Abstract
Many potential inhaled medicines fail during development due to the induction of a highly vacuolated or “foamy” alveolar macrophage phenotype response in pre-clinical studies. There is limited understanding if this response to an inhaled stimulus is adverse or adaptive, and additionally if it is a transient or irreversible process. The aim of this study was to evaluate whether high content image analysis could distinguish between different drug-induced foamy macrophage phenotypes and to determine the extent of the reversibility of the foamy phenotypes by assessing morphological changes over time. Alveolar-like macrophages derived from the human monocyte cell line U937 were exposed for 24 h to compounds known to induce a foamy macrophage phenotype (amiodarone, staurosporine) and control compounds that are not known to cause a foamy macrophage phenotype in vitro (fluticasone and salbutamol). Following drug stimulation, the cells were rested in drug-free media for the subsequent 24 or 48 h. Cell morphometric parameters (cellular and nuclear area, vacuoles numbers and size) and phospholipid content were determined using high content image analysis. The foamy macrophage recovery was dependent on the mechanism of action of the inducer compound. Amiodarone toxicity was associated with phospholipid accumulation and morphometric changes were reversed when the stimulus was removed from culture environment. Conversely cells were unable to recover from exposure to staurosporine which initiates the apoptosis pathway. This study shows that high content analysis can discriminate between different phenotypes of foamy macrophages and may contribute to better decision making in the process of new drug development.
Highlights
Alveolar macrophages reside on the luminal surface of the alveolar space and are a normal feature of the healthy lung [1]
To better understand the foamy alveolar macrophage phenomenon, it is necessary to study the timeframe over which increased vacuolation occurs and whether there are morphological and/or biochemical markers, or thresholds of these markers which indicate if the foamy macrophage phenotype is transient and reversible or a permanent and irreversible process
The high content image analysis technique reported in this study demonstrates it is possible to measure detailed morphological characteristics including cellular area, report the number of vacuoles, and quantify the area of the cell occupied by vacuoles rapidly
Summary
Alveolar macrophages reside on the luminal surface of the alveolar space and are a normal feature of the healthy lung [1]. Novel inhaled medicines are often withdrawn from further development due to concerns regarding the potential toxicity manifested by the presence of these foamy macrophage observations in pre-clinical animal studies [3] These “foamy” macrophage (FM) responses can be accompanied by additional abnormal lung tissue changes such as neutrophil infiltration or lymphocyte degeneration [4]. The highly vacuolated phenotype may be induced by a variety of cellular mechanisms including phagocytosis of poorly soluble drug particles, stimulation of excess lung surfactant, impaired metabolism of intracellular phospholipids, phagocytosis of surfactant phospholipids, apoptosis, and autophagy [3,4,5] It is not clear whether increased alveolar macrophage vacuolation should be classified as a permanent an adverse or adaptive response [3,4]. To better understand the foamy alveolar macrophage phenomenon, it is necessary to study the timeframe over which increased vacuolation occurs and whether there are morphological and/or biochemical markers, or thresholds of these markers which indicate if the foamy macrophage phenotype is transient and reversible or a permanent and irreversible process
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