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Biopsy-derived colonoid monolayers reveal a range of persistent mucosal defects in patients with ulcerative colitis.

ABSTRACT: Rationale: Ulcerative colitis (UC) is characterized by colonic mucosal inflammation and barrier dysfunction. We hypothesize that UC causes persistent defects in mucosal homeostasis, evident even in the absence of active inflammation, contributing to disease chronicity. Objective: To test this, we grew patient biopsy-derived sigmoid colonoids into air-liquid interface (ALI) monolayers, characterizing them through microscopy, proteomics, bulk RNAseq, and their susceptibility to UC patient-isolated Escherichia coli pathobiont p19A. Findings: Non-IBD ALI monolayers formed uniform crypt-like structures, mature goblet cells and a thick mucus layer containing all epithelial-derived proteins previously identified in human colonic mucus. In contrast, ALI monolayers from UC patients displayed a range of impairments, from distorted crypt-like structures and a thinner and more permeable mucus layer to severe defects in cellular differentiation and crypt development. Transcriptome analysis identified activated pathways associated with extracellular matrix formation and cell signaling, including numerous cancer-associated genes in UC ALI monolayers, which also proved significantly more susceptible to E. coli p19A. Conclusions: The culturing of patient biopsies into ALI colonoid monolayers provides a powerful model to assess human mucosal development, healing, homeostasis and mucus barrier function, revealing that UC-derived colonoid monolayers display a range of developmental and functional defects that persist in the absence of inflammation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE299477 | GEO | 2025/07/01

REPOSITORIES: GEO

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Project description:Background & Aims: Genome-wide gene expression (GWGE) profiles of mucosal colonic biopsies have suggested the existence of a continuous inflammatory state in quiescent ulcerative colitis (UC). The aim of this study was to use DNA microarray-based GWGE profiling of mucosal colonic biopsies and isolated colonocytes from UC patients and controls in order to identify the cell types responsible for the continuous inflammatory state. Methods: Adjacent mucosal colonic biopsies were obtained endoscopically from the descending colon in patients with active UC (n=8), quiescent UC (n=9), and with irritable bowel syndrome (controls, n=10). After isolation of colonocytes and subsequent extraction of total RNA, GWGE data were acquired using Human Genome U133 Plus 2.0 GeneChip Array (Affymetrix, Santa Clara, CA). Data analysis was carried out by principal component analysis and projection to latent structure-discriminant analysis using the SIMCA-P11 software (Umetrics, Umeå, Sweden). Results: A clear separation between active UC, quiescent UC and control biopsies were found, whereas the model for the colonocytes was unable to distinguish between quiescent UC and controls. The differentiation between quiescent UC and control biopsies was governed by unique profiles containing gene expressions with significant fold changes. These primarily belonged to the family of homeostatic chemokines revealing a plausible explanation to the abnormal regulated innate immune response seen in patients with UC. Conclusion: This study has demonstrated the presence of a continuous inflammatory state in quiescent UC, which seems to reflect an altered gene expression profile of lamina propria cells. Keywords: Colonocytes, continuous inflammation, mucosal colonic biopsies, gene expression profiles Adjacent mucosal colonic biopsies were attained endoscopically from the descending colon in patients with active UC (n=8), quiescent UC (n=9), and in controls (n=10). After extraction of total RNA, genome-wide gene expression data were acquired using Human Genome U133 Plus 2.0 GeneChip Array (Affymetrix, Santa Clara, CA). Amplification was required to obtain sufficient amounts of labelled complementary RNA (cRNA) target for analysis with arrays. Data analysis was carried out by principal component analysis and projection to latent structure-discriminant analysis using the SIMCA-P11 software (Umetrics, Umeå, Sweden).

Project description:The intestinal mucosal barrier is a dynamic system that allows nutrient uptake, stimulates healthy microbe-host interactions, and prevents invasion by pathogens. The mucosa consists of epithelial cells connected by cellular junctions that regulate the passage of nutrients covered by a mucus layer that plays an important role in host-microbiome interactions. Mimicking the intestinal mucosa for in vitro assays, in particular the generation of a viscous mucus layer, has proven to be challenging. Here, we present a novel in vitro intestinal culture model that produces a robust mucus layer and is based on the widely used intestinal Caco-2 cell line. We investigated the effects of air-liquid interface (ALI) culturing compared to liquid-liquid interface (LLI) on Caco-2 cells grown under low-glucose conditions in Transwell plates. In addition, we determined the impact of vasointestinal peptide (VIP) on mucus secretion, epithelial barrier properties and microbe-mucus interactions. A combination of ALI culturing with VIP addition led to formation of a robust mucus layer on the apical surface of the Caco-2 confluent layer in which the intestinal secreted mucin MUC2 was a major component. RNAseq analysis demonstrated upregulation of unique gene clusters in response to ALI and VIP conditions, but the ALI-VIP combination treatment resulted in a significant upregulation of multiple mucin genes and proteins including MUC2, MUC13 and MUC17. Expression of tight junction proteins was significantly altered in the ALI-VIP condition leading to increased permeability to small molecules, more closely reflecting an intestinal epithelium permissive for nutrient uptake. In infection experiments with commensal Lactobacillus plantarum, pathogenic Salmonella enterica serovar Enteritidis, or enterotoxigenic Escherichia coli (ETEC), the ALI-VIP mucus layer separated the bacteria from the underlying epithelium. In conclusion, ALI-VIP culture of Caco-2 cells provides an attractive in vitro model to study the function of the intestinal mucosal barrier and pathogenic and commensal microbe-host interactions.

Project description:Background. Recent studies demonstrated that Aquamin®, a calcium-, magnesium-, and multiple trace element-rich natural product, improves barrier structure and function in colonoids obtained from the tissue of healthy subjects. The goal of the present study was to determine if the colonic barrier could be improved in tissue from subjects with ulcerative colitis (UC). Methods. Colonoid cultures were established with colon biopsies from 9 individuals with UC. The colonoids were then incubated for a 2-week period under control conditions (i.e., in culture medium with a final calcium concentration of 0.25 mM) or in the same medium supplemented with Aquamin® to provide 1.5 – 4.5 mM calcium. Effects on differentiation and barrier improvement were determined using several approaches: phase-contrast & scanning electron microscopy, quantitative histology & immunohistology, mass spectrometry-based proteome assessment and transmission electron microscopy. Results. While there were no gross changes in colonoid appearance, there was evidence of increased differentiation (larger lumen diameter and wall thickness) and greater expression of cytokeratin 20 (CK20) and lesser expression of Ki67 with the intervention. Aquamin®-treated colonoids demonstrated a modest up-regulation of tight junctional proteins but stronger induction of adherens junction proteins and desmosomal proteins. Increased desmosomes were seen at the ultrastructural level. Proteomic analysis also demonstrated increased expression of basement membrane proteins and hemidesmosomal components. Proteins expressed at the apical surface (mucins and trefoils) were also increased as were several additional proteins with anti-microbial activity or that modulate inflammation. Similarly, growth and cell cycle regulatory proteins (e.g., Ki67, nucleophosmin, and stathmin) were significantly down-regulated. Laminin interactions, fibronectin matrix formation and extracellular matrix organization were the top three up-regulated pathways with the intervention. Conclusion. A majority of individuals including patients with UC do not reach the recommended daily intake for calcium and other minerals. To the extent that such deficiencies might contribute to the weakening of the colonic barrier, the findings employing UC tissue-derived colonoids here suggest that adequate mineral intake might improve the colonic barrier

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Biopsy-derived colonoid monolayers reveal a range of persistent mucosal defects in patients with ulcerative colitis.

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