Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation

Abstract

Background and purpose: Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs.

Methods: EVs were quantified and analyzed in the sera of patients after an acute stroke (<24 hours; OXVASC [Oxford Vascular Study]). Isolated EV fractions were subjected to untargeted proteomic analysis by liquid chromatography mass-spectrometry/mass-spectrometry and then applied to macrophages in culture to investigate inflammatory gene expression.

Results: EV number, but not size, is significantly increased in stroke patients when compared to age-matched controls. Proteomic analysis reveals an overall increase in acute phase proteins, including C-reactive protein. EV fractions applied to monocyte-differentiated macrophage cultures induced inflammatory gene expression.

Conclusions: Together these data show that EVs from stroke patients are proinflammatory in nature and are capable of inducing inflammation in immune cells.

Keywords: C-reactive protein; acute-phase proteins; extracellular vesicles; macrophages; stroke.

Figure 1. Characterization of the extracellular vesicle fraction in the circulation of stroke patients.

Nanosight Tracking Analysis (NTA) was used to determine (A) average distribution of vesicle size and number in the sera of stroke patients <24 hours post-stroke compared to age-matched controls. This population of extracellular vesicles was of mixed size when viewed by electron microscopy (B) and expressed common markers of a mixed EV population including Tsg101, Alix and CD9. NTA revealed an increase in number of vesicles per ml (C). EV number correlated with expression levels of and C-reactive protein (CRP; D). Data are mean ±SEM (n = 38); *p<0.05.

Figure 2. Proteomic profile of extracellular vesicles in stroke and control patients.

(A) Heat map of proteins found in extracellular vesicle populations from stroke patients and age-matched controls as determined by LC-MS/MS (represented by range of colour intensities – see colour key). Proteins are arranged by hierarchical clustering (depicted by dendrogram). Blue indicates an increase compared to control patients, red indicates a decrease. Log ratio stroke/control vs average fold change (B) shows a number of significantly upregulated proteins associated with the acute phase response, including C-reactive protein (highlighted in red). (C) Confirmation of CRP in stroke EVs by Western blot. Ingenuinty pathway analysis of control vs stroke extracellular vesicle proteins indicates the acute phase response is one of the most highly altered pathways post-stroke (D). Red indicates proteins which are up-regulated in stroke, green indicates proteins which are down-regulated.

Figure 3. Cytokine and chemokine expression in monocyte-differentiated macrophage cells after treatment with extracellular vesicles from control or stroke patients.

mRNA expression of (A) TNF; (B) IL-1β; (C) CXCL-1 and (D) CCL-2 in THP-1 cells treated with EVs from stroke patients, or age-matched controls for 6 hours. Data are mean ±SEM; n=3; **p<0.001 and ***p<0.0001 vs. controls; +p<0.05, ++p<0.001 and +++p<0.0001 vs. age-matched control EVs.