Interferons and Potential Consequences for SARS-CoV-2 Infection

RATIONALE: Severe acute respiratory syndrome 2 (SARS-CoV-2) and resulting coronavirus disease 2019 (COVID-19) have been associated with several symptoms including fever, chills, cough, fatigue, loss of taste or smell, sore throat, and congestion. Moderate cases may require hospitalization and severe cases have resulted in death. Interferons (IFNs) are a class of immune molecules typically involved in the anti-viral response. Their role in SARS-CoV-2 infection is controversial as IFNs may increase angiotensin-converting enzyme 2 (ACE2) expression, the receptor for SARS-CoV-2, yet also enhance viral clearance. IFN-α2 stimulation increases gene expression of Interleukin-6 (IL-6), a cytokine that activates STAT proteins and is associated with poor hospital outcomes in COVID-19 patients. Meanwhile, surfactant protein A (SP-A), an immunomodulatory respiratory protein, is known from our work to bind to cytokine receptors such as IL-13α1 and IL-13 ligand (Francisco et al, JI 2020). Therefore, we hypothesized that SP-A binds to ACE2 and IL-6, making it a possible novel regulator of SARS-CoV-2 infection that potentially impacts disease severity.Methods: Normal human bronchial epithelial cells (NHBE) and asthmatic diseased bronchial human epithelial cells (DHBE) were cultured at air liquid interface for 2 weeks and stimulated with increasing doses of IFN-α2 (0.1, 1, and 10 ng/ml) for 48 hours. RT-PCR was performed and gene expression for ACE2 and IL-6 was assessed by normalizing to PPIA. Binding assays were performed using a 96-well, immunosorbent assay coated with either IL-6, IL-6Rα, GP130, ACE2, IFN-α2 or the negative control, EGFR and incubated with increasing concentrations of full-length SP-A (0-5000 ng/ml) and a SPA-HRP conjugated antibody. In separate experiments, western blotting was used to detect levels of total and phosphorylated STAT-1 in primary asthmatic bronchial epithelial cells treated for 30 minutes with IL-6 (10 ng/ml) in the presence and absence of SP-A2 (20 ug/ml) peptide.Results: Stimulation with 10 ng/ml IFN-α2 significantly increased ACE2 and IL-6 gene expression in NHBE and DHBE cells (p<.05). Full length SP-A was found to bind ACE2, both IL-6 receptors (IL-6Rα and GP-130), and to a lesser extent, IL-6 cytokine. SP-A did not bind to IFN-α2 or EGFR. Asthmatic primary human lung epithelial cells treated with IL-6 resulted in phosphorylation of STAT-1, and pre-treatment with SP-A2 peptides reduced this activation by approximately 45%.Conclusion: While IFNs may play a role in viral clearance, our data suggest potential dysregulation of mechanisms affecting the response to SARS-CoV-2. SP-A may be protective based upon its effects on ACE2 and IL-6.