Background: In active inflammatory bowel disease (IBD), the intestinal epithelium undergoes a process of regeneration to re-establish homeostasis at the site of injury. Transforming growth factor beta (TGF-β)-dependent epithelial mesenchymal transition (EMT) has been postulated to contribute to this regenerative process. Emerging evidence indicates that non-canonical Wnt5b could play a role in EMT. Our hypothesis is that Wnt5b could be expressed in the intestinal mucosa and contribute to the regeneration of IBD epithelium through EMT. In order to demonstrate this, we aimed to: 1) localize the areas of expression of Wnt5b in healthy gut mucosa; 2) evaluate Wnt5b expression differences in tissue samples from non-IBD controls and IBD patients; and 3) investigate Wnt5b's effects on primary intestinal epithelial cells.
Methods: Human tissue samples were used for in situ hybridization (RNAScope) and RNAseq to characterize Wnt5b expression in the intestine of non-IBD controls and Crohn's Disease (CD) and ulcerative colitis (UC) patients. Human intestinal tissue specimens were then used to generate ex vivo epithelial organoid cultures (EpOCs). EMT was assessed by stimulation of EpOCs with Wnt5b, TGF-β or the TGF-β-neutralizing antibody 1D11. Total RNA was isolated for transcriptional analysis. A migration assay (xCELLigence RTCA System) was performed to functionally check Wnt5b-induced migration changes in dissociated EpOCs.
Results: Wnt5b was found to be expressed in both the lamina propria and epithelial crypts of non-IBD intestinal samples. Transcription of Wnt5b was increased in active CD and UC colonic tissues throughout the mucosa. In EpOCs, Wnt5b addition promoted changes that closely resembled the TGF-β-induced phenotype. These included a transition towards a fibroblast-like phenotype and a significant up-regulation of EMT markers (e.g., Vimentin, Fibronectin) and other TGF-β targets (e.g., CDKN2B, Serpine1). Moreover, the addition of Wnt5b significantly increased expression of Wnt5b and TGF-β by EpOCs. In agreement with a suggested role for TGF-β in driving this Wnt5b-induced phenotype, 1D11 antibody fully suppressed the Wnt5b-mediated changes. Of note, Wnt5b stimulation promoted a migratory phenotype in dissociated EpOCs compared to un-stimulated cultures.
Conclusions: Wnt5b could contribute to epithelial regeneration in both forms of IBD by amplifying the TGF-β-dependent EMT process.