HT-29 Human Intestinal Epithelial Cells Research Articles

Inhibition of ESCRT-independent extracellular vesicles biogenesis suppresses enterovirus 71 replication and pathogenesis in mice

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), neurological complications, and even fatalities in infants. Clinically, the increase of extracellular vesicles (EVs) in EV71 patients' serum was highly associated with the severity of HFMD. EV71 boosts EVs biogenesis in an endosomal sorting complex required for transport (ESCRT)-dependent manner to facilitate viral replication. Yet, the impact of EVs-derived from ESCRT-independent pathway on EV71 replication and pathogenesis is highly concerned. Here, we assessed the effects of EV71-induced EVs from ESCRT-independent pathway on viral replication and pathogenesis by GW4869, a neutral sphingomyelinase inhibitor. Detailly, in EV71-infected mice, blockade of the biogenesis of tissue-derived EVs in the presence of GW4869 restored body weight loss, attenuated clinical scores, and improved survival rates. Furthermore, GW4869 dampens EVs biogenesis to reduce viral load and pathogenesis in multiple tissues of EV71-infected mice. Consistently, GW4869 treatment in a human intestinal epithelial HT29 cells decreased the biogenesis of EVs, in which the progeny EV71 particle was cloaked, leading to the reduction of viral infection and replication. Collectively, GW4869 inhibits EV71-induced EVs in an ESCRT-independent pathway and ultimately suppresses EV71 replication and pathogenesis. Our study provides a novel strategy for the development of therapeutic agents in the treatment for EV71-associated HFMD.

Isolated Fraction of Gastric-Digested Camel Milk Yogurt with Carao (Cassia grandis) Pulp Fortification Enhances the Anti-Inflammatory Properties of HT-29 Human Intestinal Epithelial Cells.

Functional foods have recently generated a lot of attention among consumers looking for healthy options. Studies have examined yogurt with carao to increase health benefits and probiotic characteristics. It has been determined that carao fruit and camel milk have high phenolic compound and antioxidant activity concentrations. The objective of this study was to examine if carao (0, 1.3, 2.65, and 5.3 g/L) incorporated into yogurt enhances anti-inflammatory stimulus and antioxidant activity and impacts the physio-chemical and sensory properties of camel milk yogurt. HT-29 cells were used as a model of anti-inflammatory response, including cytokine responses of IL-8 and mRNA production of IL-1β and TNF-α in gastric digested isolated fraction. In addition, pH, titratable acidity, Streptococcus thermophilus counts and Lactobacillus bulgaricus counts of camel yogurts were examined during the fermentation process in 0, 2.5, 5, and 7 h whereas viscosity, syneresis, and radical scavenging assay evaluations were determined at hour 7. Furthermore, a consumer study was performed. Compared to control samples, the incorporation of carao into yogurts did not lead to a significant (ρ > 0.05) difference in the pH. In contrast, titratable acidity (TA), viscosity, syneresis, and antioxidant capacity significantly increased with the inclusion of 2.65 and 5.3 g/L carao, while 5.3 g/L carao significantly (ρ < 0.05) increased the counts of both bacteria. The inflammatory response of IL-8 and the level of mRNA production of IL-1β and TNF-α was significantly (ρ < 0.05) lower with 2.65 and 5.3 g/L carao yogurt compared to control camel yogurt. Sensory attributes were not impacted by the addition of 1.3 and 2.65 g/L carao. Carao could be a possible ingredient to consider when improving the nutrition value of yogurt.

Multilayer Coating with Red Ginseng Dietary Fiber Improves Intestinal Adhesion and Proliferation of Probiotics in Human Intestinal Epithelial Models.

