Developing high content imaging of macrophages: potential drug discovery tool

开发巨噬细胞的高内涵成像：潜在的药物发现工具

• 批准号: 1949677
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1949677
• 关键词: Developing; content; imaging; macrophages; potential

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.

慢性阻塞性肺疾病（COPD）包括三种不同的基础病理生理学，其特征在于气流受限;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，目前所有的治疗都针对症状，但不能改善疾病。COPD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，COPD患者经常被气道病原体定殖。COPD患者巨噬细胞对细菌、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）可用于研究单细胞（包括吞噬巨噬细胞）中的特定分子缺陷。这允许使用比常规方法如流式细胞术和读板器荧光测定法更少的细胞。本项目关注细胞骨架在吞噬作用中的作用，以期更好地理解为什么一些巨噬细胞在COPD中不能吞噬。将使用先进的SRM技术研究可能导致吞噬作用降低的潜在受体缺陷。这将允许在活的荧光标记的巨噬细胞上实时观察细胞骨架重构，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后，最后一步将是筛选推定的吞噬刺激剂，目的是确定治疗这种疾病的新疗法。该技术然后可以被修改用于在其他细胞类型中作为其他细胞机制的高含量-高通量筛选工具。假设：来自COPD患者的巨噬细胞在吞噬过程中对细菌刺激表现出修饰或减少的细胞骨架重构。优化方案以在经历细菌吞噬的巨噬细胞上进行超分辨显微镜检查活细胞上的SRM是复杂的，因为抗体太大而不能进入质膜。小分子染料能够进入细胞膜，并且可能证明在用所需的光可切换荧光团对感兴趣的特定蛋白质进行染色时是有效的，从而能够进行实时超分辨显微镜检查，跟踪分子命运和蛋白质-蛋白质相互作用。研究COPD巨噬细胞中的细胞骨架重塑和重构一旦使用细胞系优化了技术，该方案将在来自健康对照、吸烟者和COPD患者的原代组织巨噬细胞和单核细胞衍生的巨噬细胞（MDM）上实施，以研究经历吞噬的细胞的活细胞骨架重构。研究吞噬过程中COPD巨噬细胞中的受体再循环由于尚不清楚在COPD中观察到的吞噬作用降低是由于巨噬细胞识别标记物的失败还是受体再循环的缺陷，因此将研究吞噬受体的参与。筛选COPD细胞中假定的吞噬“校正剂”在该项目期间，将开发高内容显微镜方法，旨在生产一种系统，该系统将允许以高分辨率对多个细胞进行成像，以筛选潜在的治疗分子，以帮助和加速药物发现过程。概述：我的项目是在生物学和物理学之间的接口，利用定制的SRM内的光子学部门调查肺部疾病在NHLI。这种独特的合作导致了一个紧凑的，模块化的和低成本的显微镜的发展，利用dSTORM，已开发，使SRM更广泛地访问（“easySTORM”）。我们正在使用这种方法来确定96孔板格式内巨噬细胞异质性的驱动因素。

项目成果

Analysis of defective phagocytosis in COPD using super-resolution microscopy and automated bacterial quantification

使用超分辨率显微镜和自动细菌定量分析 COPD 中的吞噬作用缺陷

• DOI: 10.1183/13993003.congress-2020.1058
• 发表时间: 2020
• 作者: Wysoczanski R

Robust optical autofocus system utilizing neural networks trained for extended range and time-course and automated multiwell plate imaging including single molecule localization microscopy

• DOI: 10.1101/2021.03.05.431171
• 发表时间: 2021-03
• 期刊: bioRxiv
• 作者: J. Lightley;F. Görlitz;S. Kumar;R. Kalita;A. Kolbeinsson;E. Garcia;Y. Alexandrov;V. Bousgouni;R. Wysoczanski;P. Barnes;L. Donnelly;C. Bakal;C. Dunsby;M. Neil;S. Flaxman;P. French

Towards easier, faster, super-resolved microscopy

迈向更简单、更快、超分辨率显微镜

• DOI: 10.1117/12.2550682
• 发表时间: 2020
• 作者: Görlitz F

Automated multiwell plate STORM: towards open source super-resolved high content analysis

自动化多孔板 STORM：迈向开源超分辨高内涵分析

• DOI: 10.1117/12.2526940
• 发表时间: 2019
• 作者: Görlitz F