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京都大学 / 新制・課程博士 / 博士(医学) / 甲第25175号 / 医博第5061号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 長尾 美紀, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Listeria monocytogenes (LM) and Salmonella enterica serovar Typhimurium (ST) are intracellular bacterial pathogens that cause invasive disease in immune-altered individuals, including the immunocompromised and pregnant women. The mechanisms that modulate innate immunity to intracellular infection, particularly during pregnancy, are not well-understood. Functional selectin ligands play critical roles in leukocyte recruitment during inflammation. Increased control of LM infection in functional selectin ligand-deficient (FtDKO) mice is associated with increased levels of circulating innate immune cells, despite defective leukocyte migration compared to WT mice. Adoptive transfer of WT and FtDKO bone marrow (BM) cells to irradiated WT and FtDKO recipients demonstrates that BM reconstitution and the increased neutrophil phenotype of FtDKO mice is independent of functional selectin ligand expression within the host environment. Thus, functional selectin ligand deficiency enhances inherent innate immune resistance to intracellular infection. We then examined the impact of pregnancy-associated immunological changes on maternal susceptibility to intracellular infections. ST infection in pregnant mice results in profound systemic infection, increased fetal loss and enhanced serum and placental expression of pro-inflammatory cytokines. Pregnant mice showed decreased ratios of pro-inflammatory Th17 cells relative to anti-inflammatory regulatory T cells (Tregs) when compared to non-pregnant mice during infection. Functional inactivation of Tregs in vivo restored control of infection and normal Th17-to-Treg ratios, and reduced fetal loss. These indicate that modulation of Th17 and Treg responses impacts maternal and fetal protection from ST infection. Lastly, we examined the roles of type I interferons (IFNs) in modulating innate immunity to intracellular infections during pregnancy. Type I IFN receptor deficiency (IFNAR-/-) enhances immunity to LM and ST in the non-pregnant state by limiting pathogen-induced leukocyte death. We show that pregnant IFNAR-/- mice infected with LM retain increased protection from infection relative to WT controls. In contrast, protection conferred by IFNAR deficiency against ST infection in the non-pregnant state is abrogated during pregnancy. Distinctive maternal responses to LM and ST are associated with differential regulation of leukocyte distribution and cytokine expression in maternal systemic and/or placental compartments. Taken together, modulation of key mechanisms involved in leukocyte recruitment, immune-regulation and cytokine signaling impact host susceptibility to intracellular infections.

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Ischemia followed by reperfusion, as happens in myocardial infarction, or the development of acute respiratory distress syndrome, are associated with a exaggerated extravasation of leukocytes into the surrounding tissue , which may cause severe bystander damage. In animal models of these diseases, pharmacological blockage of the leukocyte adhesion molecule, L-selectin (CD62L), has been shown to be partially protective by reduction or inhibition of leukocyte-mediated secondary tissue damage. The aim of this project was the development of an in vitro assay to investigate the relative effectiveness of potential L-selectin antagonists. Ideally, the assay should require low sample volumes and allow for measurements of larger series of reagents. The assay system investigated was based on the homotypic granulocyte aggregation under shear stress, which mimicks the L-selectin-dependent adhesion of leukocytes to previously arrested neutrophils on vascular endothelium. After optimizing numerous variables of the aggregation assay, the requirement of L-selectin for the homotypic granulocyte aggregation induced was demonstrated by inhibition experiments using soluble L-selectin or monoclonal antibodies directed against the lectin domain of L-selectin. Several carbohydrate polymers with L-selectin binding properties, such as the seaweed-derived fucose polymer fucoidin, high-molecular-weight dextran sulfate or heparin, also inhibited neutrophil aggregation in a dose-dependent fashion. However, despite employing a flow cytometry-based read-out technique, the assay remained extremely labor intensive, precluding investigations of extended series. Therefore, the homotypic aggregation experiments with freshly isolated human granulocytes remains a useful tool to further evaluate specific questions of L-selectin dependent adhesion processes, but it is not apt for transfer into routine laboratories.