Epithelial WNT2B and Desert Hedgehog are necessary for human colonoid regeneration after bacterial cytotoxin injury

Abstract

20Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt 21 signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis 22 and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using 23 the enterohemorrhagic E. coli secreted cytotoxin, EspP to induce injury to human colonoids, we 24 evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we 25 demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for 26 regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog 27 (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates 28 WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in 29 regulating human colonic regeneration after injury. 30 31Thus far, only three studies have detailed the importance of epithelial Wnts in 55 homeostasis or response to injury in the intestine (O'Connell et al., 2018;Suh et al., 2017; Zou et 56 al., 2018), with most studies focused on the role of mesenchymal Wnts in homeostasis and 57 disease (Gregorieff et al., 2005;Greicius et al., 2018;Koch, 2017; Shoshkes-Carmel et al., 2018; 58 Valenta et al., 2016). The majority of data gained on mouse intestinal injury models suggests that 59 the mesenchymal Wnts are necessary for epithelial regeneration, but did not characterize the role 60 epithelial Wnts may be playing in these processes. 61 Human colonoid cultures are a tractable, epithelial-only model that can indefinitely 62 proliferate due to the presence of adult intestinal stem cells (Sato et al., 2011), making them an 63 excellent model to study intestinal crypt injury and hyperplasia. Foodborne bacterial pathogens, 64 such as enterohemorrhagic E. coli (EHEC) or Citrobacter rodentium, a mouse-adapted bacterium 65 that affects the intestine similarly to EHEC, can cause severe damage to the intestinal epithelia, 66 resulting in hyperproliferation and crypt hyperplasia post-infection (Khan et al., 2006; Vallance 67 et al., 2003; Xicohtencatl-Cortes et al., 2007). We have previously characterized the EHEC-68 secreted serine protease cytotoxin, EspP, as an important virulence factor in EHEC infection and 69 colonic epithelial damage (In et al., 2013). Cytotoxins in the family of serine protease 70 autotransporters of Enterobacteriaceae (SPATEs) are secreted by most pathogenic E. coli and 71 have well characterized functions that aid in bacterial adherence and colonization of epithelial 72 cells (Dautin, 2010). Two SPATEs, Pet and EspC, secreted by enteroaggregative E. coli and 73 enteropathogenic E. coli, respectively, cause cytotoxicity to intestinal explants (Henderson et al., 74 1999; Mellies et al., 2001). However, whether or not EspP has cytotoxic properties on intestinal 75 cells has been controversial (Weiss and Brockmeyer, 2012).76spheroids. In contrast, at 48h, the regenerating colonoids more resembled t...

Human breast milk enhances intestinal mucosal barrier function and innate immunity in a pediatric human enteroid model

“…HIEs represent a cutting-edge technology that recapitulates the structural and functional features of the human gastrointestinal tissue. They have been used to interrogate gut physiology, host responses to microbes, drug activity, and cell-to-cell communication (24–29). A side-by-side comparison of pediatric-vs. adult-derived duodenal HIE monolayers revealed age-associated differences with the former exhibiting shorter columnar epithelial cells and reduced TER, consistent with a less mature epithelial cell phenotype.…”