Gastrointestinal digestion products of olive mill pectin modulate intestinal inflammation through transcriptional reprogramming and molecular interactions in Caco-2 cells

Olive mill semi-solid by-products (OMSbP) are an abundant source of homogalacturonan-rich pectin that can be valorised as a bioactive ingredient for gut health. However, the molecular mechanisms by which its gastrointestinal digestion products modulate intestinal inflammation remain poorly understood. Here, we investigated the transcriptomic and molecular effects of heOMSbP, an olive-derived low-methoxyl pectin and its simulated gastrointestinal digestion products (heOMSbP-dig) in lipopolysaccharide (LPS)-challenged human intestinal Caco-2 cells. Pectin was extracted from OMSbP by citric acid–assisted thermal treatment and subjected to the standardized INFOGEST 2.0 static in vitro digestion protocol. Caco-2 cells were stimulated with LPS (1 μg mL −1 ) and subsequently treated with heOMSbP-dig (500 μg mL −1 ). RNA-seq analysis identified extensive transcriptional reprogramming, with LPS inducing a strong chemokine-rich inflammatory signature, while heOMSbP-dig significantly downregulated more than 500 genes, including CXCL8, CXCL1, CXCL10 and CCL20, and promoted enrichment of metabolic and stress-adaptation pathways. Docking analyses predicted low- to moderate-affinity interactions (−3.5 to −7.5 kcal·mol −1 ) between galacturonic acid tetramers and TLR4/MD2 or TLR2/TLR1 complexes, with higher affinity associated with increased methylation degree. Altogether, these findings indicate that digestion products of olive mill pectin reprogram LPS-induced transcriptional responses in intestinal epithelial cells and suggest that pectin–TLR interactions may contribute to the immunomodulatory potential of this upcycled polysaccharide.