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

A.烟曲霉是一种广泛分布于环境中的腐殖真菌。由于它不断产生大量的分生孢子进行无性繁殖，呼吸的空气中含有1-100 m-3分生孢子，每个个体每天不断吸入数百个分生孢子。这很少会对免疫功能正常的人造成健康问题，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中或由于AIDS，大量免疫受损患者的出现使得A.烟曲霉是过去二十年来这些患者死亡的主要原因。今天，最致命的感染形式，侵袭性曲霉菌病（IA），被认为是血液病患者感染性死亡的头号原因。过去10-15年关于免疫系统在IA中作用的研究表明，吞噬细胞，尤其是肺泡巨噬细胞（AM）和中性粒细胞在IA中发挥着主要作用。烟熏防御而且适应性免疫系统已被证明对IA的结果很重要。前期工作存在的主要问题是，迄今为止对A.烟曲霉的防御仅可能是“死后”的，即在感染过程中不直接分析成功抵抗感染的细胞或死于感染的动物。因此，目前关于IA体内事件的概念主要是相关的。特别是在适应性免疫A的情况下。烟曲霉研究因缺乏适当的模型系统而受到阻碍。在本提案中，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A。烟熏。因此，我们将直接观察活细胞在与真菌相互作用的情况下，动物成功地对抗或屈服于感染。作为一种全新的方法，我们将在活体动物体内进行活体显微镜检查。第二个项目涉及一种新的A.烟曲霉菌株，其携带可被来自T细胞受体转基因小鼠系的T细胞识别的模型抗原。这种方法使我们能够监测从头发展的适应性免疫反应在体内的经典方法，如组织学和流式细胞术，也通过活体成像在活体动物。因此，这两个项目应该导致对导致IA发展或对抗的体内事件的完全理解。

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.