A análise da expressão da enzima indoleamina 2,3-dioxigenase (IDO), envolvida na regulação da resposta imune, em vacinas de células dendríticas (DCs) como imunoterapia para tratamento de indivíduos HIV+, pode fornecer informações sobre o perfil dessas células e auxiliar o aperfeiçoamento das técnicas atualmente utilizadas. A avaliação da expressão de IDO, por citometria de fluxo, foi realizada em monócitos e DCs de indivíduos sadios e HIV+. A expressão do RNAm de IDO foi analisada por PCR e a capacidade das DCs em estimular linfoproliferação e apresentar antígenos de HIV a linfócitos autólogos foi avaliada por ensaio de cocultivo. DCs ativadas de indivíduos HIV+ demonstraram expressão mais elevada de IDO tanto em relação às DCs imaturas quanto em relação às DCs dos indivíduos sadios. DCs foram capazes de induzir resposta proliferativa e polifuncional de linfócitos autólogos. Nossos resultados sugerem uma expressão diferencial de IDO entre indivíduos sadios e HIV+, indicando um importante papel da enzima no controle da resposta imune e na patogênese da AIDS. / The evaluation of indoleamine 2,3-dioxygenase (IDO) levels, a regulatory enzyme, in the context of DCs vaccines as a therapeutic alternative for the HIV+ patients, can bring information about DCs profiles that can improve current techniques of vaccine production. IDO expression was evaluated by flow cytometry in monocytes and DCs from healthy subjects and HIV+ patients. Expression of IDO mRNA was performed by real-time PCR and the ability in stimulate lymphoproliferation and presenting HIV antigens to autologous lymphocytes was evaluated in coculture assays. Comparison between immature and activated DCs showed an increased IDO expression in activated DCs in patients group. DCs derived from HIV+ patients showed an increased IDO expression when compared to healthy donors. DCs were able to induce lymphofoproliferation and polyfunctional response in autologous lymphocytes. Our results suggest a differential expression of IDO between health subjects and HIV+ patients, indicating an important role of IDO in the control of the immune response and in the HIV pathogenesis.

Dendritic cells (DCs) are the most potent professional antigen presenting cells (pAPCs) of the immune system and play a fundamental role in coordinating innate and adaptive immune responses. Through the expression of a wide array of pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), DCs recognize a variety of microbial pathogens and infectious stimuli. Stimulation of DCs through TLR ligation results in a rapid series of activation-associated events, termed "maturation," which include the upregulation of surface co-stimulatory molecule expression, inflammatory cytokine secretion, and stimulation of naïve T cells via antigen presentation by MHC molecules. Activation of DCs through TLRs is coupled with an increased metabolic demand fulfilled by a rapid change in DC glucose metabolism and characterized by increased aerobic glycolysis rates. TLR-driven glycolytic reprogramming plays an essential role in generating building blocks required for high level protein synthesis associated with maturation. Although glucose imported from extracellular environments has been broadly considered as the major driver of glycolytic metabolism in immune cells, the contributions of intracellular glucose stores to these processes are not well-defined. The role of intracellular stores of glucose, in the form of glycogen, is widely appreciated in non-immune systems. However, very little is known about the implication of glycogen metabolism in DC immune responses. This work unveils the role and potential regulatory mechanisms of glycogen metabolism in support of DC effector function. The first part of this work primarily focuses on our characterization of the role of glycogen metabolism in early DC activation responses; while in the last chapter, we describe a potential regulatory mechanism of DC glycogen metabolism by activation-associated nitric oxide (NO) production. In this work, we tested the overarching hypothesis that DC-intrinsic glycogen metabolism supports the early glycolytic reprogramming required for effector responses and that nitric oxide can regulate this metabolism. We demonstrate that DCs possess the enzymes required for glycogen metabolic machinery and that glycogen metabolism supports DC immune effector response, particularly during early activation and in nutrient-limited environments. More importantly, we uncover a very intriguing metabolic phenomenon, in which DCs engage in the differential metabolic pathways driven by carbons derived distinctively from glycogen and free glucose. Our studies present the fundamental role and regulatory mechanisms of DC-intrinsic glycogen metabolism and underline the differential utilization of glycogen and glucose metabolism to support their effector responses. Overall, this work adds to a growing field of immuno-metabolism an improved understanding of an intricate layer of metabolic mechanisms that immune cells undertake in response to immune stimuli.

