Human Intestinal Epithelial Caco-2 Cells Research Articles

Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

Virulence characterization and comparative genomics of Listeria monocytogenes sequence type 155 strains

BackgroundListeria (L.) monocytogenes strains show a high diversity regarding stress tolerance and virulence potential. Genome studies have mainly focused on specific sequence types (STs) predominantly associated with either food or human listeriosis. This study focused on the prevalent ST155, showing equal distribution among clinical and food isolates. We evaluated the virulence potential of 20 ST155 strains and performed comparative genomic analysis of 130 ST155 strains isolated from food, food processing environments and human listeriosis cases in different countries and years.ResultsThe in vitro virulence assays using human intestinal epithelial Caco2 and hepatocytic HEPG2 cells showed an impaired virulence phenotype for six of the 20 selected ST155 strains. Genome analysis revealed no distinct clustering of strains from the same source category (food, food processing environment, and clinical isolates). All strains harbored an intact inlA and inlB locus, except four strains, which had an internal deletion in the inlA gene. All strains harbored LIPI-1, but prfA was present in a longer variant in six strains, all showing impaired virulence. The longer PrfA variant resulted in lower expression of inlA, inlB, and prfA, and no expression of hly and actA.Regarding stress-related gene content, SSI-1 was present, whereas qacH was absent in all strains. 34.6% of the strains harbored a plasmid. All but one ST155 plasmids showed high conservation and harbored cadA2, bcrABC, and a triphenylmethane reductase.ConclusionsThis study contributes to an enhanced understanding of L. monocytogenes ST155 strains, being equally distributed among isolates from humans, food, and food processing environments. The conservation of the present genetic traits and the absence of unique inherent genetic features makes these types of STs especially interesting since they are apparently equally adapted to the conditions in food processing environments, as well as in food as to the human host environment. However, a ST155-specific mutation resulting in a longer PrfA variant impaired the virulence potential of several ST155 strains.

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.

The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.

Cytotoxic, Genotoxic, and Apoptotic Effects of Nickel Oxide Nanoparticles in Intestinal Epithelial Cells.

The superior properties of nickel oxide-nanoparticles (NiO-NPs) have led to their wide use in various fields. However, there is little comprehensive knowledge about their toxicity, especially after oral exposure. The toxic effect of NiO-NPs of mean size 15.0 nm was investigated in Caco-2 (human intestinal epithelial) cells as no study has been performed on their intestinal toxicity. Following identification of their particle size distribution and cellular uptake potential, the risk of exposure to NiO-NPs was evaluated by cellular morphologic changes, cyto- and genotoxic potentials, oxidative damage, and apoptotic induction. NiO-NPs induced a 50% reduction in cell viability at 351.6 μg/mL and caused DNA damage and oxidative damage at 30-150 μg/mL. It appears that apoptosis might be a main cell death mechanism in NiO-NP-exposed intestinal cells. NiO-NPs might be hazardous to the gastrointestinal system. The results should raise concerns about using NiO-NPs in food-contact appliances and about NiO-NP-containing wastes. Further in vivo and in vitro research should be conducted to explain the specific toxicity mechanism of these particles and reduce their risk to humans.

Involvement of Organic Cation Transporter 2 and a Na+-dependent active transporter in p-tyramine transport across Caco-2 intestinal cells

AimsWe have previously demonstrated that p-tyramine (TYR), an endogenous trace amine-associated receptor 1 agonist, passage across neuronal membranes involves a transporter exhibiting the pharmacological profile of Organic Cation Transporter 2 (OCT2). Since TYR is also a constituent of foodstuffs and produced by the intestinal microbiota, here we have investigated whether similar processes are involved in the passage of 100 nM TYR across apical and basolateral membranes of the Caco-2 human intestinal epithelial cell line. Materials and methods[3H]TYR transport across apical and basolateral membranes of Caco-2 cell monolayers was measured in the presence of inhibitors of TYR metabolizing enzymes. Cellular, apical, and basolateral compartments were collected at various timepoints, TYR concentrations calculated, and transport properties pharmacologically characterized. Key findingsApical transport resulted in equimolar accumulation of TYR within cells. Pentamidine (OCT1/OCT2 inhibitor) decreased apical transport (P = 0.001) while atropine (OCT1 inhibitor) had no effect, suggesting apical transport involved OCT2. In contrast, basolateral transport resulted in 500–1000 nM cellular concentrations (P < 0.0001) indicating the presence of an active transporter. Replacement of Na+ on an equimolar basis with choline resulted in loss of TYR transport (P = 0.017). Unexpectedly, this active transport was also atropine-sensitive (P = 0.020). Kinetic analysis of the active transporter revealed Vmax = 43.0 nM/s with a Kt = 33.1 nM. SignificanceWe have demonstrated for the first time that TYR is transported across Caco-2 apical membranes via facilitated diffusion by OCT2, whereas transport across basolateral membranes is by a Na+-dependent, atropine-sensitive, active transporter.

