Intestinal Organoids as a model for small intestinal epithelium alterations in Alcohol Use Disorder (AUD)

Details

Supervisors

Starkel, Peter

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

enAlcohol abuse associated gut barrier dysfunction is thought to play an important role in the development of alcohol-associated liver disease, a major cause of alcohol related mortality and morbidity worldwide. Recent clinical observations have revealed significant alterations in intestinal epithelial homeostasis in AUD patients, including hyperproliferation and impaired differentiation process, naturally designated to regenerate a healthy epithelium. However pathophysiological processes that lead to these alterations in alcohol abuse remain poorly understood, especially, the role of intestinal epithelium, a major component of the gut barrier, and this probably due to the limitations of animal models and 2D cell line- based systems in recapitulating human intestinal physiology. In this study, Intestinal Organoids (enteroids), a 3D stem cell derived model that mimic the architecture and the function of the intestinal epithelium, and 2D-organoid-derived monolayers were used to investigate the mechanisms underlying intestinal epithelial alterations associated with Alcohol Use Disorder (AUD), with a particular focus on the proliferation process. Healthy and AUD-derived organoids, as well as 2D-organoid-derived monolayers were submitted to ethanol treatment at concentrations 40, and 70 mM for five days to assess proliferation during the proliferation phase, for 11 days to assess the proliferation during the differentiation phase, and for three days for the organoid-derived monolayers. Immunostaining of Ki67 was performed to localize and quantify the proliferating cells, and the mRNA expression of Wnt/β-catenin signaling pathway target genes was assessed by RT-qPCR. Ethanol exposure did not significantly affect cell proliferation in 3D enteroids derived from healthy donors or AUD-patients, regardless of treatment duration or concentration. In contrast, in 2D organoid-derived monolayers, apical ethanol exposure increased proliferative activity in healthy organoid-derived monolayers, while AUD organoid -derived monolayers remained unresponsive. Analysis of the expression of Wnt/β-catenin target genes revealed donor-dependent and heterogeneous regulation of the expression of these genes. In 3D enteroids, ethanol did not induce consistent changes in CD44 or LGR5 expression, whereas AXIN2 was selectively downregulated in healthy enteroids after extended exposure. In 2D monolayers, ethanol had no significant effect on AXIN2 or LGR5 expression, but induced a significant upregulation of CD44 in AUD-derived monolayers. These results indicate that ethanol promotes proliferation in 2D healthy organoid-derived monolayers while having limited effects on 3D enteroids and AUD derived organoids, and selectively modulates Wnt/β-catenin signaling.