Intestinal Epithelial Caco-2 Cells Research Articles

Protective effect of Limosilactobacillus reuteri-fermented yogurt on mouse intestinal barrier injury induced by enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a pathogen that causes traveler's diarrhea, for which an effective vaccine is lacking. Previous studies showed that Limosilactobacillus reuteri could inhibit E. coli, effectively increase the expression of its tight junction protein, and reduce the adhesion of ETEC to the intestinal epithelial Caco-2 cell line. In this study, three kinds of yogurt with different starter cultures were first prepared: Lm. reuteri yogurt (fermented by Lm. reuteri alone), traditional yogurt (fermented by Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus at a ratio of 1:1) and mixed yogurt (fermented by Lm. reuteri, S. thermophilus and L. delbrueckii subsp. bulgaricus at a ratio of 1:1:1). The physiological properties, oxidative stress, intestinal barrier function, tight junction protein, pathological conditions and intestinal microbiota composition were investigated. The data showed that Lm. reuteri-fermented yogurt pregavage could effectively alleviate the intestinal barrier impairment caused by ETEC in mice. It alleviated intestinal villus shortening and inflammatory cell infiltration, decreased plasma diamine oxidase concentration and increased claudin-1 and occludin expression in the jejunum of ETEC-infected mice. In addition, Lm. reuteri-fermented yogurt significantly reduced the ETEC load in fecal samples, reversed the increase in Pseudomonadota abundance and decreased Bacteroidota abundance caused by ETEC infection. Furthermore, the composition of the intestinal microbiota could maintain a stable state similar to that in healthy mice. These findings indicate that Lm. reuteri-fermented yogurt could alleviate intestinal barrier damage, inhibit ETEC growth and maintain the stability of the intestinal microbiota during ETEC infection. © 2023 Society of Chemical Industry.

L-Alanine promotes anti-infectious properties of Bacillus subtilis S-2 spores via the germination receptor gerAA.

Bacillus species, which have two cell-type forms (vegetative cells and spores), demonstrate a variety of probiotic functions in animal feed additives and human nutrition. We previously found that the probiotic effect of Bacillus subtilis S-2 spores with high germination response to L-alanine was specifically enhanced by the L-alanine pretreatment. The germination response of Bacillus is highly associated with the germination receptors of spores. However, how L-alanine-induced germination of spores exerts anti-infectious effect in epithelial cells remains unclear. In this study, we constructed the mutant strain of B. subtilis S-2 with germination receptor gerAA knockout to further explore the role of spore germination in resisting pathogen infection to cells. The differential probiotic effects of B. subtilis S-2 and S-2ΔgerAA spores pretreated with L-alanine were evaluated in intestinal porcine epithelial cells (IPEC-J2) or Caco2 cells infected with enterotoxigenic Escherichia coli (ETEC) or following IL-1β stimulation. The results showed that the germination response of the S-2ΔgerAA spores to L-alanine was significantly reduced. Compared with the S-2ΔgerAA spores, the L-alanine-induced germination of B. subtilis S-2 spores significantly increased the activity of anti-adhesion of ETEC to IPEC-J2 cells and reduced the expression of inflammatory factors and cell receptors. L-alanine induction also significantly promoted the expression of autophagy-related proteins in the B. subtilis S-2 spores. These findings demonstrate that the gerAA germination receptor is essential for the probiotic function of Bacillus spores and that L-alanine treatment promotes the anti-infectious properties of the germinated spores in porcine intestinal epithelial IPEC-J2 cells. The result suggests the importance of germination receptor gerAA in helping spore germination and enhancing anti-infectious activity. The findings in the study benefit to screening of potential Bacillus probiotics and increasing probiotic efficacy induced by L-alanine as an adjuvant.

Maternal Epidermal Growth Factor Promotes Neonatal Claudin-2 Dependent Increases in Small Intestinal Calcium Permeability.

