Fig. 6

COVID-19–derived pEVs induced IL-1β/TNF-α and IL-8 upregulation in neutrophils through TLR8 and NF-κB activation. A and B Human neutrophils were treated with (A) plasma–derived pEVs or (B) SARS-CoV-2 spike protein-primed pEVs in the presence of a TLR7/8 specific inhibitor for 24 h. The supernatant was collected. The levels of IL-1β, TNF-α and IL-8 were measured by using ELISA. C and D The HEK-Blue hTLR7 (left panel) and HEK-Blue hTLR8 (right panel) cells were stimulated with (C) plasma–derived EVs from the indicated individuals or (D) indicated miRNA-loaded pEVs for 24 h. NF-κB activation was evaluated in terms of luciferase activity compared to control cells. E and F Human neutrophils were treated with (E) plasma–derived EVs or (F) miR-21-loaded pEVs in the presence of TLR7/8 specific inhibitors for 24 h. The intracellular TLR7/8, phosphorylation of p65 subunits and proinflammatory cytokines/chemokines (IL-1β/TNF-α and IL-8) were analyzed using immunoblotting. Immunoblotting bands from β-actin were densitometrically measured by ImageJ to determine the lane normalization factor for samples. All experiments were performed in triplicate and data are presented as the mean ± SD. The image shown is from a single experiment that is representative of at least three separate experiments. *P < 0.05, ** P < 0.01, *** P < 0.005. HC, healthy control