Modulation of the Epithelial-Immune Cell Crosstalk and Related Galectin Secretion by DP3-5 Galacto-Oligosaccharides and β-3′Galactosyllactose

Abstract

Prebiotic galacto-oligosaccharides (GOS) were shown to support mucosal immune development by enhancing regulatory-type Th1 immune polarization induced by synthetic CpG oligodeoxynucleotides (TLR9 agonist mimicking a bacterial DNA trigger). Epithelial-derived galectin-9 was associated with these immunomodulatory effects. We aimed to identify the most active fractions within GOS based on the degree of polymerization (DP), and to study the immunomodulatory capacities of DP3-sized β-3′galactosyllactose (β-3′GL) using a transwell co-culture model of human intestinal epithelial cells (IEC) and activated peripheral blood mononuclear cells (PBMC). IEC were apically exposed to different DP fractions of GOS or β-3′GL in the presence of CpG, and basolaterally co-cultured with αCD3/CD28-activated PBMC, washed, and incubated in fresh medium for IEC-derived galectin analysis. Only DP3-5 in the presence of CpG enhanced galectin-9 secretion. DP3-sized β-3′GL promoted a regulatory-type Th1 response by increasing IFNγ and IL-10 or galectin-9 concentrations as compared to CpG alone. In addition, IEC-derived galectin-3, -4, and -9 secretion was increased by β-3′GL when combined with CpG. Therefore, the GOS DP3-5 and most effectively DP3-sized β-3′GL supported the immunomodulatory properties induced by CpG by enhancing epithelial-derived galectin secretion, which, in turn, could support mucosal immunity.

Epithelial-derived galectin-9 containing exosomes contribute to the immunomodulatory effects promoted by 2’-fucosyllactose and short-chain galacto- and long-chain fructo-oligosaccharides

“…Depending on the experimental condition, the amount of exosomes isolated differed which might have hampered the detection of galectin-9 and -4 for all conditions. As was shown previously using the HT-29/PBMC model ( 27 ), NDO facilitate CpG induced galectin-9 release by IEC and this may have taken place during the first 24 h, thus during the co-culture with PBMC. The CpG and 2’FL/GF condition may therefore have already caused the exosome release which might explain a more limited signal observed after the additional 24 h of epithelial cell culture following the co-culture with PBMC.…”

“…In addition, when NDO were combined with CpG and exposed to the FHs 74 Int/PBMC co-culture, upregulated regulatory-type Th1 and downregulated Th2-type cytokines were observed, similar to what was previously observed in HT-29 or T84 and PBMC co-cultures ( 2 , 9 ). However, even though the HT-29 cells were found to release also galectin-4 and galectin-3 beyond galectin-9 ( 2 , 9 , 27 ), the primary FHs 74 Int epithelial cells only secreted galectin-9 and -3, but not galectin-4. Also others found anti-inflammatory effects in primary fetal epithelial cells using NDO.…”

“…In line with previous studies, only upon activation of PBMC, CpG-induced upregulation of Th1-type IFNγ, regulatory IL-10 and galectin-9 were found in IEC/PBMC co-culture model ( 9 , 31 ). The addition of NDO enhanced these Th1-type and regulatory responses while lowering Th2-type IL-13 secretion ( 2 , 3 , 9 , 27 ). Unlike in the HT-29 cell line, in the FHs 74 Int/PBMC co-culture, already in the absence of CpG, GF or 2’FL/GF alone were able to modulate the secretion of cytokines like Th1-type IFNγ, Th2-type IL-13 as well as regulatory IL-10 and galectin-9.…”

“…Commercially available kits were used to measure IFNγ, IL-13, TNFα (all from Thermo Fisher Scientific, Waltham, MA, USA), IL-10 (U-Cytech, Utrecht, The Netherlands), and galectin-3 (R&D systems) following manufacturer’s protocol. Human galectin-4 and -9 were measured using antibody pairs (all from R&D systems, Minneapolis, MN, USA) as described before ( 9 , 26 , 27 ). Additionally, a CD63 detection ELISA kit (System Biosciences) was used to measure the exosomes isolated from conditioned supernatant following manufacturer’s protocol.…”

Cell Biology of Galectins: Novel Aspects and Emerging Challenges

“…Although bioinformatics analysis suggests that all human galectin molecules have potential sites for O -GlcNAcylation [ 11 ], whether this mechanism works for other galectins remains to be investigated. Ayechu-Muruzabal et al [ 13 ] demonstrate that secretion of galectin-3, -4, and -9 can be stimulated by specific types of galacto-oligosaccharides and CpG oligodeoxynucleotides in a complex transwell co-culture model of intestinal epithelial cells (human colon adenocarcinoma HT-29 cell line) with primary peripheral blood mononuclear cells, which ultimately supports the role of galectins in the mucosal immune response. In this context, the authors highlight the key immunomodulatory contribution of β-3′galactosyllactose, a component of human milk [ 14 ], which reveals an interesting aspect of health benefits associated with secreted galectins.…”