Chondroitin sulfate and its nanocomposites with protamine or chitosan stabilize and deliver available nanosized iron

Nanostructured iron (III) compounds are promising candidates for iron fortification applications due to their good solubility, bioavailability, and redox inertia. The current study synthesized ferric oxyhydroxide nanoparticles (FeONPs) based on chondroitin sulfate (ChS) and its nanocomposites with protamine sulfate (PS) or chitosan (CS) in neutral aqueous solution under ambient conditions, and evaluated their iron availability to polarized human intestinal epithelial (Caco-2) cells. Ultraviolet-visible spectroscopy, dynamic light scattering, transmission electron microscopy and energy-dispersive X-ray spectroscopy showed that ChS-FeONPs were wave-like anionic particles where FeONPs attached on the polysaccharide chains and PS/ChS-FeONPs and CS/ChS-FeONPs were irregular anionic nanoparticles where FeONPs scattered across the entire region. The calcein-fluorescence-quenching assay in polarized Caco-2 cells showed good iron uptake from ChS-FeONPs, PS/ChS-FeONPs and CS/ChS-FeONPs mainly via endocytosis, with the latter two exhibiting better iron absorption. Overall, our study provides a more facile and greener alternative route to synthesize the bioavailable nano-sized iron.

Suppressive Effects of Hot-water Extract of Magnolia obovata on Clostridium perfringens Enterotoxin-induced Cytotoxicity in Human Intestinal Caco-2 Cells.

The physiological functions of the leaves of Japanese big-leaf magnolia (Magnolia obovata) against enterotoxins produced by foodborne pathogens remain unclear. In this study, we evaluated the protective effects of M.obovata leaf extract (MLE) against the cytotoxicity of Clostridium perfringens enterotoxin (CPE), which causes the symptoms of C. perfringens type A food poisoning. The protective effects of MLE against CPE-induced cytotoxicity were evaluated in human intestinal epithelial Caco-2 cells. Pre-treatment with MLE significantly suppressed the cytotoxicity induced by CPE in undifferentiated and differentiated human intestinal Caco-2 cells at a pH range of 4.0 - 7.0. This CPE-suppressive effect was due to a hydrophilic sugar-containing compound without phenolic and protein structures but not the hydrophobic biologically active neolignans, honokiol and magnolol. MLE had a protective effect against cytotoxicity caused by type A C. perfringens. Our results provide novel insight regarding the usage of M.obovata in managing food poisoning.

Microalgae aqueous extracts exert intestinal protective effects in Caco-2 cells and dextran sodium sulphate-induced mouse colitis.

Microalgae are emerging as a good source of natural nutraceuticals. Here, we examined the intestinal protective effects of microalgae aqueous extracts (MAEs) from Chlorella pyrenoidosa, Spirulina platensis, and Synechococcus sp. PCC 7002 in human intestinal epithelial Caco-2 cells and dextran sodium sulphate (DSS)-induced colitis in C57BL/6 mice. MAEs displayed intestinal barrier-protective activities in Caco-2 cells by increasing the expression of heat shock protein (Hsp)-27 and tight junction proteins of occludin and claudin-4 and attenuating the H2O2-induced intracellular reactive oxygen species production, plasma membrane impairment and apoptosis. They also showed anti-inflammatory potential in tumor necrosis factor (TNF)-α-, interleukin (IL)-1β- and H2O2-stimulated Caco-2 cells by suppressing the secretion of IL-8 and the expression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). The 8 d daily intragastric administration of MAEs during and after 4 d DSS exposure effectively alleviated colitis symptoms of weight loss, diarrhea, rectal bleeding, and colon shortening and histopathology, protected intestinal barrier function by increasing colonic Hsp-25, occludin and claudin-4, and attenuated colonic and systemic inflammation by suppressing colonic myeloperoxidase activity, production of TNF-α, IL-1β and IL-6, expression of COX-2 and iNOS, and peripheral leukocytosis, monocytosis and granulocytosis. Microalgae can thus serve as a functional food to maintain gut health.

