Orientador: Mary Luci de Souza Queiroz / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-14T06:41:55Z (GMT). No. of bitstreams: 1 Perhs_SimoneMariaCipas_M.pdf: 3198961 bytes, checksum: 7dcc68b78919337c618a237e6794be07 (MD5) Previous issue date: 2009 / Resumo: O modelo experimental de infecção por Listeria monocytogenes tem provado ser útil na investigação dos efeitos de novos compostos na resposta imunológica primária contra bactérias intracelulares. Em particular, o número de células hematopoéticas na medula óssea e baço é de fundamental importância na resistência a esta infecção. No presente estudo, investigamos o efeito da solução hidro alcoólica de própolis (SHAP), sobre o crescimento e diferenciação de precursores hematopoéticos para granulócitos e macrófagos (CFU-GM) da medula óssea e do baço de animais infectados com Listeria monocytogenes. Além disso, quantificamos os níveis de fatores estimuladores de colônias (CSFs). A eficácia da SHAP frente a uma dose letal da bactéria também foi avaliada. Demonstramos que a SHAP protegeu os animais contra uma dose letal de L. monocytogenes, quando administrada profilaticamente na dose de 4 mg/kg durante 10 dias, com aumento de 20% na sobrevida. Este tratamento também preveniu a mielossupressão e hematopoese extramedular causadas por uma dose subletal de L. monocytogenes, devido a um aumento no número de CFU-GM na medula óssea. Além disso, observamos que os animais somente tratados (não infectados) não apresentaram diferenças significativas em relação ao grupo controle. Adicionalmente, a investigação da produção de fatores estimuladores de colônias no soro dos animais revelou um aumento de CSA nos grupos infectados após 48 e 72h pré-tratados com SHAP, sugerindo que a estimulação da mielopoese pelo produto seja mediada pela produção de fatores de crescimento. Além disso, observamos o aumento da citocinas interferon- gamma IFN-g no grupo infectado/tratado com o produto nas 48 e 72h. / Abstract: Infection with Listeria monocytogenes has proven to be a useful model to investigate early effects of new compounds on the immune response to intracellular bacteria. In particular, the number of hematopoietic cells in the bone marrow and spleen is critically important to resistance to this infection. In this study, we demonstrated the protective effects of hydro alcoholic solution of propolis in mice infected with a lethal dose of Listeria monocytogenes, when administered prophylactically at 4mg/kg for 10 days, with survival rates up to 20%. This dose also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with Listeria monocytogenes, due to the maintenance of granulocytemacrophage progenitors (CFU-GM) numbers in the bone marrow. Non-infected mice treated with hydro alcoholic solution of propolis presented similar numbers of CFU-GM in the bone marrow when compared to the controls. Furthermore, investigation of the production of colony-stimulating factors (CSF) revealed increased colony-stimulating activity (CSA) in the serum of infected mice pre-treated with hydro alcoholic solution of propolis 48 and 72 hours after the infection, suggesting that its effects on the production of growth factors mediate stimulation of myelopoiesis by hydro alcoholic solution of propolis. Moreover, we observed increased level of IFN-g in infected/treated mice. These results sustain our proposal of hydro alcoholic solution of propolis use as an adjuvant agent in the prophylaxis of opportunistic infections and reduction of side effects caused by immunosuppressive therapies. / Mestrado / Farmacologia / Mestre em Farmacologia

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.

