An Optical Toolbox for Multiplexed Imaging in Immune Cells

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Although infectious diseases are predicted to account for more than 3 million deaths worldwide by 2030, current therapeutic methods are still largely dependent on anti-microbials. This dependency is worrying due to rising antibiotic resistance, creating a need to develop other therapeutic methods to treat lung-based infections. The process of creating therapies for respiratory infections requires both an understanding of the responses that respiratory-borne microbes trigger in the host immune system, and an understanding of host-pathogen interactions in the lung. Currently, extensive research has been carried out on the former but thorough knowledge of the latter is still lacking. One way of studying host-pathogen interactions is through imaging tools, which not only provide a visual insight into these interactions, but also give a quantitative readout on responses to drugs in immune cells in situ. As such, this project aims to develop a toolbox of fluoroprobes to monitor immune cell function in inflammation models in response to infection.To achieve this, a palette of fluorophores will first be developed. They will be designed with the aim of being environmentally sensitive, giving a change in fluorescence readout in response to changes in pH and/or hydrophobicity. Furthermore, the attachment of warheads or metabolically reactive fragments to these probes enables activity monitoring and targeted imaging. The development of a family of fluorophores would thus allow for the monitoring of activity-based biomarkers in immune cells, giving insight to immune cell function and host-pathogen interactions.With these probes, we would then be able to image immune cell activity in biological models of increasing complexity. To provide a more holistic overview of host immune response to lung infections, T-cells, neutrophils and macrophages will be studied. Drug-resistant Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis, the three most common bacterial pathogens in upper and lower respiratory tract infections, will be cultured. Live cultures of these pathogens and human primary cells will then be used as a biological model to study host-pathogen interactions, using the developed toolbox of fluorophores. Assays will be designed to monitor changes in the physiological environment, such as the release of enzymes in response to inflammation. The best-performing biosensors will then be validated on established pre-clinical models, such as lung-on-chip models that simulate physiological lung conditions. Lastly, imaging of ex vivo lung tissue will be carried out to validate our findings.The development of a toolbox of optical biosensors and testing them in biological models of varying complexity would therefore provide a novel multiplexed live-cell imaging platform to study the interactions between host immune response and pathogens in the lung. This platform would accelerate the validation of new therapeutic strategies for infectious diseases, a growing burden to today's healthcare system.