Ileal and Colonic Enteroids as a Model for Studying FXR Regulation of the Enterohepatic Circulation in Humans

Abstract

IntroductionThe nuclear bile acid receptor, farnesoid x receptor (FXR), plays an important role in the enterohepatic circulation (EHC) of bile acids. Dysregulation of EHC signalling is associated with a number of intestinal disorders including inflammatory bowel disease, colorectal cancer and chronic diarrhoeal diseases, however the mechanisms involved are not clearly understood. Limitations of currently‐available cell culture or animal models, necessitates the development of more physiologically relevant models to study regulation of the EHC.AimsTo investigate the suitability of human intestinal enteroids as a model for studying the FXR regulation of the EHC.MethodsColonic and ileal enteroids derived from healthy human biopsies were either grown as undifferentiated crypt‐like cells (UD) or were induced to differentiate to villus‐like cells (DF) by removal of WNT3A from the culture medium for 5 days. DF and UD enteroids were treated with the FXR agonist obeticholic acid (OCA; 10 μM) for 6 – 24 hrs. Changes in gene expression were analysed by bulk RNASeq and were expressed as fold change in Reads Per Kilobase of Transcript (RPKM) compared to untreated controls (n = 3 throughout).ResultsWnt3A removal from the culture medium induced enteroid differentiation as indicated by increased expression of villus‐like markers (alkaline phosphatase and NHE3) and decreased crypt‐like markers (LGR5, NKCC1 and CFTR). In ileal enteroids FXR expression increased 3.6 ± 0.8 fold upon differentiation, whereas in colonic enteroids it decreased by 0.49 ± 0.08 fold. Under basal conditions, mRNA for proteins involved in the EHC of bile acids were more abundantly expressed in ileal than colonic enteroids. Treatment with OCA (10 μM; 6–24 hrs) altered expression of these proteins as shown in Table 1. Data are expressed as fold change ± SEM. Statistical analysis was performed using one way ANOVA with Tukey’s multiple comparisons test.ConclusionsHuman enteroids grown as undifferentiated crypt‐like or differentiated villus‐like cells demonstrate features characteristic of the EHC in native intestinal epithelium. Activation of FXR altered expression of mRNA for several proteins known to be important in the EHC of bile acids. Importantly, differential responses to FXR activation occurred in villus and crypt‐like enteroids from both ileum and colon, indicating that the impact of FXR on the EHC has a broader tissue specificity than previously appreciated. Our data establish human intestinal enteroids as a model for studying FXR regulation of the EHC, provide novel insights into the regulatory roles of the receptor, and point to FXR as a promising target for development of new therapeutics for intestinal diseases associated with dysregulation of the EHC.Support or Funding InformationThis work was funded by a Science Foundation Ireland Principal Investigator Award to SJK Expression of genes involved in the EHC following treatment with OCA (10 μM; 6–24 hrs) in both colonic and ileal enteroids Gene Colon Ileum Differentiated Undifferentiated Differentiated Undifferentiated OCA 6h OCA 24h OCA 6h OCA 24h OCA 6h OCA 24h OCA 6h OCA 24h FXR 0.53 ± 0.04 * 0.55 ± 0.08 * 0.53 ± 0.09 0.55 ± 0.07 * 0.31 ± 0.01 *** 0.37 ± 0.05 * 0.64 ± 0.02 ** 1.05 ± 0.24 FGF19 49.28 ± 9.05 33.08 ± 7.63 135.87 ± 98.22 81.81 ± 40.8 507.06 ± 120.6 960.42 ± 348.3 167.88 ± 49.84 183.66 ± 37.89 OSTα 2.27 ± 0.37 3.53 ± 0.86 2.73 ± 0.56 5.4 ± 0.74 * 3.45 ± 0.51 5.51 ± 0.06 *** 2.97 ± 0.18 * 5.11 ± 0.84 OSTβ 3.84 ± 0.04 *** 4.47 ± 1.25 6.88 ± 2.14 10.08 ± 2.4 4.19 ± 0.22 ** 4.96 ± 0.41 * 6.16 ± 0.42 * 7.96 ± 0.13 *** FABP6 9.92 ± 3.94 35.16 ± 11.42 16.1 ± 5.94 133.42 ± 70.03 2.14 ± 0.18 * 7.44 ± 2.22 5.37 ± 1.7 25.58 ± 7.5 ASBT 0.77 ± 0.04 1.02 ± 0.27 0.86 ± 0.12 2.44 ± 1.43 0.88 ± 0.11 0.76 ± 0.14 1.6 ± 0.18 1.09 ± 0.09 MDR1 1.14 ± 0.01 ** 0.93 ± 0.19 1.08 ± 0.18 1.24 ± 0.17 1.14 ± 0.02 * 1.01 ± 0.04 1.37 ± 0.06 * 2.93 ± 0.49 MRP2 1.23 ± 0.42 2.83 ± 1.35 1.78 ± 0.34 7.03 ± 2.03 2.93 ± 0.2 * 3.23 ± 0.07 *** 2.79 ± 0.2 * 5.18 ± 1.27 MRP3 1.15 ± 0.45 0.71 ± 0.21 1.10 ± 0.12 0.96 ± 0.15 0.86 ± 0.23 0.49 ± 0.05 * 1.34 ± 0.11 1.23 ± 0.14 Data are expressed as fold change compared to untreated controls ± SEM. Statistical analysis was performed using one way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001