Human Intestinal Cell Line Caco-2 Research Articles

Acute effects of apple extracts with different polyphenol compositions on glucose transport in human intestinal cell line Caco-2/TC7

AbstractDiets with a high-glycaemic load may increase the risk of type 2 diabetes. Food and beverages containing large amounts of highly bioaccessible starch and sugars induce pronounced postprandial blood glucose concentrations. Chronic exposure to sharp blood glucose peaks can lead to oxidative stress and pancreatic beta-cell dysfunction. Fruit polyphenols may help to limit the glucose excursion following a high-carbohydrate meal.Our previous research showed that 1.8 g polyphenol-rich apple extract (AE) mixed into a drink consumed before a high starch + sucrose meal, inhibited the incremental area under the curve of plasma glucose during the early absorption-driven postprandial phase (0–30 min) by 52% relative to a placebo drink and meal. Our latest dose-response trial showed that the glycaemic response to starch and sucrose was modified by 0.9 g AE. Possible mechanisms include activity inhibition of carbohydrate digestive enzymes and/or glucose absorption (SGLT1/GLUT2 transport). The AE is uniquely high in phlorizin (10.6%) – a strong inhibitor of SGLT1. However, evidence shows that other apple polyphenols may also inhibit SGLT1.The primary aim of our work was to explore the effect of different mixtures of apple polyphenols on gut transporters involved in glucose uptake. Acute dose-response effect of the AE and three fractions of AE (20%, 40% and 80% methanol elution during fractionation) on total glucose transport were investigated in Caco-2/TC7 cells. Uptake media (10min) contained, except for controls, increasing concentrations of AE/fractions (0–4.5mg/ml) incorporating the physiological range estimated to be present in the small intestine after ingestion of clinical trial test drinks. Glucose transport under sodium-dependent conditions was determined by radiochemical detection of Glucose D-[14C(U)] and Glucose L-[1-14C].A 47% decrease in total glucose uptake was observed with 1.12mg/ml AE (i.e. clinical trial highest physiological dose) v control (P = 0.03). Dose-response experiments showed IC50were: 1.29 mg/ml, 0.31 mg/ml and 0.88 mg/ml for AE (mg/g: 79 phlorizin; 26 epicatechin; 4 quercetin), 80% fraction (mg/g: 101 phlorizin; 26 epicatechin; 38 quercetin), 40% fraction (mg/g: 83 phlorizin; 20 epicatechin; 7 quercetin), respectively. The 20% fraction (mg/g: 4 phlorizin; 12 epicatechin; 2 quercetin) had no effect on glucose transport.We have gained greater understanding of a potential mechanism for the anti-hyperglycaemic properties of apple polyphenols, supporting potential applications of the AE in functional foods/beverages. Our in vitro data suggest that a combination of higher amounts of phlorizin and quercetin may be important to maximise intestinal glucose uptake inhibition.

Ochratoxin A challenges the intestinal epithelial cell integrity: results obtained in model experiments with Caco-2 cells

Contamination of human and animal diets with different mycotoxins have gained significant attention over the past decade. The intestinal barrier is the first site of exposure and a primary target for nutritional contaminants and hazardous substances including mycotoxins. In this study, the potential impact of ochratoxin A (OTA) on intestinal barrier integrity was highlighted using a human intestinal Caco-2 cell line. Cell viability following OTA exposure was determined by lactate dehydrogenase release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, markers of barrier integrity, such as transepithelial electrical resistance (TEER) as well as the permeability of Lucifer Yellow (LY) and fluorescein isothiocyanate (FITC)-dextran, were assessed. Furthermore, the protein expression of different tight junction (TJ) proteins, as main constituents of barrier integrity, was evaluated by Western blot. Results show that OTA reduces TEER values in a concentration- and time-dependent manner and increase the permeability of LY through the intestinal epithelial layer, while the cell viability did not change significantly. However, the damage was not severe enough to change the permeability to larger molecules, such as FITC-dextran. OTA exposure down-regulated the expression of TJ proteins claudin-1, -3 and -4 and up-regulated the expression of zona occludens 1. The observation that OTA can disrupt the epithelial barrier is of clinical importance as it may lead to an increased passage of luminal antigens into the systemic circulation.

