P111 Dietary emulsifier κ-carrageenan does not directly affect intestinal permeability in organoid-derived epithelial monolayers from patients with Crohn’s disease

Abstract

Abstract Background The dietary emulsifier carrageenan (CGN) is widely used to optimise the texture of processed foods such as ice cream, plant-based milk and processed meat products. However, CGN is suggested to play a role in IBD development by exerting pro-inflammatory effects and increasing intestinal permeability, as shown in immortalized cell lines and murine models. Specifically kappa-carrageenan (κ-CGN) has been shown to disrupt barrier function of intestinal epithelial cells. In this study, we investigated if κ-CGN can directly increase intestinal permeability in organoid-derived epithelial monolayers from patients with Crohn’s disease (CD). Methods Organoid cultures were derived from colonic biopsies of 5 patients with CD (Table 1) and seeded on Transwell® inserts to obtain epithelial monolayers. Two independent experiments were performed per patient (n = 10). Once a confluent monolayer was formed, non-inflamed and inflamed conditions were established by adding regular culture medium or inflammatory stimuli (100 ng/mL TNF-α, 20 ng/mL IL-1β, 1 µg/mL flagellin) to the basolateral side, respectively. The next day, both inflamed and non-inflamed monolayers were stimulated with 100 µg/mL κ-CGN on the apical side, or with regular culture medium as control. The basolateral side was also renewed with either the inflammatory stimuli or regular culture medium. Transepithelial electrical resistance (TEER) was measured after 24 and 48 hours of κ-CGN exposure to study permeability of the epithelium. Relative TEER (% change compared to baseline) was compared between controls and κ-CGN stimulated conditions (repeated measures ANOVA, Šídák's multiple comparisons correction). Results As expected, exposure to inflammatory stimuli on the basolateral side resulted in a significant reduction of TEER of the monolayers after 48 hours (+ 24 hours pre-stimulation), compared to the non-inflamed conditions (-22.54 ± 4.557 %, p<0.0001, paired t-test) (Figure 1A). After 24 hours of stimulation with κ-CGN, TEER did not significantly change in both the non-inflamed (p=0.90) and inflamed conditions (p=0.80), compared to the control conditions. Also after 48 hours, exposure to κ-CGN did not significantly affect the TEER in both non-inflamed (p=0.94) and inflamed (p=0.74) conditions (Figure 1B). RNA analysis of the organoid-derived monolayers is ongoing to explore the inflammatory potential of κ-CGN and possible effects on mucus production. Conclusion The dietary emulsifier κ-CGN does not directly alter the permeability of the intestinal epithelium of CD patients. Further analysis of gene expression of inflammatory markers and mucus production is ongoing. Possible indirect effects, through alterations of the microbiota or immune cells, should still be explored.