Human Milk Oligosaccharides Mediate the Crosstalk Between Intestinal Epithelial Caco-2 Cells and Lactobacillus Plantarum WCFS1in an In Vitro Model with Intestinal Peristaltic Shear Force

Abstract

Background The intestinal epithelial cells, food molecules, and gut microbiota are continuously exposed to intestinal peristaltic shear force. Shear force may impact the crosstalk of human milk oligosaccharides (hMOs) with commensal bacteria and intestinal epithelial cells. Objectives We investigated how hMOs combined with intestinal peristaltic shear force impact intestinal epithelial cells and crosstalk with a commensal bacterium. Methods We applied the Ibidi system to mimic intestinal peristaltic shear force. Caco-2 cells were exposed to a shear force (5 dynes/cm2) for 3 d, and then stimulated with the hMOs, 2′-fucosyllactose (2′-FL), 3-FL, and lacto-N-triose II (LNT2). In separate experiments, Lactobacillus plantarumWCFS1 adhesion to Caco-2 cells was studied with the same hMOs and shear force. Effects were tested on gene expression of glycocalyx-related molecules (glypican 1 [GPC1], hyaluronan synthase 1 [HAS1], HAS2, HAS3, exostosin glycosyltransferase 1 [EXT1], EXT2), defensin β-1 (DEFB1), and tight junction (tight junction protein 1 [TJP1], claudin 3 [CLDN3]) in Caco-2 cells. Protein expression of tight junctions was also quantified. Results Shear force dramatically decreased gene expression of the main enzymes for making glycosaminoglycan side chains (HAS3 by 43.3% and EXT1 by 68.7%) (P <0.01), but did not affect GPC1 which is the gene responsible for the synthesis of glypican 1 which is a major protein backbone of glycocalyx. Expression of DEFB1, TJP1, and CLDN3 genes was decreased 60.0–94.9% by shear force (P <0.001). The presence of L. plantarumWCFS1 increased GPC1, HAS2, HAS3, and ZO-1 expression by 1.78- to 3.34-fold (P <0.05). Under shear force, all hMOs significantly stimulated DEFB1 and ZO-1, whereas only 3-FL and LNT2 enhanced L. plantarumWCFS1 adhesion by 1.85- to 1.90-fold (P <0.01). Conclusions 3-FL and LNT2 support the crosstalk between the commensal bacterium L. plantarumWCFS1 and Caco-2 intestinal epithelial cells, and shear force can increase the modulating effects of hMOs.

Effects of Human Milk Oligosaccharides on the Adult Gut Microbiota and Barrier Function

“…Another parameter to consider is the influence of the mechanical forces applied to the gut-on-chip system as compared with the Caco2 cells under static culture. A recent study highlighted that applying shear force equivalent to that experienced during intestinal peristalsis to Caco2 cells increased the modulating effects of HMOs on the gene expression of tight junction proteins [65], further supporting the importance of mechanical forces, such as flow and stretch to emulate the dynamic cellular microenvironment.…”

Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip

“…For example, gut-chip analysis of dimethyloxaloylglycine, a small molecule drug candidate for the prevention of radiation-induced gut permeability, predicted clinical outcomes in humans, while these results were not reproduced by rodent models 23 . Furthermore, evaluation of milk oligosaccharides and fermentate from SHIME microbial cultures displayed protein- and metabolite-dependent activation of claudin tight-junctional proteins, limiting small molecule transport as seen in longitudinal human studies involving B. infantis treatment of IBD 34 , 35 . To the best of our knowledge, gut-chip models have not previously been used to validate the in vivo function of synthetic biotics, making this study a foundational case study for evaluating dynamic processes such as strain activity, biomarker production, and host tissue responses, as well as for extrapolation of in vitro data to provide in vivo predictions.…”

Regulation of hBD-2, hBD-3, hCAP18/LL37, and Proinflammatory Cytokine Secretion by Human Milk Oligosaccharides in an Organotypic Oral Mucosal Model

“…No other study has examined the interactions between 3-FL and hBDs in an oral mucosa model, preventing us from comparing our results with the literature. Kong et al evaluated the effects of shear forces on the intestinal epithelial barrier with Caco-2 cells and, in addition, stimulated Caco-2 cells with 2 -FL, 3-FL, and lacto-N-triose II (LNT2) [13]. They found that shear force exposure downregulated antimicrobial peptide beta defensin (BD)-1 but that, under static and shear force conditions, LNT2-not 2 -FL or 3-FL-upregulated BD-1 [13].…”

“…Kong et al evaluated the effects of shear forces on the intestinal epithelial barrier with Caco-2 cells and, in addition, stimulated Caco-2 cells with 2 -FL, 3-FL, and lacto-N-triose II (LNT2) [13]. They found that shear force exposure downregulated antimicrobial peptide beta defensin (BD)-1 but that, under static and shear force conditions, LNT2-not 2 -FL or 3-FL-upregulated BD-1 [13]. However, with Lactiplantibacillus plantarum WCFS1, all three HMOs upregulated BD-1 under shear force conditions, and 2 -FL and LNT2 did so under static conditions [13].…”

“…They found that shear force exposure downregulated antimicrobial peptide beta defensin (BD)-1 but that, under static and shear force conditions, LNT2-not 2 -FL or 3-FL-upregulated BD-1 [13]. However, with Lactiplantibacillus plantarum WCFS1, all three HMOs upregulated BD-1 under shear force conditions, and 2 -FL and LNT2 did so under static conditions [13].…”