We have recently demonstrated in a model of influenza A virus (IAV) infection that the absence of specific pulmonary DC subsets, including plasmacytoid DC (pDC) and CD8a+ DC, from the lungs leads to a significant decrease in the number of virus-specific CD8 T cells. Reconstitution of the lungs with physiologic numbers of pDC or CD8a+ DC is able to restore the pulmonary IAV-specific CD8 T cell response to near normal levels via a mechanism that is dependent upon direct DC:T cell interactions, DC-expressed MHC I and the presence of viral antigen. Interestingly, however, this rescue is DC subset specific, as reconstitution with purified alveolar and airway DC or alveolar macrophages was unable to rescue the virus-specific CD8 T cell response. Following IAV infection there is an abundance of IAV antigen and MHC I expressing cells present in the lungs, including infected epithelial cells. Given this fact and the inability of all DC subsets to rescue the virus-specific CD8 T cell response, it suggested that there were additional, undefined requirements for pDC- and CD8a+ DC-mediated rescue of the T cell response in the lungs. Further, although it was known that the reduction in virus-specific CD8 T cells in the lungs was a result of increased T cell apoptosis, it remained unclear what pathways of apoptosis were contributing to the increased cell death, and what mechanism pulmonary DC subsets were utilizing to rescue this defect. Here, we demonstrate that in the absence of lung-resident DC subsets, virus-specific CD8 T cells undergo significantly increased levels of apoptosis via both extrinsic activation induced cell death and intrinsic activated cell-autonomous death pathways. Reconstitution of aDC depleted lungs with pulmonary pDC and CD8a+ DC promotes increased T cell expression of the pro-survival molecule Bcl-2 and hence, increased T cell survival and accumulation in the lungs. Our studies herein demonstrate that pulmonary DC subsets utilize a variety of mechanisms to promote the rescue of virus-specific CD8 T cells in the lungs. Blockade of the costimulatory molecules CD70, and in some cases, 4-1BBL and OX40L, ablates the pulmonary DC mediated rescue of CD8 T cell numbers in the lungs, suggesting that late costimulation is one essential mechanism that pulmonary DC use to regulate CD8 T cell immunity following IAV infection. Further, we demonstrate that the absence of DC following IAV infection results in significantly reduced levels of IL-15 in the lungs and that pulmonary DC-mediated rescue of virus-specific CD8 T cell responses in the lungs requires the trans-presentation of IL-15 via DC-expressed IL-15Ra. In addition to the role of pulmonary DC mediated costimulation and IL-15 trans-presentation, we further demonstrate a previously unrecognized role for viral antigen in regulating the accumulation of both pulmonary DC and virus-specific CD8 T cells in the lungs, suggesting that viral load can dictate the nature of the inflammatory environment in the lungs and thus, regulate the character of the ensuing IAV-specific immune response. Collectively, the results detailed here demonstrate a previously unrecognized role for pulmonary DC in regulating primary IAV-specific CD8 T cell immunity, and hence, promoting enhanced viral clearance and recovery from disease.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

In experimentally exposed mice, the environmental contaminant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) produces significant suppression of adaptive immune responses at low doses. However, the underlying biochemical and cellular mechanisms of TCDD-induced immunotoxicity have remained elusive since the identification of these effects nearly 30 years ago. Antigen-presenting cells (APC) constitute various populations of cells essential for the initiation and maintenance of adaptive immune responses, and represent a potential target of TCDD toxicity. Thus, the studies presented here address the ability of TCDD to directly affect APC. The underlying objectives of these studies focus on the investigation of molecular signaling pathways and cellular processes potentially affected by TCDD. In order to eliminate conflicting variables found in vivo, we used ex vivo and in vitro models to address these objectives. Initial studies investigated the status and behavior of the aryl hydrocarbon receptor (AhR), a transcription factor recognized as the principal mediator of TCDD-induced immunotoxic effects, in the two main APC populations, macrophages and dendritic cells (DC). The results demonstrated that both APC populations expressed AhR. However, TCDD induced binding of AhR to dioxin response elements only in macrophages, and not DC. Because TCDD has been shown to alter DC function and survival in vivo, the possibility that TCDD altered other signaling pathways was addressed. Specifically, activation of the transcription factor NF-kB/Rel, integral in DC generation and function, was found to be suppressed by TCDD. This suppression was apparently mediated by a physical association between the AhR and proteins of NF-kB/Rel. Additional studies demonstrated that TCDD enhances the maturation of DC and appears to sensitize DC to apoptosis. These data establish that TCDD directly affects DC on the molecular and cellular levels and support several potential mechanisms of TCDD-induced immunotoxicity. / Graduation date: 2002

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 1999. / Includes bibliographical references. Also available on the Internet.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.