Dendritic cells (DCs) are antigen presenting cells (APCs), a subtype of immune cells that present cellular information to T cells in the immune system. Hypoxia inducible factor 1 alpha (HIF-1 alpha) is an important transcription factor that facilitates dendritic cell metabolism by upregulating glycolysis in activated DCs. In this project, we examined the effects of HIF-1 alpha inhibition on metabolic processes of dendritic cells. Using techniques such as flow cytometry, western blotting, and extracellular flux analyzers, we used a selective inhibitor of HIF-1 alpha to test the hypothesis that HIF-1 alpha promotes glycolytic dependent processes such as glucose production, survival, and maturation. The results revealed that HIF-1 alpha impacts oxygen consumption rates in DCs, but does not affect survival, maturation rates, and glycolytic rates under the conditions studied. Dendritic cell secretion of IL-12, a proinflammatory cytokine upregulated during metabolism, decreased in a dose dependent manner under HIF-1 alpha inhibition. Understanding the effects of HIF-1 alpha can provide insight on how dendritic cells utilize their fuel source to facilitate immunological tasks and how in the future, we can optimize these sources to improve immune system functionality.

Systemic lupus erythematosus (SLE) develops when genetically susceptible individuals lose tolerance to autoantigens, likely as a result of an environmental insult. The list of identified genetic susceptibilities is expansive, however variants in the interferon regulatory factor 5 (IRF5) gene have consistently and convincingly been shown to be associated with an increased risk of developing SLE across all ethnic and racial groups examined. These genetic variants are hypothesized to produce a gain-of-function phenotype due to increased IRF5 mRNA and increased stability of the IRF5 protein; however, definitive functional studies examining these polymorphisms in primary human cells are not possible given the genetic variation from patient to patient. IRF5 is a transcription factor that is constitutively expressed in a number of immune cells including B cells and dendritic cells. IRF5 has cell type specific roles; in dendritic cells, it primarily controls a proinflammatory program which directs T cell polarization. Dysfunctional conventional dendritic cells (cDCs) have been implicated in the onset and development of SLE due to their high capacity to activate and interact with autoreactive lymphoid cells via a number of different pathways; the exact type of dysfunction and mechanisms underlying it are still debated. Study of primary cDCs either from SLE patients or healthy controls is complicated by the low frequency of cDCs in peripheral blood (<0.1%). To better evaluate the role IRF5 plays in cDC dysfunction in SLE, I developed a method for generating cDCs from induced pluripotent stem cells (iPSCs). The cDCs derived from this protocol are similar in many respects to primary human cDCs based on their gene expression profiles, cytokine production, and ability to act as antigen presenting cells to activate T cells. I also generated a library of iPSCs with and without the IRF5 risk haplotype to enable future studies to delineate the role of IRF5 polymorphisms in human cDCs. To facilitate these future studies, I also made an IRF5 deficient iPSC line which will be essential in discerning the role of IRF5 in cDC function. More broadly, we describe herein a platform to study gene function in an isogenic model of human conventional dendritic cells.

Pathogenic mechanisms that account for the dramatic differences between the HIV-1 and HIV-2 epidemics remain unknown. Myeloid dendritic cells (DCs) are sentinels of the immune system, which sense invading pathogens and initiate immune responses. I hypothesize that failure of HIV-2 to overcome DC-intrinsic defense mechanisms results in diminished virus replication and reduced pathogenesis in vivo. Recent studies from our laboratory have identified capture of HIV-1 by CD169 (Siglec1), which results in preservation of virus infectivity in peripheral non-lysosomal compartments and transfer to CD4+ T cells, a mechanism of DC-mediated trans infection. HIV-1 interaction with CD169 was dependent on incorporation of a ganglioside, GM3, in the virus particle membrane. We hypothesized that reduced interaction of HIV-2 with CD169 is crucial for its attenuated pathogenic phenotype in vivo. Interestingly, HIV-2 virion assembly sites were divergent from HIV-1, which correlated with reduced incorporation of GM3 in HIV-2 virions, and a significant decrease in capture of HIV-2 compared to HIV-1 by mature DCs. Furthermore, reduced CD169-dependent HIV-2 capture by DCs attenuated access of HIV-2 to DC-mediated trans infection. In contrast to the trans infection pathway, HIV-2 could establish productive infection in DCs, though productive infection of DCs by HIV-2 resulted in innate immune activation, induction of IFN-α production and attenuated spread of virus in DC – CD4+ T cell co-cultures. As opposed to HIV-2, productive infection of DCs by HIV-1 was attenuated and failed to trigger type I IFN responses, thus allowing for efficient spread of HIV-1 in DC – CD4+ T cell co-cultures. These results suggest that immune sensing of HIV-2 in productively infected DCs limits viral spread. Finally, we investigated GM3-expressing nanoparticles (GM3-NPs) for delivery of therapeutics that trigger innate immune responses in CD169+ myeloid cells as a novel strategy to mimic myeloid cell-intrinsic virus control observed in HIV-2 infection. We tested the ability of GM3-coated nanoparticles that incorporated a TLR2 ligand, Pam3CSK4, to activate CD169+ cells. Interestingly, Pam3CSK4 containing GM3-NPs robustly activated CD169+ cells. These results suggest that induction of dendritic cell-intrinsic type I IFN responses might be a fruitful therapeutic strategy to restrict HIV-1 replication in vivo.