A higher concentration of calcium in breast milk than blood favors paracellular calcium absorption enabling growth during postnatal development. We aimed to determine whether suckling animals have greater intestinal calcium permeability to maximize absorption and to identify the underlying molecular mechanism. We examined intestinal claudin expression at different ages in mice and in human intestinal epithelial (Caco-2) cells in response to hormones or human milk. We also measured intestinal calcium permeability in wildtype, Cldn2 and Cldn12 KO mice and Caco-2 cells in response to hormones or human milk. Bone mineralization in mice was assessed by μCT. Calcium permeability across the jejunum and ileum of mice were 2-fold greater at 2 wk than 2 mo postnatal age. At 2 wk, Cldn2 and Cldn12 expression were greater, but only Cldn2 KO mice had decreased calcium permeability compared to wildtype. This translated to decreased bone volume, cross-sectional thickness, and tissue mineral density of femurs. Weaning from breast milk led to a 50% decrease in Cldn2 expression in the jejunum and ileum. Epidermal growth factor (EGF) in breast milk specifically increased only CLDN2 expression and calcium permeability in Caco-2 cells. These data support intestinal permeability to calcium, conferred by claudin-2, being greater in suckling mice and being driven by EGF in breast milk. Loss of the CLDN2 pathway leads to suboptimal bone mineralization at 2 wk of life. Overall, EGF-mediated control of intestinal claudin-2 expression contributes to maximal intestinal calcium absorption in suckling animals.

Metabolism of Acetaminophen by Enteric Epithelial Cells Mitigates Hepatocellular Toxicity In Vitro.

The gut-liver axis is defined by dietary and environmental communication between the gut, microbiome and the liver with its redox and immune systems, the overactivation of which can lead to hepatic injury. We used media preconditioning to mimic some aspects of the enterohepatic circulation by treating the human Caco-2 intestinal epithelial cell line with 5, 10 and 20 mM paracetamol (N-acetyl-para-aminophenol; APAP) for 24 h, after which cell culture supernatants were transferred to differentiated human hepatic HepaRG cells for a further 24 h. Cell viability was assessed by mitochondrial function and ATP production, while membrane integrity was monitored by cellular-based impedance. Metabolism by Caco-2 cells was determined by liquid chromatography with tandem mass spectrometry. Caco-2 cell viability was not affected by APAP, while cell membrane integrity and tight junctions were maintained and became tighter with increasing APAP concentrations, suggesting a reduction in the permeability of the intestinal epithelium. During 24 h incubation, Caco-2 cells metabolised 64-68% of APAP, leaving 32-36% of intact starting compound to be transferred to HepaRG cells. When cultured with Caco-2-preconditioned medium, HepaRG cells also showed no loss of cell viability or membrane integrity, completely in contrast to direct treatment with APAP, which resulted in a rapid loss of cell viability and membrane integrity and, ultimately, cell death. Thus, the pre-metabolism of APAP could mitigate previously observed hepatotoxicity to hepatic tight junctions caused by direct exposure to APAP. These observations could have important implications for the direct exposure of hepatic parenchyma to APAP, administered via the intravenous route.

Optimizing THP-1 Macrophage Culture for an Immune-Responsive Human Intestinal Model.

Previously established immune-responsive co-culture models with macrophages have limitations due to the dedifferentiation of macrophages in long-term cultures. This study is the first report of a long-term (21-day) triple co-culture of THP-1 macrophages (THP-1m) with Caco-2 intestinal epithelial cells and HT-29-methotrexate (MTX) goblet cells. We demonstrated that high-density seeded THP-1 cells treated with 100 ng/mL phorbol 12-myristate 13-acetate for 48 h differentiated stably and could be cultured for up to 21 days. THP-1m were identified by their adherent morphology and lysosome expansion. In the triple co-culture immune-responsive model, cytokine secretions during lipopolysaccharide-induced inflammation were confirmed. Tumor necrosis factor-alpha and interleukin 6 levels were elevated in the inflamed state, reaching 824.7 ± 130.0 pg/mL and 609.7 ± 139.5 pg/mL, respectively. Intestinal membrane integrity was maintained with a transepithelial electrical resistance value of 336.4 ± 18.0 Ω·cm2. Overall, our findings suggest that THP-1m can be effectively employed in models of long-term immune responses in both normal and chronic inflammatory states of the intestinal epithelium, making them a valuable tool for future research on the association between the immune system and gut health.

