Infectious diseases remain a threat to critically ill patients, particularly with the rise of antibiotic-resistant bacteria. Septic shock carries a mortality of up to ∼40% with no compelling evidence of promising therapy to reduce morbidity or mortality. Septic shock survivors are also prone to nosocomial infections. Treatment with toll-like receptor 4 (TLR4) agonists have demonstrated significant protection against common nosocomial pathogens in various clinically relevant models of infection and septic shock. TLR4 agonists are derived from a bacteria cell wall or synthesized de novo, and more recently novel small molecule TLR4 agonists have also been developed. TLR4 agonists augment innate immune functions including expansion and recruitment of innate leukocytes to the site of infection. Recent studies demonstrate TLR4-induced leukocyte metabolic reprogramming of cellular metabolism to improve antimicrobial function. Metabolic changes include sustained augmentation of macrophage glycolysis, mitochondrial function, and tricarboxylic acid cycle flux. These findings set the stage for the use of TLR4 agonists as standalone therapeutic agents or antimicrobial adjuncts in patient populations vulnerable to nosocomial infections.

Das human pathogene Varicella-Zoster Virus (VZV) gehört zur Familie der Herpesviren und ist weltweit verbreitet. Die Primärinfektion verursacht Varicellen, welche durch einen bläschenartigen Hautausschlag charakterisiert ist. Im Anschluss daran etabliert VZV eine lebenslange Latenz und verursacht nach Reaktivierung Herpes Zoster. Seit 2004 ist der Lebendimpfstoff aus attenuierten Virionen des VZV-Stammes V-Oka in Deutschland empfohlen. Im Gegensatz zur Infektion mit zirkulierenden virulenten VZV Stämmen tritt nach Verimpfung des Vakzin-Stammes V-Oka kein Exanthem auf. Die Haut ist der Hauptreplikationsort von VZV und immunologische Unterschiede zwischen virulentem VZV und dem Vakzin-Stamm treten hier am deutlichsten auf. In der vorliegenden Arbeit konnte eine neue Immunevasionsstrategie virulenter VZV Stämme aufgedeckt werden, welche erklären könnte, wie virulente VZV Stämme frühe antivirale Immunantworten umgehen. In Hautläsionen von Herpes Zoster Patienten konnte eine massive Infiltration von myeloiden inflammatorischen Dendritischen Zellen beobachtet werden. In vitro Studien mit Monozyten abgeleiteten Dendritischen Zellen (DC), welche inflammatorische DC repräsentieren, zeigten, eine signifikant erhöhte Expression von CD1c Molekülen nach Infektion mit dem Vakzin-Stamm, sowie virulentem VZV. Funktionelle Untersuchungen mit intraepithelialen CD1c-restringierten gamma delta T Zellen zeigten, dass DC nach Infektion mit dem Vakzin-Stamm phänotypisch und funktionell reiften und somit die T Zellen zur IFN-gamma Sekretion stimulierten. Im Gegensatz dazu wurde die funktionelle Reifung von DC, die mit virulentem VZV infiziert waren, geblockt. Folglich wurde kein bioaktives IL-12 sezerniert, welches als entscheidendes Cytokin zum Aufbau einer antiviralen T-Helfer 1 Immunantwort beiträgt. Darüber hinaus konnte gezeigt werden, dass virulentes VZV die Signalkaskade des Toll-like Rezeptors 2 (TLR2) in DC inhibiert und somit die IL-12 Produktion verhindert. / Varicella-zoster virus (VZV) which belongs to the family of herpesviruses is restricted to humans and distributed worldwide. Primary infection of VZV causes chickenpox characterized by a disseminated rash. Thereafter, VZV establishes a lifelong latency and can be reactivated to cause herpes zoster. Since 2004 the attenuated strain V-Oka of VZV was licensed for Germany to immunize children against VZV infection. In contrast to infection by circulating virulent VZV strains, vaccination with V-Oka remains asymptomatic. The skin is the major replication site of VZV and immunological differences between virulent VZV and the vaccine should become most apparent within this immune organ. In summary, this study discovered a new immune evasion strategy of virulent VZV strains which might explain how virulent VZV strains overcome innate antiviral responses. A strong infiltration of myeloid-derived inflammatory DCs has been detected in skin lesions of herpes zoster patients. In vitro studies with monocyte-derived dendritic cells (DCs), reflecting inflammatory DCs, showed that they were efficiently infected by both, the vaccine and a virulent VZV strain. Intriguingly, a significant upregulation of CD1c molecules on VZV-infected DCs was observed. Functional investigations using intraepithelial CD1c-restricted gamma delta T cells revealed that DCs infected with the vaccine virus were fully instructed to mature, thereby promoting IFN-gamma secretion of gamma-delta T cells. In striking contrast, DCs infected with virulent VZV strains were efficiently blocked to mature functionally. In detail, they did not secrete bioactive IL-12 which is an instrumental cytokine for generation of antiviral T helper 1 responses. Moreover, virulent VZV blocked Toll-like receptor 2 (TLR2) signaling in DCs thereby preventing production of bioactive IL-12 which in turn inhibited IFN-gamma secretion by gamma-delta T cells.

