Systems Analysis of Innate Immune Responses to Fungal-Derived Carbohydrates

 描述（由申请方提供）：烟曲霉是一种嗜盐丝状真菌，其称为分生孢子的无性孢子在环境中广泛分布，通过吸入获得，并且足够小以到达远端气道。大约10%的造血干细胞移植或实体器官移植患者会发生侵袭性曲霉病，这是一种危及生命的感染。尽管开发了有效的杀真菌剂，但播散性感染的预后相当差，这表明了解 控制宿主对A的防御。烟曲霉对开发新的预防和治疗策略至关重要。先天免疫机制在真菌防御中的重要性的证据越来越多。真菌β-1，3葡聚糖受体Dectin-1在肺防御A.在烟曲霉中表达，并与TLR 2和TLR 4合作提供协调细胞因子分泌和适应性免疫发展的关键免疫信号。真菌细胞壁是病原体与宿主关系的初始接触点。尽管对真菌细胞壁的拓扑结构进行了精细的超微结构研究，但我们对与哺乳动物细胞受体相互作用的多糖成分的理解却知之甚少。β-1，3葡聚糖是一种在许多致病真菌中发现的促炎多糖，是研究得最好的真菌碳水化合物。其他真菌碳水化合物（包括几丁质、甘露聚糖、半乳糖氨基半乳聚糖、半乳甘露聚糖和α-1，3葡聚糖）引发的精确反应仍不完全清楚。为了更好地理解这些真菌多糖对先天免疫应答的贡献，我们进行了以下关键观察，这些观察是我们提出的工作的基本原理：1）当存在于吞噬体中时，真菌触发多种免疫学感兴趣的蛋白质的特异性募集，包括CD 63、CD 82、II类MHC、LC 3、LAMP-1和TLR 9。与此形成鲜明对比的是，聚苯乙烯珠不能做到这一点。2)我们产生了大小匹配的聚苯乙烯珠，其具有共价连接的单一真菌衍生的碳水化合物的均匀涂层以探测免疫应答。这些真菌样颗粒具有生物活性并触发细胞因子应答3）使用真菌样颗粒，我们在巨噬细胞中产生吞噬体并通过质谱法确定吞噬体蛋白质组。我们推测，吞噬体的内容物触发特定的哺乳动物蛋白质组装到这个隔间，并且这种蛋白质组装协调了该细胞的净免疫输出。我们建议：1]确定募集到含有不同真菌样颗粒的吞噬体的哺乳动物蛋白质，和2]将人单核细胞中的细胞因子特征描绘为真菌样颗粒。我们将应用先进的蛋白质组学和多重细胞因子检测来实现这些目标。关于碳水化合物识别机制的知识，其对吞噬体形成和细胞因子特征的影响对于进一步理解对A的先天免疫应答将是重要的。烟曲霉，并可能导致针对这种致命病原体的疫苗开发的新见解。

 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 ß-1,3 glucan receptor Dectin-1 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. The fungal cell wall serves as the initial point of contact in the pathogen-host relationship. Despite elegant ultra-structural studies on the topology of the fungal cell wall, our understanding of the polysaccharide constituents that interact with mammalian cellular receptors is poorly understood. ß-1,3 glucan, a proinflammatory polysaccharide found on numerous pathogenic fungi, is the best studied fungal carbohydrate. The precise response triggered by other fungal carbohydrates including chitin, mannan, galactoaminogalactan, galactomannan and α-1,3 glucan remains incompletely understood. In order to understand better the contribution of these fungal polysaccharides to the innate immune response, we have made the following key observations that are the rationale for our proposed work: 1) when present in phagosomes, fungi trigger specific recruitment of multiple proteins of immunologic interest including CD63, CD82, class II MHC, LC3, LAMP-1 and TLR9. In sharp contrast, polystyrene beads failed to do so. 2) we generated size-matched polystyrene beads that possess a uniform coat of a single fungal-derived carbohydrate covalently attached to probe the immune response. These fungal like particles are biologically active and trigger cytokine responses 3) using fungal like particle, we generated phagosomes in macrophages and determined that the phagosomal proteome by mass spectrometry. We hypothesize that the content of the phagosome triggers the assembly of specific mammalian proteins to this compartment and that this assembly of proteins orchestrates the net immunologic output from this cell. We propose to: 1] Determine the mammalian proteins recruited to the phagosome containing different fungal like particles and 2] Profile the cytokine signature in human monocytes to fungal-like particles. We will apply advanced proteomics and multiplex cytokine assay to achieve these aims. Knowledge gained regarding the mechanism of carbohydrate recognition, its influence on the phagosome formation and cytokine signature will be important in furthering our understanding of the innate immune response to A. fumigatus, and could lead to novel insights to vaccine development against this deadly pathogen.