Orientador: Antonio Condino Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T09:19:02Z (GMT). No. of bitstreams: 1 Prando_CarolinaCardosodeMello_D.pdf: 7021288 bytes, checksum: e93bf24a96be00102eb5a19f5d7c8880 (MD5) Previous issue date: 2008 / Resumo: O sistema NADPH oxidase fagocítico humano possui um papel importante na defesa contra microorganismos intracelulares, incluindo micobactérias. Mutações nas subunidades deste sistema resultam na Doença Granulomatosa Crônica (DGC). O gene CYBB, localizado no cromossomo X, codifica a subunidade gp91phox, e mutações neste gene são responsáveis por cerca de 60% dos casos de DGC. Cerca de 40 anos depois da identificação de DGC, foi identificado o primeiro dos 13 defeitos genéticos associados à Susceptibilidade Mendeliana à Micobacteriose, participantes do eixo IL12/23-IFN-?. Baseado no fato de que ambas as doenças predispõem a infecções por micobactérias e que o IFN-? is é um importante ativador do gene CYBB os autores se propuseram a estudar as características clinicas de pacientes latino-americanos com DGC e o sistema NADPH oxidase e expressão do gene CYBB em pacientes com defeitos no eixo IL-12/23-IFN-?. Em relação às características clínicas: história familiar de infecções graves e/ou de repetição, bem como reação adversa à vacina BCG, linfadenopatia, abscessos de pele e profundos estavam associados à DGC, em comparação com não-DGC avaliados pelo laboratório. Defeitos nos receptores IFNGR1 e IFNGR2 e cadeia B1 do receptor de IL-12 podem apresentar expressão do gene CYBB e atividade do sistema NADPH oxidas e diminuída ou abolida, chegando a níveis comparáveis a um paciente com DGC. O IFN- ? e seus receptores são essenciais para o desenvolvimento e ativação do sistema NADPH oxidase, e pacientes com comprometimento da função deste sistema devem também ser avaliados para defeitos do eixo IL12/23-IFN-? afetando secundariamente o sistema NADPH oxidase. / Abstract: The NADPH phagocytic oxidase system plays a crucial role in host defense against intracellular microorganisms, including mycobacteria. Mutations affecting subunits of this system result in Chronic Granulomatous Disease (CGD). The CYEE gene, located in the X chromosome, encodes gp91 phox, and mutations on this gene account for more than 60% of CGD cases. Almost 40 years after, the first of 13 different genetic disorders associated with Mendelian Susceptibility to Mycobacterial Diseases (MSMD), was identified. The genes responsible for MSMD are part of the IL12/23-IFN-? axis. Based on the fact that both the diseases predispose to mycobacterial infections and that IFN-? is an important activator of CYBB gene, the authors aimed to describe clinical aspects of Latin American CGD patients and investigate ifthe NADPH oxidase system function and gp91phox expression would be affected in patients with defects in the IL-12/23-IFN-? axis. Regarding clinical features familial history of recurrent and sever infections, as well as adverse reactions to BCG vaccine, lymphadenopathy, skin and profound abscess were associated to DGC when compared to clinical features of non-CGD evaluated in the laboratory. Defects of IFNGR1 and IFNGR2 and IL12RB1 may present diminish.ed or abolished gene expression of CYBB and activity of NADPH oxidase system ate levels of a CGD patient. Based on that, we can conclude that IFN- ? and its receptor are essential for development and activation 9f NADPH oxidase system. In addition, patients who present an impaired superoxide release and/or failure on expressing gp91phox should also be evaluated for IL12/23-IFN-? axis affecting secondarily the NADPH oxidase system. / Doutorado / Doutor em Farmacologia

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

The data presented within this thesis shows the generation and characterisation of a complete-, macrophage/granulocyte- and T cell-specific IFNγR2 deficient mouse mutant. This mutant mouse is a valuable tool in dissecting the mechanism of action of the pleiotrophic cytokine IFNγ.The global mutant mouse was tested in three models in vivo - DSS induced colitis, Trichuris muris infection and EAE. The aim of the DSS-induced colitis model was to test the role of IFNγ in the innate immune system and, despite previous reports demonstrating IFNγ deficient mice are protected from DSS-colitis, our IFNγR2 deficient mice displayed equal or more severe colitis than control mice. We hypothesise that this discrepancy is due to differences in the gut microbiota.The Trichuris muris model was utilised as a method of examining the role of IFNγ in the adaptive immune system. The complete IFNγR2 mutant was resistant to a low dose T. muris infection; however, neither the T cell specific nor the macrophage/granulocyte specific mutant duplicated the resistant phenotype observed in the global knock-out mice. Analysis of a double conditional T cell and macrophage/granulocyte specific IFNγR2 mutant produced inconsistent results. Initial experiments suggested that, in combination, these deficiencies are sufficient to duplicate the resistant phenotype observed in the global mutant mice, but this was not reproducible.The final in vivo model that we used to analyse IFNγR2 mutant mice was EAE. This model was chosen as, for a long time, the mechanism of action and the involvement of IFNγ in EAE has been a matter of uncertainty. These results demonstrated that global IFNγR2 mutant mice demonstrate an atypical phenotype, with no signs of recovery. In contrast, control mice develop classical EAE symptoms with almost complete recovery prior to the termination of the experiment. The IFNγ receptor mutant mouse generated will be of great value to the scientific community as IFNγ has been demonstrated to play a role in multiple diseases and this tool allows the mechanism of action of this cytokine to be unravelled.

