Cytokine-induced barrier dysfunction and lipid signaling in a gut-on-chip model

Morelli, M.; Savova, M.V.; Queiroz, K.; Harms, A.C.; Hankemeier, T.

doi:doi:10.1096/fj.202501685R

The intestinal epithelial barrier is crucial for gut homeostasis, with dysfunction linked to inflammatory disorders like inflammatory bowel disease (IBD). While cytokines are established mediators of barrier disruption, the role of lipid signaling remains poorly understood. Using a microfluidic platform, we cultured epithelial tubules and exposed their luminal (apical) side to TNF-alpha, IL-1 beta, and IFN-gamma (0-200 ng/mL) under serum-containing or serum-free conditions. Barrier function was assessed primarily via transepithelial electrical resistance (TEER), with DRAQ7 staining and actin cytoskeletal analysis providing complementary indicators of membrane integrity and structural disruption. In parallel, a targeted liquid chromatography-tandem mass spectrometry approach was used to profile lipid mediators across apical and basolateral compartments. Cytokine exposure significantly impaired barrier integrity, as indicated by reduced TEER, alongside associated cell damage and...Show moreThe intestinal epithelial barrier is crucial for gut homeostasis, with dysfunction linked to inflammatory disorders like inflammatory bowel disease (IBD). While cytokines are established mediators of barrier disruption, the role of lipid signaling remains poorly understood. Using a microfluidic platform, we cultured epithelial tubules and exposed their luminal (apical) side to TNF-alpha, IL-1 beta, and IFN-gamma (0-200 ng/mL) under serum-containing or serum-free conditions. Barrier function was assessed primarily via transepithelial electrical resistance (TEER), with DRAQ7 staining and actin cytoskeletal analysis providing complementary indicators of membrane integrity and structural disruption. In parallel, a targeted liquid chromatography-tandem mass spectrometry approach was used to profile lipid mediators across apical and basolateral compartments. Cytokine exposure significantly impaired barrier integrity, as indicated by reduced TEER, alongside associated cell damage and structural changes reflected by DRAQ7 staining and actin remodeling. The remodeling effect was lower in serum-free medium. Lipid profiling revealed inflammatory signatures characterized by an increase in prostaglandins in the luminal compartment, particularly under serum-free conditions. PGF1 alpha increased under both media conditions, whereas the rest of the changes were condition-specific, with a rise in PGE1, PGE2, and PGD2, among others, particularly under serum-free conditions. Simultaneously, changes in other eicosanoids, but not in prostaglandins, were detected in the basolateral compartment under serum-free conditions. This proof-of-principle study demonstrates how medium composition significantly influences inflammatory responses and lipid signaling patterns in a physiologically relevant gut-on-chip model. Our integrated approach reveals the complex spatial organization of lipid mediators during cytokine-induced barrier dysfunction and provides a valuable framework for investigating the interplay between inflammation, barrier integrity, and lipid metabolism in intestinal pathophysiology.Show less

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Cytokine-induced barrier dysfunction and lipid signaling in a gut-on-chip model