Fig 2: COVID-19 positive individuals with high viral load show increased immunomodulatory lncRNAs and the secretory mucin MUC5AC expression(A–D) Relative expression of immunomodulatory lncRNAs, (A) LASI, (B) NEAT1, (C) MALAT1, and (D) WAKMAR2 in Hi-VL compared with Lo-VL individuals’ swab samples.(E) The dual-FISH analysis detected colocalization of SARS-CoV-2 viral RNA (vRNA) and LASI lncRNA in nasal swab samples. Representative micrographs from Lo-VL and Hi-VL swab samples display detection of SARS-CoV-2 nucleocapsid (CoV-2) vRNA (green) and LASI lncRNA (red) along with DAPI-stained nuclei (blue).(F) Nasal swab cells showing immunoreactive MUC5AC expression (shown in white) in the dual-FISH labeled cells; scale bar: 2 µm.(G) H-score quantitation of vRNA and LASI lncRNA in COVID-19-positive individuals.(H) Relative quantitation of mean fluorescence intensity (MFI) of MUC5AC expression in Lo-VL and Hi-VL swab samples. (n = 10/gp for data in A, B, C, & D; and n = 7/gp for data in G & H with 10 cells analyzed per sample; *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test). There was no change in MUC5B expression levels in Lo-VL and Hi-VL samples (Figure S4).

Fig 3: Blocking LASI lncRNA expression reduces the SARS-CoV-2 viral load and suppresses the MUC5AC mucin expression(A–E) Relative expression of (A) LASI lncRNA and (B) SARS-CoV-2 vRNA in cells transfected with siRNA targeting LASI (siLASI) compared with control siRNA (siCTRL)-transfected cells and infected with one MOI of SARS-CoV-2. Relative expression of (C) IRF3, (D) DDX58, and (E) MUC5AC mRNA following SARS-CoV-2 infection in siLASI-transfected cells compared with siCTRL cells. (n = 4/gp from two independent experiments; *p < 0.05; **p < 0.01; by Student’s t test).(F) The micrographs of SARS-CoV-2-infected cells following siCTRL and siLASI transfection show MUC5AC (green) immunoreactivity and DAPI-stained nuclei (blue), scale bar: 5 µm.(G) Percentage of MUC5AC+ cells within each treatment group (n = 10/gp; **p < 0.001 by Student’s t test).(H) MUC5AC mucin protein levels in the apical wash of CoV-2-infected cells and transfected with siLASI or siCTRL as determined by ELISA assay. (n = 4/gp from two independent experiments; *p < 0.05 by Student’s t test). Expression of MUC2 and MUC4 mucin mRNAs were also suppressed in siLASI cells with no change in expression of IL-6, ICAM-1, and other lncRNAs (Figure S8).

Fig 4: Expression levels of transcripts encoding inflammatory factors, airway secretory mucins, associated transcription factors, and select lncRNAs in the RNA-seq database of nasopharyngeal swabs of SARS-CoV-2 positive individuals(A–D) Relative expression of (A) airway inflammatory factors IL-6, ICAM-1, and CXCL-8 mRNAs; (B) airway mucins MUC5AC and MUC5B mRNAs; (C) transcription factors SPDEF, FOXA3, and FOXJ1 mRNAs; and (D) lncRNAs LASI, NEAT1, and MALAT1 in SARS-CoV-2 positive (CoV-2+) individuals compared with SARS-CoV-2 negative (CoV-2-) control individuals. The expression levels were calculated from the transcript reads per million (RPM) RNA-seq data (Table S1) of nasopharyngeal swabs from CoV-2+ subjects (n = 26) compared with CoV-2- control subjects (n = 8) as reported recently (Cheemarla et al., 2021). (Data shown as violin plots with individual data points, medians, and quartiles; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by Student’s t test). Similar increased expression of IFIH1 and SCGB1A1 mRNAs was observed in CoV-2+ subjects (Figure S1).

Fig 5: COVID-19 positive individuals with high nasopharyngeal viral load show increased mucoinflammatory phenotype compared with low viral load individuals(A–F) Total RNA from nasopharyngeal swab samples of COVID-19 positive individuals (n = 20) was analyzed. Based on the SARS-CoV-2 nucleocapsid viral RNA expression, the individuals with high viral load (Hi-VL) had average CT values of 25.9 ± 1.3 (n = 10), whereas those with low viral load (Lo-VL) had average CT values of 35.5 ± 0.7 (n = 10). Relative mRNA expression of (A) SARS-CoV-2 viral RNA (CoV-2 vRNA); innate inflammatory factors, (B) IL-6, and (C) ICAM-1; airway mucins (D) MUC5AC and (E) MUC5B; and (F) mucin regulatory transcriptional factor SPDEF in Hi-VL compared with Lo-VL patient swab samples. (Data shown as box and whisker plots with minimum to maximum range; n = 10/gp; *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test). Increased expression of MUC4 and SCGB1A1 mRNAs was also observed in Hi-VL subjects with no changes in select viral entry facilitating host factor mRNAs and IFN1B mRNA expression (Figure S2) and select host mucoinflammatory genes and lncRNAs strongly correlated with CoV-2 viral load (Figure S3).