently, stimulation of organoids with IFNγ impairs BMP signaling and the BMP2 feed-forward loop, and thereby blocks terminal differentiation. Together, this study shows that the fate of a cell migrating into the surface of the gastric gland is determined by an induction of BMP signaling and stabilized by an auto-paracrine BMP signaling enhancement. Reduction of this signaling by IFNγ revealed a mechanism which contributes to altered cellular differentiation and development of premalignant epithelial alterations in the context of H. pylori infection.

Studien utvärderar egenskaper hos mesenkymala stromaceller (MSC) i passage 2 och 3. I ett samarbete mellan Cellcolabs och Karolinska Institutet (KI) genomfördes projektet med Katarina Le Blancs forskargrupp. Genom att studera membranmarkörer från tre friska MSC-donatorer, tillsammans med deras förmåga att differentiera till osteoblaster, adipocyter och kondrocyter, samt deras förmåga att inhibera profileringen av CD8+ och CD4+ T-lymfocyter, och slutligen deras möjlighet att öka uttrycket av Indoleamine 2,3-dioxygenase 1 (IDO1) och interleukin 6 (IL6), kunde en jämförelse mellan passage 2 och 3 göras. I korta drag kunde enbart en tydlig skillnad göras mellan de två passagerna. Skillnaden sågs i förmågan att differentiera till osteoblaster, där passage 3 inte kunde prestera på samma nivå som passage 2. Utöver detta var resultaten för de två typerna jämförbara och antydde inte till några större förändringar mellan passage 2 och 3. För att stärka tillförlitligheten av resultatet bör dock fler MSC-donatorer jämföras. / The study evaluates the characteristics and consistency of mesenchymal stromal cells (MSCs) in two different passages, specifically 2 and 3. In a collaboration between Cellcolabs and Karolinska Institutet (KI), the project was conducted with Katarina Le Blanc’s research group. Through studying the surface expression on cells from three distinct MSC donors, along with their differentiation ability into osteoblasts, adipocytes and chondrocytes, their capability to suppress the proliferation of CD8+ and CD4+ T lymphocytes, and finally their possibility to increase the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and interleukin 6 (IL6), a comparison between passage 2 and 3 could be done. It was seen that a clear distinction could be made between the two passages while looking at their ability to differentiate into osteoblasts. The remaining results showed comparable outcomes between passage 2 and 3, suggesting minor changes with the increased passage number. However, to strengthen the reliability of the outcome, more MSC donors should be compared.

Die Epithelzellen des Dünndarms bilden die Hauptinfektionsroute für viele Protozoen wie zum Beispiel Toxoplasma gondii und Giardia duodenalis. Jedoch sind die Mechanismen dieser Infektionswege unbekannt, da geeignete Modelle fehlen, welche das Darmepithel nachbilden. In der folgenden Studie wurde eine in-vitro Plattform mit Darmorganoiden (organoid derived monolayers oder ODMs) etabliert, welche man für vergleichende Studien zu Parasit-Wirt-Interaktionen anwenden kann. Das ODM-System wurde angewendet, um die Anfangsphase einer T. gondii-Infektion zu modellieren, wobei der Schwerpunkt auf die Rolle von Interferon gamma (IFNγ) und immunitätsbezogenen GTPasen (Irgs) lag. Es wurde gezeigt, dass sich die Irg-abhängige Kontrolle virulenter Toxoplasma-Stämme zwischen dem Labormausmodell und anderen wildlebenden Nagetierarten unterscheidet. Aus diesem Grund wurden Vergleiche mit Organoiden verschiedener Labormausstämme und der Rötelmaus Myodes glareolus durchgeführt. Myodes glareolus ist eine Nagetierart, von der angenommen wird, dass sie eine höhere Resistenz gegen T. gondii aufweist. Basierend auf die Resultate der quantitativen Immunofluoreszentests und qPCR dieser These, führt die Stimulation mit IFNγ zu einer tendenziell verringerten Replikation der Parasitenstämme RH und Prugniaus in M. glareolus ODMs im Vergleich zu Mus ODMs. In dieser Studie, wurde zum ersten Mal die Rolle von Irgs bei intestinalen T. gondii-Infektionen identifiziert. Zu diesem Zweck wurden Organoide von M. glareolus mit einem fluoreszierend markierten Irgb10-Protein transfiziert, wodurch gezeigt werden konnte, dass Irgb10 T. gondii-Vakuolen dekoriert, was auf eine Beteiligung des Irg-Systems hindeutet. Schließlich wurde ein Koinfektionsmodell für