Occludin endocytosis is involved in the disruption of the intestinal epithelial barrier in a mouse model of alcoholic steatohepatitis.

We aimed to investigate the involvement of the endocytosis of occludin, a key component of tight junction (TJ), in the ethanol-induced disassembly of TJ in a model of alcoholic steatohepatitis. Wild-type mice were fed an ethanol-containing or isocaloric liquid diet for 8 weeks and then assessed for liver injury (histopathology and measurement of serum enzymes), gut permeability (in vivo lactulose/mannitol and ex vivo dye leakage assays), intestinal epithelium ultrastructure (transmission electron microscopy), and intestinal occludin localization (immunofluorescence microscopy). The human intestinal epithelial cell line Caco-2 was also analyzed in vitro for the effects of ethanol on the barrier function (transepithelial electrical resistance), occludin localization (immunofluorescence microscopy and Western blotting), and endocytosis pathways (double-labeling immunofluorescence microscopy with selective pathway inhibitors). The ethanol-fed mice developed steatohepatitis and displayed intestinal barrier dysfunction, the disruption of intestinal TJ, and enhanced intestinal endocytosis of occluding compared with the control mice. In the Caco-2 monolayers, ethanol treatment decreased transepithelial electrical resistance, disrupted TJ formation, and enhanced occludin endocytosis in a dose- and time-dependent manner. These deleterious events were reversed by pretreating the Caco-2 cells with a selective pharmacological inhibitor of macropinocytosis, but not with the inhibitors of clathrin or caveolin-mediated endocytic pathways. Chronic ethanol exposure may increase intestinal permeability by inducing the micropinocytosis of occludin, resulting in the disruption of intestinal TJ.

Human intestinal Caco-2 cell line in vitro assay to evaluate the absorption of Cd, Cu, Mn and Zn from urban environmental matrices

The Caco-2 cell line is derived from a human colon adenocarcinoma and is generally used in toxicity assays. The ingestion of soil or dust is a significant route of human exposure to potential harmful elements (PHE), and assays of bioaccessibility or bioavailability can be used to measure the potential hazard posed by exposure to toxic substances. The in vitro digestion (UBM method) and Caco-2 cell model were used to investigate the bioaccessibility and absorption by intestinal cells of the PHE in four matrices (two urban soils and two soils with lead (Pb)-mining tailings) along with the guidance material for bioaccessibility measurements, BGS 102. The gastrointestinal (GI) compartment was simulated, and the resulting material added to Caco-2 cells. In the GI, the average bioaccessibility was 24% for cadmium (Cd), 17% for copper (Cu), 0.2% for Pb, 44% for manganese (Mn) and 6% for zinc (Zn). The poor reproducibility was attributed to the pH (6.3) and the highly complex GI fluid that formed PHE precipitates and complexes. In 2h, Caco-2 cells absorbed 0.2ngmg-1 of cellular protein for Cd, 13.4ngmg-1 for Cu, 5ngmg-1 for Mn and 31.7µgmg-1 for Zn. Lead absorption was lower than the limit of quantification (< 2µgL-1). Cd was presented in the cell monolayer and could interfere in the intracellular accumulation of Cu, Mn and Zn. The use of in vitro assays allowed for an estimation of the absorption of Cd, Cu, Mn and Zn from environmental matrices to be made, and except for Mn, it had a positive correlation with bioaccessible concentration, suggesting a common association of these elements in the cellular environment.

