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).

Introduction

Cardiovascular disease (CVD) remains one of the leading causes of mortality in the developed countries [1]. With improvement in people’s living standards, increasing aging of populations and alterations of people’s life style, the prevalence of CVD is gradually increasing. Atherosclerosis (AS), characterized by endothelial dysfunction, lipid deposition in arterial walls and infiltration of inflammatory cells, is the main pathological basis of CVD [2]. Immune cells and inflammatory cytokines play important roles in the pathogenesis of AS.

Neutrophils are the most abundant immune cells involving the development of AS. In addition to phagocytosis, neutrophils play a vital role in defense against pathogenic microorganisms by releasing neutrophil extracellular traps (NETs) [3]. The process of NETs generation, called NETosis, is a specific type of cell death and different from necrosis and apoptosis. NETs is an extracellular reticular fiber structures expelled from active neutrophils in response to a variety of inflammatory stimulation, which is composed of decondensed DNA complexed with histones, nuclear chromatin and granule proteins, such as elastase, cathepsin-G and myeloperoxidase (MPO) [4]. NETs have cytotoxic and thrombotic effects and can trigger atherosclerotic plaque formation and arterial thrombosis [5]. Endogenous or exogenous danger signals such as cholesterol crystals can stimulate neutrophils to release NETs, which in turn prime macrophages for cytokine secretion, amplifying immune cell recruitment in atherosclerotic plaques [6,7].

Interleukin (IL)-8, also known as C-X-C motif ligand 8 (CXCL8), is a pro-inflammatory chemokine [8]. IL-8 produces a variety of biological functions including promoting inflammatory response, angiogenesis, mitosis and proliferation through binding to its two receptors, CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2) [9]. Compelling evidence has delineated the involvement of IL-8 and its receptors CXCR1/2 in the pathogenesis of several diseases including rheumatoid arthritis, inflammatory bowel disease, chronic obstructive pulmonary diseases, asthma, and cancers [10]. Recently, IL-8 has been identified as a critical regulator in the function of endothelial cells (ECs) and vascular smooth muscles cells (VSMCs); therefore, it is likely to participate in the development of AS [11]. A previous study reported that lymphoma-derived IL-8 induced NETs formation via interacting with CXCR2; NETs formation further promoted tumor cell proliferation and migration [12]. Thus, the current study focused on investigating the functional role of IL-8-mediated NETs formation in AS progression.

Materials and methods

Patients and controls

A total of 22 AS patients and 18 healthy volunteers who underwent physical examinations during the same period were recruited from our hospital between May 2016 and July 2018. Peripheral blood was collected from each subject. AS was diagnosed if brachial-ankle pulse wave velocity (baPWV) >1400 cm/s. The exclusion criteria were as follows: severe arrhythmia, valvular heart disease, malignant tumor, diabetes, and severe liver and kidney dysfunction. All subjects were signed informed consent and all our experimental procedures were approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University.

Neutrophils isolation and treatment

Human peripheral neutrophils were isolated from normal controls. Briefly, peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation, rinsed twice with PBS and layered over a Percoll gradient (78%/69%/52%). The dense band at the 69%/78% interface was collected as the neutrophil fraction.

The isolated neutrophils were resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Carlsbad, CA, USA) containing AS patients-derived plasma for 8 h followed by the additional of anti-IL-8 or anti-IgG neutralizing antibody (1 μg/mL; R&D Systems, Minneapolis, MN, USA) for 1 h of incubation. Meanwhile, the harvested neutrophils were seeded in serum-free medium supplemented with human recombinant IL-8 (50 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) with or without the pretreatment of CXCR1/2 or IgG antibody (5 μg/mL; R&D Systems) for 1 h. In order to investigate the downstream signaling pathways of CXCR2 that mediated NETosis, human neutrophils were pretreated for 30 min with the Src inhibitor PP2 (10 µM), the p38 inhibitor SB203580 (10 µM), the ERK inhibitor19 PD98059 (20 µM), the PI3K inhibitor wortmannin (100 nM) (all from Selleckchem, Munich, Germany) or the vehicle, respectively, and then stimulated for 8 h with 50 ng/mL recombinant human IL-8.

