Influence of hypoxia on the antiviral functions of human intestinal epithelial cells

Enteric viruses infect or initiate their lifecycle through the gastro-intestinal tract. Pathogenic enteric viruses (e.g. rotavirus, norovirus) are of major clinical importance as they cause a great fraction of pediatric diarrheal cases globally and can lead to life threatening infection in immune compromised patients. The intestinal epithelial cells constitute the primary barrier that enteric viruses need to face to initiate infection. They are organized in discrete crypt-villus structures with the stem cells located in the crypt region and the most differentiated cells (e.g. enterocytes) at the tip of the villi. Along the crypt/villus structure a steep oxygen gradient exists placing the crypts under “normal” oxygen level (normoxia) and the tips of the villi under very low oxygen levels (hypoxia). While hypoxia is well known to be critical for a healthy commensal microbiota and for regulating inflammation during inflammatory bowel disease flare-ups, how hypoxia impacts infection of intestinal epithelial cells by enteric viruses remains unknown. In this application, we will employ a set of human enteric viruses, human intestinal organoids, and gut-on-a-chip technologies to fill this gap of knowledge. In preparation for this research proposal, we have discovered that hypoxia negatively impacts the intrinsic innate immune response generated by primary non-transformed human intestinal epithelial cells upon viral infection. We have shown that hypoxia impairs interferon production, as such favoring enteric virus replication. Using an organ-on-a-chip, we reproduced the oxygen gradient present in the crypt/villus structures, and have shown that hypoxic regions, representing the villus tips, are more susceptible to viral infection due to a curtailed immune response in intestinal epithelial cells. Additionally, we have gathered evidence that stem cells and differentiated cells (e.g. enterocytes) differently respond to hypoxia. As such we hypothesize that the gut hypoxic milieu generates a proviral environment in intestinal epithelial cells by dampening the innate immune response, thereby favoring enteric virus infection, replication, and spread. To challenge these hypotheses, we will first build on our preliminary data and refine our molecular understanding of the hypoxia-mediated inhibition of immune response. Second, we will define how the physiological oxygen gradient creates distinct microenvironments within the crypt/ villus axis with the hypothesis that these microenvironments have distinct capacities to control viral infection. Finally, we will characterize at the single cell level how hypoxia induces cell type-specific inhibition of immune response (stem cells vs enterocytes) and spatially integrate these findings into the crypt/villi structure using organ-on-a-chip technologies. Our findings will inform the development of novel therapeutics targeting cellular responses to hypoxia not only to treat enteric viruses, but also other enteric pathogens, as well as for the treatment of inflammatory bowel diseases which is accompanied by oxygen-dysregulation in the gut. Project Number: 5R01AI185510-02 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Steeve Boulant | Institution: UNIVERSITY OF FLORIDA, GAINESVILLE, FL | Award Amount: $646,188 | Activity Code: R01 | Study Section: Viral Dynamics and Transmission Study Section [VDT] View on NIH RePORTER: https://reporter.nih.gov/project-details/5R01AI18551002