Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

Animals

Eight- to 12-week-old healthy C57BL/6 J mice were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, China). All animal protocols were approved by the Animal Research Committee of Southern Medical University and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Blood samples collection

Coronary blood samples of fifteen patients with STEMI were collected from Xiangdong Hospital affiliated to Hunan Normal University (10 males, 5 females, Supplemental Table 1). Fifteen angina patients served as the control group (11 males, 4 females, Supplemental Table 2). All samples were collected consecutively in the order of admission. Blood samples were immediately processed after collection. Plasma was obtained by centrifugation at 2,000 rpm for 15 min and stored at -80 °C. This study was approved by the Ethics Committee of Nanfang Hospital of Southern Medical University and the Ethics Committee of the Xiangdong Hospital affiliated to Hunan Normal University. All patients were informed about the experimental nature of this study and gave their consent to participation.

Ferric chloride (FeCl3)-induced thrombosis model

Male C57BL/6 J mice aged 8–12 weeks were used for the FeCl3-induced LCCA thrombosis model. The mice were anesthetized with 0.5% pentobarbital sodium via intraperitoneal injection, and a midline incision was made from the manubrium to the hyoid bone. The fascia was bluntly dissected and a section of LCCA at least 5 mm length was isolated. A small plastic piece was placed under the isolated part of the common carotid artery. To induce thrombosis, a piece of filter paper (1 × 2 mm) saturated with 10% (w/v) FeCl3 (Sigma, USA) solution was applied to the adventitia for 3 min. The intraperitoneal injection of the selective c5aR1 receptor inhibitor PMX53(1 µg/g [61, 62], Millipore, USA) was performed 30 min before FeCl3 exposure. For “rescue” experiments, AG490 (a STAT3 pathway inhibitor, 3 µg/g, MedChemExpress, USA) was injected intraperitoneally 15 min before the injection of PMX53. Thrombi were harvested up to 4–6 h after FeCl3 exposure.

ELISA

Blood samples collected into EDTA K3 tubes were processed for routine blood counts and to obtain platelet-poor plasma. Plasma concentrations of C5a were determined by a commercially available C5a ELISA kit (Camilo, China). The test was performed as recommended by the manufacturer’s instructions in duplicate.

Blood flow velocity by Doppler ultrasonography

Doppler ultrasonography was performed on anesthetized mice 4–6 h after FeCl3 exposure using a VEVO2100 Imaging System (Visual Sonics, ON, Canada). Firstly, LCCA was located in B-Mode, with the presence of the bifurcation, left internal carotid artery (LICA) and left external carotid artery (LECA) as the standard view. After switching to color Doppler Mode, the region of the LICA 5 mm from the bifurcation was selected as the measurement point, then the mode was switched to pulsed-wave Doppler mode to assess blood flow velocity. Images were acquired for subsequent measurements and calculations.

Histology and immunofluorescence staining of thrombi

The artery wall containing thrombi were removed and embedded vertically in optimal cutting temperature compound (Sakura, USA). Serial 10-µm frozen sections were cut and placed on slides. For histology, the slides were stained with haematoxylin and eosin (H&E). From each vessel, sections were taken at every 200 μm intervals parallel to the direction of flow. Images were acquired at room temperature with an Olympus BX53 microscope (Center Valley, PA, USA). The area of the thrombus was analysed using ImageJ software (National Institutes of Health, Bethesda, MD). For immunofluorescence staining, samples were incubated with 10% goat serum (Solarbio, China) for 1 h at room temperature. The samples were then incubated overnight at 4 ℃ with rabbit anti-mouse citrullinated histone H3 (CitH3) (1:100, Abcam, ab5103, UK) and rat anti-mouse Ly6G (1:100, BD Pharmingen, Pure 1A8, USA). After washing, the samples were incubated with an Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (1:200, Bioss, China) and an Alexa Fluor 488-conjugated goat anti-rat IgG antibody (1:200, Biolegend, China) for 1 h at room temperature. The nuclei were labelled with DAPI (BestBio, China). Images were obtained with a Leica (TCS Sp8) confocal microscope (Leica, Germany) at room temperature.

Neutrophil isolation and NET formation in vitro

Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions. Neutrophils (5 × 105) were incubated with PMX53 (10 μM) or Colivelin (5 μM, Selleck, USA) for 1 h and then stimulated with recombinant murine C5a (0.1 μM, Peprotech, USA) for 3 h. In the AG490-induced NET experiments, neutrophils (5 × 105) were incubated with AG490 (5 μM) for 3 h. The samples were incubated with 10% goat serum for 1 h at room temperature, and then incubated with rabbit anti-mouse CitH3 (1:100, Abcam, ab5103, UK) and rat anti-mouse Ly6G (1:50, BD Biosciences, BV421, clone 1A8, USA) overnight at 4 ℃. After washing, the samples were incubated with an Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (1:200, Bioss, China) and SYTOX Green Nucleic Acid Stain (167 nM, Thermo, USA). Images were obtained with a Leica (TCS Sp8) confocal microscope (Leica, Germany). For quantification of NET formation in thrombi, 3 distinct criteria needed to be fulfilled: (1) extracellular DNA projections had to emerge, (2) the projections had to originate from cells staining positive for Ly6G, and (3) the structures had to be decorated with CitH3. Only if all of these parameters were met could a structure be defined as NET and included in the quantification.