Bacillus cereus extracellular vesicles act as shuttles for biologically active multicomponent enterotoxins

BackgroundExtracellular vesicles (EVs) from Gram-positive bacteria have gained considerable importance as a novel transport system of virulence factors in host–pathogen interactions. Bacillus cereus is a Gram-positive human pathogen, causing gastrointestinal toxemia as well as local and systemic infections. The pathogenicity of enteropathogenic B. cereus has been linked to a collection of virulence factors and exotoxins. Nevertheless, the exact mechanism of virulence factor secretion and delivery to target cells is poorly understood.ResultsHere, we investigate the production and characterization of enterotoxin-associated EVs from the enteropathogenic B. cereus strain NVH0075-95 by using a proteomics approach and studied their interaction with human host cells in vitro. For the first time, comprehensive analyses of B. cereus EV proteins revealed virulence-associated factors, such as sphingomyelinase, phospholipase C, and the three-component enterotoxin Nhe. The detection of Nhe subunits was confirmed by immunoblotting, showing that the low abundant subunit NheC was exclusively detected in EVs as compared to vesicle-free supernatant. Cholesterol-dependent fusion and predominantly dynamin-mediated endocytosis of B. cereus EVs with the plasma membrane of intestinal epithelial Caco2 cells represent entry routes for delivery of Nhe components to host cells, which was assessed by confocal microscopy and finally led to delayed cytotoxicity. Furthermore, we could show that B. cereus EVs elicit an inflammatory response in human monocytes and contribute to erythrocyte lysis via a cooperative interaction of enterotoxin Nhe and sphingomyelinase.ConclusionOur results provide insights into the interaction of EVs from B. cereus with human host cells and add a new layer of complexity to our understanding of multicomponent enterotoxin assembly, offering new opportunities to decipher molecular processes involved in disease development.3ynbExxhMbqkgCYjDtonVSVideo

Bacterial Lysate from the Multi-Strain Probiotic SLAB51 Triggers Adaptative Responses to Hypoxia in Human Caco-2 Intestinal Epithelial Cells under Normoxic Conditions and Attenuates LPS-Induced Inflammatory Response.

Hypoxia-inducible factor-1α (HIF-1α), a central player in maintaining gut-microbiota homeostasis, plays a pivotal role in inducing adaptive mechanisms to hypoxia and is negatively regulated by prolyl hydroxylase 2 (PHD2). HIF-1α is stabilized through PI3K/AKT signaling regardless of oxygen levels. Considering the crucial role of the HIF pathway in intestinal mucosal physiology and its relationships with gut microbiota, this study aimed to evaluate the ability of the lysate from the multi-strain probiotic formulation SLAB51 to affect the HIF pathway in a model of in vitro human intestinal epithelium (intestinal epithelial cells, IECs) and to protect from lipopolysaccharide (LPS) challenge. The exposure of IECs to SLAB51 lysate under normoxic conditions led to a dose-dependent increase in HIF-1α protein levels, which was associated with higher glycolytic metabolism and L-lactate production. Probiotic lysate significantly reduced PHD2 levels and HIF-1α hydroxylation, thus leading to HIF-1α stabilization. The ability of SLAB51 lysate to increase HIF-1α levels was also associated with the activation of the PI3K/AKT pathway and with the inhibition of NF-κB, nitric oxide synthase 2 (NOS2), and IL-1β increase elicited by LPS treatment. Our results suggest that the probiotic treatment, by stabilizing HIF-1α, can protect from an LPS-induced inflammatory response through a mechanism involving PI3K/AKT signaling.

