Figure 1.

Lung epithelial cells release exosomes containing Nsp12 of SARS-CoV-2 that enhances the inflammatory response in lung

(A) Schematic representation of the treatment schedule for the effect of lung epithelial cell-derived exosomes containing SARS-CoV-2 proteins on lung immune cells. (B) SARS-CoV-2 protein expression plasmids transfected into lung epithelial A549 cells. (Left and middle panels) Representative blots of viral proteins in exosomes and cells as well as exosomal marker CD63 by western blot using Strep-Tactin-HRP conjugate and antibody to CD63. (Right panel) Intensity of GFP fused with spike (S) protein expressed in exosomes (Exos) and cells using BioTek’s Synergy microplate reader. (C) Representative western blot of exosomes from Vero E2 cells transfected with Nsp12 and Nsp13 plasmids. (D) Cytokines in the medium assessed by ELISA. (E) Schematic representation of intratracheal injection (left panel) and a mouse undergoing laryngoscopy to expose the vocal cords (right panel). (F and G) Exosomes from mouse lung LLC1 cells transfected with SARS-CoV-2 plasmids administered to C57BL/6 mice (5 × 10⁸/kg, body weight, n = 5) by intratracheal injection. After 24 h, the frequencies of F4/80⁺ cells (F), Gr-1⁺ cells (G), and PKH26-labled exosomes in the lung from C57BL/6 mice were assessed using flow cytometry. Numbers in boxes indicate the percentage of exosome/PKH26⁺ cells. (H) Quantification of percentage of exosome/PKH26⁺ in F4/80⁺ cells and Gr-1⁺ cells. (I) (Top panel) Assessment of cytokines in the lungs using ELISA. (J) Cytokines in the F4/80⁺ cells assessed by flow cytometry. (Bottom panel) Quantification of data from flow cytometry. (K) Representative hematoxylin and eosin (H&E)-stained sections of formalin-fixed, paraffin-embedded lungs (original magnification, ×400; scale bars, 200 μm) from C57BL/6 mice. (L) A549 cells co-transfected with the plasmids of pAcGFP1-C-Nsp12-FLAG and pLVX-Nsp13-Strep. At 72 h after transfection, Nsp12/13 complex pull-down by Strep-Tactin XT magnetic beads and immunoblot analysis with anti-FLAG antibody are shown. Data are representative of three independent experiments (error bars, SD). ∗p < 0.05, ∗∗p < 0.01 (two-tailed t test).