Human milk oligosaccharides and non-digestible carbohydrates prevent adhesion of specific pathogens via modulating glycosylation or inflammatory genes in intestinal epithelial cells

Human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) are known to inhibit the adhesion of pathogens to the gut epithelium, but the mechanisms involved are not well understood. Here, the effects of 2 '-FL, 3-FL, DP3-DP10, DP10-DP60 and DP30-DP60 inulins and DM7, DM55 and DM69 pectins were studied on pathogen adhesion to Caco-2 cells. As the growth phase influences virulence, E. coli ET8, E. coli LMG5862, E. coli O119, E. coli WA321, and S. enterica subsp. enterica LMG07233 from both log and stationary phases were tested. Specificity for enteric pathogens was tested by including the lung pathogen K. pneumoniae LMG20218. Expression of the cell membrane glycosylation genes of galectin and glycocalyx and inflammatory genes was studied in the presence and absence of 2 '-FL or NDCs. Inhibition of pathogen adhesion was observed for 2 '-FL, inulins, and pectins. Pre-incubation with 2 '-FL downregulated ICAM1, and pectins modified the glycosylation genes. In contrast, K. pneumoniae LMG20218 downregulated the inflammatory genes, but these were restored by pre-incubation with pectins, which reduced the adhesion of K. pneumoniae LMG20218. In addition, DM69 pectin significantly upregulated the inflammatory genes. 2 '-FL and pectins but not inulins inhibited pathogen adhesion to the gut epithelial Caco-2 cells through changing the cell membrane glycosylation and inflammatory genes, but the effects were molecule-, pathogen-, and growth phase-dependent

Human milk oligosaccharides and non-digestible carbohydrates reduce pathogen adhesion to intestinal epithelial cells by decoy effects or by attenuating bacterial virulence

This work investigated the effects of different chemical structures of human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) on pathogen adhesion by serving as decoy receptors. Pre-exposure of pathogens to inulins and low degree of methylation (DM) pectin prevented binding to gut epithelial Caco2-cells, but effects were dependent on the molecules' chemistry, pathogen strain and growth phase. Pre-exposure to 3-fucosyllactose increased E. coli WA321 adhesion (28%, p < 0.05), and DM69 pectin increased E. coli ET8 (15 fold, p < 0.05) and E. coli WA321 (50%, p < 0.05) adhesion. Transcriptomics analysis revealed that DM69 pectin upregulated flagella and cell membrane associated genes. However, the top 10 downregulated genes were associated with lowering of bacteria virulence. DM69 pectin increased pathogen adhesion but bacterial virulence was attenuated illustrating different mechanisms may lower pathogen adhesion. Our study illustrates that both hMOs and NDCs can reduce adhesion or attenuate virulence of pathogens but that these effects are chemistry dependent

Acetate and Butyrate Improve β-cell Metabolism and Mitochondrial Respiration under Oxidative Stress

Islet dysfunction mediated by oxidative and mitochondrial stress contributes to the development of type 1 and 2 diabetes. Acetate and butyrate, produced by gut microbiota via fermentation, have been shown to protect against oxidative and mitochondrial stress in many cell types, but their effect on pancreatic β-cell metabolism has not been studied. Here, human islets and the mouse insulinoma cell line MIN6 were pre-incubated with 1, 2, and 4 mM of acetate or butyrate with and without exposure to the apoptosis inducer and metabolic stressor streptozotocin (STZ). Both short-chain fatty acids (SCFAs) enhanced the viability of islets and β-cells, but the beneficial effects were more pronounced in the presence of STZ. Both SCFAs prevented STZ-induced cell apoptosis, viability reduction, mitochondrial dysfunction, and the overproduction of reactive oxygen species (ROS) and nitric oxide (NO) at a concentration of 1 mM but not at higher concentrations. These rescue effects of SCFAs were accompanied by preventing reduction of the mitochondrial fusion genes MFN, MFN2, and OPA1. In addition, elevation of the fission genes DRP1 and FIS1 during STZ exposure was prevented. Acetate showed more efficiency in enhancing metabolism and inhibiting ROS, while butyrate had less effect but was stronger in inhibiting the SCFA receptor GPR41 and NO generation. Our data suggest that SCFAs play an essential role in supporting β-cell metabolism and promoting survival under stressful conditions. It therewith provides a novel mechanism by which enhanced dietary fiber intake contributes to the reduction of Western diseases such as diabetes

