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

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.

Critically ill patients suffer systemic inflammation and simultaneous failure of anti-microbial functions, which leads to secondary infections including pneumonia. I have demonstrated that complement component C5a, released in large quantities during critical illness, can impair neutrophil phagocytosis and is associated with an increased risk of secondary infections. During my early post-doctoral fellowship I developed a novel multi-parameter assay of neutrophil function and combined it with phosphoproteomic profiling tools. Using these I demonstrated impairment of phagosomal maturation in neutrophils from critically ill patients, and how this could be driven by C5a. I also demonstrated a role for the phosphoinositide 3-kinase VPS34 in phagosomal maturation. My findings imply that the defect in patients is in the recently characterised LC3-associated phagocytosis (LAP) process, of which VPS34 is a key signalling node.

My objective is to understand mechanisms that drive neutrophil phagosomal maturation and LAP, how this is disrupted by C5a and the consequences of this disruption on bacterial killing. I will use primary human neutrophils and gene-edited stem cell-derived neutrophils to establish mechanisms by which the proteins I have identified affect phagocytosis and phagosomal maturation. I will use subcellular fractionation and hyper localization of organelle proteins by isotope tagging (hyperLOPIT) proteomics to map protein locations within neutrophils, using confocal microscopy validate these findings. Using a neutrophil-conditional VPS34 knockout mouse I have developed, I will investigate the relevance of these findings in models of bacterial pneumonia. My existing neutrophil phosphoproteomic profile is detailed, and I will use computational methods to identifying further pathways for investigation. Using these tools I aim to identify targets for future non-antibiotic therapy, enhancing neutrophil functions in bacterial infections.