Introduction:
In addition to gluten and a genetic predisposition, other external factors influence coeliac disease (CeD) onset.. These, including the microbiota, have not yet been well characterised, but could explain why the disease occurs later in life in a large proportion of patients. Microbiota-associated barrier defects may contribute to this process. We therefore investigated whether microorganisms alter epithelial polarity and barrier function, particularly epithelial gluten peptide uptake.
Methods:
The study was designed in two phases. In the screening phase, Caco2-Bbe and Caco2-PIP2 cells were exposed to different microorganisms. Caco2-PIP2 stably expressed a GFP-labelled PIP2 binding unit for analysis of cell polarity. Read-outs included transepithelial resistance (TER), tight junction (TJ) integrity and epithelial polarity (IF and confocal imaging) and paracellular permeability by sandwich assay. In phase II, selected microorganisms were further analysed in human duodenal organoid 2D monolayers from healthy individuals and CeD patients. Read-outs included: TER, TJ integrity, epithelial polarity, 33mer-gliadin translocation and differential transcriptome analysis of bulk-RNA isolated from microbiota-exposed duodenal organoids.
Results:
Pseudomonas aeruginosa and Candida albicans were selected in phase I. They caused a decrease in TER by 90% and 95% of the original TER, respectively. Escherichia coli K12 and Saccharomyces cerevisiae were used as controls for P. aeruginosa and C. albicans, respectively. In phase II, both candidates revealed a reduced TER, an abolition of epithelial polarity and disrupted TJs as well as an increased epithelial 33mer uptake in CeD and control 2D organoids. Candida invaded – in an ALS3-dependent manner – the 2D organoid duodenal epithelia and thereby interacted with epithelial dynamin. On the other hand, differential gene expression of P. aeruginosa-exposed epithelia revealed altered apoptosis, barrier function and ion transport.
Conclusion:
Our results suggest that distinct microorganisms can cause an epithelial barrier defect and increase 33mer transport in epithelial monolayers, thereby contributing to CeD immune pathology.