Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. Here, the use of single cell RNA sequencing to profile IEC during infection revealed an increased proportion of mid-villus enterocytes during infection and induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells. These analyses were complemented by in vivo studies, which demonstrated that IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ showed the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ signalling to uninfected enterocytes is important for control of Cryptosporidium. Single-cell RNA sequencing (scRNA-seq) data were generated in this study. Intestinal epithelial cells and intraepithelial lymphocytes were isolated form the ileum of the small intestine for two mice per group, which were pooled following removal of dead cells and prior to processing for scRNA-seq. For the C57BL/6 dataset, mice were uninfected, or infected with 50,000 oocysts of mouse-adapted C. parvum (maCp) for 4 or 10 days. For the IFNg KO dataset, mice were infected with 50,000 oocysts of maCp and on day 5 post-infection, groups of infected and uninfected mice were treated with either PBS or IFNg. 12 hours post-treatment, mice were euthanized for processing and scRNA-seq.