Fig. 3

SARS-CoV-2 spike protein-triggered platelet–derived EVs enhanced virus-primed NETs formation by TLR7/8 activation. A Increased platelet–derived EVs (pEVs) were released in the plasma of patients with COVID-19 and are associated with disease severity. Mod, Moderate. B and C The human platelets were treated with viral spike protein (2 μg/ml) for 1 h, (B) the intracellular CD62p expression was analyzed and quantified using flow cytometry. C The levels of pEVs production were detected by immunoblotting. TNFα treatment was used as positive control. D Human platelets were treated with the indicated concentrations of spike protein or TNFα for 24 h. The pEVs were isolated and quantified with the CD63 antibody using flow cytometry. E Human neutrophils were treated with spike protein-triggered pEVs for 4 h, NETs formation was observed using confocal microscopy with the IFA assay (left panel) and intracellular TLR7/8 expression was analyzed using flow cytometry. F Human neutrophils were stimulated with the SARS-CoV-2 spike protein in the presence or absence of spike protein-triggered pEVs (S-pEV), TLR7-, or TLR8-specific inhibitors for 4 h; NETs formation was observed using confocal microscopy (upper panel) and quantified by the MPO-DNA PicoGreen assay (lower panel). G Human neutrophils were transfected with control siRNA, TLR7 siRNA, or TLR8 siRNA (30 nM) for 24 h. The SARS-CoV-2 spike protein in the absence or presence of S-pEVs was added to control cells, TLR7-, or TLR8-knockdown cells, respectively. After 24 h, the expression of intracellular TLR7/8/9 and NETs-associated proteins was analyzed by using immunoblotting. The scale bar in the IFA image represents 5 μm. All experiments were performed in triplicate and data were presented as the mean ± SD. **P < 0.01, ***P < 0.005