Development of an Inflammation-Triggered In Vitro "Leaky Gut" Model Using Caco-2/HT29-MTX-E12 Combined with Macrophage-like THP-1 Cells or Primary Human-Derived Macrophages.

The "leaky gut" syndrome describes a damaged (leaky) intestinal mucosa and is considered a serious contributor to numerous chronic diseases. Chronic inflammatory bowel diseases (IBD) are particularly associated with the "leaky gut" syndrome, but also allergies, autoimmune diseases or neurological disorders. We developed a complex in vitro inflammation-triggered triple-culture model using 21-day-differentiated human intestinal Caco-2 epithelial cells and HT29-MTX-E12 mucus-producing goblet cells (90:10 ratio) in close contact with differentiated human macrophage-like THP-1 cells or primary monocyte-derived macrophages from human peripheral blood. Upon an inflammatory stimulus, the characteristics of a "leaky gut" became evident: a significant loss of intestinal cell integrity in terms of decreased transepithelial/transendothelial electrical resistance (TEER), as well as a loss of tight junction proteins. The cell permeability for FITC-dextran 4 kDa was then increased, and key pro-inflammatory cytokines, including TNF-alpha and IL-6, were substantially released. Whereas in the M1 macrophage-like THP-1 co-culture model, we could not detect the release of IL-23, which plays a crucial regulatory role in IBD, this cytokine was clearly detected when using primary human M1 macrophages instead. In conclusion, we provide an advanced human in vitro model that could be useful for screening and evaluating therapeutic drugs for IBD treatment, including potential IL-23 inhibitors.

Bioavailability of Citrulline in Watermelon Flesh, Rind, and Skin Using a Human Intestinal Epithelial Caco-2 Cell Model

Watermelon produces many byproducts (watermelon rind and skin) even though those components contain various bioactive compounds, including citrulline. This study evaluated the citrulline concentration, total phenolic content, and antioxidant activity (DPPH and FRAP assays) of different parts of watermelon and investigated the bioavailability of citrulline from different parts of watermelon using an in vitro human intestinal epithelial Caco-2 cell monolayer model. Solid-phase extracted watermelon flesh, rind, and skin samples were treated on a Caco-2 cell monolayer for 1, 2, and 4 h. The collected basolateral solution at each time point was analyzed for the percentage of citrulline transport. Watermelon flesh had the highest citrulline content, but the watermelon skin had the highest total phenolic content and antioxidant activity compared to other watermelon parts. The citrulline bioavailability showed greater % transport in watermelon skin than in watermelon flesh, rind, and L-citrulline standard. It may be due to the different food matrices of watermelon parts. This suggests that the utilization of watermelon by-products such as skin would help develop value-added products with better bioavailability of citrulline. However, since this study was conducted with an in vitro cell model, more extensive research with in vivo studies will be needed.

Does flow culture impact upon gut-probiotic interactions: A comparison with static culture

Conventional tissue culture models often lack physiological relevance. In this exploratory study, dynamic flow conditions were applied to 21-day cultured Caco-2 intestinal epithelial cells and their response compared with statically cultured counterparts. Host:microbiome interactions were also explored through co-incubation with Gram positive bacteria (Lab4 probiotics) or the Gram positive cell wall component, lipoteichoic acid (LTA).Under flow, Caco-2 cells displayed increased viability, mucin production and pH tolerance compared to cells under static conditions. Measurement of secretion rates of the pro-inflammatory cytokine, interleukin-8, showed that cells under flow were unresponsive towards stimulation with Lab4 or LTA that was in complete contrast to inductions observed under static conditions. Given that Gram positive bacteria are well tolerated within the healthy gastrointestinal tract, these data suggest that flow conditions may improve the physiological relevance of Caco-2 cells. This preliminary study encourages further work exploring the impact of flow on cell-based in vitro models.