No description available.

Gegenstand dieser Arbeit ist die Untersuchung zellulärer Testsysteme bei Patienten mit Verdacht auf latente Infektion mit M. tuberculosis. Eine latente Tuberkulose kann unter Immunsuppression zu einer aktiven Tuberkulose werden. Deshalb wird bei immunsuppressiven Therapien insbesondere mit TNF-alpha-Blockern eine Chemoprävention empfohlen. Daher ist es sehr wichtig, latente Infektionen zu erkennen. Ist ein Patient mit M. tuberculosis infiziert, reagieren seine T-Zellen auf Stimulation mit Antigenen wie ESAT-6 und CFP-10. Diese Immunantwort ist die Grundlage der modernen IFN-γ release Assays (IGRA). ESAT-6 und CFP-10 fehlen bei allen BCG-Stämmen und bei den meisten nicht-tuberkulösen Mykobakterien mit Ausnahme von M. kansasii, M. szulgai und M. marinum (Andersen et al. 2000; Behr et al, 1999; Lalvani 2003). Im Gegensatz dazu haben Personen, die mit M. tuberculosis-Komplex-Organismen infiziert sind, in der Regel T-Zellen im Blut, die diese und andere mykobakterielle Antigene erkennen. Ziel der vorliegenden Arbeit war es, die Nutzung von IGRA in der medizinischen Labordiagnostik dahingehend zu analysieren, ob es Unterschiede zwischen ELISPOT-basierten Tests und Röhrchen-Tests als Testformat gibt, inwieweit diese Tests im klinischen Alltag verlässlich sind und ob sich mit einem anderen Auswertealgorithmus eine sicherere Aussage zum Vorliegen einer latenten Tuberkulose treffen lässt. Im Einzelnen wurden dabei drei Ansätze verfolgt: 1. In einer retrospektiven Studie wurden die Ergebnisse von 2686 Patienten ausgewertet, die im Labor des Instituts für Klinische Immunologie des Universitätsklinikums Leipzig von 2013 bis 2016 als Routineuntersuchungen erhoben wurden. Bei klinisch unplausiblen Ergebnissen wurden bei einem Teil der Patienten eine Wiederholungsuntersuchungen durchgeführt. Die analytische Sensitivität und Spezifität sowie den positiven und negativen prädiktiven Wert konnten wir nur unter der Annahme abschätzen, dass der Ausfall in der Wiederholungsuntersuchung einen Hinweis auf falsch- oder richtig-positive oder -negative Werte zulässt. Wir kommen damit zu einer Sensitivität von nur 28 %, einer Spezifität von immerhin 91 %, einem positiven prädiktiven Wert von 32 % und einem negativen prädiktiven Wert von 90 %. Damit sind 68 % der positiven Werte falsch positiv und 10 % der negativen Werte falsch negativ. Unsere Untersuchungen zur Wiederholbarkeit der ELISPOT-Tests im eigenen Labor bestätigen, dass negative Ergebnisse meist wiederholbar sind, positive Werte jedoch skeptisch betrachtet werden müssen. 2. Seit 2012 sendet Instand e.V. zweimal jährlich Ringversuchsproben für den IGRA aus (Ringversuch 650, https://www.instand-ev.de/). Wir analysierten, wie viele Labors bei Teilnahme an der externen Qualitätssicherung (sogenannten Ringversuchen) für IGRA ein korrektes Ergebnis erzielt hatten und ob Unterschiede zwischen ELISPOT-Assay und Röhrchen-Test bestehen. Im Ringversuch waren die Ergebnisse von ELISPOT (z.B. TB-Spot, Oxford Immunotec) und Röhrchentest (Quantiferon Gold bzw. Gold-Plus, Qiagen oder Diasorin) bis auf den 2. Ringversuch 2019 vergleichbar und unterschieden sich nicht. 