Na Sun, Yagiz Pat, Huseyn Babayev, Sena Ardıçlı, İsmail Öğülür, Xianting Bu, Manru Li, Shuqi Jia, Beate Rückert, Raja Dhir, Kari C. Nadeau, Mübeccel Akdiş, Cezmi A. Akdis, Duygu Yazici
BACKGROUND: The epithelial barrier theory has been proposed to link the onset of many chronic inflammatory diseases to the damaged epithelial barrier induced by numerous environmental factors, including food emulsifiers. This study aimed to investigate the effects of food emulsifiers kappa-, iota- and lambda-carrageenan on intestinal epithelial barrier integrity and the underlying mechanisms. METHODS: The cytotoxicity and apoptosis of carrageenans were determined using Hoechst/propidium iodide staining and FITC annexin V/propidium iodide staining, respectively. The gut-on-a-chip was established and transepithelial electrical resistance measurement, paracellular flux assay, immunofluorescence staining of tight junction proteins, RNA-sequencing and targeted proteomics were performed. RESULTS: Kappa-, iota- and lambda-carrageenans induced dose-dependent cytotoxicity, with lambda-carrageenan showing a more severe cytotoxic effect in relatively lower doses. All three carrageenans elicited a dose- and time-dependent decrease of gut epithelial barrier strength, a significant increase in the paracellular flux and irregular and heterogeneous staining of occludin and ZO-1 compared to the untreated group, suggesting the disruption of the intestinal epithelial barrier integrity. Transcriptomics data revealed that iota- and lambda-carrageenan induced more severe pro-inflammatory responses, which were associated with the upregulation of genes involved in TNF signalling, IL-17 signalling cellular response to chemokines, cholesterol metabolism and NF-kappa B signalling pathways. Targeted proteomics data from exposed gut-on-a-chips indicated an upregulation of inflammation- and immune response-related proteins for all three carrageenans. CONCLUSIONS: The present study provides direct evidence for the detrimental effects of kappa-, iota- and lambda-carrageenans on gut epithelial cell activation and barrier integrity. The underlying mechanism of epithelial barrier disruption was largely attributed to the activation of innate immune responses by carrageenans, resulting in a pro-inflammatory response.