Abstract
The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is, however, not very well established. Based on Nile Red (NR) neutral LD imaging, using confocal microscopy and computational analysis, we developed NR Quantifier (NRQ), a novel quantification method to assess LD content in monocytes. Circulating monocytes were isolated from blood and used for the NR staining procedure. In monocytes stained with NR, we clearly distinguished, based on 3D imaging, phospholipids and exclusively intracellular neutral lipids. Next, we developed and validated NRQ, a semi-automated quantification program that detects alterations in lipid accumulation. NRQ was able to detect LD alterations after ex vivo exposure of isolated monocytes to freshly isolated LDL in a time- and dose-dependent fashion. Finally, we validated NRQ in patients with familial hypercholesterolemia and obese subjects in pre- and postprandial state. In conclusion, NRQ is a suitable tool to detect even small differences in neutral LD content in circulating monocytes using NR staining.
Highlights
The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability
In order to distinguish between membrane-bound cholesterol [32] and neutral lipid droplet (LD), the fluorescent intensity of membrane-bound cholesterol was reduced until LDs alone were visible
To test whether Nile Red (NR) staining was equivalent to Oil Red O (ORO), we examined lipid accumulation in isolated monocytes and stained LDs using both ORO and NR
Summary
The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy J. This article is available online at http://www.jlr.org [6], as well as increased monocyte-derived production of tumor necrosis factor and interleukin 1 [7] In this context, circulating monocytes may serve as a biomarker for atherosclerotic plaque vulnerability [1, 8, 9], whereas lipid accumulation in these cells may be an important driver for this systemic pro-inflammatory phenotype [6, 7, 10]. Lipid uptake must be tightly controlled in order to maintain cellular homeostasis by preventing lipid overload and subsequent cellular activation [14]
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