In vivo experiments

Male ApoE ^−/− mice on C57BL/6 J background purchased from Experimental Animal Central Laboratory of Harbin Medical University were maintained under a 12-h light/dark cycle at room temperature. ApoE ^−/− mice were fed on high-fat diet (HFD) for 12 weeks to develop a visible AS model. The HFD prescription: 2% cholesterol, 8% egg yolk powder, 10% lard, 0.2% bile salt, and 80% basic forage. All animal protocols were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Harbin Medical University. Anti-CXCR2 antibody or IgG antibody (90 µg/mouse) was injected via the tail vein during AS induction, twice a week for 12 weeks [13]. The blood samples and the whole aorta were collected after euthanasia.

Cell line and culture

THP-1 macrophages (American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained in high glucose-Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (all from Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere of 5% CO₂ at 37°C for serial passaging. THP-1 cells in the logarithmic phase were stimulated with 20 μg/mL lipopolysaccharide (LPS), or co-cultured with phorbol myristate acetate (PMA; 30 ng/mL)-treated human peripheral neutrophils for 24 h together with or without TLR9 antagonist IRS869 (1 μM; InvivoGen, San Diego, CA, USA), TLR9 agonist ODN1826 (1 μM; InvivoGen), NF-κB inhibitor BAY11-7082 (5 μM; Calbiochem, La Jolla, CA, USA) for another 24 h, respectively.

Enzyme-linked immunosorbent assay (ELISA)

The quantification of pro-inflammatory cytokines IL-8, IL-6 and IL-1β in human or mouse serum and conditioned medium from THP-1 cells, was conducted by commercial ELISA kit (Sigma-Aldrich) according to the manufacturer’s instructions.

NETs-associated myeloperoxidase (MPO)-DNA complexes were quantified using the Cell Death ELISA kit (Roche Diagnostics, Indianapolis, IN, USA). Serum samples or cell culture supernatant were added into 96-well plates coated with mouse anti-human MPO antibody after blocking with 1% bovine serum albumin, followed by incubation with peroxidase-labeled anti-DNA monoclonal antibody. The optical absorbance was measured at 405 nm in an ELISA reader (Bio-Rad Laboratories, Tokyo, Japan).

Immunofluorescence staining

NETs formation was examined by immunofluorescence staining. Peripheral neutrophils were fixed in 4% paraformaldehyde and permeabilized with Triton X-100. Then, fixed neutrophils and formalin-fixed, paraffin-embedded atherosclerotic tissues were stained with rabbit polyclonal anti-citrullinated histone H3 (CitH3) rabbit antibody (1:50 dilution; Abcam, Cambridge, UK) and AlexaFluor 488-conjugated anti-LYG6 antibody (1:100 dilution; eBioscience, San Diego, CA, USA). These slides were incubated with goat anti-rabbit IgG labeled-secondary antibodies for 2 h and the DNA was stained with 4ʹ,6-diamidino-2-phenylindole (DAPI) for 5 min. Images were captured with a confocal microscope (Zeiss, Jena, Germany).

Histopathological examination

To analyze the atherosclerotic lesions, aorta specimens were fixed in 10% formaldehyde, embedded in paraffin and cut into 5 µm sections for oil red O staining.

RNA extraction and quantitative real-time PCR

Total RNA was extracted from THP-1 cells using Trizol reagent (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions and then reverse-transcribed to cDNA using PrimeScript^TM 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The relative expression levels of TLR9 and IL-8 were normalized to GAPDH using the Primer-ScriptTM one step RT-PCR kit (Takara) on 7900 HT Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) and analyzed by 2^−ΔΔCt method.

Western blot

THP-1 cells were lysed by RIPA buffer (Santa Cruz Biotechnology). Total protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked overnight at 4°C with primary antibodies targeting phospho-NF-κB p65 (1:1000 dilution; Cell Signaling Technology) and β-actin (1:1000 dilution; Beyotime, Shanghai, China) as a loading control, washed with TBS containing 0.25% Tween 20 at 37°C for 1 h, followed with incubation with anti-mouse horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Boston, MA, USA) for 2h at room temperature. Detection was performed using an enhanced chemiluminescence reagent (Beckman Colter, Brea, CA, USA).