Quantifications of NETsin vitro

SYTOX green images of neutrophils were analysed using ImageJ software. The percentage of NET-releasing cells was determined by examining 100–200 cells per sample randomly. Briefly, the DNA area of each cell (circles) was automatically measured in 200-μm2 steps, and the distribution of the number of cells across the range of DNA area was obtained. An area larger than 400 μm2 was defined as NET. The data were transformed to the percentage of SYTOX-positive cells by dividing the SYTOX-positive counts by the total number of cells as determined from corresponding phase contrast images. The results were plotted as the percentage of cells that were positive for SYTOX for each DNA area range.

Detection of mitochondria-derived ROS

Mitochondria-derived ROS were detected using MitoSOX Red (Yeasen, Shanghai, China) following the manufacturer’s instructions. Briefly, neutrophils (5 × 105) were incubated in tissue culture plates with MitoTEMPO (10 µM, APExBio, USA) or Colivelin (10 nM) for 1 h. Then, recombinant murine C5a (0.1 μM) was added to the plates, and the cells were incubated for 2 h. After washing, the neutrophils were incubated with probes at 37 ℃ for 10 min and then washed with preheated PBS 3 times for 5 min each time. The Mito-ROS images were obtained with a Leica (TCS Sp8) confocal microscope (Leica, Germany) at room temperature and quantification of the fluorescence signal was performed using ImageJ software.

Extracellular DNA release measurement by the plate reader assays

For the plate reader assay, neutrophils (3 × 104) were seeded onto 96-well plates in the presence of cell-impermeable SYTOX Green Nucleic Acid Stain (500 nM, Thermo, USA). Fluorescence was measured using a SpectraMax i3X fluorescence microplate reader (Molecular Devices, USA) up to 4 h after the activation of the cells. The fluorescence readout obtained from cells lysed with 0.5% (vol/vol) Triton X-100 (Sigma USA) was considered to represent 100% DNA release, and the NETotic index was calculated as the percentage of the total value.

Mitochondrial separation

The human HL-60 cell line was purchased from Procell (China), and the cells were incubated with 1.25% dimethyl sulfoxide (DMSO) for 4 days to induce their differentiation into neutrophil-like cells. Recombinant human C5a (0.1 μM, Novoprotein, China) was used to stimulate the neutrophil-like cells for 2 h. Mitochondrial separation was performed using the Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer’s instructions. Briefly, neutrophil-like cells (5 × 107) were centrifuged at 100 g for 10 min at room temperature to collect the cells. Then the cells were homogenized approximately 10–30 times with a homogenizer and centrifuged at 600 g for 10 min at 4 ℃. The obtained supernatant was centrifuged at 11,000 g at 4 ℃ for 10 min, and the pellet was collected as the mitochondrial fraction. Mitochondrial lysis buffer (Beyotime, C3601-4, China) was added to the the pellet, and mitochondrial protein was used for subsequent experiments. Protein concentration of mitochondrial lysates was determined using the Bicinchoninic Acid protein assay (FDbio, Hangzhou, China). The purity of the preparation was verified by immunoblotting.

Western blotting

An equal amount of mitochondrial protein was subjected to 12% SDS–PAGE and then transferred onto a PVDF membrane (Millipore, Billerica, USA). The membranes were incubated at room temperature for 1 h in blocking buffer. After blocking, the PVDF membranes, which contained the proteins transferred from the gels, were cut into strips (~ 5 mm wide) according to the location (molecular weight) of the protein of interest. Each tailored PVDF membrane strip containing the target protein was then separately incubated over night at 4 ℃ with the corresponding primary antibody: STAT3 rabbit mAb (1:1000, Cell Signaling Technology, Boston, USA), phospho-STAT3 (Ser727) rabbit antibody (1:1000, Cell Signaling Technology, Boston, USA), VDAC rabbit mAb (1:1000, Cell Signaling Technology, Boston, USA) and actin mouse mAb (Bioss, Beijing, China). After washing, they were incubated with goat anti-rabbit IgG-HRP (1:5,000, FDbio, Hangzhou, China) or goat anti-mouse IgG-HRP (1:5,000, FDbio, Hangzhou, China). The immunoreactive bands were detected by ECL Substrate (FDbio, Hangzhou, China) and visualized with a GeneGnome XRQ chemiluminescence imager (Syngene, MD, UK). The grey values of the bands were obtained using ImageJ software.

Statistics

GraphPad Prism (v.8) was used for graphing and statistical analysis. Data are presented as means ± SD (standard deviation). All data performed the test of normal distribution and homogeneity of variance. Two-tailed Student’s t-tests were used for comparisons between two groups. For all experiments involving three or more groups, one-way ANOVA was used. Differences were considered significant at P < 0.05.

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