Modulation of Intestinal Epithelial Glycocalyx Development by Human Milk Oligosaccharides and Non-Digestible Carbohydrates

Scope The epithelial glycocalyx development is of great importance for microbial colonization. Human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) may modulate glycocalyx development. Methods and results The effects of hMOs and NDCs on human gut epithelial cells (Caco2) are investigated by quantifying thickness and area coverage of adsorbed albumin, heparan sulfate (HS), and hyaluronic acid (HA) in the glycocalyx. Effects of hMOs (2 '-FL and 3-FL) and NDCs [inulins with degrees of polymerization (DP) (DP3-DP10, DP10-DP60, DP30-DP60) and pectins with degrees of methylation (DM) (DM7, DM55, DM69)] are tested using immunofluorescence staining at 1 and 5 days stimulation. HMOs show a significant enhancing effect on glycocalyx development but effects are structure-dependent. 3-FL induces a stronger albumin adsorption and increases HS and HA stronger than 2 '-FL. The DP3-DP10, DP30-60 inulins also increase glycocalyx development in a structure-dependent manner as DP3-DP10 selectively increases HS, while DP30-DP60 specifically increases HA. Pectins have less effects, and only increase albumin adsorption. Conclusion Here, it is shown that 2 '-FL and 3-FL and inulins stimulate glycocalyx development in a structure-dependent fashion. This may contribute to formulation of effective hMO and NDC formulations in infant formulas to support microbial colonization and gut barrier function

Protective effects of lactic acid bacteria on gut epithelial barrier dysfunction are Toll like receptor 2 and protein kinase C dependent

Lactic acid bacteria (LAB) are recognized for support of host gut homeostasis but the precise mechanisms remain to be identified. LABs interact with Toll-like receptors (TLRs) which might stimulate barrier function of gut epithelial cells. We previously identified six TLR2-signalling LAB strains. As TLR2 is involved in barrier-function enhancement in gut-epithelium, the epithelial barrier-protective effect of these TLR2-signalling strains was studied by using T84 human colorectal cancer cell monolayer as an in vitro gut epithelial barrier model. The protein kinase C (PKC) dependent barrier disruptor A23187 and mitogen-activated protein kinase dependent barrier stressor deoxynivalenol were tested to determine which pathways LAB influenced. We found that exclusively the PKC dependent disruption was prevented by the selected TLR2-signalling LAB strains. This study suggests that TLR2 is a pivotal epithelial barrier modulator, and provides novel insight in the molecular mechanisms by which LAB contribute to intestinal health

Efficacy of pectins with different degrees of methyl-esterification and of blockiness in preventing gut epithelial cell barrier disruption and the impact on sodium-glucose co-transporter expression under low and high glucose conditions

Pectins support intestinal barrier function and have anti-diabetic effects, and can differ in the degree of methyl-esterification (DM) and the distribution of non-esterified galacturonic acid residues (DB). The mechanisms and effects of pectin type at different glucose levels are unknown. Pectins with different DM/DB on T84 cells were tested in the presence and absence of the barrier disruptor A23187 at 5 mM and 20 mM glucose. DM19 and DM43 pectins with high DB do rescue the intestinal barrier from disruption. Their effects were as strong as those of the barrier-rescuing anti-diabetic drug metformin, but effects with metformin were restricted to high glucose levels while pectins had effects at both low and high glucose levels. At high glucose levels, DM43HB pectin, which enhanced trans-epithelial electrical resistance, also increased the expressions of claudin1, occludin, and ZO-1. Low and high DM pectins decrease the apical expression of the sodium-glucose co-transporter (SGLT-1) and thereby influence glucose transport, explaining the anti-diabetogenic effect of pectin. Higher DB pectins had the strongest effect. Their impact on SGLT-1 was stronger than that of metformin. Pectin's rescuing effect on barrier disruption and its impact on glucose transportation and anti-diabetogenic effects depend on both the DB and the DM of pectins.</p

