High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

Abstract

IntroductionLung diseases are an increasing global health burden affecting millions of people worldwide. Only a few new inhaled medicines have reached the market in the last 30 years, in part due to foamy alveolar macrophage (FAM) responses observed in pre-clinical rat studies. The induction mechanism and signaling pathways involved in the development of highly vacuolated ‘foamy’ phenotype is not known. Furthermore, it has not been determined if these observations are adaptive or adverse responses.AimTo determine if high content image analysis techniques can distinguish between alveolar macrophage activation (LPS/IFN-γ activated and IL-4 activated macrophages) and if this could be applied to understanding the generation of ‘foamy’ macrophage phenotypes.MethodsNR8383 rat alveolar macrophages were stimulated with a mix of cytokines (LPS/IFN-γ or IL-4) for 24 h. The cells were further exposed to FAM inducing-compounds amiodarone and staurosporine. Following 24 h incubation, phagocytosis and lipid accumulation were measured using flow cytometry and high content image analysis techniques. The alveolar macrophages responses after exposure to cytokines were assessed by evaluation: (i) cell surface and biochemical markers such as: nitric oxide production, arginase-1 activity and MRC-1 receptor expression (ii) cellular morphology (iii) cellular functionality (phagocytic activity and lipids accumulation).ResultsMacrophages activated with LPS/IFN-γ showed distinct morphological (increased vacuolation) features and functionality (increased lipidosis, decreased phagocytic activity). Foamy macrophage phenotypes induced by amiodarone also displayed characteristics of proinflammatory macrophages (significantly increased nitric oxide production, increased vacuolation and lipidosis and decreased phagocytosis). In contrast, staurosporine treatment resulted in increased NO production, as well as arginase-1 activity.ConclusionHigh content image analysis was able to determine distinct differences in morphology between non-activated and LPS/IFN-γ activated macrophages, characterized by increased vacuolation and lipidosis. When exposed to compounds that induce a FAM phenotype, healthy non-activated macrophages displayed proinflammatory (amiodarone) or pro-apoptotic (staurosporine) characteristics but these responses were independent of a change in activation status. This technique could be applied in early drug discovery safety assessment to identify immune responses earlier and increase the understanding of alveolar macrophage responses to new molecules challenge in development of new inhalation therapies, which in turn will enhance decision-making in an early safety assessment of novel drug candidates.

Macrophage microvesicle‐derived circ_YTHDF2 in methamphetamine‐induced chronic lung injury

“…Alveolar macrophages (AMs) are important inflammatory and defensive cells in the lung, and are widely distributed in alveoli and airways. AMs are also primary regulatory cells that can positively influence the functions of other cell populations (Hoffman et al., 2021; Zhang et al., 2021; Zhang, Wang et al., 2019). Alveolar epithelial cells (AECs) are important regulatory cells and interact with inflammatory cells by secreting cytokines.…”

“…Alveolar macrophages (AMs) are important inflammatory and defensive cells in the lung, and are widely distributed in alveoli and airways. AMs are also primary regulatory cells that can positively influence the functions of other cell populations (Hoffman et al, 2021;Zhang et al, 2021;. Alveolar Sixty male BALB/C mice (23 ± 0.2 g) from the Animal Resource Centre, China Medical University (certificate number: Liaoning SCXK [Beijing] 2019-0010) were divided into two groups: control group (n = 27) and MA group (n = 33).…”