Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis

Ewelina Hoffman,Abigail Martin,Rhamiya Mahendran,Abhinav Kumar,Jan Klapwijk,Lea Ann Dailey,Victoria Hutter,Val Millar,Ben Forbes,Doug Ball,Aateka Patel

doi:10.1007/s11095-017-2176-5

Abstract

PurposeProgress to the clinic may be delayed or prevented when vacuolated or “foamy” alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures.MethodsHuman (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation.ResultsCell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed.ConclusionsA high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Highlights

Airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are an area of unmet clinical need despite considerable investment in developing new therapeutics in this area [1, 2]
Individual cell data for each parameter derived from the images were plotted and morphology and lipid content histograms assessed for normality (Fig. 1)
Whilst a significant difference (p < 0.05) was observed for cell area between NR8383 and U937 cells for both time points tested, U937 cell area was not significantly different (p > 0.05) from rodent alveolar macrophages obtained from bronchoalveolar lavage (BAL)

Summary

Introduction

Airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are an area of unmet clinical need despite considerable investment in developing new therapeutics in this area [1, 2]. New inhaled medicines require extensive nonclinical safety assessment before progressing to the clinic for evaluation of safety and efficacy in humans [1,2,3,4,5,6] It is not uncommon for alveolar macrophage responses to be observed in histological lung slices of animals during non-clinical inhalation toxicology studies: these are typically characterised by a highly vacuolated appearance and larger cell size [2, 3]. As the mechanism for induction of this alveolar macrophage phenotype and its relation to lung pathophysiology are not well understood, safe exposure levels are set without knowing whether these observations are truly an adverse response or an adaptation to high doses [2,3,4]. Due to these concerns over safety, inhaled compounds may fail in human studies due to lack of efficacy at the doses permitted [3,4,5]

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