MicroRNAs (miRNAs) sind endogenene kurze RNA-Moleküle, die zentrale Aufgaben bei der Regulation der eukaryotischen Zellhomöostase erfüllen. MiRNAs wurden bereits als potente Immunregulatoren beschrieben. Trotz dieser Erkenntnisse blieb die Rolle dieser kurzen RNA Moleküle in Infektionen mit Mycobacterium tuberculosis weitgehend unerforscht. Im Rahmen dieser Arbeit wurde ein miRNA-Expressionsprofil von Makrophagen generiert, die mit Mycobacterium tuberculosis infiziert waren. Dies ermöglichte die Identifizierung von miRNAs, welche bei der Infektion differenziell reguliert waren. Anhand eines ex-vivo-Modells von Todesrezeptor-induzierter Apoptose konnte gezeigt werden, dass miRNA-133b apoptoseresitente Zellen empfindlich gegen Tumornekrosefaktor-alpha (TNFalpha), TNF-related apoptosis-inducing ligand (TRAIL) oder CD95 ligand (Fas/APO1 ligand) induzierte Zytotoxizität machte. Eine umfassende Studie führte zur Identifizierung der anti-apoptotischen Proteine Fas apoptosis inhibitory molecule (FAIM) und glutathione-S-transferase pi (GSTP1) als direkte Zielgene für miRNA-133b. Desweiteren zeigte sich die Expression von Osteoprotegerin (OPG) und Fettsäuresynthase (FASN), als miRNA-133b abhängig. Dies unterstrich die pleiotrope Art der pro-apoptotischen Aktivität dieser miRNA. Die Expression von miRNA-133b wurde durch Mitglieder der Toll-like Rezeptor (TLR)-Familie aktiviert. MiRNA-133b Transfektion führte zu einer verstärkten Aktivierung des Transkriptionsfaktors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). Diese resultierte in erhöhten Mengen an Interleukinen 6 und 8 (IL6/8). Diese Ergebnisse stellen die erste detaillierte Charakterisierung von miRNA-133b im Zusammenhang der Todesrezeptor-vermittelten Apoptose und der angeborenen Immunität dar. Die erforschten molekularen Wechselwirkungen ergänzen und bereichern das Verständnis über die regulatorischen molekularen Mechanismen, die mit der Tumorentstehung und Entzündung verbunden sind. / MicroRNAs (miRNAs) are endogenous short RNA molecules which perform essential tasks in the regulation of eukaryotic cell homeostasis. During the past few years miRNAs have emerged as very potent immune regulators. Despite the consequences of this discovery for our understanding of immune response regulation hitherto virtually nothing is known about miRNA function during innate immunity to Mycobacterium tuberculosis. Herein, a miRNA expression profile of human macrophages infected with Mycobacterium tuberculosis was generated. This led to the identification of miRNAs being differentially regulated during infection. By using an experimental ex-vivo model of death receptor (DR)-induced apoptosis it could be demonstrated that miRNA-133b rendered apoptosis-resistant cells sensitive to tumor necrosis factor-alpha (TNFalpha)-, TNF-related apoptosis-inducing ligand (TRAIL)- or CD95 ligand (Fas/APO1 ligand)-activated cytotoxicity. Comprehensive analysis led to the discovery of the anti-apoptotic proteins Fas apoptosis inhibitory molecule (FAIM) and glutathione-S-transferase pi (GSTP1) as direct miRNA-133b targets. Moreover, underlining the pleiotropic and synergistic nature of miRNA activity, the expression of osteoprotegerin (OPG) and fatty acid synthase (FASN) could be further proven as miRNA-133b dependent. Expression of miRNA-133b increased following innate immune activation by members of the Toll-like receptor (TLR) family. MiRNA-133b enhanced the activity of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). This translated into increased levels of the pro-inflammatory interleukins 6 and 8 (IL6/8). The results presented in this work represent the first detailed characterization of miRNA-133b in the context of DR-mediated apoptosis and innate immunity. The molecular interactions dissected herein improve the understanding of the regulatory processes associated with tumorigenesis and the immune response.