Lipopolysaccharide-induced alterations in barrier integrity and inflammatory signaling in porcine small intestinal organoids

Inflammation-associated diseases in pigs impair the intestinal tract and impair its function, thereby posing a major threat to their health and productivity. However, current in vitro cell models fail to fully replicate porcine physiological conditions. In this study, we aimed to develop a novel inflammation model using porcine small intestinal organoids derived from adult jejunum stimulated with lipopolysaccharide (LPS). The organoids were exposed to LPS to induce inflammation, and the responses were evaluated by quantifying cytokine mRNA expression, assessing intestinal epithelial barrier integrity, and analyzing changes in the expression and localization of proteins involved in nutrient transport, receptor signaling, and enteroendocrine function using quantitative real-time PCR and immunofluorescence staining. Transcriptome analysis was performed to identify LPS-responsive differentially expressed genes and enriched pathways in porcine small intestinal organoids. LPS stimulation effectively induced inflammation in porcine small intestinal organoids, as evidenced by increased TLR4 and cytokine gene expression, compromised epithelial barrier integrity, and altered expression and localization of proteins involved in nutrient transport, bile acid signaling, and enteroendocrine function. Furthermore, transcriptome profiling revealed LPS-induced broad transcriptional changes in porcine small intestinal organoids, including enrichment of immune- and inflammation-related pathways, as well as functional categories associated with barrier maintenance, membrane function, and adaptation to inflammatory stress. Collectively, these findings demonstrate that porcine small intestinal organoids provide a physiologically relevant in vitro model for studying intestinal inflammation. This model offers a valuable platform for investigating the mechanisms underlying porcine inflammatory bowel diseases and for screening novel therapeutic agents, supporting the 3Rs principle by potentially reducing reliance on animal testing in translational research.