A human Caco-2-based co-culture model of the inflamed intestinal mucosa for particle toxicity studies

The intestinal barrier is a complex interface of the human body, possessing the largest contact surface to nutrients and antigens and containing a major part of the immune system. It has to deal with continuous exposure to a broad mixture of essential, harmful, or useless substances and particles. In the context of plastic pollution and the ubiquitous occurrence of micro- and nanoplastics, oral exposure to such particles is of particular interest. Standard intestinal in vitro models, however, are unable to mimic the role of the immune system in the particle-exposed intestine. To allow for a closer look on the effect of particles on the intestinal immune system, we here developed a co-culture model to enable investigation of the epithelial brush border monolayer in a healthy and inflamed state. The model is based on well-established Caco-2 intestinal epithelial cells cultured in a Transwell™ system. Intraepithelial immune cells were mimicked by THP-1-derived M0-macrophages and MUTZ-3-derived dendritic cells. To fulfill the requirements needed for the investigation of particles, the co-culture system was developed without an additional matrix layer. Cell–cell contacts were established between interstitial and immune cells, and the Caco-2 standard cell culture medium was used, which is well-characterized for its role in defining the identity of particle dispersions. The model was characterized using confocal microscopy, membrane integrity measurements, and cytokine release assays from inflamed and healthy cells. Finally, the new co-culture model was used for investigation on polylactic acid, melamine formaldehyde resin, and polymethylmethacrylate plastic micro- and nanoparticles.

Skatole-induced p38 and JNK activation coordinately upregulates, whereas AhR activation partially attenuates TNFα expression in intestinal epithelial cells

Increased tumor necrosis factor α (TNFα) expression in intestinal epithelial cells (IECs) plays a major role in the development and progression of inflammatory bowel disease (IBD) and colorectal cancer (CRC). The present study aimed to clarify the relationship between TNFα and skatole, a tryptophan-derived gut microbiota metabolite. The aryl hydrocarbon receptor (AhR) antagonist CH223191 promoted, whereas the p38 inhibitor SB203580 suppressed the increase in TNFα mRNA and protein expression induced by skatole in intestinal epithelial Caco-2 cells. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 repressed only the increased TNFα protein expression, whereas the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 did not affect increased TNFα expression at any level. A neutralizing antibody against TNFα partially inhibited skatole-induced cell death. Overall, these results suggested that TNFα expression is increased by the concerted actions of skatole-activated p38 and JNK, and that TNFα exerts autocrine/paracrine actions on IECs despite partial suppression by activated AhR. Therefore, skatole might play an important role in the development and progression of IBD and CRC via increased TNFα expression.

Lactic acid bacteria strains reduce in vitro mercury toxicity on the intestinal mucosa.

A bicameral model consisting of Caco-2 and HT29-MTX intestinal epithelial cells and THP-1-derived macrophages has been used to test the ability of two strains of Lactobacillus to protect from damage caused by mercury. Exposure to 1 mg/ml mercury [Hg(II) or methyl-Hg] for seven days in this model resulted in an inflammatory and pro-oxidant response mainly driven by macrophages. This led to an impairment in the intestinal barrier, defective tight-junctions, increased permeability and mucus hypersecretion. In addition, the wound-healing capacity of the epithelial monolayer was also diminished. However, the presence of heat-killed Lactobacillus intestinalis or Lactobacillus johnsonii cells during Hg exposure reverted these effects, and most of the parameters recovered values similar to control cells. Both lactobacilli showed the capacity to bind Hg(II) and methyl-Hg under the cell culture conditions. This points to Hg sequestration as a likely mechanism that counteracted Hg toxicity. However, differences in the Hg binding capacity and in the effects between both strains suggest that other probiotic-mediated mechanisms may play a role in the alleviation of the damage elicited by Hg. These results show the potential of the bicameral intestinal epithelial model for screening of effective strains for their use in later in vivo studies.

Exopolysaccharide-producing Lacticaseibacillus paracasei strains isolated from kefir as starter for functional dairy products.