T. gondii und/oder G. Duodenalis in Maus-ODMs etabliert. In diesem Modell wurde gezeigt, dass T. gondii weder die Induktion der Barrierestörung durch G. duodenalis noch die Replikation von T. gondii durch G. duodenalis beeinflusst. / The small intestinal epithelium is the primary route of infection for many protozoan parasites such as Toxoplasma gondii and Giardia duodenalis. Understanding the mechanisms of infection with such parasites, has been hindered due to the lack of appropriate models mimicking the complexity of the intestinal epithelium. Here, an in vitro platform was established, using intestinal organoids (organoid derived monolayers or ODMs) for comparative studies on parasite-host interactions. The ODM system was used to model the events during the early phase of a T. gondii infection, focusing on the role of Interferon gamma (IFNγ) and Immunity Related GTPases (Irgs). Irg dependent control of virulent Toxoplasma strains has been shown to differ between the laboratory mouse model and other wild-derived rodent strains. How these responses occur in rodent species that do not belong to the murine family, is yet to be determined. For this reason, comparisons were made with organoids from different laboratory mice strains and the bank vole Myodes glareolus, a non-muridae rodent species assumed to be more resistant to T. gondii. Based on this thesis, stimulation with IFNγ leads to a trend of reduced replication in M. glareolus ODMs compared to Mus ODMs for both Type I parasite strain RH and for Type II strain Prugniaud, based on quantitative immunofluorescence assays and qPCR. Analysis of the role of Irgs in intestinal T. gondii infections was performed, by transfecting organoids from M. glareolus with a fluorescently labelled Irgb10 protein, showing that Irgb10 decorates T. gondii vacuoles, suggesting Irg-system involvement. Finally, a co-infection model in murine ODMs was established for T. gondii and/or G. Duodenalis. Here, it could be observed that T. gondii did not influence G. duodenalis induction of barrier breakdown nor did G. duodenalis influence T. gondii replication.

Rhodococcus equi (R. equi) is one of the most serious causes of pneumonia in young foals. The clinical disease is of great concern to breeding farms worldwide due to the impact of mortality on economic losses. While adult horses are resistant to R. equi, foals exhibit a distinct age-associated susceptibility. The mechanism underlying this susceptibility in foals is not well understood. Interferon-gamma (IFNg) plays an important role in the clearance of R. equi, but its expression is impaired in neonatal foals. Moreover, the regulation of this age-related IFNg expression in foals remains unknown. In humans, IFNg expression has been shown to be regulated by DNA methylation, lymphoproliferation, and influenced by environmental exposure. Therefore, we hypothesized that environmental exposure promotes IFNg expression through regulation of DNA methylation and lymphoproliferation. The objectives were: (1) to estimate the relevance of IFN-g production and R. equi infection in foals; (2) investigate the role of lymphoproliferation and DNA methylation in the regulation of IFN-g expression in foals; (3) to evaluate the effect of environmental exposure on IFN-g expression by housing foals in a barn environment verses pasture.; (4) to investigate the effect of environment exposure on antigen-presenting cells (APC), which sensor the environmental antigens and modulate IFN-g production by T cells. The results demonstrated that the IFN-g expression was inversely correlated with the age-related susceptibility to R. equi infection. lymphoproliferation promoted IFN-g expression in foals, whereas, DNA methylation repressed IFN-g expression. The IFN-g expression was augmented in foals exposed to the barn air which contained higher numbers of aerosol miroorganisms. DNA on the IFN-g promoter was demethylated and the lymphoproliferative activity was elevated in foals with barn-air exposure. The barn-air exposure also promoted the maturation and activation of APC to prime IFN-g expression by T cells in foals. Overall, this body of work demenstrated a relationship between IFN-g expression and R. equi infection, provided novel information on mechanisms that regulate IFN-g expression, and identified the effect of environment on mechanisms responsible for IFN-g expression.

