A Leaky Human Colon Model Reveals Uncoupled Apical/Basal Cytotoxicity in Early Clostridioides difficile Toxin Exposure

Abstract

Background & AimsClostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis and increase morbidity and mortality. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop pre-clinical models for new therapies. Properties of cancer cell lines and 3D organoids inherently limit these efforts. Here, we develop adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigate the impact of toxin application to apical/basal aspects of monolayers, and evaluate whether a leaky epithelial barrier enhances toxicity. MethodsSingle-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to cell lineages across the human gut. Transcriptomics informed timing of stem cell differentiation to achieve in vitro colonocyte maturation like that observed in vivo. Transepithelial electrical resistance (TEER) and fluorescent dextran permeability assays measured cytotoxicity as barrier loss post-toxin exposure. Leaky epithelial barriers were induced with diclofenac. ResultsscRNAseq demonstrated broad and variable toxin receptor expression across the human gut lineages. Absorptive colonocytes displayed generally enhanced toxin receptor, Rho GTPase, and cell junction expression. 21-day differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes. hCE monolayers exhibited high barrier function after 1-day differentiation. Basal TcdA/B application to monolayers caused more toxicity and apoptosis than apical exposure. Diclofenac induced leaky hCE monolayers and enhanced toxicity of apical TcdB exposure. ConclusionsApical/basal toxicities are uncoupled with more rapid onset and increased magnitude of basal toxicity. Leaky paracellular junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive culture system to evaluate the impact of microbial toxins on gut epithelium.