Journal: iScience

Article Title: Immunomodulatory LncRNA on antisense strand of ICAM-1 augments SARS-CoV-2 infection-associated airway mucoinflammatory phenotype

doi: 10.1016/j.isci.2022.104685

Figure Lengend Snippet: SARS-CoV-2 infection of human respiratory epithelial cells induces robust mucoinflammatory response in a 3D airway tissue model (A–F) Respiratory airway epithelial cells differentiated on air-liquid interface were infected with one MOI of SARS-CoV-2 clinical isolate (USA-WA1/2020 isolate) and analyzed at 0, 1, 4, 24, and 48 h postinfection (hpi). Viral loads were determined in (A) the apical washes and (B) the total cellular RNA. Relative expression levels of the inflammatory factors, (C) IL-6 , and (D) ICAM-1 mRNA; and (E) airway mucin MUC5AC ; and (F) SPDEF transcriptional factor in the total cellular RNA was analyzed by qRT-PCR. (n = 4/gp; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 by ANOVA). (G) Representative micrographs of uninfected control (CTRL) and SARS-CoV-2-infected (CoV-2+) cells showing MUC5AC (shown in green) immunoreactivity along with the DAPI stained nuclei (shown in blue); scale bar: 5 μm. (H) Percentage of MUC5AC+ cells within each treatment group. (I–K) Secreted protein levels of (I) MUC5AC mucin in apical washes and (J) IL-6 and (K) ICAM-1 in culture media supernatants as determined by specific ELISA assays (n = 4/gp; ∗p < 0.05; ∗∗p < 0.01; by Student’s t test). There was a significant suppression of viral entry host factors with elevated expression of other mucin genes and SCGB1A1 mRNA following CoV-2 infection ( Figure S5 ).

Article Snippet: Primary human airway epithelial cells , MATTEK , AIR-100.

Techniques: Infection, Expressing, Quantitative RT-PCR, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Immunomodulatory LncRNA on antisense strand of ICAM-1 augments SARS-CoV-2 infection-associated airway mucoinflammatory phenotype

doi: 10.1016/j.isci.2022.104685

Figure Lengend Snippet: SARS-CoV-2 infection induces LASI lncRNA expression in human respiratory epithelial cells that potentially show direct interaction with CoV-2 spike RNA (A) Relative expression levels of LASI lncRNA in SARS-CoV-2-infected cells at 0, 1, 4, 24, and 48 hpi. (n = 4/gp; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 by ANOVA). (B) Colocalization of SARS-CoV-2 vRNA and LASI transcripts in CoV-2-infected (CoV-2 + ) cells as determined by dual-FISH staining and the structured-illumination imaging analysis. Representative micrographs of dual-FISH-stained cells showing SARS-CoV-2 N1 vRNA (in red) and LASI lncRNAs (in green) along with DAPI-stained nuclei (in blue); scale bar: 2 μm. (C and D) H-score quantitation of (C) CoV-2 vRNA and (D) LASI lncRNAs per cell in CoV-2+ and control cells. (n = 9–10 cells/gp; ∗∗p < 0.01; ∗∗∗p < 0.001 by Student’s t test). (E) Modeled 3D structure of SARS-CoV-2 spike vRNA nucleotide sequence from 1198 to 1268, and the LASI lncRNA interacting region (1227-1237) is highlighted in blue. (F) The intra-sequence base-pairing of spike nucleotides forms the hairpin stem structure. (G) Modeled 3D structure of the CoV-2 Spike vRNA duplexed with LASI lncRNA sequence 646-635 (highlighted in orange) at the end of 100 ns simulation (see at online supplemental data). (H) Inter-sequence base-pairing of CoV-2 vRNA with LASI lncRNA sequence (shown in orange). There was no significant change in interferon-related gene expression; however, expression of other immunomodulatory lncRNAs were differentially regulated following CoV-2 infection ( Figure S6 ). LASI -interacting sequence is conserved in Spike viral RNAs of CoV-2 Delta and Omicron variants ( Figure S7 ).

Article Snippet: Primary human airway epithelial cells , MATTEK , AIR-100.

Techniques: Infection, Expressing, Staining, Imaging, Quantitation Assay, Control, Sequencing, Gene Expression

Journal: iScience

Article Title: Immunomodulatory LncRNA on antisense strand of ICAM-1 augments SARS-CoV-2 infection-associated airway mucoinflammatory phenotype

doi: 10.1016/j.isci.2022.104685

Figure Lengend Snippet: Airway epithelial miRNAs are modulated by SARS-CoV-2 infection and regulated by LASI lncRNA Relative expression levels of miRNAs in si LASI -treated cells following 48 h SARS-CoV-2 infection compared with control-infected cells as analyzed by small RNA-seq analysis. (A) Heatmap of select miRNAs upregulated by CoV-2 infection and suppressed in siLASI-treated cells (∗miR-4488 was upregulated >200-fold in infected control cells); see Table S2 for the comprehensive list. (B) Expression levels of miRNAs that are downregulated by CoV-2 infection but induced in si LASI -treated cells. (C–E) Relative quantitation of miRNAs: (C) miR-4488, (D) let-7b-5p, and (E) miR-150-5p that were upregulated by SARS-CoV-2 infection in siCTRL cells but suppressed in siLASI-transfected cells. (F–H) Relative expression of miRNAs: (F) miR-6510-3p, (G) miR-200a-5p, and (H) miR-197-3p that were downregulated by SARS-CoV-2 infection in siCTRL cells but induced in siLASI-transfected cells. (∗p < 0.05; ∗∗p < 0.001 by Student’s t test). Expression levels of select miRNAs were also elevated in Hi-VL nasopharyngeal swab samples of our study cohort ( Figure S9 ).

Article Snippet: Primary human airway epithelial cells , MATTEK , AIR-100.

Techniques: Infection, Expressing, Control, RNA Sequencing, Quantitation Assay, Transfection

Journal: iScience

Article Title: Immunomodulatory LncRNA on antisense strand of ICAM-1 augments SARS-CoV-2 infection-associated airway mucoinflammatory phenotype

doi: 10.1016/j.isci.2022.104685

Figure Lengend Snippet:

Article Snippet: Primary human airway epithelial cells , MATTEK , AIR-100.

Techniques: Virus, Recombinant, Amplification, Fluorescence, Transfection, SYBR Green Assay, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Sandwich ELISA, RNAscope, Multiplex Assay, Sequencing, Knock-Out, Gene Expression, Control, Negative Control, Software, Microscopy