Systems Analysis of Innate Immune Responses to Fungal-Derived Carbohydrates

 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Dectin-1与TLR2和TLR4协同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起重要作用。在病原体-宿主关系中，真菌细胞壁是最初的接触点。尽管对真菌细胞壁的拓扑结构进行了优雅的超微结构研究，但我们对与哺乳动物细胞受体相互作用的多糖成分的了解很少。β-1，3葡聚糖是一种在多种病原真菌中发现的促炎多糖，是研究最深入的真菌碳水化合物。其他真菌碳水化合物包括甲壳素、甘露聚糖、半乳氨基半乳糖、半乳甘露聚糖和α-1，3葡聚糖引发的确切反应仍不完全清楚。为了更好地了解这些真菌多糖对先天性免疫反应的贡献，我们做了以下关键观察，这是我们工作的基础：1)当真菌存在于吞噬体内时，会触发多种免疫学感兴趣的蛋白的特异性募集，包括CD63、CD82、II类MHC、LC3、LAMP-1和TLR9。与此形成鲜明对比的是，聚苯乙烯微珠未能做到这一点。2)我们生成了大小匹配的聚苯乙烯珠子，它具有单一真菌衍生的碳水化合物的均匀涂层，共价连接以探测免疫反应。这些真菌样颗粒具有生物活性并触发细胞因子反应3)利用真菌样颗粒，我们在巨噬细胞中产生了吞噬小体，并通过质谱分析确定了吞噬小体蛋白质组。我们假设吞噬小体的内容触发了特定哺乳动物蛋白质的组装到这个隔室，并且这个蛋白质组装协调了这个细胞的净免疫输出。我们建议：1)确定被招募到含有不同真菌样颗粒的吞噬小体中的哺乳动物蛋白质，以及2]将人类单核细胞中的细胞因子特征描述为真菌样颗粒。我们将应用先进的蛋白质组学和多重细胞因子分析来实现这些目标。了解碳水化合物识别的机制及其对吞噬小体形成和细胞因子信号的影响，将有助于我们进一步了解烟曲霉菌的天然免疫反应，并可能为开发针对这种致命病原体的疫苗提供新的见解。

 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.