Exopolysaccharides (EPS) produced by lactic acid bacteria are molecules of great interest for the dairy food industry. Lacticaseibacillus paracasei CIDCA 8339, CIDCA 83123, and CIDCA 83124 are potentially probiotic strains isolated from kefir grains whose EPS-production on MRS broth is dependent on incubation temperature. The aim of the present work is to evaluate the effect of fermentation temperature on the characteristics of EPS produced in milk by L. paracasei strains and the consequent impact on the rheological properties of the fermented products. Additionally, the protective effect of these EPS against Salmonella infection was evaluated in vitro. Acid gels with each strain were obtained by milk fermentation at 20°C, 30°C, and 37°C evidencing for all the strains a reduction in growth and acidification rate at lower temperature. Lacticaseibacillus paracasei CIDCA 83123 showed low fermentation rate at all temperatures requiring between 3 and 8 days to obtain acids gels, whereas CIDCA 8339 and 83124 needed between 24 and 48 h even when the temperature was 20°C. Fermentation temperature led to changes in crude EPS characteristics of the three strains, observing an increase in the relative amount of the high molecular weight fraction when the fermentation temperature diminished. Additionally, EPS83124 and EPS83123 presented modifications in monosaccharide composition, with a reduction of rhamnose and an increase of amino-sugars as temperature rise. These changes in the structure of EPS83124 resulted in an increase of the apparent viscosity of milks fermented at 20°C (223 mPa.s) and 30°C (217 mPa.s) with respect to acid gels obtained at 37°C (167 mPa.s). In order to deepen the knowledge on EPS characteristics, monosaccharide composition of low and high molecular weight EPS fractions were evaluated. Finally, it was evidenced that the preincubation of intestinal epithelial cells Caco-2/TC-7 with EPS8339 and EPS83124 partially inhibit the association and invasion of Salmonella. In light of these results, it can be concluded that the selection of the EPS-producing strain along with the appropriate fermentation conditions could be an interesting strategy to improve the technological properties of these L. paracasei fermented milks with potential protective effects against intestinal pathogens.

Screening of heat stress-regulating active fractions in mung beans.

Heat stress caused by high temperatures has important adverse effects on the safety and health status of humans and animals, and dietary interventions to alleviate heat stress in daily life are highly feasible. In this study, the components of mung bean that have heat stress-regulating effects were characterized by in vitro antioxidant indicators and heat stress cell models. As a result, 15 target monomeric polyphenol fractions were identified based on untargeted analysis on an ultra performance liquid chromatography coupled with high field quadrupole orbit high resolution mass spectrometry (UHPLC-QE-HF-HRMS) platform and available reports. The results of DPPH and ABTS radical scavenging showed that mung bean polyphenols (crude extract) and 15 monomeric polyphenols had better antioxidant activity, followed by oil and mung bean peptides, while protein and polysaccharides had relatively poor antioxidant activity. Qualitative and quantitative assays for 20 polyphenols (15 polyphenols and 5 isomers) were then established based on platform targets. Vitexin, orientin, and caffeic acid were identified as monomeric polyphenols for heat stress control in mung beans based on their content. Finally, mild (39°C), moderate (41°C), and severe (43°C) heat stress models were successfully constructed based on mouse intestinal epithelial Mode-k cells and human colorectal adenocarcinoma Caco-2 cell lines, all with an optimal heat stress modeling time of 6 h. Screening of mung bean fractions using HSP70 mRNA content, a key indicator of heat stress. As a result, HSP70 mRNA content was significantly up-regulated by different levels of heat stress in both cell models. The addition of mung bean polyphenols (crude extract), vitexin, orientin, and caffeic acid resulted in significant down-regulation of HSP70 mRNA content, and the higher the level of heat stress, the more significant the regulation effect, with orientin having the best effect. Mung bean proteins, peptides, polysaccharides, oils and mung bean soup resulted in increased or no change in HSP70 mRNA levels after most heat stresses. The polyphenols were shown to be the main heat stress regulating components in mung bean. The results of the validation experiments confirm that the above three monomeric polyphenols may be the main heat stress regulating substances in mung bean. The role of polyphenols in the regulation of heat stress is closely linked to their antioxidant properties.