Improvement of antimalarial activity of a 3-alkylpiridine alkaloid analog by replacing the pyridine ring to a thiazole-containing heterocycle: Mode of action, mutagenicity profile, and Caco-2 cell-based permeability

The development of new antimalarial drugs is urgent to overcome the spread of resistance to the current treatment. Herein we synthesized the compound 3, a hit-to‑lead optimization of a thiazole based on the most promising 3-alkylpyridine marine alkaloid analog. Compound 3 was tested against Plasmodium falciparum and has shown to be more potent than its precursor (IC50 values of 1.55 and 14.7 μM, respectively), with higher selectivity index (74.7) for noncancerous human cell line. This compound was not mutagenic and showed genotoxicity only at concentrations four-fold higher than its IC50. Compound 3 was tested in vivo against Plasmodium berghei NK65 strain and inhibited the development of parasite at 50 mg/kg. In silico and UV–vis approaches determined that compound 3 acts impairing hemozoin crystallization and confocal microscopy experiments corroborate these findings as the compound was capable of diminishing food vacuole acidity. The assay of uptake using human intestinal Caco-2 cell line showed that compound 3 is absorbed similarly to chloroquine, a standard antimalarial agent. Therefore, we present here compound 3 as a potent new lead antimalarial compound.

Additive effects of Kothala himbutu (Salacia reticulata) extract and a lactic acid bacterium (Enterococcus faecalis YM0831) for suppression of sucrose-induced hyperglycemia in an in vivo silkworm evaluation system.

Using a silkworm evaluation system, we previously evaluated various substances that suppress postprandial hyperglycemia. Enterococcus faecalis YM0831, a lactic acid bacterium that inhibits glucose uptake by the human intestinal Caco-2 cell line, exhibited hyperglycemia-suppressing effects in the silkworm system. In the present study, we found that Kothala himbutu (Salacia reticulata) extract, a traditional medicine containing α-glucosidase inhibitors, suppressed sucrose-induced hyperglycemia in the silkworm system. Moreover, combined oral administration of lactic acid bacteria YM0831 with Kothala himbutu extract had stronger suppressive effects on sucrose-induced hyperglycemia than single administration of either component. These findings suggest that the silkworm system provides a simple way to evaluate the effects of supplements on the suppression of blood glucose level induced by sucrose ingestion.

Iron Supplements and Oncogenic Effects in Human Intestinal Cells (P05-011-19)

ObjectivesWe have previously shown that different iron supplements affect mechanisms for cell growth and viability in different ways and specifically we observed that two common iron supplements/fortificants, ferric citrate and ferric EDTA, promoted induction of the oncogenic growth factor Amphiregulin whilst ferrous sulfate did not. This difference is important and raises the question if some iron compounds should not be used as dietary supplements or for iron fortification of foods. MethodsTherefore, we have been screening iron compounds on the safe list of the European Food Safety Authority (EFSA) using human intestinal cell lines (Hutu-80 and Caco-2) and a proteomic immunoassay for 92 known markers related to cancer. ResultsPreliminary data show that a few more iron compounds, than the previously observed, have oncogenic properties and that amphiregulin protein levels are up-regulated. ConclusionsThese properties need further investigation and translation into the in vivo situation to evaluate if these iron compounds should be avoided as dietary supplements. Funding SourcesFORMAS.

Uptake and effects of orally ingested polystyrene microplastic particles in vitro and in vivo.

Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1µm (4.55 × 107 particles), 4µm (4.55 × 107particles) and 10µm (1.49 × 106 particles) microplastics at a volume of 10mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.

Enterococcus faecalis YM0831 suppresses sucrose-induced hyperglycemia in a silkworm model and in humans

Hyperglycemia caused by excessive intake of sucrose leads to lifestyle-related diseases such as diabetes. Administration of a lactic acid bacterial strain to mice suppresses sucrose-induced hyperglycemia, but evidence for a similar effect in humans is lacking. Here we show that Enterococcus faecalis YM0831, identified using an in vivo screening system with silkworms, suppressed sucrose-induced hyperglycemia in humans. E. faecalis YM0831 also suppressed glucose-induced hyperglycemia in silkworms. E. faecalis YM0831 inhibited glucose uptake by the human intestinal epithelial cell line Caco-2. A transposon insertion mutant of E. faecalis YM0831, which showed decreased inhibitory activity against glucose uptake by Caco-2 cells, also exhibited decreased inhibitory activity against both sucrose-induced and glucose-induced hyperglycemia in silkworms. In human clinical trials, oral ingestion of E. faecalis YM0831 suppressed the increase in blood glucose in a sucrose tolerance test. These findings suggest that E. faecalis YM0831 inhibits intestinal glucose transport and suppresses sucrose-induced hyperglycemia in humans.

