Tri-pentamers in galacto-oligosaccharide mixture induce galectin-9 release in CpG dnaprimed activated human intestinal epithelial cells

Introduction: A diet containing short chain galacto- and long chain fructo-oligosaccharides (scGOS/lcFOS, 9:1), and Bifidobacterium breve (containing CpG-DNA, TLR-9 ligand) suppresses allergic symptoms in mice and humans in association with enhanced local intestinal and/ or systemic galectin-9 levels.1 In mice as well as a co-culture model of human intestinal epithelial cells (IEC) and activated peripheral blood mononuclear cells (PBMC), IEC were identified as a source of galectin-91,2. Galectin-9 inhibits IgE mediated mast cell degranulation. In addition, galectin-9 was shown to enhance regulatory IL-10 secretion and Th1 polarization in the co-culture model upon IEC exposure to synthetic CpG-DNA, which was further enhanced by scGOS/lcFOS. Objectives: These findings prompted us to identify the most bioactive components in the scGOS mixture. Methods: scGOS consists of galactose chains, starting with glucose, with a degree of polymerization (DP) between 2 to 10 (GOSDP). Different oligomers were separated according to these DP from the scGOS mixture by size exclusion chromatography. To determine the most bioactive oligomer, various GOS-DP were tested in combination with synthetic CpG-DNA through apical exposure of HT-29 cells (IEC) co-cultured with anti-CD3/CD28-activated PBMC, derived from buffy coats. The basolateral medium was collected after 24 hours and IEC were used for qPCR to measure galectin-9 mRNA expression. In the supernatant, galectin-9, IL-10 and Th1 and Th2 associated cytokines were measured. Results: Only in the presence of CpG DNA all GOS-DP enhanced galectin-9, IFN-γ and IL-10 secretion after 24 hours in the co-culture model. Especially the trimer-pentamer mixture of scGOS (GOS-DP3- 5) further enhanced galectin-9 and regulatory IL-10 secretion when compared to exposure with CpG-DNA alone (P