The role of TLR9 on Aspergillus fumigatus phagosomes

描述(申请人提供)：烟曲霉是一种腐生丝状真菌，其无性孢子称为分生孢子在环境中广泛分布，通过吸入获得，小到足以到达远端呼吸道。大约10%的接受造血干细胞移植或实体器官移植的患者会患上侵袭性曲霉病，这是一种威胁生命的感染。尽管开发了有效的杀菌剂，但播散性感染的预后相当差，这表明对规则的了解 控制寄主对烟曲霉菌的防御对于开发新的预防和治疗策略至关重要。越来越多的证据表明先天免疫机制在真菌防御中的重要性。真菌β-1，3-葡聚糖受体Dectin1与TLR2和TLR4合作提供关键的免疫信号，协调细胞因子的分泌和适应性免疫的发展，在肺部对烟曲霉的防御中是必不可少的。当受到烟曲霉菌的攻击时，TLR9缺陷小鼠不能建立抗原特异性T细胞反应，这直接暗示TLR9参与了宿主对这种真菌病原体的防御。然而，TLR9介导的烟曲霉菌免疫应答的细胞生物学过程在很大程度上仍未解决。为了更好地了解TLR9在宿主防御烟曲霉菌中的作用，我们做了以下关键观察到，这是我们提出的工作的基本原理：1)烟曲霉噬菌体在巨噬细胞中的存在导致TLR9的亚细胞分布发生了戏剧性的变化，从ER到真菌周围明亮的环状隔室2)缺乏Dectin-1的巨噬细胞未能招募TLR9，这表明Dectin-1的存在是TLR9正确运输到真菌吞噬小体所必需的3)带有纯化的真菌衍生β-1，3-葡聚糖的聚苯乙烯微球可以在吞噬过程中模拟真菌颗粒。我们推测，烟曲霉细胞壁的一个成分介导了TLR9的募集，Dectin-1控制着TLR9向真菌吞噬小体的运输。我们建议：1)确定Dectin-1在APC中TLR9募集到烟曲霉噬菌体中的作用。2)确定TLR9募集到含有真菌样颗粒的吞噬体所需的分子条件。3)用光学捕捉法分析巨噬细胞对RC、SC、H、β-1，3葡聚糖和半乳甘露聚糖微球免疫应答的差异。我们将应用先进的成像方法，包括使用旋转圆盘的活细胞成像、共聚焦显微镜和光学捕获来控制宿主细胞和病原体之间的空间相互作用。关于TLR9转运到真菌噬菌体的调控机制的知识将有助于我们进一步理解对烟曲霉菌的天然免疫反应，并可能导致对如何调节宿主对这种致命病原体的防御的新见解。

DESCRIPTION (provided by applicant): Aspergillus fumigatus is a saprophytic filamentous fungus whose asexual spores called conidia are widespread in the environment, acquired through inhalation and small enough to reach the distal airways. Approximately 10% of patients with hematopoietic stem cell transplants or solid organ transplants will develop invasive aspergillosis, a life-threatening infection. Despite the development of effective fungicidal agents the prognosis for disseminated infection is quite poor, indicating that knowledge of the rules that govern the host defense against A. fumigatus is critical for development of new prevention and therapeutic strategies. Evidence for the importance of innate immune mechanisms in fungal defense is mounting. The fungal ¿-1,3 glucan receptor Dectin1 is essential in pulmonary defense against A. fumigatus and collaborates with TLR2 and TLR4 in providing critical immune signals that coordinate cytokine secretion and development of the adaptive immunity. When challenged with A.fumigatus, TLR9-deficient mice are incapable of mounting an antigen-specific T cell response, directly implicating the involvement of TLR9 in the host defense against this fungal pathogen. However, the cell biological processes underlying TLR9-mediated A.fumigatus immune responses are still largely unresolved. In order to understand better the role of TLR9 in host defense against A. fumigatus, we have made the following key observations that are the rationale for our proposed work: 1) the presence of A. fumigatus phagosomes in macrophages results in a dramatic change of the subcellular distribution of TLR9 from the ER to a bright, ring-shaped compartment around the fungus 2) macrophages lacking Dectin-1 fail to recruit TLR9, indicating that the presence of Dectin-1 is required for proper TLR9 trafficking to the fungal phagosome 3) polystyrene beads with purified, fungal-derived ¿-1,3-glucan attached covalently to the surface can mimic fungal particles during phagocytosis. We hypothesize that a component of the A. fumigatus cell wall mediates TLR9 recruitment and that Dectin-1 controls the trafficking of TLR9 to the fungal phagosome. We propose to: 1) determine the role of Dectin-1 in TLR9 recruitment to A. fumigatus phagosomes in APCs. 2) determine the molecular requirement for TLR9 recruitment to phagosomes containing fungal-like particles. 3) dissect the differences in immunological responses to RC, SC, H, ¿-1,3 glucan and galactomannan beads by macrophages using optical trap. We will apply advanced imaging modalities including live cell imaging using spinning disk confocal microscopy and optical trapping to control spatial interactions between host cell and pathogen. Knowledge gained regarding the mechanism of regulation of TLR9 trafficking to fungal phagosomes will be important in furthering our understanding of the innate immune response to A. fumigatus, and could lead to novel insights on how to modulate the host defense against this deadly pathogen.