Akkermansia muciniphila improves heat stress-impaired intestinal barrier function by modulating HSP27 in Caco-2 cells

ObjectiveHeat stress causes an elevation of intestinal epithelial barrier permeability and leads to multiple organ dysfunction in heatstroke. Akkermansia muciniphila (A. muciniphila) plays a role in maintaining intestinal integrity and improving the inflammatory state. This study aimed to investigate whether A. muciniphila could alleviate heat stress-induced dysfunction of intestinal permeability in Caco-2 monolayers and have the preventive effects on heatstroke. MethodsHuman intestinal epithelial Caco-2 cells were preincubated with live or pasteurized A. muciniphila then exposed to heat stress at 43 °C. Transepithelial electrical resistance (TEER) and the flux of horseradish peroxidase (HRP) across cell monolayers were measured to determine intestinal permeability. The levels of the tight junction proteins Occludin, ZO-1 and HSP27 were analyzed by Western blotting. These proteins were immunostained and localized by fluorescence microscopy. TJ morphology was observed using transmission electron microscopy (TEM). ResultsBoth live and pasteurized A. muciniphila effectively attenuated the decrease in TEER and impairment of intestinal permeability in HRP flux induced by heat exposure. A. muciniphila significantly elevated the expression of Occludin and ZO-1 by promoting HSP27 phosphorylation. The distortion and redistribution of tight junction proteins and disruption of morphology were also effectively prevented by pretreatment with A. muciniphila. ConclusionThis study indicates for the first time that both live and pasteurized A. muciniphila play an important protective role against heat-induced permeability dysfunction and epithelial barrier damage.

In vitro-based prediction of human plasma concentrations of food-related compounds.

Efforts have been made to replace animal experiments in safety evaluations, including in vitro-based predictions of human internal exposures, such as predicting peak plasma concentration (Cmax) values for xenobiotics and comparing these values with in vitro-based toxicity endpoints. Herein, the authors predicted the Cmax values of food-related compounds in humans based on existing and novel in vitro techniques. In this study, 20 food-related compounds, which have been previously reported in human pharmacokinetic or toxicokinetic studies, were evaluated. Human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIEC) and Caco-2 cells, HepaRG cells, equilibrium dialysis of human plasma, and LLC-PK1 cell monolayer were used to assess intestinal absorption and availability, hepatic metabolism, unbound plasma fraction, and secretion and reabsorption in renal tubular cells, respectively. After conversion of these parameters into human kinetic parameters, the plasma concentration profiles of these compounds were predicted using in silico methods, and the obtained Cmax values were found to be between 0.017 and 183 times the reported Cmax values. When the in silico-predicted parameters were modified with in vitro data, the predicted Cmax values came within 0.1-10 times the reported values because the metabolic activities of hiPSC-SIECs, such as uridine 5’-diphospho-glucuronosyl transferase, are more similar to those of human primary enterocytes. Thus, combining in vitro test results with the plasma concentration simulations resulted in more accurate and transparent predictions of Cmax values of food-related compounds than those obtained using in silico-derived predictions alone. This method facilitates accurate safety evaluation without the need for animal experiments.

IPA and its precursors differently modulate the proliferation, differentiation, and integrity of intestinal epithelial cells.

