Rice-derived arabinoxylan fibers are particle size-dependent inducers of trained immunity in a human macrophage-intestinal epithelial cell co-culture model.

Arabinoxylans have been identified for a wide range of purported health-promoting applications, primarily attributed to its immunomodulatory effects. Previously, we have reported the ability of arabinoxylans to induce non-specific memory in innate immune cells, commonly referred to as "trained innate immunity". In the present study, we investigated the effect of particle size on innate immune training and resilience in primary human macrophages as well as in a more physiologically relevant macrophage-intestinal epithelial cell co-culture model. We demonstrated that smaller (>45 & < 90 µm) compared to larger (>90 µm) particle size fractions of rice bran-derived arabinoxylan preparations have a higher enhancing effect on training and resilience in both models. Smaller particle size fractions elevated TNF-α production in primary macrophages and enhanced Dectin-1 receptor activation in reporter cell lines compared to larger particles. Responses were arabinoxylan source specific as only the rice-derived arabinoxylans showed these immune-supportive effects. This particle size-dependent induction of trained immunity was confirmed in the established co-culture model. These findings demonstrate the influence of particle size on the immunomodulatory potential of arabinoxylans, provide further insight into the structure-activity relationship, and offer new opportunities to optimize the immune-enhancing effects of these dietary fibers.

Dietary fat induced chylomicron-mediated LPS translocation in a bicameral Caco-2cell model.

BACKGROUND: There is increasing evidence that dietary fat, especially saturated fat, promotes the translocation of lipopolysaccharide (LPS) via chylomicron production in the gut. Chylomicrons can subsequently transport LPS to other parts of the body, where they can induce low-grade chronic inflammation that is linked to various metabolic and gut-related diseases. To identify promising (food) compounds that can prevent or ameliorate LPS-related low-grade inflammation, we developed and optimized a bicameral in vitro model for dietary fat-induced LPS translocation that closely mimics the in vivo situation and facilitates high-throughput screening. METHODS: Caco-2 cells were cultured in monolayers and differentiated to a small intestinal phenotype in 21 days. Thereafter, optimal conditions for fat-induced chylomicron production were determined by apical exposure of Caco-2 cells to a dilution range of in vitro digested palm oil and sunflower oil, optionally preceded by a 1-week apical FBS deprivation (cultured without apical fetal bovine serum). Chylomicron production was assessed by measuring basolateral levels of the chylomicron-related marker apolipoprotein B. Next, LPS was coincubated at various concentrations with the digested oils, and fat-induced LPS translocation to the basolateral side was assessed. RESULTS: We found that dietary fat-induced LPS translocation in Caco-2 cells was optimal after apical exposure to digested oils at a 1:50 dilution in combination with 750 ng/mL LPS, preceded by 1 week of apical FBS deprivation. Coincubation with the chylomicron blocker Pluronic L81 confirmed that fat-induced LPS translocation is mediated via chylomicron production in this Caco-2 cell model. CONCLUSION: We developed a robust Caco-2 cell model for dietary fat-induced LPS translocation that can be used for high-throughput screening of (food) compounds that can reduce LPS-related low-grade inflammation.

Inhibition of α-glucosidases by tea polyphenols in rat intestinal extract and Caco-2 cells grown on Transwell.

Inhibition of maltase, sucrase, isomaltase and glucoamylase activity by acarbose, epigallocatechin gallate, epicatechin gallate and four polyphenol-rich tea extract from white, green, oolong, black tea, were investigated by using rat intestinal enzymes and human Caco-2 cells. Regarding rat intestinal enzyme mixture, all four tea extracts were very effective in inhibiting maltase and glucoamylase activity, but only white tea extract inhibited sucrase and isomaltase activity and the inhibition was limited. Mixed-type inhibition on rat maltase activity was observed. Tea extracts in combination with acarbose, produced a synergistic inhibitory effect on rat maltase activity. Caco-2 cells experiments were conducted in Transwells. Green tea extract and epigallocatechin gallate show dose-dependent inhibition on human sucrase activity, but no inhibition on rat sucrase activity. The opposite was observed on maltase activity. The results highlighted the different response in the two investigated model systems and show that tea polyphenols are good inhibitors for α-glucosidase activity.

A combined microphysiological-computational omics approach in dietary protein evaluation.

