Journal: Molecular Therapy

Article Title: Plant-derived exosomal microRNAs inhibit lung inflammation induced by exosomes SARS-CoV-2 Nsp12

doi: 10.1016/j.ymthe.2021.05.005

Figure Lengend Snippet: 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 × 10 8 /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).

Article Snippet: The mouse C57BL/6 lung carcinoma LLC1 and macrophage cell lines, monkey kidney Vero E6 cells, and human alveolar basal epithelial A549 and monocytic U937 cell lines (American Type Culture Collection, Rockville, MD, USA) were grown at 37°C in 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) and RPMI 1640 medium (Life Technologies), respectively.

Techniques: Derivative Assay, Expressing, Transfection, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Flow Cytometry, Staining, Formalin-fixed Paraffin-Embedded, Magnetic Beads, Two Tailed Test

Journal: Molecular Therapy

Article Title: Plant-derived exosomal microRNAs inhibit lung inflammation induced by exosomes SARS-CoV-2 Nsp12

doi: 10.1016/j.ymthe.2021.05.005

Figure Lengend Snippet: aly-miR396a-5p reduces NF-κB activated by Nsp12/13 through phosphorylation of IKKβ (A) Western blot analysis showing the phosphorylation (p) of IKKβ, IκBα, and NF-κB (p65). JNK as well as total NF-κB (p65) are shown in macrophages of the lung in C57BL/6 mice (n = 5) inoculated by intratracheal administration with exosomes (5 × 10 8 /kg, body weight) from LLC1 cells transfected with Nsp12 and/or Nsp13 as well as aly-miR396a-5p. Arrows mark the positions of p54 and p46 subunits of p-JNK. GAPDH served as a loading control. Numbers below western blots represent densitometry values normalized to the loading control. (B) Pretreatment with p-JNK inhibitor (SP600125, 5 mg/kg/day, body weight) and p-IκBα inhibitor (Bay 11-7821, 10 mg/kg/day, body weight) (n = 5) by intraperitoneal injection 3 days following intratracheal administration of exosomes. Western blot analysis shows p-IκBα, p-JNK, and p-p65 in lung macrophages. (C) Western blot analysis of cleaved (c-)caspase-3, c-caspase-7, and c-PARP in the lungs of mice. (D) Analysis of apoptosis by TUNEL staining in lung tissues. The TUNEL assay revealed apoptotic-positive cells in lung marked by GFP staining. The blue DAPI stain marks intact DNA. Original magnification, ×400 (left panel). (Right panel) Quantification of TUNEL-positive cells. The data were collected by counting positive cells from three lung sections of specimens and are shown as mean ± SD versus vehicle. ∗∗p < 0.01. NS, not significant. (E) Analysis of apoptosis by flow cytometry using annexin V-FITC staining in EpCAM + cells of lungs from mice. (Top panel) Numbers in boxes indicate a representative percentage of EpCAM + apoptotic cells. The adjunct histograms display the univariate plots that correspond to the EpCAM in the bivariate plot. (Bottom panel) Quantification of percentage of EpCAM + annexin V + 7-aminoactinomycin D (7-AAD) − cells. Data are representative of three independent experiments (error bars, SD). ∗p < 0.05, ∗∗p < 0.01 (two-tailed t test). (F) Analysis of apoptosis by flow cytometry in lung epithelial A549 cells presented to Nsp12/13 and Bay 11-7821 (top panel), or culture supernatant from U937 macrophages treated with A549-derived Nsp12/13 exosomes with or without Bay 11-7821 (middle panel), and anti-TNF-α, anti-IL-1β, and anti-IL-6 antibodies (10 ng/mL, bottom panel), respectively. Numbers in boxes indicate a representative percentage of annexin V + 7-AAD − apoptotic cells. (Right panel) Quantification of percentage of annexin V + 7-AAD − cells. Data are representative of three independent experiments (error bars, SD); versus Nsp12/13 group: ∗p < 0.05, ∗∗p < 0.01 (two-tailed t test). (G) Proposed model for the crosstalk between GELN miR396a-5p that regulates cytokine expression mediated by SARS-CoV-2 Nsp12 in a manner dependent on NF-κB signaling.

Article Snippet: The mouse C57BL/6 lung carcinoma LLC1 and macrophage cell lines, monkey kidney Vero E6 cells, and human alveolar basal epithelial A549 and monocytic U937 cell lines (American Type Culture Collection, Rockville, MD, USA) were grown at 37°C in 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) and RPMI 1640 medium (Life Technologies), respectively.

Techniques: Phospho-proteomics, Western Blot, Transfection, Control, Injection, TUNEL Assay, Staining, Flow Cytometry, Two Tailed Test, Derivative Assay, Expressing