Statistical analysis

Statistical analysis was performed using SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation (SD). Statistical significance among groups was compared using a Student’s t-test or one-way analysis of variance (ANOVA) test. P < 0.05 indicated a statistically significant difference.

Results

Nets formation was significantly increased in AS patients

As shown in Figure 1, AS patients exhibited higher serum levels of pro-inflammatory cytokines including IL-8 (Figure 1(a)), IL-6 (Figure 1(b)) and IL-1β (Figure 1(c)) as well as MPO-DNA complexes, a specific marker of NETs (Figure 1(d)) compared to normal individuals. We subsequently isolated peripheral blood neutrophils from patients with AS and normal subjects. The ex vivo generation of NETs as measured by MPO-DNA complexes (Figure 2(a)) and immunofluorescence microscopy (Figure 2(b)) was observably increased in neutrophils of AS patients than that of normal controls. Together, these results indicated that NETs formation was significantly increased in AS patients.

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.

IL-8-CXCR2 axis induced NETosis through the activation of Src and MAPK pathways

We then used AS patients’ plasma to stimulate healthy donors’ neutrophils. Our data showed that NETosis induced by AS patients’ plasma was abolished by the addition of neutralizing antibody to IL-8 (Figure 3(a-b)). We further performed in vitro experiments to understand whether IL-8 played a vital role in NETosis. Immunofluorescence confocal microscopy confirmed NETs formation after stimulation of human recombinant IL-8, while the antibody to CXCR2, not CXCR1 inhibited IL-8-induced NETs formation (Figure 3(c)). Next, we investigated the downstream signaling pathways of CXCR2 that mediated NETosis. The treatment of Src, ERK and p38 inhibitor, but not the PI3K inhibitor led to the abrogation of IL-8-stimulated NETosis (Figure 3(d)). These data suggested that IL-8-CXCR2 axis contributed to NETosis through the Src and MAPK signaling pathways.

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.

CXCR2 depletion inhibited NETosis and alleviated AS progression

To determine whether IL-8 in vivo induced NET formation through CXCR2 signaling pathway to influence AS development, ApoE ^−/− mice were fed with HFD for 12 weeks to induce experimental AS model. We observed that serum levels of MPO-DNA complexes (Figure 4(a)), IL-8, IL-6 and IL-1β (Figure 4(b)) were memorably reduced after the intravenous injection of anti-CXCR2 antibody. We then examined atherosclerotic plaques by H&E staining and found that the administration of anti-CXCR2 antibody significantly decreased lesion areas (Figure 4(c)). As demonstrated by Figure 4(d), CitH3-positive areas, the marker of NETs formation, were markedly decreased within atherosclerotic lesions in ApoE ^−/− mice. Collectively, these results indicated that CXCR2 knockdown in vivo could reduce NETosis, resulting in the recovery of AS in the ApoE ^−/− mouse model.

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.

NETs increased IL-8 mRNA expression in macrophages via TLR9-mediated NF-κB activation

As expected, healthy donors-derived neutrophils exhibited increased NETosis in response to PMA treatment, as detected by fluorescence microscopy (Figure 5(a)), since PMA is a well-known inducer of NETs. Finally, we studied the molecular mechanism by which NETosis triggered AS deterioration. LPS or PMA-stimulated neutrophils led to elevated secretion of IL-8, IL-6 and IL-1β from THP-1 macrophages (Figure 5(b)). The mRNA expression of TLR9 was also signally increased in THP-1 cells after co-cultured with PMA-treated neutrophils (Figure 5(c)). Further investigation revealed that TLR9 antagonist IRS869 suppressed, but TLR9 agonist ODN1826 promoted NETosis-induced increase in IL-6 and IL-1β protein levels, IL-8 protein and mRNA levels (Figure 5(d-e)), and NF-κB p65 phosphorylation (Figure 5(f)). NF-κB inhibitor BAY11-7082-driven anti-inflammatory effect (Figure 6(a-b)) were similar to the effect of TLR9 antagonist via decreasing phosphorylation levels of NF-κB p65 (Figure 6(c)), even in combination with TLR9 agonist ODN1826. These findings uncovered that NETs in vitro licensed macrophages for cytokine production via TLR9-NF-κB pathway.