Nanoplastics as a potential environmental health factor: effects of polystyrene nanoparticles on human intestinal epithelial Caco-2 cells

Our results show that both nPS are easily internalized by Caco-2 cells in a concentration-dependent manner, but no relevant toxic effects are observed under the analyzed exposure conditions.

Determination and prediction of permeability across intestinal epithelial cell monolayer of a diverse range of industrial chemicals/drugs for estimation of oral absorption as a putative marker of hepatotoxicity

Apparent permeability coefficients (Papp) across a human intestinal epithelial Caco-2 cell monolayer were measured for a range of industrial/drug chemicals. A predictive equation for determining in vitro Papp values of fifty-six substances was set up using multivariate regression analysis based on in silico-estimated physicochemical properties (molecular weights and water distribution coefficients for apical and basal pH environments) (r = 0.77, p < 0.01). Predicted logPapp values of a secondary set of 34 compounds were correlated with the measured values. Under the medicinal logPapp values associated with their reported fraction absorbed, a significant inverse non-linear correlation was found between the logarithmic transformed values of observed Papp values and reported hepatic no-observed-effect levels of industrial chemicals (r = –0.55, p < 0.01, n = 29). In vitro determination and/or in silico prediction of permeability across intestinal cells could be effective for estimating oral absorption as a putative indicator for hepatotoxicity.

PSVI-11 Antioxidative effects of red-osier dogwood extract on H2O2-treated human intestinal epithelial Caco-2 cells

Abstract Red-osier dogwood is a species of flowering plant native in North America. Its extract may exhibit the anti-oxidative and anti-bacterial properties because it contains plenty of phenolic compounds, polysaccharides, and bioactive peptides. Hence, red-osier dogwood extract (RDE) could be a potential alternative to antibiotics. In the present study, an in vitro oxidative damage model via using Caco-2 cells was established to evaluate the antioxidative effects of RDE. The results showed that 1 mM H2O2 significantly decreased the cell viability, and increased interleukin 8 (IL-8) secretion and reactive oxygen species (ROS) production (P &amp;lt; 0.05). The mRNA abundance of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and IL-8 also increased in cells treated by H2O2. Caco-2 cells exposed to H2O2 for 6 h were then treated with 100 µg/mL RDE for 24 h. The IL-8 secretion and ROS production induced by H2O2 was significantly blocked by RDE (P &amp;lt; 0.05). RDE reduced the leakage of Fluorescein isothiocyanate (FITC)-dextran, and increased transepithelial resistance (TEER) value compared with the control (H2O2 treatment). Moreover, RDE increased Zonula occludens-1 (ZO-1) and β-actin staining in the cells and tight junction proteins expression including ZO-1, claudin 1, claudin 3 and occludin. These results suggested that RDE enhanced barrier functions and reduced ROS production in the epithelial cells under H2O2-induced oxidative damage.

Production of functional mimics of human milk oligosaccharides by enzymatic glycosylation of bovine milk oligosaccharides

Consumption of mothers' milk is associated with reduced incidence and severity of enteric infections, leading to reduced morbidity in breastfed infants. Fucosylated and sialylated human milk oligosaccharides (HMO) are important for both direct antimicrobial action – likely via a decoy effect – and indirect antimicrobial action through commensal growth enhancement. Bovine milk oligosaccharides (BMO) are a potential source of HMO-mimics as BMO resemble HMO; however, they have simpler and less fucosylated structures. BMO isolated at large scales from bovine whey permeate were modified by the addition of fucose and/or sialic acid to generate HMO-like glycans using high-yield and cost-effective one-pot multienzyme approaches. Quadrupole time-of-flight LC/MS analysis revealed that 22 oligosaccharides were synthesized and 9 had identical composition to known HMO. Preliminary anti-adherence activity assays indicated that fucosylated BMO decreased the uptake of enterohemorrhagic Escherichia coli O157:H7 by human intestinal epithelial Caco-2 cells more effectively than native BMO.