Suppression of pyrimidine biosynthesis by targeting DHODH enzyme robustly inhibits rotavirus replication

Rotavirus infection remains a great health burden worldwide especially in some developing countries. It causes severe dehydrating diarrhea in infants, young children, as well as immunocompromised and organ transplanted patients. Viral replication heavily relies on the host to supply nucleosides. Thus, host enzymes involved in nucleotide biosynthesis represent potential targets for antiviral development. Dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. In this study, we demonstrated that two specific DHODH enzyme inhibitors, brequinar (BQR) and leflunomide (LFM) robustly inhibited rotavirus replication in conventional human intestinal Caco2 cell line as well as in human primary intestinal organoids. The antiviral effect is conserved in both laboratory strain SA11 and rotavirus strain 2011K isolated from clinical sample. Mechanistic study indicated that BQR and LFM exerted their anti-rotavirus effect through targeting DHODH to deplete pyrimidine nucleotide pool. Therefore, targeting pyrimidine biosynthesis represents a potential approach for developing antiviral strategies against rotavirus.

Absorption Characteristics of Chitobiose and Chitopentaose in the Human Intestinal Cell Line Caco-2 and Everted Gut Sacs.

The everted gut sacs and Caco-2 cell models were used to investigate the intestinal absorptive characteristics and subcellular localization of chitobiose and chitopentaose in this study. In everted gut sacs, the absorptive pattern showed no concentration-dependent manner when the concentration was lower than 10 mM. In the presence of phlorizin (100 μM) and phloretin (100 μM), the chitobiose absorption rates decreased by (4.97 ± 0.89)% and (19.2 ± 2.77)%, and they were (10.4 ± 2.43)% and (27.5 ± 1.68)% for chitopentaose. In Caco-2 cells, the concentration showed influences similar to those with the everted gut sacs results. After adding phlorizin and phloretin in the apical side, the PappAP-BL of chitobiose and chitopentaose significantly decreased. Considering the translocation, they were enriched in endoplasmic reticulum and mitochondrion. This study indicated that concentration and active transporter were capable of mediating the absorption of chitobiose and chitopentaose, and the subcellular localization of them could help to study the mechanisms of their effects.

In Vitro Immunomodulatory Effect of Food Supplement from Aloe vera.

Food industries typically use Aloe vera as concentrated (100× to 200×) and dried powders in their final products. These powders are obtained by extrusion of Aloe inner leaf gel (ILG) or Aloe whole leaf (WLP); the juice is filtered through diatomaceous earth and activated carbon before spray drying at temperatures below 70 °C. In another process, Aloe inner leaf gel was dried at ~80 °C and mashed to a powder rich in high molecular weight fibres and soluble polysaccharides (ILF). In contrast to ILG and WLP, the ILF sample was cytotoxic for the human intestinal cell line Caco-2 (CC50 = 1 g/l), even at concentrations below the recommended dose for human consumption. At lower concentrations (250 mg/l) with LPS challenged macrophage-like THP-1 cells decreased by 40% the release of the anti-inflammatory cytokine IL-10, whereas the release of the proinflammatory cytokine IL-1β increased by 35% (compared to untreated but challenged macrophage-like THP-1 cells). Unexpectedly, under the same conditions, the less cytotoxic ILG and WLP, both samples with a lower fibre content, significantly increased (up to 2.4 times) the release of IL-10, while the concentration of IL-1β remained unaltered and of TNFα decreased by 35%. Even more interesting is that a treatment of the ILF sample with activated carbon reduced its cytotoxicity and increased the IL-10 release (3.1 times). Based on these results, we suggest applying an activated carbon treatment on Aloe-starting products, which have high fibre content and have received high temperature treatment, in order to reduce their cytotoxicity and improve their immunomodulatory properties.

Probiotic Properties of Enterococcus Isolated From Artisanal Dairy Products.