3. Obwohl die meisten Labore an den Ringversuchen erfolgreich teilnehmen, ist die recht häufige Anzahl vor allem falsch positiver Ergebnisse im diagnostischen Alltag problematisch. Wir haben daher in einem dritten Untersuchungsschritt die Validierungsdaten eines Labors detailliert untersucht, um ein definiertes Verfahren zur Ermittlung positiver Testergebnisse vorzuschlagen. Zwischen 2011 und 2013 erfolgte im Labor Ettlingen eine umfangreiche Qualitätssicherung des ELISPOT unter Nutzung von 70 Proben in Doppelbestimmung. Dabei wurden jeweils die Messungen für die Positiv- und die Negativkontrolle sowie die Werte nach Stimulation mit ESAT-6 und CFP-10 analysiert. Um relevante Unterschiede zwischen Negativkontrolle und der eigentlichen Messung zu bewerten, wurden die Unterschiede mit der Wiederholpräzision in Beziehung gesetzt: Die Unterschiede sollten größer sein als die daraus abgeleitete Ungenauigkeit (Impräzision) der Messungen. Daraus wurde ein Cut-off-Wert kalkuliert, der unmittelbar auf den Daten des Labors beruht. Hierzu ist die Homogenität der Standardabweichungen über alle Proben erforderlich. Sie wird durch die Wurzeltransformation aller Daten erreicht. Für die verwendeten Daten ist sie anwendbar (Altman 1991, Bland 2000). Dies bedeutete bei den untersuchten Daten, dass bei Doppelbestimmungen die Unterschiede zwischen dem Testergebnis immer um den Faktor 0,76 größer sein muss als die Negativkontrolle. Dazu sind allerdings zwei Voraussetzungen zu erfüllen: 1. die Werte müssen vor Analyse wurzeltransformiert werden. 2. benötigt werden mindestens Doppelbestimmungen. Da keine Doppelbestimmungen vorliegen, konnte das Verfahren nicht an einem eigenen Datensatz verifiziert werden. Wir schlussfolgern daraus zusammenfassend: 1. in der Praxis gibt es keine wesentlichen Unterschiede zwischen ELISPOT und Röhrchen-Test als Testformat, was Sensitivität und Spezifität anbelangt 2. IGRAs sind ein verlässliches Werkzeug im klinischen Alltag, insbesondere wenn es um den Ausschluss einer latenten Tuberkulose geht 3. die Auswertung von ELISPOT-Daten lässt sich über Mehrfachbestimmung und quadratwurzeltransformierte Auswertung optimieren (wobei das noch in einer Folgearbeit zu beweisen ist).:Inhalt Abkürzungsverzeichnis 3 1 EINFÜHRUNG 5 1.1 Epidemiologie der Tuberkulose 5 1.2 Pathogenese der Tuberkulose 8 1.3 Prävention der Tuberkulose 10 1.4 Diagnostik der Tuberkulose 11 1.4.1 Radiologische Untersuchungen 12 1.4.2 Mikrobiologische Untersuchungen 12 1.4.3 Molekularbiologischer Nachweis 13 1.5 Nachweis der Immunreaktion gegenüber M. tuberculosis 14 1.5.1 Tuberkulin-Hauttest 14 1.5.2 Serologische Tests auf M. tuberculosis-Infektion 16 1.5.3 Zelluläre Labortests auf M. tuberculosis-Reaktivität 18 2 AUFGABENSTELLUNG 22 3 MATERIAL UND METHODEN 23 3.1 Patienten 23 3.2 Blutentnahme und Zellpräparation 23 3.3 ELISPOT 25 3.4 Ringversuch zur externen Qualitätssicherung 26 3.5 Retrospektive Analyse der Daten des eigenen Labors und der Ringversuchsdaten 26 3.6 Analyse der Validierungsdaten aus Ettlingen 27 4 ERGEBNISSE 29 4.1 Analyse der ELISPOT-Daten des eigenen Labors 29 4.2 Ergebnisse des Instand-Ringversuchs 33 4.3 Analyse der ELISPOT-Wiederholungsmessungen 35 5 DISKUSSION 41 6 Zusammenfassung 48 Literaturverzeichnis 52 Lebenslauf 63 Danksagung 64 Erklärung über die eigenständige Abfassung der Arbeit 65

