Intravital imaging of the interaction of the pathogenic fungus Aspergillus fumigatus with cells of the innate and adaptive immune system

烟曲霉菌是一种腐生真菌，广泛分布于环境中。由于它不断地产生大量的分生孢子用于无性繁殖，所以呼吸的空气中含有1-100m-3的分生孢子，每个人每天不断地吸入100个分生孢子。这很少对免疫能力强的人造成健康问题，因为大量的分生孢子也很容易被先天免疫系统的细胞清除。然而，由于临床干预，例如在移植医学或肿瘤学或由于艾滋病，出现了大量免疫缺陷患者，使烟曲霉菌成为过去20年这些患者的主要死亡原因。今天，侵袭性曲霉病(IA)是最致命的感染形式，被认为是血液病患者感染性死亡的头号原因。近10-15年来对免疫系统在IA中的作用的研究表明，吞噬细胞，特别是肺泡巨噬细胞(AM)和中性粒细胞在烟曲霉菌防御中起主要作用。此外，适应性免疫系统已被证明对IA的结果很重要。以前工作的主要问题是，到目前为止，对烟曲霉菌防御的调查只可能是“死后”，即在感染过程中没有直接分析成功抵抗感染的细胞或屈服于感染的动物。因此，目前关于IA体内事件的概念主要是相互关联的。特别是在适应性免疫的情况下，由于缺乏适当的模型系统，烟曲霉菌的研究一直受到阻碍。在这项提案中，我们打算弥合这些差距。在第一个项目中，我们将分析先天免疫系统与烟曲霉菌的相互作用和反应。因此，在动物成功抵抗或屈服于感染的情况下，我们将直接观察活细胞与真菌相互作用的过程。作为一种全新的方法，我们将通过活着的动物体内显微镜来完成这项工作。第二个项目涉及开发一种新的烟曲霉菌株，它携带一种模式抗原，可以被来自T细胞受体转基因小鼠品系的T细胞识别。这种方法使我们能够通过组织学和流式细胞术等经典方法以及活体动物的活体成像来监测体内适应性免疫反应的从头发展。因此，这两个项目应该导致对导致IA发展或斗争的活体事件的全面了解。

A. fumigatus is a saprophytic fungus, which is ubiquitously dispersed in the environment. Since it is constantly producing large amounts of conidia for asexual reproduction, the breathing air contains 1-100 m-3 conidia and every individual is constantly inhaling several 100 conidia per day. This rarely causes health problems for immunocompetent persons, as also huge amounts of conidia are easily cleared by cells of the innate immune system. However, the appearance of large cohorts of immunecompromised patients due to clinical intervention, e.g. in transplantation medicine or oncology or due to AIDS has made A. fumigatus a major cause of death among these patients in the last two decades. Today, the most fatal form of infection, invasive Aspergillosis (IA), is considered as the number one cause for infectious death in hematological patients. Research of the last 10-15 years on the role of the immune system in IA has shown that phagocytes, especially alveolar macrophages (AM) and neutrophils play a major role in A. fumigatus defense. But also the adaptive immune system has been shown to be important for the outcome of IA. The major problem with the previous work is that so far investigation of A. fumigatus defense has only been possible "post mortem", i.e. the cells, which have successfully fought an infection or the animals, which have succumbed to it, were not analyzed directly during the process of infection. Therefore, current concepts as to the in vivo events of IA are mainly correlative. Especially in the case of adaptive immunity A. fumigatus research has been hampered by the lack of an appropriate model system. In this proposal we intend to close these gaps. In a first project we will analyze the interaction and response of the innate immune system with A. fumigatus. Thereby, we will directly observe living cells during their interaction with the fungus under circumstances, where the animal successfully fights or succumbs to an infection. As a completely new approach we will do this by intravital microscopy in living animals. The second project deals with the development of a new A. fumigatus strain, which carries a model antigen, that can be recognized by T cells from T cell receptor transgenic mouse lines. This approach allows us to monitor the de novo development of an adaptive immune response in vivo by classical methods such as histology and flow cytometry and also by intravital imaging in the living animal. Thus, these two projects should lead to a completely understanding of the in vivo events leading to the development or fight of IA.