Structure-function relationship between homogalacturonan pectins and intestinal immunity:Microbiota-(in)dependent effects on the gastrointestinal immune barrier

Pectins are dietary fibers that have been recognized to reduce the incidence of lifestyle related disease. Pectins can contain several chemical structures, including homogalacturonan regions which consist of a galacturonan backbone. These galacturonans can be methyl-esterified causing a difference in degree and distribution of methyl-esters in pectins. It is known that pectins can influence the intestinal immune system, but which specific structures are responsible for these effects is unknown. Therefore, the immunomodulatory role of the degree of methyl-esterification (DM) and the distribution of methyl-esters in homogalacturonan pectins on the intestinal immune system was investigated. Current studies demonstrate that low DM pectins and pectins with an intermediate DM and blockwise distribution of methyl-esters inhibit the activation of Toll-like receptor (TLR) 2-1 and prevent the development of TLR2-mediated inflammation of the small intestine in mice. Besides, administration of a low DM pectin that contains a more blockwise distribution of methyl-esters and intermediate DM pectins influence T cell immunity and the microbiota composition in the intestine of healthy mice. In addition, pectins inhibit the growth of Citrobacter rodentium and prevent inflammation caused by this pathogen in the large intestine. These effects of pectins on C. rodentium were independent of structural differences. In conclusion, homogalacturonan pectins can influence the intestinal immune system through direct effects on TLR2, modulation of intestinal microbiota composition or by inhibiting the growth of enteric pathogens in the intestine. The degree and distribution of methyl-esters of pectins appear to play an important role in these effects

The effects of different dietary fiber pectin structures on the gastrointestinal immune barrier:impact via gut microbiota and direct effects on immune cells

Pectins are dietary fibers with different structural characteristics. Specific pectin structures can influence the gastrointestinal immune barrier by directly interacting with immune cells or by impacting the intestinal microbiota. The impact of pectin strongly depends on the specific structural characteristics of pectin; for example, the degree of methyl-esterification, acetylation and rhamnogalacturonan I or rhamnogalacturonan II neutral side chains. Here, we review the interactions of specific pectin structures with the gastrointestinal immune barrier. The effects of pectin include strengthening the mucus layer, enhancing epithelial integrity, and activating or inhibiting dendritic cell and macrophage responses. The direct interaction of pectins with the gastrointestinal immune barrier may be governed through pattern recognition receptors, such as Toll-like receptors 2 and 4 or Galectin-3. In addition, specific pectins can stimulate the diversity and abundance of beneficial microbial communities. Furthermore, the gastrointestinal immune barrier may be enhanced by short-chain fatty acids. Moreover, pectins can enhance the intestinal immune barrier by favoring the adhesion of commensal bacteria and inhibiting the adhesion of pathogens to epithelial cells. Current data illustrate that pectin may be a powerful dietary fiber to manage and prevent several inflammatory conditions, but additional human studies with pectin molecules with well-defined structures are urgently needed

The influence of calcium on pectin's impact on TLR2 signalling

High intake of dietary fibres and calcium has been correlated to a lower frequency of Western disease such as allergy, asthma and obesity. How the combined higher intake of dietary fibres and calcium reduces the incidence of these diseases is unknown. Dietary fibre pectin can interact with Toll-like receptor (TLR) 2 and calcium in a degree of methyl-esterification (DM)-dependent manner. Low DM pectins interact stronger with TLR2 than high DM pectins. Since low DM pectin are known to bind calcium strongly, we investigated how calcium influences the DM-dependent impact of pectins on TLR2 signalling. We tested TLR2 activating, inhibiting and binding properties of pectins with DM18, DM52 and DM69 under 0 mM, 1 mM and 10 mM calcium conditions. None of the pectins activated TLR2, but pectins inhibited TLR2. Under 0 mM calcium conditions, especially DM18 and DM52 strongly inhibited TLR2 and bound strongly to TLR2. Addition of 1 and 10 mM calcium to these pectins reduced TLR2 inhibition and TLR2 binding. Our study shows that calcium reduces inhibition of TLR2 by low and intermediate DM pectins, but calcium has lower impact on TLR2 inhibition by high DM pectins. Calcium may therefore beneficially influence the impact of pectin on TLR2 signalling and contribute to an improved intestinal barrier function. A combined higher intake of pectin and calcium may therefore contribute to a lower incidence of Western diseases.</p

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

Revealing methyl-esterification patterns of pectins by enzymatic fingerprinting:Beyond the degree of blockiness

Citrus pectins were studied by enzymatic fingerprinting using a simultaneous enzyme treatment with endo-polygalacturonase (endo-PG) from Kluyveromyces fragilis and pectin lyase (PL) from Aspergillus niger to reveal the methyl-ester distribution patterns over the pectin backbone. Using HILIC-MS combined with HPAEC enabled the separation and identification of the diagnostic oligomers released. Structural information on the pectins was provided by using novel descriptive parameters such as degree of blockiness of methyl-esterified oligomers by PG (DBPGme) and degree of blockiness of methyl-esterified oligomers by PL (DBPLme). This approach enabled us to clearly differentiate citrus pectins with various methyl-esterification patterns. The simultaneous use of PG and PL showed additional information, which is not revealed in digests using PG or PL alone. This approach can be valuable to differentiate pectins having the same DM and to get specific structural information on pectins and therefore to be able to better predict their physical and biochemical functionalities

2 '-Fucosyllactose impacts the expression of mucus-related genes in goblet cells and maintains barrier function of gut epithelial cells