No description available.

Diarrheal disease caused by attaching and effacing pathogens, such as enteropathogenic E. coli (EPEC), is a worldwide health concern. As the second leading cause of diarrheal-related death in young children, new investigations into host defense against EPEC, as well as future therapeutics, is greatly needed. To elucidate the host immune responses to these enteric pathogens, the attaching and effacing (A/E) murine pathogen, Citrobacter rodentium, has been widely used. It is well understood that C. rodentium infection induces a robust Th1 response within the host. Yet how these pleiotropic IFNγ immune responses are initiated, propagated, and the accessory immune cell types involved remains poorly understood. In this thesis, I investigated how innate immune cell types such as natural killer cells, which are significant producers of IFNγ, mediate these Th1 directed responses. This work identified that both NK and NK-like innate lymphoid type 1 cells (ILC1s) are capable of producing IFNγ in response to C. rodentium, and NK cells rapidly increase in numbers within the colon during the early stages of infection. Depletion of these cell types causes a delayed Th1 CD4+ T cell response within the colon, resulting in increased bacterial load, and greater degree of colonic pathology at later time points. Additionally, depletion of these cells results in decreased CXCL9 chemokine expression in mice. I later determined that CXCL9 exhibited direct antimicrobial action against Citrobacter in vitro. Depletion of this chemokine in vivo, in the absence of adaptive immune responses, or its receptor CXCR3, results in increased mortality rates, elevated bacterial loads, greater degree of pathology, and deeper penetration of bacteria within the colonic crypts. These data indicate a potential direct antimicrobial role for this IFNγ-induced chemokine, independent of its known properties for the homing of T cells to the site of infection. These findings demonstrate the importance of accessory IFNγ-producing immune cells in not only mediating Th1 CD4+ T cells responses, but also other innate host defense mechanisms against A/E pathogens. / Thesis / Doctor of Philosophy (PhD)

Class II cytokine receptors are important pleiotropic regulators of the immune system that play a central role in pathogen defense, tumor surveillance and immune system homeostasis. Most of these activities are very promising for biomedical applications, which, however, have so far failed to succeed due to severe undesired side effects resulting from the pleiotropic nature of these cytokine receptors. Controlling the functional plasticity of class I/II cytokine receptor signaling by engineered cytokines has recently emerged as a promising approach to selectively reduce such side effects. In this context, systematic studies on the IFNalpha/beta receptor and other systems have identified that the binding kinetics of the ligand-receptor interaction play an important role in defining signaling specificity. This has been explained by altered equilibrium and dynamics of the signaling complex in the plasma membrane. In this work, I have investigated how the spatiotemporal organization and dynamics of signaling complexes regulate activation and signaling specificity of other members of the class II cytokine receptors. I focused on the type II IFN and IL-10 systems that supposedly form hexameric ligand-receptor signaling complexes in the plasma membrane. To this end, we developed an orthogonal multicolor anti-GFP nanobody-based labeling strategy, that allowed imaging of up to four different class II cytokine receptor subunits simultaneously. Using this labeling strategy, I investigated the spatiotemporal dynamics of IFNGR and IL-10R complex assembly by co-localization and co-tracking of single receptor subunits. Thereby, I did show that unliganded receptor subunits of IFNGR and IL-10R remain monomeric at the cell surface, whereas binding of the ligand led to fast and efficient receptor homo- and hetero-dimerization, verifying a ligand-induced receptor complex assembly model for both cytokine receptors. Moreover, I verified the hexameric ligand-receptor complex structure in cellulo. Analysis of single molecule trajectories and co-trajectories revealed a decrease in mobility and diffusion of IFNGR and IL-10R subunits upon ligand stimulation indicating receptor confinement and endocytosis. In this context, I identified an abnormal diffusion behavior of IL-10R2 that was dependent on the length of its transmembrane helix. We used partial agonists for both receptor complexes to systematically alter receptor binding stoichiometry and complex stability in the plasma membrane and correlated these with downstream signaling responses. Our analysis revealed a minor contribution of the second low affinity receptor subunit and its associated kinase to the overall signaling activity. However, the second high affinity binding subunit was indispensable to acquire full signaling potential. We managed to obtained decoupling of gene expression for both hexameric class II cytokine receptors by utilizing engineered ligands with altered receptor binding affinities. Our findings could pave the way for new biomedical approaches with engineered IFNgamma and IL-10 in the future. Furthermore, we uncovered pathogenic mechanisms behind the IFNGR2-T168N mutant and auto-IFNgamma antibodies, both of which prominently cause the Mendelian Susceptibility to Mycobacteria Disease (MSMD) syndrome, showing that both interfere with IFNGR activation by preventing recruitment of IFNGR2 into receptor complexes.