Exacerbated pro‐inflammatory immune response contributes to COVID‐19 pathology. However, despite the mounting evidence about SARS‐CoV‐2 infecting the human gut, little is known about the antiviral programs triggered in this organ. To address this gap, we performed single‐cell transcriptomics of SARS‐CoV‐2‐infected intestinal organoids. We identified a subpopulation of enterocytes as the prime target of SARS‐CoV‐2 and, interestingly, found the lack of positive correlation between susceptibility to infection and the expression of ACE2 . Infected cells activated strong pro‐inflammatory programs and produced interferon, while expression of interferon‐stimulated genes was limited to bystander cells due to SARS‐CoV‐2 suppressing the autocrine action of interferon. These findings reveal that SARS‐CoV‐2 curtails the immune response and highlights the gut as a pro‐inflammatory reservoir that should be considered to fully understand SARS‐CoV‐2 pathogenesis.

Human intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a framework combining single‐cell RNA‐Seq and highly multiplex RNA FISH and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages and induces expression of the cell proliferation marker MKI67. Intriguingly, each intestinal epithelial cell lineage exhibits a unique basal expression of interferon‐stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon‐stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our framework is applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis.

Rotavirus is one of the most common causes of gastroenteritis worldwide. In developing countries, rotavirus infections lead to more than 200,000 deaths in infants and children.

Type-III interferons (or IFNλs) play important roles in antiviral defense and intestinal epithelial barrier integrity. While interferon expression has been primarily studied in response to pathogens, basal interferon expression also occurs in pathogen-free environments. However, the mechanisms regulating basal IFN-λ expression and their functions have not yet been elucidated. Here, we show that basal IFN-λ2/3 expression is linked to the development of an intact cellular epithelium characterized by formation of tight junctions and establishment of barrier function. Our findings indicate that basal IFN-λ2/3 expression depends on cGAS-STING-mediated mitochondrial DNA detection, while it is inhibited by the Hippo mechanotransduction pathway at low cellular densities. Cells lacking basal IFN-λ2/3 expression fail to develop proper tight junctions and establish normal barrier function. Mechanistically, IFN-λ2/3 suppresses Claudin-2 expression, thereby promoting barrier formation as cells become confluent. These results demonstrate a previously unknown function of basal IFNλ expression 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.

Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to neighboring cells) of epithelial cells affects their response to interferons. We identified that cells at the edge of cellular colonies are more responsive than cells embedded within colonies. We determined that this spatial heterogeneity in interferon response resulted from the polarized basolateral interferon receptor distribution, making cells located in the center of cellular colonies less responsive to ectopic interferon stimulation. This was conserved across cell lines and primary cells originating from epithelial tissues. Importantly, cells embedded within cellular colonies were not protected from viral infection by apical interferon treatment, demonstrating that the population context-driven heterogeneous response to interferon influences the outcome of viral infection. Our data highlights that the behavior of isolated cells does not directly translate to their behavior in a population, placing the population context as one important factor influencing heterogeneity during interferon response in epithelial cells.

Rotavirus is an enteric virus that leads to 200,000 deaths worldwide every year. The live-cell imaging evaluating rotavirus infection of MA104 cells revealed that rotavirus replication and spread occurs in a spatially controlled manner. Specifically, following initial rotavirus infection, the infected cells die, and the second round of infection occurs in the restricted area surrounding the initially infected cell. Interestingly, we found that the time required to establish the secondary infection is shorter compared to the time required for the initial infection. To determine if this increase in the kinetic of secondary infection was due to the early release of viruses or priming of the cells that are infected during the secondary infection, we used a combination of live-cell microscopy, trypsin neutralization assays, and the pharmacological inhibition of calcium signaling. Together, our results show that the second round of infection required rotavirus to be released and accessible to extracellular proteases. In addition, we found that the calcium wave induced upon rotavirus infection was critical for initial infection but did not play a role in the establishment of a secondary infection. Finally, we uncovered that high viral titers released from the initial infection were sufficient to accelerate the rate of the secondary infection.