Fig. 5

pEVs-carried miR-21/let-7b enhanced NETs formation by TLR7/8 activation. A Human platelet–derived EVs were loaded with Cy5-labeled miR-21/let-7b mimic (red) or mimic control (red) and then co-cultured with dHL-60 cells for 4 h. Endogenous TLR7 or TLR8 was stained with TLR7 or TLR8 antibodies (green), respectively. The miR-21/let-7b transmission and endogenous TLR7/8 in dHL-60 cells was detected by using the immunofluorescence assay. The scale bar in the image represents 10 μm. B Human neutrophils were transfected with control siRNA, TLR7, or TLR8 siRNA (30 nM) for 24 h; MiR-21/let-7b mimic-loaded pEVs were added to control cells, TLR7-, or TLR8-knockdown cells, respectively. After 24 h, NETs formation was observed using confocal microscopy (left panel) and quantified by MPO-DNA PicoGreen assay (right panel). C Human neutrophils were treated with IRS661 (TLR7 specific inhibitor), or Cu-CPT9a (TLR8-specific inhibitor) for 4 h. PBS was used as solvent control (SC). MiR-21/let-7b mimic-loaded pEVs were added to individual treating cells, respectively. After 24 h, NETs formation was observed using confocal microscopy (left panel) and quantified by MPO-DNA PicoGreen assay (right panel). D MiR-21 mimic-loaded pEVs were added to control cells, TLR7-, or TLR8-knockdown cells, respectively. The levels of cytosolic ROS in were detected using flow cytometry with dihydrorhodamine (DHR) 123 staining. E Human neutrophils were treated with miR-21 mimic-loaded pEVs in the presence of TLR7/8-specific inhibitors for 24 h. The expression of intracellular TLR7/8/9 and NETs-associated proteins was analyzed by using immunoblotting. F and G Human neutrophils were treated with SARS-CoV-2 spike protein (S protein) or/and spike protein-primed platelets–derived EVs (S-pEVs) in the presence of a miRNA inhibitor control or an miR-21 inhibitor for 24 h. F NETs formation was observed using confocal microscopy. G The expression of intracellular TLR7/8/9 and NETs-associated proteins was analyzed using immunoblotting. Immunoblotting bands from β-actin were densitometrically measured by ImageJ to determine the lane normalization factor for samples. The scale bar in the IFA image represents 5 μm. The image shown is from a single experiment that is representative of at least three separate experiments. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005. SC, solvent control. IC, microRNA inhibitor control