Basal IFNλ2/3 expression regulates tight junction formation in human epithelial cells

Type III interferons (IFNλs) play crucial roles in antiviral defense and intestinal epithelial barrier integrity. While interferon expression has been primarily studied in response to pathogens, basal interferon expression occurs in pathogen-free environments. However, the mechanisms regulating basal IFNλ expression and its function have not yet been elucidated. Our findings reveal that basal IFNλ2/3 expression correlates with the development of an intact cellular epithelium characterized by formation of tight junctions and establishment of barrier function. We discovered that basal IFNλ2/3 expression depends on cGAS/STING-mediated mitochondrial DNA detection and is inhibited by the Hippo mechanotransduction pathway at low cellular densities. Notably, cells lacking basal IFNλ2/3 fail to develop proper tight junctions and barrier function. Mechanistically, we found that IFNλ2/3 negatively regulates claudin-2 expression, thereby promoting barrier formation as cells become confluent. These results demonstrate a previously unknown function of basal IFNλs in regulating epithelial cell junction formation and highlight their importance not only during pathogen challenges but also in maintaining epithelial cell function under steady-state conditions. To investigate the interplay between cellular density and basal IFNλ signaling in human intestinal epithelial cells, we generated T84 IFNλ2/3 KO and IFNLR KO cells. T84 WT, IFNλ2/3 KO and IFNLR KO were seeded at high and low density. One day pos-seeding of low density and two-days post-seeding of high density, cells were harvested and sent for RNA sequencing.