An Optical Toolbox for Multiplexed Imaging in Immune Cells

尽管预计到2030年，传染病将导致全球300多万人死亡，但目前的治疗方法仍在很大程度上依赖于抗微生物药物。由于抗生素耐药性的上升，这种依赖性令人担忧，需要开发其他治疗方法来治疗肺部感染。创建呼吸道感染疗法的过程需要了解病原微生物在宿主免疫系统中引发的反应，以及了解肺部的宿主-病原体相互作用。目前，对前者的研究已相当深入，但对后者的认识还很欠缺。研究宿主-病原体相互作用的一种方法是通过成像工具，这不仅提供了对这些相互作用的视觉洞察，而且还提供了原位免疫细胞对药物反应的定量读数。因此，本项目旨在开发一个荧光探针工具箱，以监测炎症模型中免疫细胞对感染的反应。为此，将首先开发一个荧光团调色板。它们的设计目的是对环境敏感，响应pH和/或疏水性的变化而改变荧光读数。此外，将弹头或代谢反应性碎片连接到这些探针上，可以进行活动监测和靶向成像。因此，开发一系列荧光团将允许监测免疫细胞中基于活性的生物标志物，从而深入了解免疫细胞功能和宿主-病原体相互作用。有了这些探针，我们将能够在日益复杂的生物模型中对免疫细胞活性进行成像。为了提供对肺部感染的宿主免疫应答的更全面的概述，将研究T细胞、中性粒细胞和巨噬细胞。将培养耐药肺炎链球菌、流感嗜血杆菌和卡他莫拉菌，这三种上呼吸道和下呼吸道感染中最常见的细菌病原体。这些病原体和人类原代细胞的活培养物将被用作生物模型，使用开发的荧光团工具箱研究宿主-病原体相互作用。检测将被设计用于监测生理环境的变化，例如炎症反应中酶的释放。然后将在已建立的临床前模型上验证性能最佳的生物传感器，例如模拟生理肺部条件的肺芯片模型。最后，离体肺组织的成像将进行验证我们的研究结果，因此，光学生物传感器的工具箱的开发和测试它们在不同的复杂性的生物模型将提供一个新的多路复用的活细胞成像平台，研究宿主免疫反应和病原体之间的相互作用在肺部。该平台将加速传染病新治疗策略的验证，这是当今医疗保健系统日益增长的负担。

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.