Investigating interactions between endothelial cells, type I interferon, and infection, highlighting a novel tonic IFN response enhanced by coculture with specific cell types

Vasculature networks stretch the entirety of the body, moving nutrients and oxygen to tissues and expelling their wastes. The endothelial cells that make up the inner lining of the vasculature are highly involved in mechanisms including immune cell mobility, inflammation, infection, and clotting. Endothelial cells regulate these pathways with a complex mix of signaling factors, communicating with each other, immune cells, and the cells of neighboring tissues. In regards to infection, endothelial cells act as sentinels, guarding against pathogens attempting to enter and exit circulation. To play this role, endothelial cells convey inflammatory responses such as type I interferon signaling. However, the relationship between the endothelium and inflammation is complex, with Inflammatory dysregulation in the endothelium being associated with disease. To study the vasculature in vitro, the complex environment of endothelial cells in vivo is mimicked using coculture with relevant cell types and advanced culture methods under shear stress. In this study, the interactions between infection and the endothelium were explored using physiologically relevant cell lines, with particular focus on type I interferon (IFN) signaling. IFN was observed to impair the functions of endothelial cells in the angiogenic assays of tube formation and spheroid outgrowth. The relevant endothelial pathogen human cytomegalovirus (HCMV) was observed to alter the angiogenic functions of healthy, bystander endothelial cells, as well as disrupting morphological differentiation under media flow. To investigate the effects of IFN on a transcriptional level, a bulk RNA sequencing of the endothelial cells was performed. A key insight that was revealed in this data was the apparent presence of constitutive IFN signaling even in absence of infection, i.e. at homeostasis. This constitutive IFN provided the endothelial cells with a baseline of resistance against infection, blocking the viral entry. Notably, this constitutive IFN signaling conveyed selective protection to other cell types in coculture with the endothelial cells providing protection against HCMV infection to bronchial epithelial cells but not fibroblasts. This protection even extended against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with coculture fully protecting the vulnerable epithelial cells. Strikingly, quantification of IFN in the supernatants of endothelial/epithelial coculture revealed a significant increase in secreted IFN compared to both monocultures. It was found that the endothelial cells in coculture were the primary source of IFN transcripts in coculture. In order to interrogate the mechanisms behind this coculture-stimulated IFN, an endothelial/epithelial coculture method was utilized that compared communication via close cell-cell contact or via indirect contact mediated by shared media. It was found that this coculture-stimulated increase in IFN was strictly cell-cell contact dependent. Further interrogation using small molecule inhibitors found the cellular response to be toll-like receptor (TLR) 7/8/9 independent, but partially stimulator or interferon genes (STING) dependent. However, STING inhibition did not reverse the protective effect of coculture against SARS-CoV-2. Strikingly, gap junction inhibition resulted in both a loss of detectable IFN in coculture and a reduction of the coculture’s protective effect against SARS-CoV-2. Overall, this study highlighted a novel cellular response of constitutive IFN signaling in endothelial cells, providing protection to the cells against viral entry. This IFN signaling is increased in coculture with certain cell types, capable of conveying antiviral effects to vulnerable cell types. This increase is dependent on close cell-cell contact, and is dependent on functional gap junction communication.