Background: Migrant farmworkers are among the highest-risk populations for latent TB infection (LTBI) in the United States with numerous barriers to healthcare access and increased vulnerability to infectious diseases. LTBI is usually diagnosed on the border using the tuberculin skin test (TST). QuantiFERON-TB Gold In-Tube (QFT-GIT) also measures immune response against specific Mycobacterium tuberculosis antigens. The objective of this study is to assess the comparability of TST and QFT-GIT to detect LTBI among migrant farmworkers on the border, as well as to examine the effects of various demographic and clinical factors on test positivity. Methods: Participants were recruited using mobile clinics on the San Luis US-Mexico border and tested with QFT-GIT and TST. Demographic profiles and clinical histories were collected. Kappa coefficients assessed agreement between TST and QFT-GIT using various assay cutoffs. Logistic regression examined factors associated with positive TST or QFT-GIT results. Results: Of 109 participants, 59 of 108 (55 %) were either TST (24/71, 34 %) or QFT-GIT (52/106, 50 %) positive. Concordance between TST and QFT-GIT was fair (71 % agreement,kappa= 0.38, 95 % CI: 0.15, 0.61). Factors associated with LTBI positivity included smoking (OR = 1.26, 95 % CI-1.01-1.58) and diabetes/high blood sugar (OR = 0.70, 95 % CI = 0.51-0.98). Discussion: Test concordance between the two tests was fair, with numerous discordant results observed. Greater proportion of positives detected using QFT-GIT may help avoid LTBI under-diagnosis. Assessment of LTBI status on the border provides evidence whether QFT-GIT should replace the TST in routine practice, as well as identifies risk factors for LTBI among migrant populations.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Manuel Borca / Stephen Higgs / African swine fever (ASF) is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus. ASFV is endemic in Sardinia and Saharan Africa and has been recently expanded from the Caucasus to Eastern Europe. There is no vaccine to prevent the disease and current control measures are limited to culling and restricted animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pret4 virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus) is completely attenuated. Swine infected with Pret4 Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 dpi showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi). This animal model was used to associate the presence of host immune response and protection against the challenge. Anti-ASFV antibodies and cytokines in serum, as well as ASFV-specific IFN-γ production in PBMCs, were assessed in each group. Interestingly, with the exception of ASFV-specific antibodies in the surviving swine challenged at 21 and 28 dpi, no solid association between any of the parameters assessed and the extent of protection could be established. These results were corroborated using a similar model based on the use of a rationally attenuated derivative of the highly virulent strain Georgia 2007. These results, encompassing data from 114 immunized swine, underscore the complexity of the system under study where it is very plausible that protection against disease or infection relies heavily on the concurrence and or interaction of different host immune mechanisms.