Impact of exposure to cold and cold-osmotic stresses on virulence-associated characteristics of Listeria monocytogenes strains

The objective of this work was to investigate the effect of stress conditions frequently encountered in food-associated environments on virulence-associated characteristics of eight strains of Listeria monocytogenes. Strains were grown at low (11 °C, cold stress) and optimal (37 °C) temperatures and in high NaCl concentrations (6% NaCl, 11 °C; cold-osmotic stress) and tested for their ability to invade the human intestinal epithelial Caco-2 cells. Results demonstrate that the correlation between exposure to cold stress and increased invasion phenotype is strain-dependent as strains investigated exhibited different behaviours, i.e. exposure to cold stress conditions resulted in a significant increase of invasion levels in five out of the eight strains tested, when compared to growth under optimal conditions. On the other hand, when these cold-adapted cells were subsequently submitted to high salt concentrations and low temperature, their enhanced ability to invade Caco-2 was lost. Surprisingly, saturated fatty acids (SFA) and branched chain fatty acids (BCFA) decreased when L. monocytogenes were exposed to stress conditions as opposed to what has been observed in other studies, therefore highlighting that further studies will need to deepen in the understanding of the lipid metabolism of these strains. The effect of stress conditions on the survival of three selected L. monocytogenes strains through an in vitro gastrointestinal (GI) tract digestion model was further investigated. The exposure to cold-osmotic stress increased the survival of one strain through the GI tract.

Evaluation of Incompatibility Group I1 (IncI1) Plasmid-Containing Salmonella enterica and Assessment of the Plasmids in Bacteriocin Production and Biofilm Development.

Mobile genetic elements, such as plasmids, can potentially increase the ability of bacteria to infect and persist in vertebrate host cells. IncI1 plasmids are widely distributed in Salmonella from food animal sources and associated with clinically important strains. These plasmids often encode antimicrobial resistance; however, little is known about their impact on the virulence of Salmonella strains. To assess the potential impact of the plasmids on virulence, 43 IncI1-positive Salmonella isolates from human and animal sources were subjected to whole genome sequence (WGS) analyses and evaluated for their abilities to invade and persist for 48 h in Caco-2 human intestinal epithelial cells, form biofilms and encode bacteriocins. Draft WGS data were submitted to predict the presence of virulence and antimicrobial resistance genes, plasmid replicon types present, conduct plasmid multilocus sequence typing (pMLST), and core genome MLST (cgMLST) in the isolates. Caco-2 cells were infected with Salmonella strains and incubated for both one and 48 h for the invasion and persistence assays, respectively. Additionally, Salmonella isolates and IncI1 plasmid carrying transconjugants (n = 12) generated in Escherichia coli were assessed for their ability to produce biofilms and bacteriocin inhibition of growth of other bacteria. All Salmonella isolates infected Caco-2 cells and persisted in the cells at 48 hrs. Persistent cell counts were observed to be significantly higher than invasion assay cell counts in 26% of the isolates. Among the IncI1 plasmids, there were 18 pMLST types. Nearly 35% (n = 15) of Salmonella isolates produced biofilms; however, none of the IncI1-positive transconjugants produced increased biofilms compared to the recipient. Approximately 65% (n = 28) of isolates and 67% (n = 8) of IncI1-positive transconjugants were able to inhibit growth of at least one E. coli strain; however, none inhibited the growth of strains from species other than E. coli. The study characterized IncI1 positive Salmonella isolates and provided evidence about the potential contributions of IncI1 plasmids virulence phenotypes and areas where they do not. These findings should allow for more focused efforts to assess the impact of plasmids on bacterial pathophysiology and human health.

Cholera Toxin B Subunit and Peptide LKEKK Inhibit TNF-α Signaling in Intestinal Epithelial Cells and Reduce Inflammation in a Mouse Model of Colitis

Cholera toxin B subunit (CT-B) and synthetic peptide LKEKK corresponding to the sequence 16-20 of thymosin-α1 and the sequence 131-135 of interferon-α2 (the concentration range of 100-5000 µM) significantly reduced TNF-α-stimulated pro-inflammatory cytokine expression and increases the expression of the anti-inflammatory cytokine IL-10 in human Caco-2 intestinal epithelial cells. In a mouse model of dextran sodium sulfate-induced colitis CT-B and peptide, LKEKK (20 mg/kg body weight orally for 14 days) decreased the production of TNF-α and IL-6, as well as the severity of inflammation. Thus, CT-B and peptide LKEKK are able to suppress inflammation in vitro and in vivo.

