A leaky human colon model reveals uncoupled apical/basal cytotoxicity in early Clostridioides difficile toxin exposure

Abstract

Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis in part by disrupting epithelial barrier function. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxins on apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human lineages. Transcriptomics compared hCE to Caco-2, informed timing of in vitro stem cell differentiation, and revealed transcriptional responses to TcdA. Transepithelial electrical resistance (TEER) and fluorescent permeability assays measured cytotoxicity. Contribution of TcdA/B toxicity was evaluated in a diclofenac-induced leaky gut model. scRNAseq demonstrated broad and variable toxin receptor expression. Absorptive colonocytes in vivo displayed increased toxin receptor, Rho GTPase, and cell junction gene expression. Advanced TcdA toxicity generally decreased cytokine/chemokine and increased tight junction and death receptor genes. Differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes in vivo. Basal exposure of TcdA/B caused greater toxicity and apoptosis than apical exposure. Apical exposure to toxins was enhanced by diclofenac. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude post-basal toxin exposure. Leaky junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive system to evaluate the impact of microbial toxins on gut epithelium.

A Colonic Organoid Model Challenged with the Large Toxins of Clostridioides difficile TcdA and TcdB Exhibit Deregulated Tight Junction Proteins

“…Another recent study confirmed our observations of a barrier defect after exposure to TcdA or TcdB [42]. In this study, the redistribution of ZO-1 within the monolayer was held solely responsible for the measured barrier dysfunction.…”

Milk‐derived extracellular vesicles functionalized with anti‐tumour necrosis factor‐α nanobody and anti‐microbial peptide alleviate ulcerative colitis in mice

“…To measure cellular uptake of EV TAT‐GFP , Caco‐2 cells (1 × 10 5 ), a cell line derived from colon tumour with physiological similarity to small intestine (Ok et al., 2023 ), were seeded and cultured in 24‐well plates (Corning, USA) for 24 h. Free TAT‐GFP (20 µg), DiR‐labelled EV (60 µg) and EV TAT‐GFP (60 µg EV and 20 µg TAT‐GFP) after or before digestion in the simulated gastrointestinal fluid were added to cells, followed by incubation for 24 h. Cellular uptake and localization of EV TAT‐GFP , EV or TAT‐GFP were analysed by flow cytometry (FACSVerse, BD, USA) and confocal fluorescence microscope (FV1000, Olympus, Japan), respectively.…”

Design of 8-mer peptides that block Clostridioides difficile toxin A in intestinal cells

“…Liberated crypts were expanded as a monolayer on a neutralized collagen hydrogels. Crypts were placed on the top of 1 mg/mL collagen hydrogels (1 ml into each well of 6-well plate) at a density of 10,000 crypts/well, overlaid with 3 mL of Expansion Media (EM) 61 containing 10 mmol/L Y-27632 (S1049; SelleckChem, Houston, TX), and incubated at 37 °C in 5% CO 2 . EM was used to expand the epithelial cell numbers as monolayers; media was changed the day after seeding and every 48 hours afterwards.…”

“…EM was used to expand the epithelial cell numbers as monolayers; media was changed the day after seeding and every 48 hours afterwards. When the cell coverage was greater than 80% (typically 4 to 6 days), the epithelium was dissociated to fragments 62 to seed onto either 6-well tissue-culture plates coated with collagen hydrogels for continued expansion, or onto 12-well Transwell inserts (3460; Corning, Corning, NY) coated with 1% Matrigel for experiments 61 . To generate differentiated enterocytes for the toxicity assays, 2 wells of a 6-well colonic ISC expansion plate, where cells were ~90% confluent, were dissociated 61 as described above and plated on 12-well transwell inserts coated with 1% Matrigel.…”

“…Colonic ISCs were expanded in colon expansion media (EM, see Ref. 61 for all media formulations) until confluent (~4-days). After colonic ISCs were confluent, the media was changed to differentiation media (DM) and transepithelial electrical resistance (TEER) was measured every 24-hours using the EVOM2 (World Precision Instruments, FL).…”