O objetivo central desta dissertação de mestrado foi analisar se, pela sua resposta ao interferon-g (IFNg), as células não leucocitárias contribuem ao controle dos protozoários Trypanosoma cruzi, Plasmodium chabaudi AS e Plasmodium berghei ANKA. O IFNg é uma citocina que promove a ativação de diversos tipos de leucócitos, a sua ação sobre as células mononucleares fagóciticas merece um destaque especial. Apesar de conhecermos os pormenores do papel desta citocina na ativação dos leucócitos, desconhecemos se o IFNg exerce ação ativadora sobre as células estruturais (não leucocitárias), ou seja, sobre as células não profissionais da resposta imune. A nossa hipótese de trabalho é que, no caso dos parasitas intracelulares, o IFNg poderia reforçar a ação sinalizadora e efetora das células estruturais infectadas. Por outro lado, em ambas as situações de parasitas intracelulares e extracelulares, o IFNg, ao agir sobre diversas células estruturais, poderia induzir a produção de mediadores inflamatórios (citocinas, quimiocinas, etc) que contribuiriam direta ou indiretamente à remoção/controle do parasita. A nossa abordagem tem sido o estudo da infecção por estes protozoários em quimeras de medula óssea B6/IFNgRKO, nas quais as células não leucocitárias são deficientes em receptores para IFNg e as células leucocitárias são normais. A análise por imunofluorescência de cortes histológicos mostrou uma alta expressão de IFNgR pelas células estruturais do coração dos animais B6 e quimeras controle B6/B6, mas nenhuma expressão deste receptor nos cortes histológicos correspondentes de animais IFNgRKO e quimeras experimentais B6/IFNgRKO, apesar de grande parte dos leucócitos dos animais deste último grupo ter se tornado IFNgR+. Após infecção pelo T. cruzi, o coração e músculo esquelético dos animais quiméricos B6/IFNgRKO mostraram maior carga parasitária e menor intensidade dos infiltrados 8 inflamatórios do que aqueles dos animais quiméricos B6/B6, resultados que sugerem o envolvimento das células estruturais no controle do parasita e promoção do recrutamento leucocitário. Na infecção pelo Plasmodium chabaudi AS a análise comparativa dos grupos IFNgRKO e quimera B6/IFNgRKO mostrou que no início da infecção as curvas de parasitemia destes grupos são idênticas sugerindo que nesta fase da infecção a presença do IFNgR nos leucócitos em pouco contribui na evolução da parasitemia. Por outro lado, a análise comparativa dos grupos quimera B6/IFNgRKO e quimera B6/B6 mostrou níveis mais elevados de parasitemia e maior índice de mortalidade nos animais B6/IFNgRKO, sugerindo que as células estruturais participam no controle do parasita através da sua resposta ao IFNg. Entretanto, em uma experiência preliminar de infecção pelo Plasmodium berghei ANKA não observamos grandes diferenças entre os animais dos grupos B6/IFNgRKO e B6/B6, não somente no que se refere à curva de parasitemias, como também na indução de morte precoce decorrente de malária cerebral. / The main purpose of our work was to analyze if by their response to Interferon-g (IFN-g), the non-leucocyte cells are able to control Trypanosoma cruzi, Plasmodium chabaudi AS and Plasmodium berghei ANKA protozoans. IFNg was described as a cytokine that promote activation on different types of leucocytes, its action on mononuclear phagocytic cells is important. Despite the fact that this cytokine activate leucocytes, it is unknown whether IFNg activates the structural cells (non-leucocytes), that is, the non-professional cells of the immune response. Our hypotheses suggest that in the case of intracellular parasites, IFNg could help the infected structural cells by increasing their signaling and effect actions. In addition, during the response against intracellular and extracellular parasites, IFNg could induce the production of inflammatory mediators by these cells guaranteeing direct or indirectly the parasite clearance. In the present study, we analyzed the infection of diferente protozoans on bone marrow B6/IFNgRKO chimeras, in which the non-leukocyte cells are deficient in IFNg receptor and the leukocyte cells are normal. Immunofluorescence analyses of histological sections revealed a high expression of IFNgR on the structural cells from the heart of B6 and control chimeras B6/B6 animals, but non-expression of this receptor on histological sections from IFNgRKO and experimental chimeras B6/IFNgRKO, despite the fact that a great part of leucocytes from the last group of animals express the receptor. After T. cruzi infection, the cardiac and skeletal muscle from B6/IFNgRKO chimera animals showed a huge amount of parasite and less infiltration inflammatory than B6/B6 animals, suggesting that the structural cells are involved in the parasite control and leukocyte recruitment. During Plasmodium chabaudi AS infection, comparative analyses from IFNgRKO and 10 B6/IFNgRKO groups showed that parasitemia curves at the early phase are similar, suggesting that during this phase IFNgR expression on leukocytes are not important. On the other hand, parasitemia and mortality levels on B6/IFNgRKO and B6/B6 groups were higher than those on B6/IFNgRKO animals, determining that structural cells participate during the course of infection through their response to IFNg. However, when B6/IFNgRKO and B6/B6 animals were infected with Plasmodium berghei ANKA no significantly difference was observed between these groups related to the course of parasitemia and cerebral malaria.