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Obesidade é fator de risco para a hipertensão arterial e os mecanismos envolvidos nesta doença não são totalmente esclarecidos. Camundongos C57BL/6J e transgênicos com com deleção em neurônios e glia da via inflamatória do receptor toll-like-NFκB foram submetidos à dieta hiperlipídica (HL) por 8 e 15 semanas e avaliados parâmetros metabólicos, pressão arterial, frequência cardíaca, atividade do sistema nervoso autônomo, fatores inflamatórios e neuropeptídeos no hipotálamo e no tronco encefálico. Os camundongos expostos HL desenvolveram hipertensão arterial acompanhada de disfunção autonômica e aumento de CART no DMH. Os animais transgênicos quando submetidos à dieta HL desenvolveram um quadro de obesidade, porém não apresentaram hipertensão arterial e disfunção autonômica. Além disso, o grupo de animais HL aumentou o RNAm de CCL5 no hipotálamo e de CD86 no tronco-encefálico e a densidade de microglia no NTS caudal. Os resultados sugerem novos mecanismos para a hipertensão e disfunção autonômica secundárias à ingestão de dieta hiperlipídica mostrando o papel do CART o DMH e o envolvimento da via inflamatória do TLR-NFκB em neurônios e glia nos mecanismos desta patologia. / Obesity is a risk factor for high blood pressure and the mechanisms involved in this disease are not fully clarified. C57BL/6J and transgenic mice with toll-like-NFκB receptor inflammatory- pathway deletion in neurons and glia were fed with high-fat diet (HL) for 8 or 15 weeks and assessed metabolic parameters, blood pressure, heart rate, autonomic nervous system activity, inflammatory factors and neuropeptides in the hypothalamus and brainstem. The HL mice developed hypertension accompanied with autonomic dysfunction and increased CART in DMH. Transgenic animals when submitted to HL diet developed obesity, but not showed high blood pressure and autonomic dysfunction. In addition, HL animals had increased CCL5 mRNA in hypothalamus, CD86 mRNA in brainstem and micróglia density in caudal NTS. The results suggest new mechanisms for hypertension and autonomic dysfunction secondary to intake of high-fat diet by showing CART role in DMH and the involvement of the inflammatory pathway TLR-NFκB in neurons and glia.

Obesidade é fator de risco para a hipertensão arterial e os mecanismos envolvidos nesta doença não são totalmente esclarecidos. Camundongos C57BL/6J e transgênicos com com deleção em neurônios e glia da via inflamatória do receptor toll-like-NFκB foram submetidos à dieta hiperlipídica (HL) por 8 e 15 semanas e avaliados parâmetros metabólicos, pressão arterial, frequência cardíaca, atividade do sistema nervoso autônomo, fatores inflamatórios e neuropeptídeos no hipotálamo e no tronco encefálico. Os camundongos expostos HL desenvolveram hipertensão arterial acompanhada de disfunção autonômica e aumento de CART no DMH. Os animais transgênicos quando submetidos à dieta HL desenvolveram um quadro de obesidade, porém não apresentaram hipertensão arterial e disfunção autonômica. Além disso, o grupo de animais HL aumentou o RNAm de CCL5 no hipotálamo e de CD86 no tronco-encefálico e a densidade de microglia no NTS caudal. Os resultados sugerem novos mecanismos para a hipertensão e disfunção autonômica secundárias à ingestão de dieta hiperlipídica mostrando o papel do CART o DMH e o envolvimento da via inflamatória do TLR-NFκB em neurônios e glia nos mecanismos desta patologia. / Obesity is a risk factor for high blood pressure and the mechanisms involved in this disease are not fully clarified. C57BL/6J and transgenic mice with toll-like-NFκB receptor inflammatory- pathway deletion in neurons and glia were fed with high-fat diet (HL) for 8 or 15 weeks and assessed metabolic parameters, blood pressure, heart rate, autonomic nervous system activity, inflammatory factors and neuropeptides in the hypothalamus and brainstem. The HL mice developed hypertension accompanied with autonomic dysfunction and increased CART in DMH. Transgenic animals when submitted to HL diet developed obesity, but not showed high blood pressure and autonomic dysfunction. In addition, HL animals had increased CCL5 mRNA in hypothalamus, CD86 mRNA in brainstem and micróglia density in caudal NTS. The results suggest new mechanisms for hypertension and autonomic dysfunction secondary to intake of high-fat diet by showing CART role in DMH and the involvement of the inflammatory pathway TLR-NFκB in neurons and glia.