Food security is under increased pressure due to the ever-growing world population. To tackle this, alternative protein sources need to be evaluated for nutritional value, which requires information on digesta peptide composition in comparison to established protein sources and coupling to biological parameters. Here, a combined experimental and computational approach is presented, which compared seventeen protein sources with cow's whey protein concentrate (WPC) as the benchmark. In vitro digestion of proteins was followed by proteomics analysis and statistical model-based clustering. Information on digesta peptide composition resulted in 3 cluster groups, primarily driven by the peptide overlap with the benchmark protein WPC. Functional protein data was then incorporated in the computational model after evaluating the effects of eighteen protein digests on intestinal barrier integrity, viability, brush border enzyme activity, and immune parameters using a bioengineered intestine as microphysiological gut system. This resulted in 6 cluster groups. Biological clustering was driven by viability, brush border enzyme activity, and significant differences in immune parameters. Finally, a combination of proteomic and biological efficacy data resulted in 5 clusters groups, driven by a combination of digesta peptide composition and biological effects. The key finding of our holistic approach is that protein source (animal, plant or alternative derived) is not a driving force behind the delivery of bioactive peptides and their biological efficacy.

Novel Dietary Proteins Selectively Affect Intestinal Health In Vitro after Clostridium difficile-Secreted Toxin A Exposure.

Bacterial gastroenteritis forms a burden on a global scale, both socially and economically. The Gram-positive bacterium Clostridium difficile is an inducer of gastrointestinal bacterial infections, often triggered following disruption of the microbiota by broad-spectrum antibiotics to treat other conditions. The clinical manifestatiaons, e.g., diarrhea, are driven by its toxins secretion, toxin A (TcdA) and toxin B (TcdB). Current therapies are focused on discontinuing patient medication, including antibiotics. However, relapse rates upon therapy are high (20-25%). Here, eighteen dietary proteins were evaluated for their capacity to restore gut health upon C. difficile-derived TcdA exposure. We used bioengineered intestinal tubules to assess proteins for their beneficial effects by examining the epithelial barrier, cell viability, brush-border enzyme activity, IL-6 secretion, IL-8 secretion and nitric oxide (NO) levels upon TcdA challenge. TcdA effectively disrupted the epithelial barrier, increased mitochondrial activity, but did not affect alkaline phosphatase activity, IL-6, IL-8 and NO levels. Intervention with dietary proteins did not show a protective effect on epithelial barrier integrity or mitochondrial activity. However, bovine plasma and potato protein increased alkaline phosphatase activity, egg-white protein increased IL-6 and IL-8 release and wheat, lesser mealworm and yeast protein increased NO levels after TcdA exposure. Hence, dietary proteins can influence parameters involved in intestinal physiology and immune activation suggesting that supplementation with specific dietary proteins may be of benefit during C. difficile infections.

Peptide Release after Simulated Infant In Vitro Digestion of Dry Heated Cow's Milk Protein and Transport of Potentially Immunoreactive Peptides across the Caco-2 Cell Monolayer.

Dry heating of cow's milk protein, as applied in the production of "baked milk", facilitates the resolution of cow's milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow's milk protein powder was heated at low temperature (60 °C) and high temperature (130 °C) and applied to simulated infant in vitro digestion. Digestion-derived peptides after 10 min and 60 min in the intestinal phase were measured using LC-MS/MS. Moreover, digests after 10 min intestinal digestion were applied to a Caco-2 cell monolayer. T-cell epitopes were analysed using prediction software, while specific immunoglobin E (sIgE) binding epitopes were identified based on the existing literature. The largest number of sIgE binding epitopes was found in unheated samples, while T-cell epitopes were equally represented in all samples. Transport of glycated peptide indicated a preference for glucosyl lysine and lactosyl-lysine-modified peptides, while transport of peptides containing epitope structures was limited. This showed that the release of immunoreactive peptides can be affected by the applied heating conditions; however, availability of peptides containing epitopes might be limited.

Investigating the transport dynamics of anthocyanins from unprocessed fruit and processed fruit juice from sour cherry (Prunus cerasus L.) across intestinal epithelial cells.

Anthocyanins can contribute to human health through preventing a variety of diseases. The uptake of these compounds from food and the parameters determining uptake efficiency within the human body are still poorly understood. Here we have employed a Caco-2 cell based system to investigate the transport of key antioxidant food components from sour cherry (Prunus cerasus L.) across the intestinal epithelial barrier. Anthocyanins and (-)-epicatechin were supplied in three contrasting matrices: fruit, processed fruit cherry juice, and polyphenolic fractions obtained by solid-phase extraction. Results show that both compound types behave differently. Fruit or juice matrices display comparable transport across the epithelial cell layer. The juice supplements sucrose and citric acid, which are regularly added to processed foods, have a positive effect on stability and transport. Polyphenolic fractions display a lower transport efficiency, relative to that of the fruit or juice, indicating the importance of food matrix components for intestinal absorption of polyphenols.

Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation.

An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.