Human Milk Oligosaccharides Mediate the Crosstalk Between Intestinal Epithelial Caco-2 Cells and Lactobacillus Plantarum WCFS1 in an In Vitro Model with Intestinal Peristaltic Shear Force

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 plantarum WCFS1 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. plantarum WCFS1 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. plantarum WCFS1 adhesion by 1.85- to 1.90-fold (P <0.01). Conclusions: 3-FL and LNT2 support the crosstalk between the commensal bacterium L. plantarum WCFS1 and Caco-2 intestinal epithelial cells, and shear force can increase the modulating effects of hMOs

Selective Modification of Streptozotocin at the C3 Position to Improve Its Bioactivity as Antibiotic and Reduce Its Cytotoxicity towards Insulin-Producing β Cells

With the increasing resistance of bacteria to current antibiotics, novel compounds are urgently needed to treat bacterial infections. Streptozotocin (STZ) is a natural product that has broad-spectrum antibiotic activity, albeit with limited use because of its toxicity to pancreatic β cells. In an attempt to derivatize STZ through structural modification at the C3 position, we performed the synthesis of three novel STZ analogues by making use of our recently developed regioselective oxidation protocol. Keto-STZ (2) shows the highest inhibition of bacterial growth (minimum inhibitory concentration (MIC) and viability assays), but is also the most cytotoxic compound. Pre-sensitizing the bacteria with GlcNAc increased the antimicrobial effect, but did not result in complete killing. Interestingly, allo-STZ (3) revealed moderate concentration-dependent antimicrobial activity and no cytotoxicity towards β cells, and deoxy-STZ (4) showed no activity at all

L. plantarum WCFS1 enhances Treg frequencies by activating DCs even in absence of sampling of bacteria in the Peyer Patches

Probiotics such as L. plantarum WCFS1 can modulate immune responses in healthy subjects but how this occurs is still largely unknown. Immune-sampling in the Peyer Patches has been suggested to be one of the mechanisms. Here we studied the systemic and intestinal immune effects in combination with a trafficking study through the intestine of a well-established immunomodulating probiotic, i.e. L. plantarum WCFS1. We demonstrate that not more than 2-3 bacteria were sampled and in many animals not any bacterium could be found in the PP. Despite this, L. plantarum was associated with a strong increase in infiltration of regulatory CD103+ DCs and generation of regulatory T cells in the spleen. Also, a reduced splenic T helper cell cytokine response was observed after ex vivo restimulation. L. plantarum enhanced Treg cells and attenuated the T helper 2 response in healthy mice. We demonstrate that, in healthy mice, immune sampling is a rare phenomenon and not required for immunomodulation. Also in absence of any sampling immune activation was found illustrating that host-microbe interaction on the Peyer Patches was enough to induce immunomodulation of DCs and T-cells

Two Patients with Extremely Elevated Tumor Markers: Where Is the Malignancy?

Serum tumor markers are useful to evaluate a cancer's response to treatment, for early detection of cancer relapse, and, in some cases, to diagnose malignancy. In this paper, we present two patients with significantly elevated serum tumor markers without evidence of malignant disease. An 18-year-old patient suffering from autoimmune hepatitis had markedly increased alpha-fetoprotein (aFP) levels (2,002 μg/L; normal <10 ug/L). Extensive imaging showed no signs of hepatocellular carcinoma or other cancer, and treatment with Prednisone led to rapid normalization of both liver enzymes and aFP. The second patient, a 60-year-old female with painless jaundice due to biliary stone disease, had very high serum levels of CA19-9 (18,000 kU/L, normal <27 kU/L). Liver biochemistry and serum CA19-9 concentration decreased to almost normal values (45 kU/L) after biliary stenting. These cases demonstrate that serum tumor markers can be elevated in benign disease and are therefore not appropriate to diagnose cancer