Hepatitis C virus (HCV) has chronically infected an estimated 170million people worldwide. There are many impediments to the development of an effective vaccine for HCV infection. Dendritic cells (DC) remain the most important antigen-presenting cells for host immune responses, and are capable of either inducing productive immunity or maintaining the state of tolerance to self and non-self antigens. Researchers have recently explored the mechanisms by which DC function is regulated during HCV infection, leading to impaired antiviral T-cell responses and so to persistent viral infection. Recently, DC-based vaccines against HCV have been developed. This review summarizes the current understanding of DC function during HCV infection and explores the prospects of DC-based HCV vaccine. In particular, it describes the biology of DC, the phenotype of DC in HCV-infected patients, the effect of HCV on DC development and function, the studies on new DC-based vaccines against HCV infection, and strategies to improve the efficacy of DC-based vaccines.

The definition of Natural Killer (NK) cells has undergone dramatic modification with the advance of immunological research tools. NK cells can no longer be classified only as indiscriminate killer innate immune cells. NK cells are now known to form functional relationships with accessory cells to shape and promote the adaptive immune response. Recently, antigen specific and cytokine induced memory NK cells have been demonstrated to exhibit the characteristic phases of an adaptive immune cell, including extended persistence and robust function in response to reexposure. New findings in basic NK immunobiology indicate that the role of NK cells is underappreciated and these cells may potentially be manipulated for the development of anti-viral therapies.

Pneumocystis jirovecii causes fungal pneumonia in immunocompromised patients and can be fatal if left untreated. The global mortality rate is estimated to be over 200 000 in AIDS patients. In non-AIDS patients there is an estimated mortality rate of 50 000 cases. This rate is increasing in developed countries, attributed to an increase in disorders which require immunotherapy. These include hematologic malignancies, organ transplant, inflammatory disorders and pre-existing lung disease. Immediate immunity is initiated by receptors that recognize pathogen associated molecular patterns on the surface of pathogenic fungi. Specifically, C-type lectin receptors (CLRs) have been shown to be the principal initiators of innate immune response during fungal infection. Limited studies have focused on the role of CLRs in Pneumocystis infection. Dectin1and Mincle have been shown to recognise Pneumocystis surface antigens with Dectin-1 recognizing β-glucans on the Pneumocystis cell wall leading to an effective immune response. However, the role of a newly described CLR, the Dendritic Cell Immunoactivating Receptor (DCAR) remains undefined. For this reason, we investigated the potential role of this receptor in a mouse model of Pneumocystis murina infection. Wild type and DCAR-deficient C57BL/6 mice were infected with P. murina organisms via intratracheal instillation. Fungal burden was measured in the lung using quantitative Polymerase Chain Reaction. DCAR-deficient mice had a significantly reduced burden compared to wild type mice at Day 7 and 14 post-infection. To identify the immune components involved in pathogen clearance in these mice we measured cellular recruitment and cytokine production at both early (48 hours) and late (7, 14 and 21 days) time points. Flow cytometry analysis showed an increase in alveolar macrophage, dendritic cells, inflammatory monocytes, eosinophils and T cell recruitment to the lung. While ELISA showed increased levels of IL-1β and IFN-γ at 48 hours, and later on in infection IL-1β and IL-12p40 levels were also elevated. Histology analysis determined the localization of the recruited cells, and v interestingly showed an increase in mucus production at day 21 in DCARdeficient mice. In conclusion, we have identified DCAR deficiency as a potential driver of protective immunity in mice during P. murina infection. This may be associated with increased levels of IL-1β in DCAR-deficient mice. Furthermore, DCAR may also be important in adaptive inflammatory response regulation, as DCAR-deficient mice have increased cellular recruitment and mucus production later in infection. The mechanism by which the deletion of this receptor affords these mice the ability to efficiently clear P. murina remains to be determined.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18849号 / 医博第3960号 / 新制||医||1007(附属図書館) / 31800 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 坂井 義治, 教授 長澤 丘司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18892号 / 医博第4003号 / 新制||医||1009(附属図書館) / 31843 / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 鈴木 茂彦, 教授 長澤 丘司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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