Antiviral Immunomodulatory Signaling of Inducible Epithelial Resistance Recalibrates Host Response to Coronavirus and Influenza Infection

Abstract

Rationale: The ongoing COVID-19 pandemic highlights the need to develop novel anti-pneumonia interventions against emerging and established pathogens. We previously reported a therapeutic dyad of immunostimulatory small molecules that induces innate immunity, termed inducible epithelial resistance, against a wide range of pneumonia-causing pathogens, including coronaviruses and influenza viruses. This combination (“Pam2-ODN”) is comprised of a Toll-like receptor (TLR)-2/6 agonist, Pam2CSK4, and a TLR-9 agonist, ODN M362, that stimulate protective responses from lung epithelial cells and promotes synergistic survival benefits and microbicidal effects that exceeds the additive effects of treatment with individual ligands. Here, we investigate the immunomodulatory signaling mechanism of Pam2-ODN that reduces susceptibility to viral infection in lung epithelial cells. Methods: Transcriptional responses of human and mouse lung epithelial cells to influenza A H1N1 or SARS-CoV-2 (GSE147507) or Pam2-ODN (GSE289984) were analyzed using R and IPA software to build host-based antiviral innate immune pathways to infections and identify relevant transcriptional factors (TFs) involved. Isolated human or mouse lung epithelial cells were stimulated with PBS or Pam2-ODN and challenged with influenza A H3N2 or coronavirus OC43 to study transcriptional control of relevant TFs by high-throughput methods of immunofluorescence (IF) staining, in-cell western blotting and imaging flow cytometry (IFC). Results: Network-based enrichment analysis reduced infection of epithelial cells with Pam2-ODN against both coronavirus and influenza through inhibition of viral budding and viral RNA replication. In silico prediction of pathogen-specific host-based responses with Pam2-ODN included disruption of SARS-CoV-2's IL-1, 6 and 8 signaling, and inhibition of influenza A's anti-interferon mechanisms. Functional enrichment analysis revealed activation of these host innate immune responses by Pam2-ODN prior to viral exposure through activity of NF-kB/RelA and AP-1/cJun. IFC and IF confirmed an NF-kB-dependent transcriptional cooperation of RelA and cJun with Pam2-ODN in both mouse and human lung epithelial cells. Phospho-kinetic studies revealed an early transient activity of RelA-(pS536), followed by a sustained cJun-(pS73) signal in lung epithelial cells after Pam2-ODN. Upon viral infection, Pam2-ODN treated cells activated cJun-(pS73) signaling more rapidly in response to both influenza and coronavirus infection. Coronavirus-induced activation of RelA-(pS536) after infection was suppressed in Pam2-ODN treated cells. Conclusion: Inducible epithelial resistance by Pam2- ODN enhances broad host-based antiviral innate immunity responses through signaling pathways of RelA and cJun, which also abrogate virus-specific pathogenic mechanisms. Furthermore, phospho-signaling control of these transcriptional responses in lung epithelium suggest a novel pathogen-dependent antiviral response immune mechanism to infection.