Fig. 2

COVID-19 patient–derived EVs induced NETs formation through TLR7/8 activation and NADPH oxidase-dependent ROS production. A COVID-19 patient–derived EV-induced NETs formation was inhibited in the presence of the vacuolar type H⁺-ATPase inhibitor bafilomycin A1 (BafA1, 100 nM). The levels of NETs formation were observed (left panel) and quantified (right panel). SC, solvent control. B Human neutrophils were treated with COVID-19 patient- or healthy control (HC) subject–derived EVs in the presence or absence of the TLR7/8 specific inhibitor (10 μM). The levels of TLR7 (left panel) or TLR8 (right panel) were analyzed using flow cytometry. IRS661, TLR7-specific inhibitor; Cu-CPT9a, TLR8-specific inhibitor. C The levels of intracellular TLR7, TLR8 and TLR9 in the neutrophils of COVID-19 patients and HC subjects were analyzed using flow cytometry. MFI, mean fluorescence intensity. D Comparison of NETs formation induced by COVID-19 patient–derived EVs in TLR7- or TLR8-knockdown neutrophils. NETs formation was observed using a confocal microscope (left panel) and quantified by the MPO-DNA PicoGreen assay (right panel). E Human neutrophils were transfected with control siRNA, TLR7, or TLR8 siRNA (30 nM) for 24 h. The COVID-19 patient– or HC–derived EVs were 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. Immunoblotting bands from β-actin were densitometrically measured by ImageJ to determine the lane normalization factor for samples. The image shown is from a single experiment that is representative of at least three separate experiments. F Human neutrophils were treated with COVID-19 patient–derived EVs in the presence or absence of the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 25 μM). NETs formation was observed using a confocal microscope (left panel) and quantified by the MPO-DNA PicoGreen assay (right panel). G Human neutrophils were treated with COVID-19 patient–derived EVs in the presence or absence of the TLR7-, TLR8- and NADPH oxidase inhibitors. The levels of cytosolic ROS were detected using flow cytometry with dihydrorhodamine (DHR) 123 staining. The scale bar in the IFA image represents 5 μm. All experiments were performed in triplicate and data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005