Improving Transient Transfection Efficiency in a Differentiated, Polar Epithelial Cell Layer.

Polar, differentiated epithelial cell culture models (especially at confluence) are difficult to transfect compared with the higher transfection efficiencies that one obtains with relatively less differentiated, nonpolar cell culture models. Here, we sought to develop a strategy to enhance the efficiency of transfecting polar, differentiated epithelial cells. We found that chemically abrading the differentiated CACO-2 human intestinal epithelial cell layer by a trypsin and EDTA pretreatment (before the use of detergent-like transfection reagents) dramatically improved transfection efficiency in this polar, differentiated model. Although this treatment did improve the transfection efficiency, it also induced leakiness in the epithelial barrier by both opening tight junctional complexes and by creating holes in the cell layer because of low-level cell death and detachment. Thus, this approach to enhance the transfection efficiency of polar, differentiated cells will be useful for assessment of the effect of the transfected/expressed protein on (re)formation of an epithelial barrier rather than on a functional barrier itself.

Characterization of Antibiotic Resistance Genes, Plasmids, Biofilm Formation, and In Vitro Invasion Capacity of Salmonella Enteritidis Isolates from Children with Gastroenteritis.

Objectives: The primary aim of this study was to investigate the presence of antibiotic resistance genes (ARGs) and plasmid replicon types for 75 multidrug-resistant (MDR) Salmonella Enteritidis isolates from children with gastroenteritis. We also evaluated the association among biofilm formation, in vitro invasion capacity, and antibiotic resistance phenotypes. Materials and Methods: Twenty-two ARGs and 18 different plasmid incompatibility types were investigated using polymerase chain reaction (PCR) and DNA sequencing. In vitro invasion capacity of S. Enteritidis isolates possessing different antibiotic resistance patterns was assessed using the Caco2 human intestinal epithelial cell line and biofilm formation was performed in a 96-well polystyrene well format using crystal violet detection. Results: The presence of plasmid-mediated quinolone resistance genes and β-lactamase genes was established using PCR amplification. All the tested S. Enteritidis isolates that were fluoroquinolone resistant possessed gyrA mutations and 50% also possessed mutations in parC. MDR S. Enteritidis isolates containing three (29/75) or four (21/75) plasmid replicon types were predominant and 71/75 carried both FIIs and FIC replicon-type plasmids. MDR isolates were strong or moderate biofilm producers and a significant positive association (p < 0.05) between antibiotic resistance and biomass of biofilms was observed in the strains assayed. A ceftiofur-resistant strain was significantly more invasive (p < 0.01) than the other isolates. Conclusions: We observed a high incidence of ARGs and diversity of plasmids in S. Enteritidis isolates from children. Biofilm formation and invasion capacity highlight a significant hazard to public health.

Effects of Protein-Derived Amino Acid Modification Products Present in Infant Formula on Metabolic Function, Oxidative Stress, and Intestinal Permeability in Cell Models.

Proteins present in infant formulas are modified by oxidation and glycation during processing. Modified amino acid residues released from proteins may be absorbed in the gastrointestinal tract, and pose a health risk to infants. In this study, the markers of glycation furosine (1.7-3.5 μg per milligram of protein) and Nε-(carboxymethyl)lysine (28-81 ng per milligram of protein) were quantitated in infant formulas. The effects of these species, and other amino acid modifications, at the levels detected in infant formulas, on 3T3-L1 (murine preadipocyte) and Caco-2 (human intestinal epithelial) cells were assessed. Incubation of 3T3-L1 cells for 48 h with amino acid side chain oxidation and glycation products (1 and 10 μM) resulted in a loss (up to 40%, p < 0.05) of cell thiols and decreased metabolic activity compared with those of the controls. In contrast, Caco-2 cells showed a stimulation (10-50%, p < 0.05) of cellular metabolism on exposure to these products for 24 or 48 h. A 28% ( p < 0.05) increase in protein carbonyls was detected upon incubation with 200 μM modified amino acids for 48 h, although no alteration in transepithelial electrical resistance was detected. Oxidation products were detected in the basolateral compartments of Caco-2 monolayers when modified amino acids were applied to the apical side, consistent with limited permeability (up to 3.4%) across the monolayer. These data indicate that modified amino acids present in infant formulas can induce effects on different cell types, with evidence of bioavailability and induction of cellular stress. This may lead to potential health risks for infants consistently exposed to high levels of infant formulas.

