Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium

Abstract

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions.

Fig. 1. Hypercholesterolaemia promotes NMV release and accumulation in the vessel wall.

NMVs were prepared from stimulated human (a–c) or mouse (d–f) neutrophils. a, d Transmission electron micrograph of an NMV pellet. b, e Transmission electron micrograph of a negatively stained NMV sample on carbon-coated copper grids. Magnification of both micrographs ×28,500. Scale bars = 0.25 µm (a), 0.2 µm (b), 0.25 µm (c), 0.2 µm (d). Representative histogram showing size distribution of human (c) and mouse (f) NMVs analysed using Tunable Resistive Pulse Sensing and Nanoparticle Tracking Analysis, respectively. g NMVs were detected in human plasma samples before and after high-fat diet using flow cytometry by staining with FITC-anti-CD66b (n = 15). h Total plasma MVs in ApoE^−/− mice fed chow (n = 6) or high-fat diet for 6 weeks (n = 5) and i in ApoE^−/− mice fed high-fat diet for 6 weeks with and without neutrophil depletion (n = 5) were quantified by flow cytometry. Numbers were normalised to the mean of control samples (filled circles, g–i). j NMVs were detected in aortic arch homogenates by staining with FITC-anti-mouse Ly6G. The number of NMVs in the aortic arch of ApoE^−/− mice on western diet for 6 (n = 7) or 20 weeks (n = 4) was compared to that of ApoE^−/− mice on chow (dotted line) using flow cytometry. Data are presented as mean ± SEM and statistical significance evaluated using a paired (g) or unpaired (h–j) t-test. *P < 0.05, **P < 0.01. All n numbers represent independent participants/animals. Source data are provided as a Source Data file.

Fig. 2. NMVs preferentially adhere to atheroprone regions in vivo.

Fluorescently labelled NMVs (green) were injected via the tail vein into ApoE^−/− mice that had been fed a Western diet for 6 weeks. After 2 h, mice were culled and en face immunostaining of the mouse aortic arch was performed. Representative en face images of NMV adhesion in atheroprotected (outer curvature, a, b) and atheroprone (inner curvature c, d) regions of the aorta, visualised by confocal fluorescence microscopy. Endothelial cells were identified by staining with anti-CD31 antibody (red) and cell nuclei were identified using TO-PRO Iodide (magenta). Outer and inner curvature of the ascending aorta were identified by anatomical landmarks and confirmed by characterising the phenotype of endothelial cells; those at the outer curvature were aligned, elongated and uniform—a characteristic of cells under high shear, whereas cells in the inner curvature had a disorganised appearance. Samples were visualised using a ×100 objective at ×1 zoom (a, c) and at ×6 zoom (b, d). Scale bars = 20 µm (a, c), 5 µm (b, d). e Quantification of adherent NMVs presented as mean ± SEM (n = 4 animals) and statistical significance evaluated using a paired t-test test. *P < 0.05. Source data are provided as a Source Data file.

Fig. 3. NMVs adhere to HCAEC under flow and enhance monocyte migration.

a, b HCAEC were cultured (72 h) under static, high laminar (HSS) or low oscillatory shear stress (OSS). Fluorescently labelled NMVs (red) were added for 2 h. Cells co-stained for CD31 (green) and TO-PRO-3 (blue). a Representative images (scale bar = 50 µm) and b quantification of NMV adhesion (n = 3). c HCAEC cultured as for a were incubated with fluorescently labelled anti-ICAM-1, changes analysed by flow cytometry and displayed as mean fluorescence intensity (MFI) (n = 5). d, e HCAEC cultured under OSS (72 h) were incubated with anti-ICAM-1 or isotype control antibody (100 µg mL⁻¹) for 1 h before addition of fluorescently labelled NMVs. d Representative images (scale bar = 100 µm) and e quantification of NMV (red) adhesion. Data expressed as percentage of mean of isotype sample (n = 3). f, g After 72 h exposure to OSS, NMVs were perfused over conditioned HCAEC for 2 h. Fluorescently labelled monocytes (red) were added to perfusion media for 2–4 h. f Representative images (scale bar = 100 µm) and g quantification of adherent monocytes (n = 3). Data expressed as percentage of mean number of adherent monocytes per HCAEC in control samples (−NMV). h–j HCAEC cultured on transwells were incubated with (+NMV) or without (−NMV) NMVs for 30 min followed by addition of monocytes. Monocyte transmigration toward CCL2 (5 nmol/L) was measured after 90 min (n = 5). i Monocyte transmigration was repeated in absence of HCAEC (n = 3). j NMVs were treated with anti-CD18 or isotype control for 20 min, washed and the transmigration experiment repeated (n = 4). h–j Data expressed as percentage of mean of control samples (−NMV/isotype) and presented as mean ± SEM. Statistical significance evaluated using a paired t-test (e, i, j) or one-way ANOVA followed by Tukey’s (b, c) or Dunnettʼs (g, h) test. NS not statistically significant, *P < 0.05, **P < 0.01. All n numbers represent independent experiments. Source data are provided in Source Data file.