Indole-3-propionic acid (IPA) is a tryptophan-derived microbial metabolite that has been associated with protective effects against inflammatory and metabolic diseases. However, there is a lack of knowledge regarding the effects of IPA under physiological conditions and at the intestinal level. Human intestinal epithelial Caco-2 cells were treated for 2, 24, and/or 72 h with IPA or its precursors - indole, tryptophan, and propionate - at 1, 10, 100, 250, or 500 μM to assess cell viability, integrity, differentiation, and proliferation. IPA induced cell proliferation and this effect was associated with a higher expression of extracellular signal-regulated kinase 2 (ERK2) and a lower expression of c-Jun. Although indole and propionate also induced cell proliferation, this involved ERK2 and c-Jun independent mechanisms. On the other hand, both tryptophan and propionate increased cell integrity and reduced the expression of claudin-1, whereas propionate decreased cell differentiation. In conclusion, these findings suggested that IPA and its precursors distinctly contribute to the proliferation, differentiation, and barrier function properties of human intestinal epithelial cells. Moreover, the pro-proliferative effect of IPA in intestinal epithelial cells was not explained by its precursors and is rather related to its whole chemical structure. Maintaining IPA at physiological levels, e.g., through IPA-producing commensal bacteria, may be important to preserve the integrity of the intestinal barrier and play an integral role in maintaining metabolic homeostasis.

Cytoskeletal Responses and Aif-1 Expression in Caco-2 Monolayers Exposed to Phorbol-12-Myristate-13-Acetate and Carnosine.

The dis(re)organization of the cytoskeletal actin in enterocytes mediates epithelial barrier dys(re)function, playing a key role in modulating epithelial monolayer's integrity and remodeling under transition from physiological to pathological states. Here, by fluorescence-based morphological and morphometric analyses, we detected differential responses of cytoskeletal actin in intestinal epithelial Caco-2 cell monolayers at two different stages of their spontaneous differentiation, i.e., undifferentiated cells at 7 days post-seeding (dps) and differentiated enterocyte-like cells at 21 dps, upon challenge in vitro with the inflammation-mimicking stimulus of phorbol-12-myristate-13-acetate (PMA). In addition, specific responses were found in the presence of the natural dipeptide carnosine detecting its potential counteraction against PMA-induced cytoskeletal alterations and remodeling in differentiated Caco-2 monolayers. In such an experimental context, by both immunocytochemistry and Western blot assays in Caco-2 monolayers, we identified the expression of the allograft inflammatory factor 1 (AIF-1) as protein functionally related to both inflammatory and cytoskeletal pathways. In 21 dps monolayers, particularly, we detected variations of its intracellular localization associated with the inflammatory stimulus and its mRNA/protein increase associated with the differentiated 21 dps enterocyte-like monolayer compared to the undifferentiated cells.

Motility increase of adherent invasive Escherichia coli (AIEC) induced by a sub-inhibitory concentration of recombinant endolysin LysPA90.

Endolysins are bacteriophage enzymes required for the eruption of phages from inside host bacteria via the degradation of the peptidoglycan cell wall. Recombinant endolysins are increasingly being seen as potential antibacterial candidates, with a number currently undergoing clinical trials. Bacteriophage PBPA90 infecting Pseudomonas aeruginosa harbors a gene encoding an endolysin, lysPA90. Herein, recombinant LysPA90 demonstrated an intrinsic antibacterial activity against Escherichia coli in vitro. It was observed that a sub-inhibitory concentration of the recombinant protein induced the upregulation of genes related to flagella biosynthesis in a commensal E. coli strain. Increases in the number of bacterial flagella, and in motility, were experimentally substantiated. The treatment caused membrane stress, leading to the upregulation of genes rpoE, rpoH, dnaK, dnaJ, and flhC, which are upstream regulators of flagella biosynthesis. When adherent invasive Escherichia coli (AIEC) strains were treated with subinhibitory concentrations of the endolysin, bacterial adhesion and invasion into intestinal epithelial Caco-2 cells was seen to visibly increase under microscopic examination. Bacterial counting further corroborated this adhesion and invasion of AIEC strains into Caco-2 cells, with a resultant slight decrease in the viability of Caco-2 cells then being observed. Additionally, genes related to flagella expression were also upregulated in the AIEC strains. Finally, the enhanced expression of the proinflammatory cytokine genes TNF-α, IL-6, IL-8, and MCP1 in Caco-2 cells was noted after the increased invasion of the AIEC strains. While novel treatments involving endolysins offer great promise, these results highlight the need for the further exploration of possible unanticipated and unintended effects.