Initiation of the Immune Response to Aspergillus fumigatus

描述(申请人提供)：呼吸免疫系统每天清除数百个空气传播的烟曲霉孢子(分生孢子)。肺部无节制的孢子萌发会导致侵袭性曲霉病(IA)，这是免疫受损宿主感染发病率和死亡率的主要原因。除了常驻的肺泡巨噬细胞和招募的中性粒细胞外，我们发现在肺部烟曲霉菌攻击后，表达趋化因子受体CCR2的单核细胞迅速涌入。招募的单核细胞形成单核细胞来源的CD11b+树突状细胞，将真菌抗原输送到引流淋巴结，并促进肺部真菌特异性CD4T细胞的启动。切除表达CCR2的细胞会导致真菌清除延迟和丧失真菌特异性的CD4T细胞反应。为了研究直接接触真菌细胞触发的单核细胞介导的宿主防御机制，我们开发了一种新的荧光烟曲霉菌株来可视化真菌摄取，并区分肺内宿主白细胞内活的和灭活的真菌细胞。用这种方法，我们检验了一个单核细胞功能模型，该模型将细胞激活和效应机制与真菌摄取联系起来，并通过适配蛋白CARD9和MyD88整合来自C型凝集素(CTL)和Toll样受体(TLRs)的信号，以及来自细胞内Nod样受体(NLR)NLRP3的信号。这项拟议工作的基本原理是，它将全面了解单核细胞及其衍生物在宿主防御吸入真菌孢子方面的作用。支持这一建议的假设是，单核细胞形成一个细胞抗真菌效应系统，通过与真菌细胞的直接相互作用和CTL、TLR和NLR信号通路的输入来指导肺内的先天和适应性抗真菌免疫反应。这些目标将(1)确定免疫活性和中性粒细胞减少宿主中单核细胞激活和对真菌细胞杀伤的贡献的机制，(2)确定依赖CARD9、MyD88和NLRP3的信号对感染结局、单核细胞依赖的先天和获得性免疫功能以及协调中性粒细胞快速募集到受感染的呼吸道的相对贡献。实验设计将使我们能够将单核细胞与其他免疫细胞亚群进行功能比较，并描述启动对烟曲霉菌免疫反应的基本步骤。拟议的研究为体内基于荧光的方法提供了一个模型，该方法剖析了宿主与病原体相遇的双边细胞结果。

DESCRIPTION (provided by applicant): The respiratory immune system clears hundreds of airborne Aspergillus fumigatus spores (conidia) daily. Unchecked spore germination in the lung leads to invasive aspergillosis (IA), a major cause of infectious morbidity and mortality in immune compromised hosts. Beyond resident alveolar macrophages and recruited neutrophils, we identified a rapid influx of chemokine receptor CCR2-expressing monocytes following pulmonary A. fumigatus challenge. Recruited monocytes form monocyte-derived CD11b+ DCs, transport fungal antigen to draining lymph nodes, and facilitate the priming of fungus-specific CD4 T cells in the lung. Ablation of CCR2-expressing cells results in delayed fungal clearance and loss of fungus-specific CD4 T cell responses. To interrogate monocyte-mediated host defense mechanisms triggered by the direct encounter with fungal cells, we developed a novel fluorescent A. fumigatus strain to visualize fungal uptake and distinguish viable and inactivated fungal cells within host leukocytes in the lung. With this approach, we examine a model of monocyte function that links cell activation and effector mechanisms to fungal uptake and that integrates signals from C-type lectin (CTL) and Toll-like receptors (TLRs) via the adaptor proteins CARD9 and MyD88 and from the intracellular NOD-like receptor (NLR) NLRP3. The rationale for the proposed work is that it will provide a comprehensive view of monocytes and their derivatives in host defense against inhaled fungal spores. The hypothesis that underlies this proposal is that monocytes form a cellular antifungal effector system shaped by direct interactions with fungal cells and input from CTL, TLR, and NLR signaling pathways to direct innate and adaptive antifungal immune responses in the lung. The aims will (1) define the mechanism of monocyte activation and contribution to fungal cell killing in immune competent and neutropenic hosts and (2) determine the relative contribution of CARD9-, MyD88-, and NLRP3-dependent signals on the outcome of infection, on monocyte-dependent innate and adaptive immune functions, and on orchestrating rapid neutrophil recruitment to infected airways. The experimental design will enable us to compare monocytes functionally with other immune cell subsets and to describe essential steps in the initiation of the immune response to A. fumigatus. The proposed studies serve as a model for in vivo fluorescence-based approaches that dissect the bilateral cellular outcomes of host-pathogen encounters.