Initiation of the Immune Response to Aspergillus fumigatus

描述（由申请人提供）：呼吸免疫系统每天清除数百个空气中的烟曲霉孢子（分生孢子）。肺部未经检查的孢子萌发导致侵袭性曲霉病（IA），这是免疫受损宿主感染性发病率和死亡率的主要原因。除了常驻肺泡巨噬细胞和募集的中性粒细胞外，我们还发现在肺部烟曲霉感染后，表达趋化因子受体ccr2的单核细胞迅速涌入。募集的单核细胞形成单核细胞来源的CD11b+ dc，将真菌抗原运输到引流淋巴结，并促进肺部真菌特异性CD4 T细胞的启动。ccr2表达细胞的消融导致真菌清除延迟和真菌特异性CD4 T细胞反应的丧失。为了探究由直接接触真菌细胞引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状芽孢杆菌菌株，以观察真菌的摄取情况，并区分肺部宿主白细胞内活的和灭活的真菌细胞。通过这种方法，我们研究了一个单核细胞功能模型，该模型将细胞活化和效应机制与真菌摄取联系起来，并通过适配器蛋白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。这项工作的基本原理是，它将提供一个全面的观点，单核细胞及其衍生物在宿主防御吸入真菌孢子。这一假说的基础是单核细胞形成了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部的先天和适应性抗真菌免疫反应。目的将：(1)确定单核细胞激活的机制，以及在免疫能力强和中性粒细胞减少的宿主中对真菌细胞杀伤的贡献；(2)确定CARD9-、MyD88-和nlrp53依赖性信号在感染结果、单核细胞依赖性先天和适应性免疫功能以及协调中性粒细胞快速募集到感染气道中的相对贡献。实验设计将使我们能够比较单核细胞与其他免疫细胞亚群的功能，并描述对烟曲霉免疫反应启动的基本步骤。提出的研究可作为基于体内荧光的方法的模型，该方法可解剖宿主-病原体遭遇的双边细胞结果。

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.