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.

Clostridioides difficile toxin A-mediated Caco-2 cell barrier damage was attenuated by insect-derived fractions and corresponded to increased gene transcription of cell junctional and proliferation proteins.

Pathogenesis of C. difficile in the intestine is associated with the secretion of toxins which can damage the intestinal epithelial layer and result in diseases such as diarrhoea. Treatment for C. difficile infections consists of antibiotics which, however, have non-specific microbiocidal effects and may cause intestinal dysbiosis which results in subsequent health issues. Therefore, alternative treatments to C. difficile infections are required. We investigated whether different black soldier fly- and mealworm-derived fractions, after applying the INFOGEST digestion protocol, could counteract C. difficile toxin A-mediated barrier damage of small intestinal Caco-2 cells. Treatment and pre-treatment with insect-derived fractions significantly (p < 0.05) mitigated the decrease of the transepithelial electrical resistance (TEER) of Caco-2 cells induced by C. difficile toxin A. In relation to these effects, RNA sequencing data showed an increased transcription of cell junctional and proliferation protein genes in Caco-2 cells. Furthermore, the transcription of genes regulating immune signalling was also increased. To identify whether this resulted in immune activation we used a Caco-2/THP-1 co-culture model where the cells were only separated by a permeable membrane. However, the insect-derived fractions did not change the basolateral secreted IL-8 levels in this model. To conclude, our findings suggest that black soldier fly- and mealworm-derived fractions can attenuate C. difficile induced intestinal barrier disruption and they might be promising tools to reduce the symptoms of C. difficile infections.

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.

In Vitro Studies Toward the Use of Chitin as Nutraceutical: Impact on the Intestinal Epithelium, Macrophages, and Microbiota.

SCOPE: Chitin, the most abundant polysaccharide found in nature after cellulose, is known for its ability to support wound healing and to lower plasma-oxidized low-density lipoprotein (LDL) levels. Studies have also revealed immunomodulatory potential but contradicting results are often impossible to coalesce through usage of chitin of different or unknown physicochemical consistency. In addition, only a limited set of cellular models have been used to test the bioactivity of chitin. METHODS AND RESULTS: Chitin is investigated with well-defined physicochemical consistency for its immunomodulatory potency using THP-1 macrophages, impact on intestinal epithelial barrier using Caco-2 cells, and fermentation by fecal-derived microbiota. Results show that chitin with a degree of acetylation (DA) of ≈83%, regardless of size, does not affect the intestinal epithelial barrier integrity. Large-sized chitin significantly increases acetic acid production by gut microbiota without altering the composition. Exposure of small-sized chitin to THP-1 macrophages lead to significantly increased secretion of IL-1ß, IL-8, IL-10, and CXCL10 in a multi-receptor and clathrin-mediated endocytosis dependent manner. CONCLUSIONS: These findings indicate that small-sized chitin does not harm the intestinal barrier nor affects SCFA secretion and microbiota composition, but does impact immune activity which could be beneficial to subjects in need of immune support or activation.

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.

Heat treatment of ß-lactoglobulin affects its digestion and translocation in the upper digestive tract.

Heat treatment is a commonly applied unit operation in the processing of ß-lactoglobulin containing products. This does, however, influence its structure and thereby impacts its activity and digestibility. We describe how various heat-treatments of ß-lactoglobulin change the digestibility using a modified version of the current consensus INFOGEST protocol. Additionally, protein was investigated for its translocation over the intestinal epithelial barrier, which would bring them in contact with immune cells. The extent of gastric digestibility was higher when the protein structure was more modified, while the influence of glycation with lactose was limited. Translocation studies of protein across Caco-2 cell monolayers showed a lower translocation rate of protein heated in solution compared to the others. Our study indicates that structural modifications after different heat-treatments of ß-lactoglobulin increase in particular gastric digestibility and the translocation efficiency across intestinal epithelial cells.

Release of Major Peanut Allergens from Their Matrix under Various pH and Simulated Saliva Conditions-Ara h2 and Ara h6 Are Readily Bio-Accessible.

The oral mucosa is the first immune tissue that encounters allergens upon ingestion of food. We hypothesized that the bio-accessibility of allergens at this stage may be a key determinant for sensitization. Light roasted peanut flour was suspended at various pH in buffers mimicking saliva. Protein concentrations and allergens profiles were determined in the supernatants. Peanut protein solubility was poor in the pH range between 3 and 6, while at a low pH (1.5) and at moderately high pHs (>8), it increased. In the pH range of saliva, between 6.5 and 8.5, the allergens Ara h2 and Ara h6 were readily released, whereas Ara h1 and Ara h3 were poorly released. Increasing the pH from 6.5 to 8.5 slightly increased the release of Ara h1 and Ara h3, but the recovery remained low (approximately 20%) compared to that of Ara h2 and Ara h6 (approximately 100% and 65%, respectively). This remarkable difference in the extraction kinetics suggests that Ara h2 and Ara h6 are the first allergens an individual is exposed to upon ingestion of peanut-containing food. We conclude that the peanut allergens Ara h2 and Ara h6 are quickly bio-accessible in the mouth, potentially explaining their extraordinary allergenicity.

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.

Daffodil flowers delay senescence in cut Iris flowers.

Visible symptoms of tepal senescence in cut Iris x hollandica (cv. Blue Magic) flowers were delayed by placing one cut daffodil flower (Narcissus pseudonarcissus, cv. Carlton) in the same vase. Addition of mucilage, exuded by daffodil stems, to the vase water had the same effect as the flowering daffodil stem. The active compound in the mucilage was identified as narciclasine (using LC/MS, GC/MS, 1H and 13C-NMR, and comparison with an authentic sample of narciclasine). The delay of senescence, either by mucilage or purified narciclasine, was correlated with a delayed increase in protease activity, and with a considerable reduction of maximum protease activity. Narciclasine did not affect in vitro protease activity, but is known to inhibit protein synthesis at the ribosomal level. Its effects on senescence and protease activity were similar to those of cycloheximide (CHX), another inhibitor of protein synthesis, but the effective narciclasine concentration was about 100 times lower than that of CHX. It is concluded that the delay of Iris tepal senescence by daffodil stems is due to narciclasine in daffodil mucilage, which apparently inhibits the synthesis of proteins involved in senescence.