Scope: Cost-effective microbial biosynthesis of 2′-fucosyllactose (2′-FL) allows its application in infant formula. The specific effects of 2′-FL on the gastrointestinal immune barrier are still largely unknown. Methods/results: Here, we quantified and compared the effects of HMOs isolated from human milk, 2′-FL/lactose (Lac), and 2′-FL on the expression of the mucus associated genes MUC2, TFF3, RETLB and the Golgi sulfotransferases, CHST5, and GAL3ST5, in human goblet cells. We also determined whether these compounds have protective effects on A23187-induced barrier disruption of human T84 gut epithelial cells in vitro. The impact of isolated HMOs and 2′-FL/Lac on the mRNA expression of the mucus-related genes was minor while pure 2′-FL significantly induced GAL3ST2 and CHST5. Isolated HMOs, 2′-FL/Lac and 2′-FL all prevented A23187-induced barrier disruption in human T84 cells. Conclusion: Our findings indicate that 2′-FL modulates the secretory function of goblet cells and protects gut epithelial cells

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

Distinct fermentation of human milk oligosaccharides 3-FL and LNT2 and GOS/inulin by infant gut microbiota and impact on adhesion of Lactobacillus plantarum WCFS1 to gut epithelial cells

Human milk oligosaccharides (hMOs) are unique bioactive components in human milk. 3-Fucosyllactose (3-FL) is an abundantly present hMO that can be produced in sufficient amounts to allow application in infant formula. Lacto-N-triaose II (LNT2) can be obtained by acid hydrolysis of lacto-N-neotetraose (LNnT). Both 3-FL and LNT2 have been shown to have health benefits, but their impact on infant microbiota composition and microbial metabolic products such as short-chain fatty acids (SCFAs) is unknown. To gain more insight in fermentability, we performed in vitro fermentation studies of 3-FL and LNT2 using pooled fecal microbiota from 12-week-old infants. The commonly investigated galacto-oligosaccharides (GOS)/inulin (9 : 1) served as control. Compared to GOS/inulin, we observed a delayed utilization of 3-FL, which was utilized at 60.3% after 36 h of fermentation, and induced the gradual production of acetic acid and lactic acid. 3-FL specifically enriched bacteria of Bacteroides and Enterococcus genus. LNT2 was fermented much faster. After 14 h of fermentation, 90.1% was already utilized, and production of acetic acid, succinic acid, lactic acid and butyric acid was observed. LNT2 specifically increased the abundance of Collinsella, as well as Bifidobacterium. The GOS present in the GOS/inulin mixture was completely fermented after 14 h, while for inulin, only low DP was rapidly utilized after 14 h. To determine whether the fermentation might lead to enhanced colonization of commensal bacteria to gut epithelial cells, we investigated adhesion of the commensal Lactobacillus plantarum WCFS1 to Caco-2 cells. The fermentation digesta of LNT2 collected after 14 h, 24 h, and 36 h, and GOS/inulin after 24 h of fermentation significantly increased the adhesion of L. plantarum WCFS1 to Caco-2 cells, while 3-FL had no such effect. Our findings illustrate that fermentation of hMOs is very structure-dependent and different from the commonly applied GOS/inulin, which might lead to differential potencies to stimulate adhesion of commensal cells to gut epithelium and consequent microbial colonization. This knowledge might contribute to the design of tailored infant formulas containing specific hMO molecules to meet the need of infants during the transition from breastfeeding to formula

Pectins that Structurally Differ in the Distribution of Methyl-Esters Attenuate Citrobacter rodentium-Induced Colitis

Introduction: Pectins have anti-inflammatory properties on intestinal immunity through direct interactions on Toll-like receptors (TLRs) in the small intestine or via stimulating microbiota-dependent effects in the large intestine. Both the degree of methyl-esterification (DM) and the distribution of methyl-esters (degree of blockiness; DB) of pectins contribute to this influence on immunity, but whether and how the DB impacts immunity through microbiota-dependent effects in the large intestine is unknown. Therefore, this study tests pectins that structurally differ in DB in a mouse model with Citrobacter rodentium induced colitis and studies the impact on the intestinal microbiota composition and associated attenuation of inflammation. Methods and Results: Both low and high DB pectins induce a more rich and diverse microbiota composition. These pectins also lower the bacterial load of C. rodentium in cecal digesta. Through these effects, both low and high DB pectins attenuate C. rodentium induced colitis resulting in reduced intestinal damage, reduced numbers of Th1-cells, which are increased in case of C. rodentium induced colitis, and reduced levels of GATA3+ Tregs, which are related to tissue inflammation. Conclusion: Pectins prevent C. rodentium induced colonic inflammation by lowering the C. rodentium load in the caecum independently of the DB

TLR 2/1 interaction of pectin depends on its chemical structure and conformation

Citrus pectins have demonstrated health benefits through direct interaction with Toll-like receptor 2. Methyl-ester distribution patterns over the homogalacturonan were found to contribute to such immunomodulatory activity, therefore molecular interactions with TLR2 were studied. Molecular-docking analysis was performed using four GalA-heptamers, GalA7Me0, GalA7Me1,6, GalA7Me1,7 and GalA7Me2,5. The molecular relations were measured in various possible conformations. Furthermore, commercial citrus pectins were characterized by enzymatic fingerprinting using polygalacturonase and pectin-lyase to determine their methyl-ester distribution patterns. The response of 12 structurally different pectic polymers on TLR2 binding and the molecular docking with four pectic oligomers clearly demonstrated interactions with human-TLR2 in a structure-dependent way, where blocks of (non)methyl-esterified GalA were shown to inhibit TLR2/1 dimerization. Our results may be used to understand the immunomodulatory effects of certain pectins via TLR2. Knowledge of how pectins with certain methyl-ester distribution patterns bind to TLRs may lead to tailored pectins to prevent inflammation.</p