Background <p>The endothelium promotes a non-adherent vascular surface that facilitates tissue perfusion, prevents clotting, and limits inflammation. Endothelial cells (ECs) execute these tissue-specific functions through the integration of signaling pathways promoted by growth factors, cytokines, extracellular matrix components, and signals from mechanosensory complexes. Furthermore, ECs secrete various molecular signals, leading to the establishment of a specific niche microenvironment. Importantly, ECs can serve as sentinels against invading viral pathogens, propagating anti-viral responses such as the secretion of type I interferons (IFNs). Identification of mechanisms that alter immunity and inflammation at this critical barrier is important to understanding endothelial dysfunctions and the endothelium’s overall role in disease.</p> Methods <p>To investigate the regulation and function of IFN signaling in endothelial cells, we used a conditionally immortalized human cell line. We analyzed IFN gene expression by RT-qPCR and used an Mx2 promoter-dependent bioassay to quantify the levels of secreted IFN during homeostatic conditions. Multiple cell types were screened for the ability to enhance tonic IFN production by endothelial cells in a direct coculture model. The role of direct cell-cell interactions in this behavior was studied using cell culture insert settings and inhibitors specifically targeting gap junction communication. The antiviral effects of endothelial tonic IFN production were determined with SARS-CoV-2 and HCMV infections.</p> Results <p>We demonstrate that endothelial cells can generate a type I IFN response in absence of infection under homeostatic conditions. These tonic IFN levels rise dramatically when endothelial cells are in direct contact with epithelial cells, though not when cultured with other cell types. The transcriptional induction of type I IFN genes occurs only in endothelial cells and requires direct cell-cell contacts. We further show that IFN induction can be blocked by interfering with gap junction communication and is partially dependent on the cGAS/STING pathway. Notably, the IFN activity derived by this cell type-specific interaction efficiently protects neighboring lung epithelial cells against SARS-CoV-2 infection. </p> Conclusions <p>We propose that the upregulation of tonic IFN production by the endothelial-epithelial cell axis can contribute directly to pathogen defense and/or strengthens the innate immune response by elevated priming. While the contributing molecular signaling pathways underlying this activation have not been fully identified, this newly described mechanism has potential relevance during acute or chronic lung injuries, as it enhances the level of tonic antiviral activity. Furthermore, excessive lung inflammation in nonviral pathologies may be dampened by elevated levels of tonic IFNs.</p>