Abstract

Background Atherosclerotic plaque formation is the underlying cause of heart attack and stroke. Macrophages play a key role in plaque progression. Neutrophils are rarely detected in plaques but have been shown to play an integral role in plaque development. We have previously shown that microvesicles released from stimulated neutrophils are present in plaques and enhance plaque formation. Neutrophil microvesicles (NMV) have been found to modulate macrophage activity in atherosclerosis and are known to contain microRNA that can influence macrophage behaviour. We hypothesise that NMV can interact with macrophages within atherosclerotic plaques and alter macrophage phenotype and function. Methods To determine whether NMV can cross the endothelium, human coronary artery endothelial cells (HCAEC) were cultured ± TNF on transwell inserts. NMV were stained with PKH lipophilic membrane dye and added to the upper compartment of the transwells at ratios of 1:10, 1:50, and 1:100 HCAEC: NMV. After 24h NMV in the basal chamber were visualised using fluorescent microscopy and quantified using Fiji. To investigate NMV internalisation by macrophages, M0 human monocyte-derived macrophages (HMDM) were treated at a ratio of 1:100 HMDM:PKH stained NMV for periods of 24, 6 and 1h. Following treatment, NMV internalisation was quantified by flow cytometry and visualised using confocal microscopy. Trypan blue was used to quench surface fluorescence and distinguish between adherent and internalised NMV. Modulation of macrophage polarisation by NMV was assessed by RT- qPCR analysis of CD68, CD86, MRC1 and CD163 expression after 24h. Results NMV were able to cross the endothelium in a dose-dependent manner. NMV also interacted with both stimulated and unstimulated HCAECs. NMV interaction with HMDM occurred rapidly with approximately 10% HMDM being identified as PKH+ve after 1h. Internalization of NMVs by HMDMs increased over time with approximately 64% of PKH+ve cells containing NMV after 24h. However, incubation of HMDM with NMVs did not result in any significant changes in polarisation marker expression after 24h. Conclusions NMV can cross the endothelial monolayer and interact with macrophages. They are internalised by macrophages but are not able to induce changes in polarisation in the absence of other polarising stimuli. Further investigation is underway to elucidate the mechanisms of NMV internalisation and determine whether NMV can act synergistically with cytokines present in the plaque to influence macrophage polarisation. Conflict of Interest None