The present study focused on probiotic characterization and safety evaluation of Enterococcus isolates from different artisanal dairy products. All the isolates exhibited inhibitory activity against several food spoilage bacteria and food-borne pathogens, including Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, Yersinia enterocolitica, Klebsiella pneumoniae, Escherichia coli, and Bacillus subtilis. The PCR results indicated the presence of at least one enterocin structural gene in all the tested strains. The Enterococcus isolates were further evaluated regarding their safety properties and functional features. The isolates were susceptible to vancomycin, gentamycin, and chloramphenicol. The results of PCR amplification revealed that all the tested isolates harbored none of the tested virulence genes except E. faecalis (ES9), which showed the presence of esp gene. The Enterococcus isolates showed cholesterol lowering properties. The selected isolates showed a high tolerance to low pH, and toward bile salts. They also demonstrated hydrophobicity activity, auto-aggregation, and adhesion ability to the human intestinal Caco-2 cell line. These properties may contribute the bacteria colonizing the gut. This study revealed that the Enterococcus isolates, especially E. durans ES11, ES20 and ES32, might be excellent candidates for production of functional foods to promote health benefits.

Mechanisms of Niemann-Pick type C1 Like 1 protein degradation in intestinal epithelial cells.

Intestinal Niemann-Pick C1 Like 1 (NPC1L1) protein plays a key role in cholesterol absorption. A decrease in NPC1L1 expression has been implicated in lowering plasma cholesterol and mitigating the risk for coronary heart disease. Little is known about the mechanisms responsible for NPC1L1 protein degradation that upon activation may lead to a reduction in NPC1L1 protein levels in intestinal epithelial cells (IECs). In current studies, the human intestinal Caco-2 and HuTu-80 cell lines expressing NPC1L1-hemagglutinin fusion protein were used to investigate the mechanisms of NPC1L1 protein degradation. Incubation with the proteasome inhibitors MG-132 and lactacystin (10 μM, 24 h) significantly increased NPC1L1 protein levels in IECs. Also, the inhibition of the lysosomal pathway with bafilomycin A1 (80 nM, 24 h) resulted in a significant increase in NPC1L1 protein levels. Immunoprecipitation studies showed that NPC1L1 protein is both a poly- and monoubiquinated polypeptide and that the inhibition of the proteasomal pathway remarkably increased the level of the polyubiquinated NPC1L1. The surface expression of NPC1L1 was increased by the inhibition of both proteasomal and lysosomal pathways. Furthermore, the pharmacological inhibition of mitogen-activated protein kinase pathway (PD-98059, 15 μM, 24 h) and siRNA silencing of ERK1/2 resulted in a significant decrease in NPC1L1 protein levels in IECs. In conclusion, our results showed that basal level of intestinal cholesterol transporter NPC1L1 protein is modulated by both ubiquitin proteasome- and lysosome-dependent degradation as well as by ERK1/2-dependent pathway. The modulation of these pathways may provide novel clues for therapeutic intervention to inhibit cholesterol absorption and lower plasma cholesterol.

Serotonin Modulates AhR Activation by Interfering with CYP1A1-Mediated Clearance of AhR Ligands.

Background/Aims:Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1).Methods:Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured.Results:We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of β-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation.Conclusion:Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.

Effect of bread making process on bioactive molecules in durum wheat bread and assessment of antioxidant properties by Caco-2 cell culture model

Effects of grain milling and bread making process on some bioactive components naturally present in three durum wheat bread samples and the study of the potential antioxidant properties of phenolic compounds by the human intestinal epithelial cell line Caco-2 were considered in this study. Lutein, the main carotenoid in these matrices, decreased from grain to bread from 50% to 70% depending on the variety while zeaxanthin and β-carotene content was quite negligible both in grain and in bread samples.Semolina generally showed the lowest extractable phenolic compounds (1280.5–1914.6 mg/kg d.m.) either respect to grain (1445.3–1923.0 mg/kg d.m.) or to bread samples (1537.4–1759.0 mg/kg d.m.). Non-extractable phenolic compounds which resulted strongly reduced in semolina, increased in bread respect to semolina (about 50%) due to the modification induced on the food matrix and, probably, to the Maillard reaction, which takes place during baking.No cellular damage was observed up to a dose of 10.0 μg/mL of polyphenolic extract. The same amount resulted able to protect the cell from the damage produced by the oxidant agent AAPH. These results underline that the potential health benefits of these compounds are dose-dependent and this aspect must be always taken into consideration.