Fig. 4. HCAEC activation by NMVs.

HCAEC were cultured for 72 h under static conditions (a) or oscillatory shear stress (OSS; b) and were then incubated with (+NMV) or without (−NMV) NMVs for 2 h (a; n = 5) and 4 h (a; n = 7, b; n = 4). Release of CCL2, CXCL8 and IL-6 into the media was analysed using cytometric bead array (a) or ELISA (b). c HCAEC were incubated with (+NMV) or without (−NMV) NMVs for 2 h under static conditions and alterations in inflammatory protein (n = 5) expression were investigated using western blotting. Samples were quantified using densitometry and normalised to GAPDH. d, e HCAEC were incubated with (+NMV) or without (−NMV) NMVs for 2 h under static conditions (n = 10; upper panel) or OSS (n = 3; lower panel) and gene expression changes measured using RT-qPCR. Samples were normalised to β-actin. Results are presented as mean ± SEM and statistical significance evaluated using a paired t-test. NS not statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001. All n numbers represent independent experiments. Source data are provided as a Source Data file.

Fig. 5. NMV internalisation by endothelial cells.

a Confocal z stack image taken at a depth of 0.8 µm from the base of an HCAEC incubated with fluorescently labelled NMVs (green) and labelled with early endosome marker (CellLight® Early Endosomes-RFP; red) and Hoechst (nuclei; blue). The arrowheads indicate colocalisation of NMV and CellLight® Early Endosomes-RFP fluorescence. Scale bar = 5 µm. b Orthogonal view (scale bar = 10 µm and c 3D reconstruction of an ApoE^−/− mouse aorta stained en face with anti-CD31 (endothelial cells; red) and TO-PRO-3 (nuclei; magenta) showing internalisation of fluorescently labelled NMVs (green) 2 h after i.v. injection. Elastin autofluorescence also appears as green. CD31 expression on the apical surface was used for orientation and the plane of view set just below. Note the misaligned endothelial cell nuclei, characteristic of an area of disturbed flow. Arrows denote NMVs. HCAEC were cultured under static conditions (d, e) or flow conditions (f) for 72 h followed by incubation with fluorescently labelled NMVs for 2 h under static (d, e) or flow (f) conditions (n = 3). Fluorescence from residual surface bound NMVs was quenched with trypan blue and data were analysed for changes in mean fluorescence intensity by flow cytometry. d The experiment was performed at 4 °C, room temperature (RT) or 37 °C. e HCAEC were incubated at 37 °C in the presence of TNF (4 h prior to the addition of NMVs) and/or anti-ICAM-1 or isotype control. Data are expressed as a percentage of the mean of the isotype control samples. Data are presented as mean ± SEM and statistical significance evaluated using one-way ANOVA followed by Tukey’s post test for multiple comparisons (d, e) or paired t-test (f). *P < 0.05, **P < 0.01, ***P < 0.001. All n numbers represent independent experiments. Source data are provided as a Source Data file.

Fig. 6. NMVs contain miRNAs that are delivered to HCAEC and alter gene expression.

a miRNA content of NMVs prepared from stimulated human neutrophils quantified by RT-qPCR (n = 5). b miR-155 and miR-223 content of MVs in human plasma pre- and post high-fat diet (HFD, n = 5), or (c) plasma from mice fed chow or Western diet (WD, 6 weeks; n = 5) was quantified by RT-qPCR. d miR-155 expression levels in NMVs isolated from mice fed chow or Western diet (n = 3) was measured by RT-qPCR. e HCAEC were incubated with (+NMV) or without (−NMV) NMVs for 2 h and miR-155 (n = 10) and BCL6 (n = 9) expression levels measured by RT-qPCR. f HCAEC were incubated with NMVs prepared from human neutrophils isolated pre- and post HFD and miR-155 expression levels quantified by RT-qPCR (n = 3). The dotted line shows the mean copy number per HCAEC in the absence of NMVs. g HCAEC were incubated with NMVs prepared from miR-155^−/− vs. wild type mouse neutrophils and miR-155 expression levels quantified by RT-qPCR (n = 3). h HCAEC were transfected with 25 ρmol of miR-155 antagomir (A) or scrambled control (Sc) and incubated with NMVs for 2 h. HCAEC expression of BCL6, RELA and its downstream targets was investigated by RT-qPCR (n = 5). Data are presented as mean ± SEM and statistical significance evaluated using a paired or unpaired t-test as appropriate. NS not statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All n numbers represent independent experiments. Source data are provided as a Source Data file.

