Human Colonic Organoids: An Epithelial Barrier Model of Crohn's Disease

Abstract

It’s unknown if the increased intestinal permeability in patients with Crohn’s Disease (CD) is a cause or consequence of inflammation. In vivo, interactions between the microbiota, epithelium and immune system prevent investigation of the intrinsic properties of each component. Therefore, in this study colonic organoids have been used to first, determine their suitability as a model of the colonic epithelium, second to model the impact of bacterial components and pro-inflammatory cytokines on the paracellular barrier and thirdly to determine if the permeability of the intestinal epithelium is altered in patients with CD independent of the effects of the immune system and luminal bacteria Colonic organoids were grown from crypts isolated from transverse colonic biopsies from healthy and patients with CD. Ultrastructure of epithelial cells was determined by transmission electron microscopy. Transcript levels of proteins were determined by qPCR and the quantification and localisation of these proteins were determined by immunoblotting and immunofluorescent microscopy. Permeability of the epithelium was determined by FITC-dextran uptake into colonic organoids. Proliferation was analysed and measured by Ki67 staining and apoptosis by TUNEL staining. Statistical significance was determined by an unpaired or paired Student’s t-test or by a one-way ANOVA with a Bonferroni posthoc test. Spontaneously maturing day 15 colonoids have a polarized well-differentiated epithelium with well-developed tight junctions (TJs), similar to the native epithelium. Four-day-old colonospheres, in which the epithelium is functionally but not structurally polarised, had a cytoplasmic localisation of TJs. Exposure to lipopolysaccharide, but not lipoteichoic acid or muramyl dipeptide (20ng/mL) induced goblet cell development but had no effect on the TJs or epithelial permeability. However, pro-inflammatory cytokines, TNF-alpha (100ng/mL) and IFN-gamma (10ng/mL) increased the epithelial permeability and this was associated with loss of barrier-forming (occludin) and adaptor TJ proteins (ZO-1) and a parallel increase in the pore-forming TJ protein (claudin-2). Colonic organoids grown from patients with CD had an inherent defect in the development of the epithelium, with an apparent loss of polarity of the epithelium, evident as a redistribution of actin and E-cadherin from the membrane into the cytoplasm and a loss of NKCC1. This was associated with a redistribution of occludin and ZO-1, from the junction to the basolateral membrane and into the cytoplasm. Additionally, claudin-2 was concentrated in the TJ of the CD colonoids, whereas in control colonoids it was distributed throughout the basolateral membrane. Significantly, the epithelium of the CD colonoids was (p<0.05) more permeable than the epithelium of control colonoids. The native epithelium and colonic organoids derived from patients with CD had a significant downregulation of p120, a cytoplasmic regulatory protein associated with the stabilization of TJs, actin and E-cadherin. These data indicate that colonic organoids are a suitable model of the colonic epithelium and demonstrate that the colonic epithelium of patients with CD has an inherent permeability defect due to redistribution of the junctional proteins possibly as a consequence of the loss of p120. This occurs independent of the bacteria and immune system, suggesting that the increased epithelial permeability is a cause not a consequence of inflammation in CD