Platelet-derived exosomes promote neutrophil extracellular trap formation during septic shock

Background Platelets have been demonstrated to be potent activators of neutrophil extracellular trap (NET) formation during sepsis. However, the mediators and molecular pathways involved in human platelet-mediated NET generation remain poorly defined. Circulating plasma exosomes mostly originating from platelets may induce vascular apoptosis and myocardial dysfunction during sepsis; however, their role in NET formation remains unclear. This study aimed to detect whether platelet-derived exosomes could promote NET formation during septic shock and determine the potential mechanisms involved. Methods Polymorphonuclear neutrophils (PMNs) were cocultured with exosomes isolated from the plasma of healthy controls and septic shock patients or the supernatant of human platelets stimulated ex vivo with phosphate buffer saline (PBS) or lipopolysaccharide (LPS). A lethal cecal ligation and puncture (CLP) mouse model was used to mimic sepsis in vivo; then, NET formation and molecular pathways were detected. Results NET components (dsDNA and MPO-DNA complexes) were significantly increased in response to treatment with septic shock patient-derived exosomes and correlated positively with disease severity and outcome. In the animal CLP model, platelet depletion reduced plasma exosome concentration, NET formation, and lung injury. Mechanistic studies demonstrated that exosomal high-mobility group protein 1 (HMGB1) and/or miR-15b-5p and miR-378a-3p induced NET formation through the Akt/mTOR autophagy pathway. Furthermore, the results suggested that IκB kinase (IKK) controls platelet-derived exosome secretion in septic shock. Conclusions Platelet-derived exosomes promote excessive NET formation in sepsis and subsequent organ injury. This finding suggests a previously unidentified role of platelet-derived exosomes in sepsis and may lead to new therapeutic approaches.


Exosome isolation and characterization
Exosomes were isolated from the plasma of healthy controls and septic shock patients or from the supernatant of platelets stimulated ex vivo using Total Exosome Isolation Reagent. Briefly, blood was collected in centrifuge tubes containing EDTA, and the culture medium was centrifuged at 2000 g for 10 min to remove cells and debris. The supernatant was centrifuged at 10,000 g for 30 min to further remove PBS was then added to the exosomes to reach a total volume of 400 μL, followed by incubation at 4°C with gentle agitation overnight and centrifuged at 1000 g for 5 min.
To determine the proportion of platelet-derived exosomes in the whole exosome quantity isolated from plasma, exosome-coated beads were stained with PE anti-human CD63, FITC anti-human CD41 antibody, or human IgG (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. To determine the expression of HMGB1 in exosomes, exosome-coated beads were stained with PE anti-human HMGB1 antibody and human IgG for 1 h at room temperature. Then, 1 mL PBS was added to wash beads twice and resuspended in 300 μL PBS.
Exosome-coated beads were gated on FSC and SSC, and analyzed by flow cytometry.

Transmission electron microscopy
After exosome isolation, the pellets were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The fixed pellets were placed on 100-mesh, carbon-coated, formvar-coated nickel grids treated with poly-L-lysine for 30 min.
After washing the samples with several drops of PBS, samples were incubated with drops of buffered 1% glutaraldehyde for 5 min and then washed several times with drops of distilled water. Afterward, samples were negatively stained with drops of Millipore-filtered aqueous 4% uranyl acetate for 5 min. The stain was blotted from the grids with filter paper, and samples were allowed to dry. The microscopy images were captured by a JEOL JEM-1400 transmission electron microscope operating at 120 kV.

Platelet purification and activation
Human platelets were isolated from EDTA-anticoagulated venous blood prepared as previously described [1]. Briefly, platelet-rich plasma was obtained by centrifugation at 250 g for 10 minutes, and platelets were sedimented at 1000 g for 15 minutes and then resuspended at 10 8 platelets/mL in HEPES-Tyrode's buffer. Platelet activation was induced upon incubation with PBS or 1 μg/mL LPS or 0.1 U/mL thrombin for 3 h at 37°C, and platelets were pelleted by centrifugation at 2000 g for 10 minutes.

Polymorphonuclear neutrophil isolation and NET induction
PMNs were isolated from the venous blood of healthy volunteers by discontinuous density gradient centrifugation with two commercially available

HL-60 cell culture and transfection
The human acute promyelocytic leukemia cell line HL-60 (ATCC-CCL-240) was maintained in phenol red-free RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% FBS in 5% CO2 at 37°C. To allow for neutrophil-like differentiation, the cells were seeded at 10 6 cells/mL in the above-mentioned cell medium supplemented with 1.25% DMSO for 3 days, as described [2]. For transfection, cells were stimulated to undergo neutrophil-like differentiation as described above and seeded in 12-well plates (500,000 cells/well).

Platelet depletion
In order to investigate the role of platelets in NET formation and ALI development, platelets were depleted as previously described [3]. Briefly, mice received 2 injections of busulfan (20 mg/kg dissolved in polyethylene glycol 400) or vehicle (poly polyethylene glycol 400) on days 0 and 3. Experiments were performed on day 14. Platelet depletion by busulfan led to 40% reduction of platelets without affecting leukocytes, which was consistent with previous study [3]. Platelet depletion was also achieved by an antibody to mouse thrombocyte serum (50 μL/mouse, i.v.) 2 h before experiments [4], which depleted circulating platelets by at least 60%.

Mouse model of CLP and in vivo exosome administration
The mouse CLP model was prepared as previously described [5]. Mice were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) via i.p. injection. After disinfection, a 1 cm midline laparotomy was made in the abdomen. The cecum was then exteriorized, ligated below the cecal valve, and punctured twice with an 18-gauge needle to induce lethal sepsis. A small drop of cecal content was extruded.
The cecum was then returned to the peritoneal cavity and the abdominal incision was To explore exosome function in vivo, mice were treated with exosomes isolated from the plasma of sham or CLP mice (300 μg/mouse) through i.p. injection using 31-gauge insulin syringes. After 24 h, BALF, venous blood, and lung tissue were harvested as described previously.

Fluorescence imaging of Dil-exosomes
Exosomes were isolated from equal plasma volumes (100 μL) from sham or

NET quantification assay
To quantify NETs in cell culture supernatants, plasma, and mouse BALF, we washes, 100 μL peroxidase substrate was added. The absorbance was measured at 405 nm after incubating for 20 min at room temperature in the dark.

Flow cytometry detection of ROS and autophagy in PMNs
After coculturing with exosomes, reactive oxygen species (ROS) generation in for 30 min, followed by fixation in 4% paraformaldehyde for 10 min at room temperature. PMNs were gated on FSC and SSC, and the mean fluorescence intensity was examined by flow cytometry.

Confocal microscopy
The PMNs were allowed to settle on glass coverslips precoated with

Luciferase assay
The 3ʹ-UTR of the PDK1 sequence containing the predicted miR-378a-3p and miR-15b-5p binding sites and its mutant were cloned into the plasmid vector and transfected into HEK293 cells. In all transfections, a Renilla luciferase vector was co-transfected to monitor transfection efficiency. All luciferase results were reported as relative light units: the average of the observed photinus pyralis firefly activity divided by the average of the activity recorded from the Renilla luciferase vector.

Sequencing of miRNA
Total RNA was extracted from exosomes using the miRNeasy Serum/Plasma Kit according to the manufacturer's instructions. Subsequently, total RNA was qualified and quantified using a NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, Waltham, MA, USA). A library was prepared with 1 μg total RNA for each sample. Total RNA was purified by electrophoretic separation on a 15% urea