京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22135号 / 生博第422号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 朝長 啓造, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20782号 / 生博第388号 / 新制||生||51(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 朝長 啓造, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

No description available.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24119号 / 生博第481号 / 新制||生||64(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 鈴木 淳, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the immune-mediated destruction of insulin-producing beta-cells of pancreatic islets, culminating in critical insulin deficiency. Both genetic and environmental factors likely orchestrate an immune-mediated functional loss of beta cell mass, leading to the clinical manifestation of disease and lifelong dependence on insulin therapy. Additional evidence suggests the role of innate and adaptive immune mechanisms leading to inflammation in beta cells mediated by proinflammatory cytokines and chemokines, activation of beta-cell-reactive T cells,and failure of immune tolerance. Viral infections have been proposed as causal determinants or initiating triggers for T1D but remain unproven. Understanding the relationship between viral infections and the development of T1D is essential for T1D prevention. Importantly, virus-induced innate immune responses, particularly type I interferon (IFN-I, IFN-a/b), have been implicated in the initiation of islet autoimmunity and development of T1D. The goal of my thesis project is to investigate how the IFN-I signaling pathway affects the development of T1D using the LEW.1WR1 rat model of autoimmune diabetes. My hypothesis is that disrupting IFN-Isignaling via functional deficiency of the IFN-I interferon receptor (IFNAR) prevents or delays the development of virus-induced diabetes.For this purpose, I generated IFNAR subunit 1(IFNAR1)-deficient LEW.1WR1 rats using CRISPR-Cas9 genome editing and confirmed the functional disruption of IFNAR1. The absence of IFNAR1 results in a significant delay in onset and frequency of diabetes following poly I:C challenge and reduces the incidence of insulitis after poly I:C treatment. The frequency of diabetes induced by Kilham rat virus (KRV) is also reduced in IFNAR1-deficient LEW.1WR1 rats. Furthermore, I observe a decrease in CD8+T cells in spleens from KRV-infected IFNAR1-deficient rats relative to that in KRV-infected wild-type rats. While splenic regulatory T cells are depleted in WT rats during KRV-infection, no such decrease is observed in KRV-infected IFNAR1-deficient rats. A comprehensive bulk RNA-seq analysis reveals a decrease of interferon-stimulated genes and inflammatory gene expression in IFNAR1-deficient rats relative to wild-type rats following KRV challenge. Collectively, the results from these studies provided mechanistic insights into the essential role of virus-induced, IFN-I-initiated innate immune responses in the early phase of autoimmune diabetes pathogenesis.

En réponse à une infection, un signal de danger, ou de cytokines inflammatoires, les cellules dendritiques subissent un programme de maturation augmentant leur capacité à activer les lymphocytes T CD4+ et CD8+. Au cours de ce travail nous avons cherché à caractériser la reprogrammation transcriptomique des DC lors de l'infection par le cytomégalovirus murin (MCMV). Nous avons identifié un programme commun de maturation entre les différentes sous-populations de DC spléniques. Nous avons mis en évidence qu'il existe un programme transcriptomique de maturation commun à toutes les sous-populations de DC, induit par tous les stimuli examinés et évolutivement conservé au sein des mammifères. Nous avons également identifié les interférons (IFN) de type I comme des cytokines majeures promouvant la maturation des DC in vivo. La perte spécifique par les DC de la capacité à répondre aux IFN de type I entraine une diminution de la survie des souris lors de l'infection par le MCMV, révélant pour la première fois l'importance des effets intrinsèques cellulaires des IFN de type I sur les DC pour la résistance à une infection virale.Le développement puis l'utilisation d'un nouveau modèle de souris mutante ciblant la sous-population de DC XCR1+ nous a permis de mettre mis en évidence pour la première fois un rôle de ces cellules pour l'activation des lymphocytes T CD8 mémoires (Tm CD8+) dans l'infection par Listeria monocytogenes, et d'identifier les mécanismes sous-jacents. Les DC XCR1+ interagissent in situ avec les Tm CD8+. La synthèse de la chimiokine CXCL9 et la production d'interleukine-12 par les DC XCR1+ attirent et activent de façon optimale les Tm CD8+ qui produisent de l'IFN-γ. / Dendritic cells (DC) sense danger, microbial and cytokine signals that drive DC maturation which in turn allows proper activation of T lymphocytes. We characterized the gene expression program of splenic DC in vivo during murine cytomegalovirus (MCMV) infection. We identified a core set of genes commonly regulated in all subsets of mouse spleen DC. This set of genes was regulated upon DC maturation irrespective of the stimuli used and of the responding DC subsets and it was conserved between mouse and human. We identified type I interferon (IFN) as a major cytokine driving the expression of this core gene set in DC subsets. The loss of type I IFN responsiveness selectively in DC resulted in an increased mortality of mice after MCMV infection, unraveling a crucial role of cell-intrinsic responses to type I IFN in DC during a viral infection in vivo.We also developed and studied a new mouse model to target the XCR1+ DC subset in vivo. We found for the first time that XCR1+ DC promote recall of memory CD8 T cells upon secondary Listeria monocytogenes infection in vivo, and we identified the underlying mechanism. XCR1+ DC attract memory CD8 T cells through the secretion of the chemokine CXCL9. This attraction leads to an increase in the IFN-γ production by memory CD8 T cells. XCR1+ DC also induce the proliferation of memory CD8 T cells. This work significantly advanced our understanding of the in vivo functions of DC during infections.

