Introduction
Chronic intestinal inflammation, increased permeability, and dysbiosis are hallmark features of cystic fibrosis (CF), yet the molecular underpinnings of gut barrier dysfunction in people with CF (pwCF) remain incompletely understood.
Aims & Methods
This study aimed to characterize epithelial markers of intestinal permeability using both immortalized and primary human intestinal epithelial cell models, with and without functional CFTR. A secondary objective was to investigate the impact of inflammatory stimuli on barrier integrity and explore lipid-based strategies for restoring epithelial function in CF. Caco-2 cells (~5000/well) were cultured in 96-well transwells (Corning) for 4 weeks to develop confluent monolayers. Healthy primary human colonic epithelial and endothelial cells (Emulate Inc.) were cultured in a microfluidic colon-on-a-chip system simulating peristalsis and blood flow. CF models were generated by CFTR gene knockdown via liposomal siRNA transfection, validated by RT-qPCR and western blotting. Epithelial permeability was assessed by transepithelial electrical resistance (TEER) and FITC-dextran flux at 48-, 72-, and 96-h post-treatment. Cells were fixed and stained for DAPI, ZO-1, and VE-cadherin, then analyzed by confocal microscopy. To assess cytoprotection, Caco-2 monolayers were pretreated with phosphatidylinositol (PI) lipids PI(16:0/18:1(9Z), PI(18:0/20:4), and LPI(20:4/0:0) (Avanti Polar Lipid and Sigma-Aldrich, 10 μM, 15 min), followed by exposure to a panel of toxins (C. difficile TcdA/B, Pseudomonas exotoxin A, Staphylococcus aureus delta-toxin) and the chemical inflammatory agent DSS (dextran sulfate sodium) for 24 h. Experiments were performed in triplicate.
Results
CFTR knockdown in Caco-2 cells (siCFTR) significantly reduced TEER compared to non-targeting controls (p < 0.05, n = 3), indicating increased epithelial permeability. This disruption was dramatically exacerbated following exposure to C. difficile toxins A and B (50 pg/mL), which caused a profound TEER decline (p < 0.0001). Immunofluorescence imaging revealed mislocalization and downregulation of ZO-1 in siCFTR cells. In the colon-on-a-chip model, CFTR depletion also disrupted ZO-1 and VE-cadherin architecture in both epithelial and endothelial layers, confirming widespread junctional compromise.
Among the tested lipid metabolites, PI (18:0/20:4) significantly preserved barrier function, by reducing FITC-dextran flux at 48 h following DSS exposure (p < 0.001) and against C. difficile toxins A/B and S. aureus delta-toxin at 96 h (p < 0.0001). Other lipids showed no significant cytoprotective effects under these conditions, suggesting a metabolic-specific and time-dependent cytoprotective effect. TEER and cytokine assays are underway.
Conclusion
Loss of CFTR function leads to impaired epithelial barrier integrity in both immortalized and primary CF gut models, with pronounced vulnerability to microbial toxins and proinflammatory stimuli. Junctional protein disruption (ZO-1, VE-cadherin) appears central to this phenotype. Notably, PI (18:0/20:4) emerged as a promising cytoprotective agent capable of restoring epithelial resistance. Importantly, PI and its derivatives are essential for cell membrane structure, intracellular signalling, and cytoskeletal dynamics. Ongoing studies will expand screening of anti-inflammatory and barrier-stabilizing therapeutics and leverage these platforms to develop patient-specific “gut avatars” for personalized therapeutic testing in CF.
Acknowledgements
This work was funded by Cystic Fibrosis Trust Award SRC 023