Developing high content imaging of macrophages: potential drug discovery tool

慢性阻塞性肺疾病(COPD)包括以气流受限为特征的三种不同的潜在病理生理：慢性支气管炎、小气道疾病和肺气肿。主要原因是吸烟，对肺部的损害在很大程度上是不可逆的，目前所有的治疗方法都针对症状，但不能改变疾病。慢性阻塞性肺疾病与慢性炎症和肺组织内吞噬细胞数量增加有关。然而，尽管如此，慢性阻塞性肺病患者往往带有呼吸道病原体。COPD患者的巨噬细胞对细菌、真菌孢子和凋亡细胞的吞噬作用减少，但其潜在机制尚不清楚。超分辨显微镜(SRM)可以研究单个细胞中的特定分子缺陷，包括吞噬作用的巨噬细胞。这允许使用比传统方法更少的细胞，如流式细胞术和平板阅读器荧光法。本项目涉及细胞骨架在吞噬作用中的作用，以期更好地了解COPD中一些巨噬细胞无法吞噬的原因。可能导致吞噬功能降低的潜在受体缺陷将使用先进的SRM技术进行研究。这将允许实时可视化活的荧光标记巨噬细胞上的细胞骨架重组，以响应细菌刺激和与细胞表面受体的潜在相互作用。在建立了量化这些变化所需的高含量技术之后，最后一步将是筛选假定的吞噬刺激因子，目的是确定治疗这种疾病的新疗法。这项技术可以被修改用于其他类型的细胞，作为其他细胞机制的高含量-高通量筛选工具。假设：COPD患者的巨噬细胞在吞噬过程中对细菌刺激的细胞骨架重构会发生改变或减少。目的：1.优化一种对吞噬细菌的巨噬细胞进行超分辨显微镜的方法。活细胞上的SRM很复杂，因为抗体太大，无法进入质膜。小分子染料能够进入细胞膜，并可能被证明有效地用所需的光可切换荧光团对特定的蛋白质进行染色，从而能够进行实时超分辨显微镜、跟踪分子命运和蛋白质-蛋白质相互作用。研究COPD巨噬细胞的细胞骨架重构和重组一旦使用细胞系优化了技术，该方案将在来自健康对照组、吸烟者和COPD患者的初级组织巨噬细胞和单核细胞来源的巨噬细胞(MDM)上实施，以研究吞噬细胞的活细胞骨架重构。研究COPD巨噬细胞吞噬过程中的受体循环由于目前尚不清楚COPD患者中观察到的吞噬功能降低是由于巨噬细胞未能识别标志物还是由于受体循环缺陷，因此将对吞噬受体的参与进行调查。筛选COPD细胞中假定的吞噬‘校正器’在这个项目期间，将开发高含量显微镜方法，目的是生产一种系统，允许以高分辨率对几个细胞进行成像，筛选潜在的治疗分子，以帮助并加速药物发现过程。概述：我的项目处于生物学和物理学的交界处，利用光子学系内定制的SRM来研究NHLI的肺部疾病。这种独特的合作导致了一种紧凑、模块化和低成本的显微镜的开发，该显微镜利用dSTORM开发，使SRM更容易获得(“easySTORM”)。我们正在使用这种方法来确定96孔板中巨噬细胞异质性的驱动因素。

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.