'Mechanisms of impaired neutrophil phagosome maturation and its impact on invasive bacterial infections

住进重症监护病房(ICU)的患者发生继发感染的风险很高，继发感染是指入院时不存在的感染，但会发展为ICU治疗的并发症。这些继发性感染给患者带来了重大负担，增加了死亡风险，延长了他们在重症监护病房的时间。我已经证明，身体免疫细胞防御系统的受损是这些感染发展的一个主要风险。参与对抗微生物的最重要的免疫细胞之一是中性粒细胞，我之前的工作已经证明，危重患者的中性粒细胞不能吞噬(吞噬细胞)细菌。最近，我证明了一旦食用细菌就会激活的杀戮机制，即所谓的吞噬小体成熟，在危重患者的中性粒细胞中也会受到损害。细菌杀灭的这种缺陷是由身体在受到严重感染和重大伤害等侮辱时产生的大量分子(称为C5a)驱动的。使用我开发的工具来描述和量化中性粒细胞用来对入侵的细菌做出反应的信号/通信分子，并实时评估细胞的抗菌功能，我已经确定了C5a可能损害这些中性粒细胞功能的一些潜在方式。这个项目的目的是研究这些信号/通信分子，以确定它们的工作机制。最终目标是找到恢复中性粒细胞功能和在不使用抗生素的情况下对抗感染的治疗方法。我之前的工作特别关注一种名为Vps34的信号酶。我将使用一系列技术，包括高分辨率细胞显微镜和绘制中性粒细胞之间的蛋白质分布图，来研究C5a和阻断Vps34如何改变蛋白质的分布。我将利用这些信息来了解这些细胞是如何导致细菌被削弱杀灭的。人类中性粒细胞的寿命太短，不能进行基因改变，但我有技术可以对寿命更长的中性粒细胞进行基因改变，这将使我们能够进一步研究关键的信号/通信媒介在细菌杀灭中的作用。我最近开发了一种中性粒细胞中没有Vps34的小鼠，我将用它来研究这种酶在一种相关疾病模型中所起的作用，即细菌性肺部感染(肺炎)。这是将培养皿中分离细胞的发现转化为对生物的影响，从而最终转化为患者的重要一步。我已经生成的数据非常详细地描述了中性粒细胞中细菌的信号分子反应，虽然这个项目专注于一种特定的途径，但还有多条进一步的途径有待识别和探索。本项目的最后部分将更详细地探索这一数据集，以确定未来调查的途径。这项工作将建立在我在剑桥大学内建立的合作基础上，使我能够接触到尖端技术和专业知识。这项工作将有许多重要的成果。首先，它将提供更多关于导致我在患者中性粒细胞中发现缺陷的机制的知识，确定治疗和预防继发性感染的新疗法的潜在靶点。其次，它将使我掌握研究这种关键免疫细胞的其他功能所需的技能和知识，这也将对其他类型的细胞有用。它将创建一个确定候选目标的平台，以及筛选这些目标的渠道。然后，最有希望的药物可以用于动物模型和人类实验药物。这将最大限度地以高效和具有成本效益的方式获得成功研究的机会。

Patients admitted to intensive care units (ICU) are at high risk of developing secondary infections, which are infections which are not present on admission but develop as a complication of ICU therapy. These secondary infections place a major burden on the patient, increasing the risk of death and prolonging their stay in intensive care. I have shown that impairment of the body's immune cell defences is a major risk for the development of these infections. One of the most important immune cells involved in the fight against microbes is the neutrophil, and my previous work has demonstrated that critically ill patients have neutrophils which fail to eat ('phagocytose') bacteria. More recently I have shown that the killing mechanisms that are activated once the bacteria are eaten, so called 'phagosomal maturation', are also impaired in neutrophils from critically ill patients. This defect in bacterial killing is driven by a molecule produced by the body in large amounts in response to insults such as severe infections and major injury (called C5a). Using tools I have developed to profile and quantify the signalling/communication molecules that neutrophils use to respond to invading bacteria, and to evaluate the cells' anti-bacterial functions in real time, I have identified a number of potential ways in which C5a may impair these neutrophil functions. The aim of this project is to examine these signalling/communication molecules to identify the mechanisms by which they work. The ultimate aim is to identify therapies to restore neutrophil function and fight infections without antibiotics.My particular focus, arising from my previous work, is on a signalling enzyme called VPS34. I will use a range of techniques, including high resolution cellular microscopy and mapping of protein distribution across neutrophils to examine how C5a and blocking VPS34 alter the distribution of proteins. I will use this information to understand how these lead to impaired bacterial killing by these cells. Human neutrophils are too short-lived to be genetically altered, but I have techniques which allow genetic alteration of longer-lived neutrophil-like cells which will allow further investigation of the role of key signalling/communication mediators in bacterial killing. I have recently developed a mouse which has no VPS34 in its neutrophils that I will use to examine the role played by this enzyme in a relevant disease model, namely bacterial lung infection (pneumonia). This is an important step in translating the findings from isolated cells in a dish into the effects into living creatures, and hence ultimately into patients. The data I have generated profiling the signalling molecule response to bacteria in neutrophils is very detailed, and whilst this project focuses on one particular pathway, there are multiple further pathways to be identified and explored. The final part of this project will explore this data set in greater detail to identify pathways for future investigation. This work will build on collaborations I have established within the University of Cambridge, allowing me access to cutting edge techniques and expertise.This work will have a number of important outputs. First, it will provide greater knowledge of the mechanisms which lead to the defect I have identified in patient neutrophils, identifying potential targets for new therapies to treat and prevent secondary infections. Second it will equip me with the skills and knowledge required to investigate other functions in this key immune cell, and which will also be of use in other cell types. It will create a platform for the identification of candidate targets, and a pipeline for screening these targets. The most promising ones can then be taken into animal models and human experimental medicine. This maximises the chances of successful studies in an efficient and cost-effective manner.