Neutrophil extracellular traps induced by IL-8 aggravate atherosclerosis via activation NF-κB signaling in macrophages

Abstract

Here, we sought to explore the underlying role of interleukin (IL)-8 in neutrophil extracellular traps (NETs) formation during atherosclerosis (AS). The concentration of pro-inflammatory cytokines IL-8, IL-6 and IL-1β was determined by enzyme-linked immunosorbent assay (ELISA). NETs formation was evaluated by immunofluorescence and myeloperoxidase (MPO)-DNA complex ELISA. The mRNA levels of IL-8 and Toll-like receptor 9 (TLR9) were measured by quantitative real-time PCR (qRT-PCR). The phosphorylation levels of NF-κB p65 were detected by western blotting. The hematoxylin and eosin (H&E) staining of atherosclerotic lesion areas was performed in ApoE-deficiency mice. Results showed that patients with AS showed higher serum levels of IL-8, a pro-inflammatory cytokine and NETs. IL-8 interacted with its receptor CXC chemokine receptor 2 (CXCR2) on neutrophils, leading to the formation of NETs via Src and extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK) signaling to aggravate AS progression in vivo. PMA-induced NETosis directly upregulated the TLR9/NF-κB pathway in macrophages and subsequently initiated the release of IL-8. Our data reveal a neutrophil-macrophage interaction in AS progression, and indicate that NETs represent as a novel therapeutic target in treatment of AS and other cardiovascular diseases (CVD).

Keywords: Atherosclerosis; IL-8; macrophages; neutrophil extracellular traps.

Figure 1.

Serum levels of pro-inflammatory cytokines and NETs in AS patients. IL-8 (a), IL-6 (b), and IL-1β (c) levels, and the quantification of MPO-DNA complexes (d) using ELISA in serum obtained from AS patients (n = 22) and normal controls (n = 18). *P < 0.05, **P < 0.01 vs. normal controls.

Figure 2.

Quantification of NETs from human serum-derived neutrophils of AS patients. MPO-DNA levels (a) and NETs formation visualized by immunofluorescence confocal microscopy (Green: Ly6G; red: CitH3; blue: DNA) (b) in neutrophils derived from AS patients (n = 22) and normal controls (n = 18). **P < 0.01 vs. normal controls.

Figure 3.

IL-8-CXCR2 axis induced NETosis through the activation of Src and MAPK pathways. MPO-DNA levels (a) and NETs formation (b) in neutrophils derived from normal controls treated with AS patients-derived plasma or plus anti-IL-8 or anti-IgG neutralizing antibody (1 μg/mL). *P < 0.05, **P < 0.01 vs. AS-plasma+IgG; NETs formation in neutrophils derived from normal controls treated with human recombinant IL-8 (500 ng/mL) along with CXCR1/2 or IgG antibody (1 μg/mL) (c) as well as the Src inhibitor PP2 (10 µM), the p38 inhibitor SB203580 (10 µM), the ERK inhibitor19 PD98059 (20 µM), the PI3K inhibitor wortmannin (100 nM) or the vehicle (d). **P < 0.01 vs. Control group or IL-8+ Vehicle; ^##P < 0.01 vs.IL-8+ IgG. n = 3.

Figure 4.

CXCR2 depletion inhibited NETosis and alleviated AS progression. Serum levels of MPO-DNA (a), IL-8, IL-6 and IL-1β (b) levels, oil red O staining of cross-sections of the proximal aorta showing atheromatous plaque (black arrow) (c) and NETs formation (d) in AS model mice injected of IgG or anti-CXCR2 antibody via tail vein. *P < 0.05, **P < 0.01 vs. AS+IgG. n = 6.

Figure 5.

NETs activated TLR9 signaling in macrophages. (a) The quantification of NETs in in neutrophils derived from normal controls treated with PBS or PMA (30 ng/mL) for 4 h. **P < 0.01 vs. PBS. IL-8, IL-6 and IL-1β levels (b) and TLR9 mRNA expression by qRT-PCR (c) in THP-1 cells stimulated with or without 20 μg/mL LPS, or co-cultured with PMA (30 ng/mL)-treated human peripheral neutrophils for 24 h. **P < 0.01 vs. NC. IL-8, IL-6 and IL-1β levels (d), IL-8 mRNA expression (e) and phospho-p65 protein levels (f) in THP-1 cells co-cultured with or without PMA (30 ng/mL)-treated human peripheral neutrophils for 24 h, together with or without TLR9 antagonist IRS869 (1 μM), TLR9 agonist ODN1826 (1 μM) for another 24 h, respectively. **P < 0.01 vs. NC; ^#P < 0.05, ^##P < 0.01 vs. NETs. n = 3.

Figure 6.

TLR9/NF-κB pathway involves in NETs-mediated IL-8 release. IL-8, IL-6 and IL-1β levels (a), IL-8 mRNA expression (b) and phospho-p65 protein levels (c) in THP-1 cells co-cultured with PMA (30 ng/mL)-treated human peripheral neutrophils for 24 h, together with or without TLR9 agonist ODN1826 (1 μM) and/or NF-κB inhibitor BAY11-7082 (5 μM) for another 24 h. *P < 0.05, **P < 0.01 vs. NETs; ^#P < 0.05, ^##P < 0.01 vs. NETs+ODN. n = 3.

Figure 7.

Schematic diagram describing the molecular signaling in the neutrophils and macrophages.