Fig. 7. NMVs increase miR-155 and reduce BCL6 expression in atheroprone regions in vivo.

a ApoE^−/− mice were injected with NMVs via the tail vein and miR-155 expression levels in the aorta were quantified by RT-qPCR 2 h after injection of saline (−NMV) or NMVs (+NMV; n = 4). All RT-qPCR samples were normalised with β-actin. b, c Carotid arteries were isolated from ApoE^−/− mice and incubated ex vivo with (+NMV) or without (−NMV) NMVs for 2 h and stained en face with anti-BCL6 antibody (black in single channel, upper panels/red in merged images, lower panels). Endothelial cells were identified by staining with anti-CD31 (green) and cell nuclei were identified using TO-PRO-3 Iodide (blue). Representative en face images of the BCL6 channel and merged channels are shown. Scale bar = 20 µm. Total fluorescence intensity of BCL6 expression was quantified using ImageJ software (n = 4) and expressed as a percentage of the mean fluorescence in the control samples (−NMV). Data are presented as mean ± SEM and statistical significance evaluated using an unpaired t-test. *P < 0.05. All n numbers represent independent animals. Source data are provided as a Source Data file.

Fig. 8. NMVs induce activation of NF-κB in atheroprone regions and enhance atherosclerosis.

ApoE^−/− mice fed a Western diet were injected with NMVs, or an equivalent volume of saline, twice weekly via the tail vein for 6 weeks. a Representative en face images of RELA (red) expression in the aorta of mice injected with saline (−NMV) or NMVs (+NMV) in atheroprone regions visualised using confocal fluorescence microscopy. Scale bar = 10 µm. Endothelial cells were identified by staining with anti-CD31 (green) and cell nuclei were identified using TO-PRO-3 Iodide (blue). Examples of nuclear staining of RelA indicated with arrowheads. b Mean fluorescence intensity was quantified using ImageJ software and data expressed as mean ± SEM (n = 5). c, d Plaque formation was measured in dissected aortae using en face Oil Red O staining and imaged by bright field microscopy. c Representative images are shown. d Areas of plaque formation were determined in the entire aorta using NIS-elements analysis software (n = 7). e, f The aortic arches of mice injected with saline (−NMV) or NMVs (+NMV) were studied by en face staining to quantify macrophages (MAC-3, red). e Endothelial cells were identified by staining with anti-CD31 antibody (green) and cell nuclei were identified using TO-PRO-3 Iodide (blue). Scale bar = 10 µm. f Mean fluorescence intensity was quantified using Image J software (n = 3). Data are expressed as a percentage of the mean of the control samples (−NMV) and presented as mean ± SEM. Statistical significance was evaluated using two-way ANOVA followed by Bonferroniʼs post hoc test (b) or an unpaired t-test (d, f). *P < 0.05, **P < 0.01. All n numbers represent independent animals. Source data are provided as a Source Data file.

Fig. 9. NMVs enhance formation and macrophage content of atherosclerotic plaques in a miR-155-dependent manner.

ApoE^−/− mice fed a Western diet for 6 weeks were injected with NMVs isolated from wild type (WT) or miR-155^−/− mouse neutrophils, or an equivalent volume of saline (control), twice weekly via the tail vein for the final 2 weeks. a Plaque formation and b macrophage content was measured in frozen aortic root sections by Oil Red O and MAC-3 staining respectively and imaged by bright field microscopy. a Representative images of Oil Red O staining from control, wild type NMV and miR-155^−/− NMV injected ApoE^−/− mice are shown. Scale bar = 200 µm. Areas of plaque formation were determined in aortic root sections using NIS-elements analysis software (n = 10). b Representative images of MAC-3 staining from control, wild type NMV and miR-155^−/− NMV injected ApoE^−/− mice are shown. Scale bar = 50 µm. MAC-3 positive staining within plaques was determined using NIS-elements analysis software (n = 10). Data are expressed as a percentage of the mean of the control samples (no NMV) and presented as mean ± SEM. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post hoc test. NS not statistically significant, *P < 0.05, ***P < 0.001 compared to control. ^###P < 0.001 wild type NMV compared to miR-155^−/− NMV injected mice. All n numbers represent independent animals. Source data are provided as a Source Data file.

Fig. 10. Proposed model of the molecular mechanism of action of NMVs in atherosclerosis.

High fat diet increases the level of circulating NMVs (i) that preferentially adhere to atheroprone regions of arteries (ii) where they become internalised by endothelial cells (iii). Delivery of miR-155 to the endothelial cells downregulates BCL6, leading to an increase in NF-κB expression and subsequent inflammatory activation. This results in an increase in the number of monocytes recruited to the vessel wall and enhanced atherosclerotic plaque formation.