Reprogramming Human Inflammatory Macrophages in Symptomatic Carotid Stenosis: Potential Mechanisms for Stabilisation of Atherosclerotic Carotid Plaques

Abstract

AbstractBackgroundInflammation is a precursor to atherosclerotic plaque destabilisation, leading to ischaemic events such as stroke. Macrophage phenotypes can be altered by the microenvironment, and certain anti-inflammatory agents may, therefore, stabilise plaques and reduce the risk of recurrent ischaemic events.MethodsThirteen carotid plaques were obtained from stroke/ Transient Ischaemic Attack (TIA) patients undergoing carotid endarterectomy. An immunofluorescence stain was used to identify common macrophage markers (pan macrophage: CD68, pro-inflammatory: CD86, anti-inflammatory: MRC1), and a novel analysis technique was used to measure the prevalence of macrophage phenotypes in carotid plaques in relation to other histological features of instability.Anin vitromodel of human blood-derived macrophages was also developed to evaluate the effect of statins and glucocorticoids on macrophage-specific markers using RT-qPCR, Western Blot and immunofluorescence stain. The physiological effect of dexamethasone was further evaluated on macrophages and human carotid plaques culturedex vivo.ResultsThe macrophage population (CD68+) in the carotid plaques was dominated by “double-positive” (CD86+MRC1+) macrophages (67.8%), followed by “M1-like” (CD86+MRC1-) (16.5%), “M2-like” (CD86-MRC1+) (8.7%) and “double-negative” (CD86-MRC1-) (7.0%) macrophages. M1-like macrophages were more prevalent in unstable plaque sections than stable ones (p=0.0022).Exposure to dexamethasone increased macrophageMRC1gene expressionin vitroandex vivo. Dexamethasone also reduced Oxidised Low-Density Lipoprotein Receptor 1 (OLR1) gene and protein expression, leading to a decreased ox-LDL uptake in foam cell assays. This was, in turn, associated with reduced lipid uptake in macrophages, as shown by Oil Red O staining.ConclusionsHuman macrophages may be “switched” to a less inflammatory phenotype by exposure to clinically relevant concentrations of glucocorticoid, potentially mediated by a reduction in Oxidised LDL uptake. This effect was not observed following macrophage exposure to statins. Glucocorticoids may have a future role in preventing ischaemic events in patients with advanced atherosclerosis.Graphical AbstractHighlightsA high prevalence (68% in this study) of carotid plaque macrophages express both pro-inflammatory (CD86) and anti-inflammatory (MRC1) markers. These may represent a novel macrophage population.Human macrophages may be “reprogrammed” to a less inflammatory phenotype following exposure to glucocorticoids.Dexamethasone increasedMRC1and decreasedOLR1expression in macrophages derived from human blood samplesin vitroand in cells derived from cultured human carotid plaque tissueex vivo.This was associated with reduced oxLDL uptake and reduced lipid accumulation in the macrophages.Dexamethasone has the potential to stabilise carotid atherosclerotic plaques in humans.