Tietze Lysann, Urbano Laura, Eisenmann Stephan, Schwarzinger Jacqueline, Kollan Julia, Forbes Ben, Dailey Lea Ann, Hädrich Gabriela
Department of Visceral, Transplant, Thoracic and Vascular Surgery, University of Leipzig Medical Center, 04103, Leipzig, Germany.
Department of Clinical, Pharmaceutical and Biological Sciences, University of Hertfordshire, College Lane, Hatfield, Hertfordshire, AL10 9AB, UK.
Pharm Res. 2025 Jan;42(1):93-108. doi: 10.1007/s11095-024-03806-y. Epub 2025 Jan 7.
In vitro screening of macrophages for drug-induced effects, such as phospholipidosis, is useful for detecting potentially problematic compounds in the preclinical development of oral inhaled products. High-content image analysis (HCIA) is a multi-parameter approach for cytotoxicity screening. This study provides new insights into HCIA-derived response patterns of murine J774A.1 cells and primary human alveolar macrophages (hAM).
Several compounds were compared with reference groups (cationic amphiphilic drugs and apoptosis inducers) at different concentrations (0.01 to 10 µM). After incubation, cells were stained with fluorescence markers and HCIA was performed (Cytation™ 5 Cell Imaging System). Ten parameters were analysed: non-adherent cells, increased or reduced mitochondrial activity, membrane permeability, cell area, nuclear area, polynucleated cells, vacuole area, neutral and phospholipid content. A new system of response categorisation was developed for data analysis.
Murine J774A.1 cells exhibited a drug-induced response pattern that was distinct to the corresponding pattern of hAM cells. Comparison with the literature revealed that primary cells (rat or human origin) have similar response patterns, while cell lines (mouse, rat or human) exhibited a different response pattern. Hierarchical clustering revealed toxicologically aligned clusters of compounds, suggesting potential use for understanding mechanisms of drug effects in cell lines and primary cells.
Valuable information for selecting a suitable cell type for HCIA screening of macrophage responses to drug compounds is provided. All cell types were suitable for screening drug-induced phospholipidosis. Still, human primary alveolar macrophages responded differently to drug treatment compared to macrophage cell lines and may be required to evaluate broader response-patterns and mechanisms of toxicity.
体外筛选巨噬细胞的药物诱导效应，如磷脂沉积症，对于在口服吸入产品的临床前开发中检测潜在问题化合物很有用。高内涵图像分析（HCIA）是一种用于细胞毒性筛选的多参数方法。本研究为源自HCIA的小鼠J774A.1细胞和原代人肺泡巨噬细胞（hAM）的反应模式提供了新见解。
将几种化合物与参考组（阳离子两亲性药物和凋亡诱导剂）在不同浓度（0.01至10 μM）下进行比较。孵育后，用荧光标记物对细胞进行染色，并进行HCIA（Cytation™ 5细胞成像系统）。分析了十个参数：非贴壁细胞、线粒体活性增加或降低、膜通透性、细胞面积、核面积、多核细胞、液泡面积、中性和磷脂含量。开发了一种新的反应分类系统用于数据分析。
小鼠J774A.1细胞表现出与hAM细胞相应模式不同的药物诱导反应模式。与文献比较表明，原代细胞（大鼠或人源）具有相似的反应模式，而细胞系（小鼠、大鼠或人源）表现出不同的反应模式。层次聚类揭示了毒理学上排列的化合物簇，表明在理解细胞系和原代细胞中药物作用机制方面有潜在用途。
提供了关于选择合适细胞类型用于HCIA筛选巨噬细胞对药物化合物反应的有价值信息。所有细胞类型都适合筛选药物诱导的磷脂沉积症。然而，与巨噬细胞系相比，人原代肺泡巨噬细胞对药物治疗的反应不同，可能需要用于评估更广泛的反应模式和毒性机制。
