Reagents and antibodies

All culture media and buffers were purchased from Gibco. The source of anti-hCLEC5A mAb (clone 3E12A2) and anti-hTLR2 mAb (# MAB2616, R and D system) were as previously described [18]. Antibodies for immunofluorescence staining are as followings: rabbit anti-citrullinated histone H3 (#NB100-57135; Novus), goat anti-human/mouse myeloperoxidase polyclonal antibody (# AF3667, R&D system). Antibodies for immunohistochemical (IHC) staining are: rabbit anti-citrullinated histone H3 (#NB100-57135; Novus), goat anti-human/mouse myeloperoxidase polyclonal antibody (#AF3667, R&D system), anti-CD11b antibody (#ab13357, Abcam), anti-CD64 antibody (#MA5-29704, Invitrogen), anti-Siglec-F antibody (#PA5-11675, Invitrogen), anti-F4/80 antibody (#ab74383, Abcam), anti-CCR2 antibody (#NBP-35334, Novus Biologicals), anti-Ly6C antibody (#SC-23080, Santa Cruz). Secondary antibodies: donkey anti-mouse IgG (H + L) Alexa 488-conjugated antibody (#715-545-151, Jackson ImmunoResearch), donkey anti-goat IgG (H + L) Alexa 647-conjugated antibody (#705-605-147, Jackson ImmunoResearch), donkey anti-Human IgG (H + L) HRP-conjugated antibody (#709-035-149, Jackson ImmunoResearch), HRP-conjugated donkey anti-rabbit IgG (H + L) (#711-035-152, Jackson ImmunoResearch), HRP-conjugated donkey anti-goat IgG (H + L) (#SC-2020, Santa Cruz), HRP-conjugated donkey anti-mouse IgG (H + L) (#715-035-150, Jackson ImmunoResearch).

Isolation of human primary neutrophils and platelets

Blood was drawn from healthy donors (unvaccinated with SARS-CoV-2 vaccine) into an anticoagulant ACD-containing syringe (ACD: blood ratio = 1:6, v/v), platelet-rich plasma was collected by centrifuge at 230 × g for 15 min. Pellet of platelets was harvested by centrifugation at 1000 × g for 10 min, then suspended in Tyrode’s buffer. For human neutrophils, whole blood was laid on the Ficoll-Paque (GE Healthcare, 45-001-748) and centrifuged at 500 × g for 15 min to get red blood cells (RBCs)-granulocytes-rich layer. After RBC lysis, neutrophils were washed and suspended in RPMI containing 10% serum from unvaccinated blood type A/B healthy donors. The protocol was approved by the Human Subject Research Ethics, Academia Sinica (AS-IRB-BM-20025).

Isolation of mouse primary neutrophils and platelets

Platelets-rich plasma from murine peripheral blood was collected into ACD-containing Eppendorf and centrifugated at 230 × g for 4 min. Platelets were washed once and harvested by centrifugation at 1000 × g for 4 min, pellet was suspended in Tyrode’s buffer. For neutrophil isolation, bone marrow was collected and incubated with RBC lysis buffer. Bone marrow cells were suspended in a 45% Percoll solution, then laid on 52%, 63%, and 81% Percoll, and were further centrifugated at 1000 × g for 30 min. Neutrophils were harvested from layer 3 of the Percoll gradient, followed by washing with HBSS twice before being suspended in RPMI containing 10% FBS.

SARS-CoV-2 propagation

SARS-CoV-2 Taiwan/4/2020 was propagated in Vero E6 cells. Viral titer was determined by observation of the cytopathic effect (CPE) in Vero cells. This strain was used in all of the experiments.

Production and purification of pseudotyped lentivirus

The pseudotyped lentivirus carrying the SARS-CoV-2 spike protein was generated as described previously [31]. In brief, HEK-293 T cells were transiently transfected with pLAS2w.Fluc.Ppuro, pcDNA3.1-2019-nCoV-S, and pCMV-ΔR8.91 using TransITR-LT1 transfection reagent (Mirus). Cell debris was removed by centrifugation at 4000 × g for 10 min, followed by passing the supernatant through a 0.45 μm syringe filter (Pall Corporation). For pseudotyped virus purification and concentration, supernatant was mixed with 0.2 × volume of 50% PEG 8,000 (Sigma) and incubated at 4 °C for 2 h. The pseudotyped lentivirus was then recovered by centrifugation at 5000 × g for 2 h, and resuspended in sterilized phosphate-buffered saline, aliquoted, and stored at − 80 °C.

Mouse model for SARS-CoV-2 infection

Virus preparation and inoculation of SARS-CoV-2 into C57BL/6 and clec5a−/−/tlr2−/− mice were as described [32]. In brief, mice were intranasally injected with 3 × 1011 vg of AAV6/hACE2 in 50 μl saline before 14 days of SARS-CoV-2 inoculation, then C57BL/6 and clec5a−/−/tlr2−/− mice (N = 3 of each group) were intranasally changed with 1 × 105 TCID50 of SARS-CoV-2 in a volume of 100 μl. Lung tissue was collected at 3 days and 5 days post-infection for further analysis. All the animal experiments followed the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at AS core (protocol ID 20-10-1521).

