Macrophage Microvesicle Production and Targeting in Atherosclerosis

Extracellular vesicles (EVs) are cell-derived membrane structures that include exosomes and microvesicles (MVs). They are found circulating in the blood of healthy individuals, but increased levels have been observed in pathological conditions such as cardiovascular disease. MVs are shed from a variety of cells including monocytes and macrophages that are involved in atherosclerotic plaque development and instability. Macrophage-derived MVs were shown previously to contain pro-inflammatory cytokines including IL-1β, with the potential to induce inflammation in atherosclerosis in their delivery of biological cargo. The original study selectively isolated phosphatidylserine (PS)-exposed MVs found to contain IL-1β, following LPS and P2X7-dependent ATP stimulation. Since the time of this study, differential centrifugation methods have been standardised for the isolation of extracellular vesicles, quantified using bead-calibrated flow cytometry where vesicles are often found to be composed of a mixed PS-positive and PS-negative population. MerTK has been identified as a key PS-receptor; preliminary work in our group using a zebrafish line lacking MerTK showed attenuated targeting of IL-1β to the head region following tail-fin injury, which was speculated to be mediated by MV delivery.
It was hypothesised that macrophages shed PS-exposed MVs containing IL-1β, which is targeted to endothelial cells via the MerTK PS receptor contributing to atherosclerosis. Macrophage-derived MVs were isolates using differential centrifugation to characterise their number, size and PS-exposure by annexin V-labelling using flow cytometry in order to determine whether production and PS-exposure was stimulus dependent. The proportion of IL-1β secretion in MVs was assessed, compared to vesicle-independent release. The dependency of targeting of IL-1β-containing MVs on MerTK expression, and whether expression of this receptor alters with stimulus and cell-type, was also assessed.
The number of macrophage-derived MVs produced in differentiated THP-1 human monocytic cells, in immortalised mouse bone-marrow derived macrophages and in human primary monocyte-derived macrophages was not found to be stimulus dependent in response to LPS and BzATP treatment. The proportion of PS-positive MVs did not alter with these stimuli compared to untreated control cells. Microvesicle numbers and PS-positivity were unaffected by P2X7 receptor and caspase-1 inhibition. Secretion of IL-1β was increased in human primary monocyte-derived macrophages following LPS and BzATP treatment. Separation of MVs using differential centrifugation showed that the highest proportion of IL-1β was in the MVs-free fraction compared to the MV pellet.
Differential expression of MerTK was shown in a variety of cell types associated with atherosclerotic plaque progression, with an absence in vascular smooth muscle cells and higher expression in endothelial cells and macrophages. MerTK expression was not induced by a variety of stimuli and conditions including inflammation, infection and altered shear stress, the latter relating to disturbed blood flow at atheroprone regions of the endothelium. Expression of MerTK was detected in epithelial cells including the HeLa cell line. The role of MerTK in regulating IL-1β targeting from MVs was tested using HeLa-cell IL-6 induction as a bioassay for IL-1β targeting. Knock-down of MerTK in HeLa cells did not inhibit MV IL-1β-targeting in this IL-6 bioassay; we wish to test this in endothelial cells going forward.
Overall, these findings indicate that macrophage MVs production is not stimulus dependent, that most IL-1β is secreted via vesicle-independent mechanisms, and that MVs do not appear to target their IL-1β-content in a Mer-TK-dependent manner.