Host-defense peptides enhance enteric viral infection in a small intestinal organoid model and in vivo

Abstract

The human small intestinal epithelium is a physical barrier to microbes, but it also produces numerous proteins and peptides that form a chemical barrier to infection. The most abundant of these peptides are the enteric α-defensins, which are produced by specialized epithelial cells called Paneth cells. Studies examining the role of α-defensins in enteric infection have been hampered by an inability to grow Paneth cells in culture. Using a recently described method for growing primary stem cell-derived small intestinal organoids, we established an infection model to examine the impact of naturally secreted α-defensins on enteric pathogens in vitro. Small intestinal organoids cultured from wild type and functional α-defensin-deficient mice (Mmp7–/–) were microinjected with enteric pathogens to mimic apical infection. Wild type organoids were shown to produce functional α-defensins, which were capable of restricting the luminal growth of Salmonella enterica serovar Typhimurium (STM). Growth restriction was demonstrated for multiple strains of STM by both microscopy and viable bacteria counts, but growth of STM was not inhibited in Mmp7–/– organoids. This model was then validated in a separate study demonstrating that mucoid Klebsiella pneumoniae variants are more resistant to mouse enteric α-defensin killing than non-mucoid variants of K. pneumoniae, which lack a capsule. The small intestinal organoid infection model was next used to examine the impact of naturally secreted α-defensins on enteric viral infection. Consistent with results using purified enteric α-defensins, infection by mouse adenovirus-2 (MAdV-2), a natural enteric pathogen of mice, was enhanced after microinjection into wild type organoids. Enhancement was not observed upon infection of colonic or Mmp7–/– organoids, which lack mature α-defensins. Establishing that results observed in small intestinal organoids are predictive of in vivo effects, wild type mice orally infected with MAdV-2 shed more virus in their feces than Mmp7–/– mice. In summary, we have created and validated a model to investigate interactions between enteric pathogens and small intestinal epithelial cells. These studies are the first to examine the impact of direct prolonged ex vivo co-culture of enteric pathogens with viable primary epithelial cells capable of naturally producing enteric α-defensins.