Enteroids Generated from Patients with Severe Inflammation in Crohn’s Disease Maintain Alterations of Junctional Proteins

Abstract

Background The mechanisms underlying loss of intestinal epithelial barrier [IEB] function in Crohn’s disease [CD] are poorly understood. We tested whether human enteroids generated from isolated intestinal crypts of CD patients serve as an appropriate in vitro model to analyse changes of IEB proteins observed in patients’ specimens. Methods Gut samples from CD patients and healthy individuals who underwent surgery were collected. Enteroids were generated from intestinal crypts and analyses of junctional proteins in comparison to full wall samples were performed. Results Histopathology confirmed the presence of CD and the extent of inflammation in intestinal full wall sections. As revealed by immunostaining and Western blot analysis, profound changes in expression patterns of tight junction, adherens junction and desmosomal proteins were observed in full wall specimens when CD was present. Unexpectedly, when enteroids were generated from specimens of CD patients with severe inflammation, alterations of most tight junction proteins and the majority of changes in desmosomal proteins but not E-cadherin were maintained under culture conditions. Importantly, these changes were maintained without any additional stimulation of cytokines. Interestingly, qRT-PCR demonstrated that mRNA levels of junctional proteins were not different when enteroids from CD patients were compared to enteroids from healthy controls. Conclusions These data indicate that enteroids generated from patients with severe inflammation in CD maintain some characteristics of intestinal barrier protein changes on a post-transcriptional level. The enteroid in vitro model represents an appropriate tool to gain further cellular and molecular insights into the pathogenesis of barrier dysfunction in CD.

Targeting desmosomal adhesion and signalling for intestinal barrier stabilization in inflammatory bowel diseases—Lessons from experimental models and patients

“…This indicated that loss of Dsg2 in CD is maintained in the intestinal regeneration niche or on a stem level respectively. In this study loss of Dsg2 was present on a protein level, whereas Dsg2 mRNA was unaltered indicating a posttranscriptional mechanism leading to loss of Dsg2 in CD 98 . While the detailed mechanisms underlying this observation remain to be elucidated this may support the view that inflammation‐induced barrier defects in CD are not just a secondary phenomenon because of an inappropriate immune response but play a primary role in the pathogenesis of CD 99 .…”

“…Interestingly, when enteroids from the same CD patients were generated from crypts that contain the intestinal stem cell niche, loss of Dsg2 was maintained under culture conditions. This phenomenom was observed without cytokine stimulation and was present over several passages of enteroid culture 98 . This indicated that loss of Dsg2 in CD is maintained in the intestinal regeneration niche or on a stem level respectively.…”

“…This phenomenom was observed without cytokine stimulation and was present over several passages of enteroid culture. 98 This indicated that loss of Dsg2 in CD is maintained in the intestinal regeneration niche or on a stem level respectively. In this study loss of Dsg2 was present on a protein level, whereas Dsg2 mRNA was unaltered indicating a posttranscriptional mechanism leading to loss of Dsg2 in CD.…”

Intestinal Epithelial Barrier Maturation by Enteric Glial Cells Is GDNF-Dependent

“…Animals were kept on a standard diet and 12-h day and night cycles. To generate organoids, intestinal epithelial cells were isolated from mice intestine as described previously [37].…”

“…Quantification of immunostaining was carried out as described previously [37]. In brief, a 10-μm line was placed orthogonal to the cell border with the cell border representing the middle of this line.…”