Developing high content imaging of macrophages: potential drug discovery tool

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Chronic obstructive pulmonary disease (COPD) comprises three, distinct underlying pathophysiologies characterised by airflow limitation; chronic bronchitis, small airways disease and emphysema. The main cause is smoking and damage to the lung is largely irreversible, with all current therapies targeting symptoms but are not disease modifying. COPD is associated with chronic inflammation and increased numbers of phagocytic cells within the lung tissue. However, despite this, COPD patients are often colonised with airway pathogens. Phagocytosis of bacteria, fungal spores and apoptotic cells is reduced in macrophages from COPD patients, but the underlying mechanism remains unclear.Super-resolved microscopy (SRM) may allow investigation of specific molecular defects in single cells, including macrophages undergoing phagocytosis. This allows for the use of fewer cells than in conventional methods such as flow cytometry and plate reader fluorimetry. This project concerns the role of the cytoskeleton in phagocytosis, with the view to better understanding why some macrophages fail to phagocytose in COPD. Potential receptor defects that may lead to reduced phagocytosis will be investigated using advanced SRM techniques. This will allow visualisation of the cytoskeleton restructuring in real-time on live fluorescently-labelled macrophages in response to bacterial stimulation and potential interactions with cell surface receptors. Having established high-content techniques required to quantify these changes, the final step would be to screen putative phagocytosis stimulators with the aim of identifying novel therapies for the treatment of this disease. This technique could then be modified for use in other cell types as a high content - high throughput screening tool for other cellular mechanisms.Hypothesis: Macrophages from COPD patients will show modified or reduced cytoskeletal restructuring to bacterial stimuli during phagocytosis.Aims:1. Optimise a protocol to perform super-resolved microscopy on macrophages undergoing phagocytosis of bacteriaSRM on live cells is complex as antibodies are too large to enter the plasma membrane. Small molecule dyes are able to enter the cell membrane and are likely to prove effective at staining specific proteins of interest with a desired photo-switchable fluorophore, giving the capability to perform live super-resolved microscopy, tracking molecule fates and protein-protein interactions.2. Investigate cytoskeleton remodelling and restructuring in COPD macrophages Once techniques have been optimised using cell lines, the protocol will be implemented on primary tissue macrophages and monocyte-derived macrophages (MDM) from healthy controls, smokers and COPD patients to investigate live cytoskeletal restructuring of cells undergoing phagocytosis.3. Investigate receptor recycling in COPD macrophages during phagocytosisAs it remains unclear as to whether decreased phagocytosis observed in COPD is due to a failure of macrophages to recognise markers or a defect in receptor recycling, the involvement of phagocytosis receptors will be investigated.4. Screen putative phagocytosis 'correctors' in COPD cells High-content microscopy approaches will be developed during this project with the aim to produce a system that will allow several cells to be imaged at high resolution, to screen potential therapeutic molecules to aid and hasten the drug discovery process.Overview: My project is at the interface between biology and physics, utilising custom-built SRM within the Photonics Department to investigate lung disease at the NHLI. This unique collaboration has led to the development of a compact, modular and low-cost microscope utilising dSTORM that has been developed to make SRM more widely accessible ("easySTORM"). We are using this approach to determine the drivers of macrophage heterogeneity within a 96-well plate format.