Celiac disease is a gluten-induced systemic autoimmune enteropathy with an estimated global seroprevalence of approximately 1.4% [1]. In genetically susceptible individuals, dietary gluten triggers a gluten-specific, T-cell-mediated immune response and is associated with intestinal epithelial barrier dysfunction. Most in vitro models used to study wheat-induced responses rely on monocultures of either epithelial or immune cells, failing to capture epithelial-immune crosstalk, while patient-derived organoids, although highly physiologically relevant, remain costly, low-throughput, and difficult to standardise. As such, there is a need for accessible, integrated and scalable in vitro models to investigate the effects of food-derived biomolecules and facilitate the identification of potential therapeutic interventions.
In this study, we developed a functionally characterised tri-culture model of the human small intestine integrating Caco-2 epithelial cells, HT-29-MTX-E12 mucus-producing cells, and THP-1 immune cells to recapitulate gliadin-induced epithelial barrier disruption and basolateral immune activation in vitro. We systematically compared the tri-culture with THP-1 monocultures and 21-days differentiated Caco-2:HT-29 co-cultures. Therefore, we characterised barrier integrity, mucus production, and immune responsiveness to the gliadin peptides α-gliadin p31-43 and p57-68, as well as to the Prolamin Working Group (PWG) standard.
α-Gliadin treatment upregulated pro-inflammatory gene expression (CXCL8, MCP-1, and IL-1β) in THP-1 cells within the tri-culture but not in THP-1 monoculture. Conversely, PWG triggered upregulation of these genes (CXCL8 and MCP-1) in THP-1 monoculture but not in tri-culture, indicating barrier-mediated immune effects. These findings emphasise the importance of studying epithelial-immune interactions in in vitro models used for celiac disease.
To summarise, the tri-culture platform enables the study of epithelial-immune crosstalk after gluten challenge while retaining standardisation, cost efficiency, and scalability required for high-throughput in vitro screening.