Collection of tissues for RNA isolation

Mice were sacrificed at 3 days and 5 days post-infection. For RNA isolation, lung was dug into TRizol-containing MagNA Lyser Green Beads (Roche) for tissue homogenization and further isolated RNA using TriRNA Pure Kit (Geneaid) according to the manufacturer’s instruction. cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit and the real-time PCR was performed as followed conditions: 95 °C for 5 min, followed by 30 cycles of 15 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. The primer sequences were listed in Additional file 2: Table S1. Data were shown as fold change compared to mock after normalized to GAPDH.

Immunohistochemistry (IHC)

Lung tissue was fixed in 10% paraformaldehyde for 48 h and embedded in paraffin subsequently. Tissue sections were deparaffined and rehydrated before H&E stain and multiple-color fluorescent staining using Opal 7-Color IHC Kits (Akoya bioscience). Samples were incubated with primary antibody (1:50) at 4 °C overnight, followed by incubation with secondary antibody (1:100) at room temperature for 1 h. The Opal fluorescent dye was applied according to the vendor’s instructions. Images were captured with a Leica confocal microscope with white light laser system (TCS SP8X-FALCON) and exported using the Leica Application Suite X software. The NET/thrombosis quantitation and cell population analysis by MetaMorph™ image software.

Collagen deposition

Lung sections were de-paraffined and re-hydrated before being stained with Picro Sirius Red Stain Kit (#ab150681, Abcam), and images were captured by a light microscope with polarized light (Nikon). Quantification of collagen was performed by MetaMorph™, and the level of collagen deposition was presented as area (μm2) of collagen under 20× and 40× magnification, repectively. For Masson’s trichrome stain, tissue sections were stained with Trichrome Stain (Masson) Kit (Sigma, # HT15-1KT), the level of pulmonary fibrosis was evaluated by the modified Ascroft scale as previously described [33].

Isolation of extracellular vesicles (EVs)

Plasma from healthy donors and COVID-19 patients with severe pneumonia were centrifugated at 3500 × g for 15 min to remove cells and debris. Supernatants were further centrifuged at 100,000 × g for 1.5 h at 4 °C. Pellets were washed with saline and centrifuged at 100,000 × g for 1.5 h at 4 °C, followed by resuspension in 1 ml of saline. The protein concentration of EVs was determined by DC protein assay (Bio-Rad) according to the manufacturer's instruction.

Surface markers measurement by flow cytometry

The EVs from healthy control (HC, n = 3) and COVID-19 patient (n = 10) plasma were suspended in saline, then stained with capture beads and detection reagents according to the manufacturer’s instruction. The surface markers level was measured by flow cytometry FACSVerse™ and presented as mean fluoresce intensity (MFI). Statistical analysis was calculated with an unpaired and nonparametric Student’s t-test with Mann–Whitney test. ns: no significant difference, *p < 0.05, **p < 0.01.

Induction of neutrophil extracellular traps (NETs)

Human neutrophils (4 × 105/ml) were seeded on poly-L-lysine-coated 12 mm coverslip in 24 well and simultaneously incubated with SARS-CoV-2 (MOI = 0.1 or 1) in presence of autologous platelets (4 × 106/ml) for 5 or 20 h at 37 °C. For SARS-CoV-2-spike pseudotyped virus stimulation, human neutrophils (4 × 105/ml) were incubated with SARS-CoV-2-spike pseudotyped virus (MOI = 0.25) and co-incubated with autologous platelets (4 × 106/ml) for 3 h at 37 °C. For blocking assay, neutrophils were preincubated with isotype (10 μg/ml), anti-CLEC5A mAb (10 μg/ml, clone 3E12A2), anti-TLR2 mAb (10 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2. For EVs stimulation assay, neutrophil was incubated with EVs from the plasma of healthy controls (HC-EVs) or COVID-19 EVs for 3 h at 37 °C.

Visualization and quantification of NET structure

Cells were immersed in fixation buffer (containing 4% paraformaldehyde) overnight, followed by permeabilization using 0.5% Triton X100 in PBS, then incubated with anti-MPO antibody (1:100), anti-citrullinated histone antibody (1:100), and Hoechst 33342 (1:100,000). The level of NETs was calculated using the histone image captured by a Leica confocal microscope with white light laser system (TCS SP8 X-FALCON), and analyzed by MetaMorph™ software.

Mass spectrometry analysis and ingenuity pathways analysis (IPA)

Mass spectrometry analysis was performed in the Mass Spectrometry Core Facility located in the Genomic Research Center, Academia Sinica. In brief, EVs samples were lysed by RIPA solution containing phosphatase and protease inhibitors. Before the mass spectrometry analysis, samples were washed in PBS, followed by trypsin digestion before subjected to LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific Inc.). Data were further analyzed by the Ingenuity Pathways Analysis (IPA) software. The significance of p-value was calculated by the right-tailed Fisher's Exact Test and shown as -log (p-values).

Statistical analysis

All the numbers of samples or mice were described in the figure legend, and the statistical significance was calculated using GraphPad Prism (version 9.0) software (GraphPad Software Inc., San Diego, CA, USA). Data were presented as mean ± SEM and the statistical significance was measured using an unpaired and nonparametric Student’s t-test with Mann–Whitney test. In all of the experiments, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.