Clostridium perfringens Delta-Toxin Damages the Mouse Small Intestine.

Clostridium perfringens strains B and C cause fatal intestinal diseases in animals. The secreted pore-forming toxin delta-toxin is one of the virulence factors of the strains, but the mechanism of intestinal pathogenesis is unclear. Here, we investigated the effects of delta-toxin on the mouse ileal loop. Delta-toxin caused fluid accumulation and intestinal permeability to fluorescein isothiocyanate (FITC)-dextran in the mouse ileal loop in a dose- and time-dependent manner. Treatment with delta-toxin induced significant histological damage and shortening of villi. Delta-toxin activates a disintegrin and metalloprotease (ADAM) 10, leading to the cleavage of E-cadherin, the epithelial adherens junction protein, in human intestinal epithelial Caco-2 cells. In this study, E-cadherin immunostaining in mouse intestinal epithelial cells was almost undetectable 1 h after toxin treatment. ADAM10 inhibitor (GI254023X) blocked the toxin-induced fluid accumulation and E-cadherin loss in the mouse ileal loop. Delta-toxin stimulated the shedding of intestinal epithelial cells. The shedding cells showed the accumulation of E-cadherin in intracellular vesicles and the increased expression of active caspase-3. Our findings demonstrate that delta-toxin causes intestinal epithelial cell damage through the loss of E-cadherin cleaved by ADAM10.

RGal Increases Intestinal Epithelial Barrier Function in a PKC‐Dependent Manner and Drives an Inflammatory Gene Expression Profile

BackgroundLoss of intestinal epithelial barrier function has been linked to inflammatory bowel disease (IBD). We previously showed that the dietary fibre, rhamnogalacturonan (RGal), reduces inflammation in a murine model of DSS colitis. Additionally, apical treatment of Caco2 human intestinal epithelial cells with RGal increases transepithelial electrical resistance (TER) and accelerates wound‐healing, but the underlying mechanisms remain unknown.AimsWe aimed to determine the mechanisms of the RGal mediated increase in epithelial barrier function. (1) Given the ability of PKC to modulate barrier permeability we aimed to determine the role of PKC in the RGal‐mediated increase in epithelial barrier function. (2) We aimed to determine changes in gene expression following RGal treatment.Methods(1) To determine the role of PKC in the RGal‐induced increase in epithelial barrier function, Caco2 monolayers were mounted in Ussing Chambers, pre‐treated with pan protein kinase C (PKC), PKCz‐specific, or classical PKC inhibitors (GFX, PKCz pseudosubstrate or Go6976 respectively) and then treated with RGal. Change in TER and FITC‐dextran flux in response to RGal were assessed. (2) To determine the transcriptionally dependent effects of RGal on epithelial barrier function, Caco2 monolayers were treated apically for 6, 12 and 24h with RGal and RNAseq was performed. Genes with changes in expression more than 3‐fold were entered into Enrichr for pathway analysis. In order to confirm changes in gene expression from RNAseq, a multi‐array immunoassay was performed.Results(1) RGal (1 mg/mL) reduced FITC‐dextran flux by 52.0% (n=5, p&lt;0.05) 30 min post‐treatment. The effect of RGal on macromolecular permeability was reduced with GFX pre‐treatment (500 nM) by 75.3% (n=5, p&lt;0.01). However, the ability of GFX to block the RGal‐mediated increase in barrier function was not mimicked in monolayers pre‐treated with PKCz pseudosubstrate inhibitor (10 mM, n=6) or Go6976 (10 nM, n=5). (2) While RGal had no significant effect on tight junction gene expression 6h post‐treatment, it upregulated the expression of inflammatory response genes including neutrophil regulatory genes such as CXCL8. Consistent with gene expression data, multiplex protein assay revealed an upregulation in CXCL8 family chemokines and G‐CSF as early as 2h in cell lysates and 6h in the supernatant.ConclusionsThe RGal‐mediated increase in intestinal epithelial barrier function is dependent on PKC. Transcriptionally, RGal upregulates the expression of inflammatory mediators such as CXCL8. Understanding the mechanism of the RGal‐induced increase in intestinal epithelial barrier function may allow for leverage of these mechanisms to treat IBD.Support or Funding InformationProject supported by the NSERC (Canada), J.S. supported by an UGREF from the American Physiological Society.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.