Optimising Innate Host Defence to Combat Antimicrobial Resistance

The treatment of bacterial infection is complicated by antibiotic resistance. The body's defence against bacteria relies on the immune system and requires blood cells, called macrophages and neutrophils that eat and kill bacteria. Despite being frequently exposed to bacteria that cause serious infections most people rarely become ill due to these bacteria. We can learn from how the immune system protects most people and develop medicines to re-engage this system if it fails. This approach is currently limited by incomplete understanding of the precise mechanisms that kill bacteria in immune cells but our consortium has made great strides to address this. We now wish to refine our understanding of mechanisms that we have already identified and supplement this with further experiments to identify the best approaches with which to modulate these responses in patients.

In the body macrophages are the first line of defence against bacteria. We will use techniques that manipulate all the macrophage's genes individually and identify which are most important in regulating bacterial killing. We have also identified that when macrophages commit cell-suicide it helps clear bacteria and we will look for genes that regulate this process. When macrophages are overwhelmed by bacteria neutrophils are important to remove bacteria. For neutrophils we cannot manipulate the cell's genes but we will use an approach that uses antibodies to target all the proteins in the cell and will perform a similar screen to identify factors influencing bacterial killing. We also have some candidates we have already identified which regulate this process. We will then study how important the mechanisms we find are in models of infection where immune cells interact with other cell types. In particular we want to ensure we not only enhance bacterial killing but also minimize the capacity of neutrophil-derived immune factors to cause bystander damage to the body's tissues.

Next we will screen panels of chemical structures to enhance the selected mechanisms of bacteria killing. We will work with industry partners to adapt these structures for medical use. In particular we will test how well these target the specific location in the cell where the killing factors are produced using new approaches, termed super-resolution microscopy (SRM), that allow us to measure their production and location in the cell with great precision. We will modify the chemical structures to ensure our medicines target the right mechanism and location in the macrophage or neutrophil. These compounds will then be tested in our models of bacterial infection, including models of bacteria resistant to multiple antibiotics.

We will also test how the bacteria respond to attempts by the immune system to kill them. This will also inform understanding of how bacteria escape immune responses and spread between species to establish reservoirs of infection in animals that contribute to human disease with antibiotic resistant bacteria.

To confirm our findings are relevant to patients and to test potential medicines that we develop we will study macrophages and neutrophils from healthy volunteers or patients at risk of bacterial infection. Our approach will be significantly enhanced by our ability to image the interaction of bacteria with macrophages and neutrophils, and specifically the factors that regulate or mediate bacterial killing, in the lung of patients. This involves new developments with unique chemical probes and fibre optical imaging. We can potentially translate our findings rapidly to patients because many of the agents we will use to manipulate the innate response are drugs licensed for other medical indications. Our approach will reduce reliance on antibiotics and provide an alternative approach based on modifying the body's immune response that will be active against a range of bacteria, irrespective of their sensitivity to antibiotics.

The SHIELD consortium will combat antimicrobial resistance by enhancing phagocyte microbicidal responses. We will investigate microbicidal mechanisms and host responses in macrophages and neutrophils in response to Streptococcus pneumoniae and Staphylococcus aureus and validate key findings with antimicrobial resistant bacteria.

Automated genetic screens (e.g. CRISPR) will be performed in macrophages to identify microbicidal mechanisms. A reverse phase proteomic array (RPPA) of core neutrophil transduction responses to bacteria will be followed by screening the effects of pathway inhibition on bacterial killing and regulation of inflammation. Screen 'hits' will be supplemented with candidate regulators we have already identified. Mechanisms will be validated in phagocytes and in genetically modified mice and zebrafish models of infection. Compound libraries will be screened in phagocytes and zebrafish to enhance microbicidal responses that maximize bacterial clearance and minimise inflammation. After lead optimization we will confirm efficacy using super-resolution microscopy and mouse models of bacterial infection.

Animal adapted strains of bacteria will be serially passaged in human phagocytes to identify bacterial mutations arising from selective pressure. Analysis of how mutations influence microbicidal function will further clarify mechanisms and we will explore if mutational 'hot-spots' can be neutralized with monoclonal antibodies to limit immune escape.

The physiological relevance of selected mechanisms and pharmacological 'hits' will be confirmed in primary tissue macrophages and neutrophils isolated from inflamed tissue in healthy adults and patients at risk of bacterial infection. We will use molecular optical imaging with SmartProbes to document key phagocyte microbicidal responses to bacteria in the alveolar space. We will also use this platform to perform initial micro-dosing of lead compounds and expedite delivery of future phase I trials.

The development of novel host-based therapeutics to bacterial infections will have far reaching cross-sector consequences.

Academic sector: Refining understanding of the key mechanisms regulating antimicrobial killing and controlling inflammatory responses will be of broad interest to those developing new approaches to bacterial infection, investigating other forms of infection or inflammatory diseases for which inappropriate responses are harmful. The basic principles will extend to veterinary medicine and aid the 'One Health' initiative. The models developed will be shared with academic colleagues and the results of screens will be freely available on public databases (short term impact, 3-5 years).

NHS: Costs related to severe infection and in particular to antimicrobial resistance are high. New approaches to lessen the burden of these infections will decrease financial costs and save lives (long-term impact, >10 years). They will help restore confidence in the health service by users worried of the burden of antimicrobial resistance. The specific results will inform clinical trials of new therapies to combat bacterial infection by antimicrobial resistant micro-organisms (medium term impact, 5-10 years).

Industry: The identification of novel targets to combat antimicrobial resistance will be a major stimulus to the pharmaceutical industry. The development of new anti-infective agents will contribute to the replenishment of a pipeline, which is depleted. The anti-infective market is a major part of the pharmaceutical industry with global reach and this would have significant impact on the industry (medium term impact, 5-10 years). There would also be indirect effects on industry, including the need to develop better diagnostic tests of early infection or of severe inflammation, which could inform use of the new treatments. A key aspect of our proposal is a strong validation platform (short-term impact, 3-5 years), including the analysis of responses in the most relevant tissue phagocytes. An exciting component is the development of our imaging methodologies, including super-resolution microscopy to define precise localisation of targeting not possible previously. Molecular optical imaging with SmartProbes, will lead to confirmation of efficacy and appropriate targeting in the alveolar space of patients for the first time. This can expedite human translation and the selection of targets for phase I studies and will be an invaluable platform for use by industry (medium term impact).

Policy makers: National and international clinical guidelines will need to be reappraised in the light of alternative strategies to enhance host defence during infection (long-term impact, > 10 years). Therapeutics targeting the host could be administered as targeted prophylaxis to high-risk groups, be administered during the early stages of infection or most likely during established infection. The use of host response modulation would represent a paradigm shift in the approach to infection and how existing agents such as antimicrobials are used. They would require personalised stratification of risk and of severe complications of infection, which would impact management (medium term impact, 5-10 years). In addition they would lead to less reliance on antimicrobial agents (long term impact). The importance of infection would mean these policies would have a global perspective (long term impact).

Society: New approaches to combat infection would reduce health costs, morbidity and mortality (long term impact, > 10 years). Increased understanding of the management of infection and of antimicrobial stewardship, facilitated by publicising this research, would remove some of the inappropriate demand for antimicrobials and encourage greater engagement between the public, health care professionals and industry to tackle infection related problems with a responsible approach (short term impact, 3-5 years).