Cucurbitacin E Inhibits Proliferation and Migration of Intestinal Epithelial Cells via Activating Cofilin.

The proliferation and migration of intestinal epithelial cell is important to the barrier integrity of intestinal epithelium. Cucurbitacin E (CuE) is one of the tetracyclic triterpenoids extracted from the cucurbitaceae that has been shown to inhibit cancer cell growth, tumor angiogenesis and inflammatory response. Nevertheless, the role of Cucurbitacin E in regulating the proliferation and migration of intestinal epithelial cells remain unclear. In this study, the human intestinal epithelial cell line Caco-2 was treated with CuE and the effects of CuE on cell cycle, proliferation, migration and actin dynamics in Caco-2 cells were investigated successively. We found that CuE significantly inhibited the cell proliferation and migration, inducing the cell cycle arrest in G2/M phase and disrupting the actin dynamic balance in Caco-2 cells. Finally, we showed that CuE inhibited cofilin phosphorylation by suppressing the phosphorylation of both LIM kinase (LIMK)1 and LIMK2 in vitro, resulting in the activation of cofilin, which is closely associated with cell proliferation and migration. Therefore, our studies provided the first evidence that CuE inhibited the proliferation and migration of intestinal epithelial cells via activating cofilin, and CuE is a potential candidate in intestinal disease therapy.

6-Thioguanine inhibits rotavirus replication through suppression of Rac1 GDP/GTP cycling

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational.

Basal interferon signaling and therapeutic use of interferons in controlling rotavirus infection in human intestinal cells and organoids

Rotavirus (RV) primarily infects enterocytes and results in severe diarrhea, particularly in children. It is known that the host immune responses determine the outcome of viral infections. Following infections, interferons (IFNs) are produced as the first and the main anti-viral cytokines to combat the virus. Here we showed that RV predominantly induced type III IFNs (IFN-λ1), and to a less extent, type I IFNs (IFN-α and IFN-β) in human intestinal cells. However, it did not produce detectable IFN proteins and thus, was not sufficient to inhibit RV replication. In contrast, we revealed the essential roles of the basal IFN signaling in limiting RV replication by silencing STAT1, STAT2 and IRF9 genes. In addition, exogenous IFN treatment demonstrated that RV replication was able to be inhibited by all types of IFNs, both in human intestinal Caco2 cell line and in primary intestinal organoids. In these models, IFNs significantly upregulated a panel of well-known anti-viral IFN-stimulated genes (ISGs). Importantly, inhibition of the JAK-STAT cascade abrogated ISG induction and the anti-RV effects of IFNs. Thus, our study shall contribute to better understanding of the complex RV-host interactions and provide rationale for therapeutic development of IFN-based treatment against RV infection.

Supplementation of chondroitin sulfate-oligosaccharides in skim bovine milk improves Fe uptake in a human intestinal Caco-2 cell line

The poor iron bioavailability of bovine milk remains a nutritional problem. This study evaluated the effect of bovine chondroitin sulfate-oligosaccharides (CS-oligos) with various skim bovine milk (SBM) fractions on Fe uptake by an in vitro digestion/Caco-2 cell model. SBM was separated into casein, whey, lactose and oligosaccharides by ultrafiltration. Modified fractions representing the absence of either casein, whey, or lactose were then prepared. Extrinsic ferrous sulfate was added at 20 µmol/L. Ferritin formation in the cells was used to assess Fe uptake. It is the first study to show the effect of milk oligosaccharides on Fe uptake, which was similar to that of whey. The ferritin synthesis rate was 1.5-fold greater in SBM after the addition of CS-oligos. The enhancing effect of CS-oligos was the most prominent with milk oligosaccharides and the least with casein. The results suggest the possibility of supplementing CS-oligos into different low iron bioavailability dairy products.