P0098 Effect of the dietary emulsifier κ-carrageenan on the inflammatory state and permeability of intestinal epithelial cells from patients with Crohn’s disease

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Abstract Background The consumption of ultra-processed foods has increased worldwide and is associated with the rise in inflammatory bowel disease (IBD)1. However, any causative factors and their underlying mechanisms are yet to be identified. Interestingly, our group found a higher consumption of the dietary emulsifier carrageenan (CGN) in Belgian IBD patients, compared to healthy controls2. This study aimed to elucidate whether different types of CGN can alter the permeability and inflammatory state of the intestinal epithelium in patients with Crohn’s disease (CD). Methods Confluent Caco-2/HT29-MTX cocultures (n=4) were exposed to either κ-, ι-, or λ-CGN (100 µg/ml) for 24 hours. Confluent epithelial monolayers derived from colonic organoids3 of 5 CD patients were exposed to κ-CGN (100 µg/ml), the most abundant subtype in foods, for 48 hours. Two independent experiments were performed per patient (n = 10). In both models, inflammation was established by adding an inflammatory mix (100 ng/mL TNF-α, 20 ng/mL IL-1β, and 1 µg/mL flagellin)4 to the basolateral side, 24 hours prior to CGN exposure. Changes in permeability were measured by transepithelial electrical resistance (TEER) at predetermined timepoints (0, 12, 24, 48 hours). In organoid-derived monolayers, cytokines were quantified in the apical and basolateral supernatant using the V-PLEX Proinflammatory Panel 1 kit (Meso Scale Diagnostics) and gene expression was analysed with RT-qPCR (Taqman probes, run on Viia7) (n = 8; 2 samples excluded due to low RNA concentrations). Results In organoid-derived monolayers, κ-CGN increased the expression of TNF, IL8 and IL1B, both in inflamed and non-inflamed monolayers (n=8; Repeated measures (RM) one-way ANOVA), compared to the control (Fig. 1A). Release of the cytokines IL-6, IL-13, IL-4, IL-2, and IL-10 in the apical supernatant was increased after 48 hours of κ-CGN stimulation (n=8; RM one-way ANOVA) (Fig. 1B). κ-CGN exposure did not affect TEER (n=10; RM one-way ANOVA) (Fig. 2B+C), but induced an upregulation of tight junction markers ZO1 and OCLN, and resulted in a downregulation of MUC5AC and upregulation of MUC5B (n=8; RM one-way ANOVA or Friedman test) (Fig. 2D). Additionally, none of the CGN subtypes altered permeability of non-inflamed or inflamed Caco-2/HT29-MTX cocultures (n=4; Kruskal-Wallis test), compared to the unexposed control (Fig. 2A). Conclusion Dietary κ-CGN caused upregulation of inflammatory markers and affected cytokine release of intestinal epithelial cells from patients with CD, while permeability remained unaltered. When inflammation was already present, this pro-inflammatory effect was more pronounced, suggesting a role for dietary CGN in perpetuating inflammation during active CD.