High Content Image Analysis of Cellular Responses of the Murine J774A.1 Cell Line and Primary Human Cells Alveolar Macrophages to an Extended Panel of Pharmaceutical Agents

Introduction: 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). Methods: 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. Results: 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. Conclusions: 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.