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.

Discussion

AS is a chronic inflammatory disorder involving innate and adaptive immune responses [14]. Hyperlipidemia and inflammation are regarded as two major research directions in the field of AS pathogenesis [15]. Neutrophils are a component of the innate immune system, which attach themselves to atherosclerotic plaques, primarily through NET formation. In our present study, we reported that pro-inflammatory cytokine IL-8 triggered neutrophils to release NETs through the IL-8/CXCR2 signaling pathway. Activated NETs further induced the production of IL-8 from macrophages via the TLR9/NF-κB pathway, thereby exacerbating AS development.

In this study, we firstly confirmed increased NET formation, as measured by circulating MPO-DNA levels and higher production of pro-inflammatory cytokines including IL-8, IL-6 and IL-1β in the serum of patients with AS. In addition, NETs generation by neutrophils from AS patients was upregulated compared to neutrophils from normal controls, suggesting a possible overproduction in NETs formation in AS patients. Consistent with our results, Borissoff et al previously showed increased levels of circulating dsDNA, nucleosomes and MPO-DNA complexes in patients with severe coronary AS [16]. Besides, exaggerated NETs formation was also observed in atherosclerotic lesions of ApoE-deficiency mice [17]. However, the sample size is relatively small, which is a limitation of this study.

IL-8 is the most intensively studied pro-inflammatory chemokine secreted by monocytes, macrophages, fibroblasts, endothelial cells, and epithelial cells through its interaction with the chemokine receptors CXCR1/2, playing a central role in inflammation and cancers [9,10]. To the best of our knowledge, IL-8 promotes monocyte adhesion to arterial endothelial cells in the early stage of lesion formation, contributing to angiogenesis in the late stage of plaque formation [18]. Our study revealed that NETosis induced by patients’ serum could be inhibited by IL-8 antibody. We further found that IL-8 promoted NETosis via interacting with CXCR2, instead of CXCR1. The pharmacologic inhibition of CXCR2-related downstream pathway Src, p38 and ERK led to the abrogation of IL-8/CXCL2-stimulated NETosis in human neutrophils. Further in vivo research proved that the administration of anti-CXCR2 antibody effectively attenuated NETs generation, cytokine production and atherosclerotic lesion in HFD-induced ApoE ^−/− mice. These data suggested the inductive effect of IL-8-CXCR2 axis on NETosis in AS progression.

In recent years, abundant studies provided strong evidence investigated the effect of NETs on cytokine production by macrophages. For example, Hu et al [19] demonstrated that LPS-induced NETs significantly upregulated the production of IL-1β from macrophages. A recent study suggested that adult-onset Still’s disease (AOSD) neutrophils released NETs to exert a potent capacity to accelerate the activation of pro-inflammatory macrophages and increased the expression of IL-1β, IL-6, and tumor necrosis factor (TNF)-α [20]. In line with previous studies, our results indicated that PMA, a potent NET activator, triggered neutrophils to release NETs, which in turn stimulated the production of pro-inflammatory cytokines IL-8, IL-6 and IL-1β from macrophages by upregulating TLR9 mRNA expression. A growing body of evidence has delineated TLRs-induced NF-κB-independent production of the pro-inflammatory cytokines in AS [21]. TLR9/NF-κB pro-inflammatory signaling pathway has been reported to promote inflammatory cell infiltration induced by NETs [22]. PMA-induced NETs combined with TLR9 agonist could more effectively promote the secretion of IL-8, IL-6 and IL-1β via inducing NF-κB phosphorylation. NETs-driven pro-inflammatory function was reversed by the addition of TLR9 antagonist or NF-κB inhibitor.

In conclusion, we identify a novel link between neutrophils and macrophages by NET formation in AS pathogenesis and provide evidence for the first time that IL-8-CXCR2 axis induces NETs generation, that induces the production of IL-8 by macrophages via the TLR9/NF-κB signaling pathway, contributing to AS deterioration (Figure 7).

Figure 7.

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

Disclosure statement

No potential conflict of interest was reported by the authors.