Les pDC représentent la première ligne de défense de l’organisme contre les pathogènes et établissent le lien essentiel entre l’immunité innée et adaptative. Les pDC endocytent et détruisent les particules virales et ainsi détectent leur matériel génétique grâce à des senseurs antiviraux de la famille des Toll-Like Receptors (TLR). L’activation des TLR7/9 induit la production massive d’interféron de type I (IFN-I), un antiviral puissant indispensable au contrôle de la propagation virale lors des phases aigues de l’infection. Cependant, l’IFN-I peut s’avérer avoir des effets délétères dans un grand nombre d’infections chroniques et de maladies auto-immunes. Ainsi, il semble indispensable de découvrir les mécanismes régulateurs des pDC ainsi que des modulateurs de l’activation des pDC. Nous avons ainsi montré que les monoamines (histamine, dopamine, sérotonine) et les polyamines (spermine et spermidine) inhibent l’activation complète des pDC stimulées par divers virus. Par la suite, nous avons identifié CXCR4 comme étant le récepteur des amines sur les pDC. Ainsi nous avons pu montrer que les amines pouvaient réguler les pDC en passant par CXCR4 et que ce récepteur était un interrupteur d’activation potentiel des pDC lors des infections virales. Afin de comprendre le mécanisme des amines, nous avons développé une nouvelle technologie : la transfection de siRNA dans les pDC primaires humaines. D’autre part, nous avons détecté des cellules géantes multinucléées en forme de roue de bicyclette lorsque les pDC sont cultivées in vitro avec de grandes quantités de virus VIH. Ainsi, comme les monocytes et les macrophages, les pDC peuvent former in vitro des cellules géantes multinucléées exprimant de hauts niveaux de protéines virales p24 de VIH-1. Cependant, les pDC ne sont que très peu infectées (moins de 5%). Nous nous sommes alors demandé si le corécepteur CXCR4 du virus VIH était aussi important que le récepteur CD4 pour la reconnaissance de ce dernier lors de l’activation des pDC. / PDC are the first line of defense of our organism against pathogens and establish the essential link between the innate and adaptive immunity. pDC endocyte and destroy the viral particles and thus, detect the genetic material with their antiviral sensors from the Toll-Like Family (TLR). The activation of TLR7/9 induces massive production of type I interferon (IFN-I), a powerful antiviral molecule, essential to control viral propagation during the acute phases of the infection. However, type I IFN can have deleterious effects in a large number of chronic infections and autoimmune diseases. Thus, it seems essential to discover the regulatory mechanism of pDC as well as pDC activation modulators. We showed that monoamines (histamine, dopamine and serotonin) and polyamines (spermine and spermidine) inhibit completely the activation of virus-stimulated pDC. Thus, we showed that amines regulated pDC activation through CXCR4 engagement and that this receptor was a potential switch "on-off" for pDC during viral infections. To better understand the mechanism of action by which amines inhibit pDC activation, we developed a new technology: siRNA transfection in human primary pDC. Furthermore, we detected multinuclear giant cells bearing the shape of a bicycle wheel when pDC are cultured in vitro with high quantities of HIV virus. Thus, on top of monocytes and macrophages, pDC can form in vitro multinuclear giant cells with high levels of p24 viral protein of HIV-1. However, pDC barely get infected (less than 5%). We then wondered if the receptors and co-receptors of the virus were important for the viral recognition during HIV-activation of pDC.

HIV remains an incurable viral infection and a significant global health concern. Despite the advent of antiretroviral therapy, there are 36.9 million recorded cases of HIV worldwide, with an additional 1.8 million new infections recorded in 2017 alone. An HIV cure is therefore one of several priorities within the field, and will require HIV “reservoir” cells—comprised of latently-HIV infected CD4+ T cells and productively-infected, tissue resident macrophages—to be selectively killed in vivo. HIV reservoir cells are rarely found within the peripheral circulation, residing instead within inaccessible tissue sanctuaries. Consequently, their characterization has been limited to in vitro laboratory models. To complicate matters further, a definitive cellular surface marker of HIV infected cells has yet to be identified. Impairment of the type I interferon (IFN1) response has been observed during HIV infection, however, making it a unique intracellular maker of HIV-infected cells. The recent development of oncolytic viruses (OV) designed to selectively kill IFN-defective cancer cells also suggests that these IFN1 defects possess therapeutic value. It was therefore hypothesized that the impairment of the IFN1 response in HIV-infected CD4+ cells and macrophages could serve as a target for oncolytic virus-mediated killing. The induction of several antiviral IFN-stimulated proteins, including PKR and ISG15, was inhibited in HIV-infected monocyte-derived macrophages (MDM) following stimulation with IFNα or a synthetic RNA. Consequently, HIV-infected MDM were more susceptible to infection and killing by the oncolytic Maraba virus, MG1. Importantly, MG1-mediated killing required the presence of replication-competent OV, and could not be potentiated by UV-inactivated MG1 or supernatants from MG1-infected cells. The ability of MG1 to target the HIV reservoir was further confirmed using alveolar macrophages collected from the lungs of cART-suppressed individuals living with HIV. These findings indicate that IFN1 defects are a feature of HIV infected cells, which can be exploited for selective killing by OV. This project is therefore unique in that it demonstrates that HIV reservoir cells can be eradicated in a targeted manner by exploiting an intracellular marker of HIV infection. As MG1-based cancer therapies are currently being explored in Phase I/II clinical trials, there is potential for this approach to be adapted for use within the HIV cure field.