TLR3-TRIF signaling in villus-like enterocytes drives type III interferon responses to enteroviruses

Enteroviruses initiate infection at the intestinal epithelium but can spread systemically to cause severe disease. Although both MDA5 and TLR3 have been implicated in enterovirus sensing, the mechanisms by which the intestinal epithelium detects these viruses remain poorly defined. To address this, we infected human intestinal organoids (enteroids) with the group B enterovirus echovirus 11 (E11) and compared responses in models differentiated to mimic either crypt-like or villus-like epithelium. Villus-like enteroids produced significantly more type III interferons following E11 infection and exhibited heightened responsiveness to type III interferon signaling. Single-cell RNA sequencing of infected enteroids revealed that E11 broadly infected epithelial cell types, but type III interferon expression was largely restricted to mature enterocytes. Notably, enterocyte differentiation was also associated with upregulation of TLR3 and other innate immune genes. Using CRISPR-Cas9 knockout enteroids, we found that TLR3-TRIF signaling was essential for intestinal type III IFN responses to E11 infection, whereas loss of MAVS, the adaptor for MDA5, had no effect. Together, these data support a model in which mature enterocytes serve as key sensors of enterovirus infection via TLR3-TRIF signaling, triggering a localized type III IFN response that may help restrict viral spread. IMPORTANCE: Enteroviruses are commonly circulating viruses that can cause a broad spectrum of disease, particularly in pediatric populations. Innate immune sensing in the intestinal epithelium likely plays a critical role in determining the outcome of enterovirus infections. Deficiencies in TLR3 signaling, type III IFN responses, or crypt-villus architecture may contribute to severe disease presentations. Our findings highlight the importance of TLR3-mediated sensing in mature enterocytes, which may also have broader implications for other intestinal viruses, as well as for inflammatory conditions like inflammatory bowel disease. A deeper understanding of how TLR3 sensing and type III IFN production are regulated in the intestine could inform new therapeutic strategies aimed at modulating mucosal sensitivity to viral nucleic acids and enhancing antiviral defense.