Severe COVID19 Patients and a 3D Airway Tissue Model of SARS-CoV-2 Infection Express High Levels of Airway Mucins and Associated Immunomodulatory Long Noncoding RNAs

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Background: Respiratory epithelial cells (RECs) lining the upper airways are primary entry-point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID19). Managing the overwhelming inflammatory response and mucus hypersecretion are among the major challenges faced in achieving effective treatment. The present study analyzes the acute inflammatory and mucous responses following SARS-CoV-2 infection. Methods: Nasal swabs from COVID19 patients with mild and severe pathologies were analyzed for the expression of viral RNA (vRNA) for nucleocapsid (N) and spike (S) proteins, viral entry regulating host factors (ACE-2 and TMPRSS-2), epithelial inflammatory factors (IL-6, ICAM-1 and CXCL-8), respiratory mucins (MUC1, 2, 4, 5AC, 5B, 7, 16 and 19), mucin regulatory transcription factors (SPDEF and FOXA3) and select long noncoding RNAs (lncRNAs) by qRT-PCR. Sub-cellular localization and association of lncRNAs and SARS-CoV-2 vRNA was depicted by Dual-fluorescence in situ hybridization (FISH), and immunostaining for epithelial cell markers. A 3D in-vitro cell culture model was developed using primary human RECs differentiated on transwells at air-liquid interface and were infected with a SARS-CoV-2 clinical isolate (USA-WI1/2020) via apical as well as basal regions. Samples were collected at 0, 1, 4, 24 and 48 h post-infection (p.i.) and the expression of aforementioned factors were analyzed in cell lysates and media. Results: Severely affected patients showed significantly higher expression of IL-6, ICAM-1, and CXCL-8 along with the respiratory mucins, MUC4, 5AC, 16, and 19 and the transcriptional regulators, SPDEF and FOXA3 compared to the mild COVID19 patients. Our recently identified novel lncRNAs, LASI (lncRNA on antisense strand to ICAM-1), and TOSL (TNFAIP3-opposite strand lncRNA) were significantly higher in severe patients whereas NEAT1 and MALAT1 levels were lower as were the ACE2 and TMPRSS2. SARS-CoV-2 clinical isolate productively infected 3D human REC model with highest expression of SARS-CoV-2 N vRNA at 24 h p.i., and showed increased expression of inflammatory factors and LASI and TOSL at 1 h p.i. The dual-FISH staining of LASI and SARS-CoV-2 N1 vRNA validated that both the transcripts were enriched in nuclear/perinuclear region of RECs and, club cells and MUC5AC+ cells of severe COVID19.Conclusions: Together, these data indicate that severely infected COVID19 patients are impacted by high respiratory mucin expression and the associated airway inflammation. Interestingly, the lncRNAs, LASI and TOSL showed associated increased expression suggesting a possible role for these innate immunomodulators in SARS-CoV-2 induced innate airway mucosal responses.


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