To exert their beneficial effects, it is essential for the commensal bacteria of probiotic supplements to be sufficiently protected as they pass through the low pH environment of the stomach, and effectively colonize the intestinal epithelium downstream. Here, we investigated the effect of a multilayer coating containing red ginseng dietary fiber, on the acid tolerance, and the adhesion and proliferation capacities of three Lactobacillus strains (Limosilactobacillus reuteri KGC1901, Lacticaseibacillus casei KGC1201, Limosilactobacillus fermentum KGC1601) isolated from Panax ginseng, using HT-29 cells, mucin-coated plates, and human pluripotent stem cell-derived intestinal epithelial cells as in vitro models of human gut physiology. We observed that the multilayer-coated strains displayed improved survival rates after passage through gastric juice, as well as high adhesion and proliferation capacities within the various gut epithelial systems tested, compared to their uncoated counterparts. Our findings demonstrated that the multilayer coat effectively protected commensal microbiota and led to improved adhesion and colonization of intestinal epithelial cells, and consequently to higher probiotic efficacy.

Transcriptional responses of human intestinal epithelial HT-29 cells to spore-displayed p40 derived from Lacticaseibacillus rhamnosus GG

BackgroundsThe aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells.Resultsp40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action.ConclusionsThis study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.

Campylobacter concisus upregulates PD-L1 mRNA expression in IFN-γ sensitized intestinal epithelial cells and induces cell death in esophageal epithelial cells

ABSTRACT Introduction: Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) and Barrett’s esophagus (BE). Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that is used by tumor cells for immune evasion and has increased expression in patients with IBD and BE. We examined whether C. concisus upregulates PD-L1 expression in intestinal and esophageal epithelial cells. Methods: Human intestinal epithelial HT-29 cells and esophageal epithelial FLO-1 cells with and without interferon (IFN)-γ sensitization were incubated with C. concisus strains. The level of PD-L1 mRNA was quantified using quantitative real-time PCR. Cytokines were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Apoptosis of HT-29 and FLO-1 cells were measured using caspase 3/7 assay. Results: We found that intestinal epithelial cells with IFN-γ sensitization incubated with C. concisus significantly upregulated PD-L1 expression and significantly increased the production of interleukin (IL)-8. Whereas, PD-L1 expression was significantly inhibited in IFN-γ sensitized FLO-1 cells incubated with C. concisus strains. Furthermore, FLO-1 cells with and without IFN-γ sensitization incubated with C. concisus strains both had significantly higher levels of cell death. Conclusion: C. concisushas the potential to cause damage to both intestinal and esophageal epithelial cells, however, with different pathogenic effects.

Escherichia coli K12 Upregulates Programmed Cell Death Ligand 1 (PD-L1) Expression in Gamma Interferon-Sensitized Intestinal Epithelial Cells via the NF-κB Pathway.

Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein which is used by tumor cells for immune evasion. PD-L1 is upregulated in inflamed intestinal tissues. The intestinal tract is colonized by millions of bacteria, most of which are commensal bacterial species. We hypothesized that under inflammatory conditions, some commensal bacterial species contribute to increased PD-L1 expression in intestinal epithelium and examined this hypothesis. Human intestinal epithelial HT-29 cells with and without interferon (IFN)-γ sensitization were incubated with six strains of four enteric bacterial species. The mRNA and protein levels of PD-L1 in HT-29 cells were examined using quantitative real-time PCR and flow cytometry, respectively. The levels of interleukin (IL)-1β, IL-18, IL-6, IL-8, and tumor necrosis factor (TNF)-α secreted by HT-29 cells were measured using enzyme-linked immunosorbent assay. Apoptosis of HT-29 cells was measured using a caspase 3/7 assay. We found that Escherichia coli K12 significantly upregulated both PD-L1 mRNA and protein in IFN-γ-sensitized HT-29 cells. E. coli K12 induced the production of IL-8 in HT-29 cells, however, IL-8 did not affect HT-29 PD-L1 expression. Inhibition of the nuclear factor-kappa B pathway significantly reduced E. coli K12-induced PD-L1 expression in HT-29 cells. The other two E. coli strains and two enteric bacterial species did not significantly affect PD-L1 expression in HT-29 cells. Enterococcus faecalis significantly inhibited PD-L1 expression due to induction of cell death. Data from this study suggest that some gut bacterial species have the potential to affect immune function under inflammatory conditions via upregulating epithelial PD-L1 expression.

Extracellular Vesicles Produced by the Probiotic Propionibacterium freudenreichii CIRM-BIA 129 Mitigate Inflammation by Modulating the NF-κB Pathway.

Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered to be a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. Among probiotic Gram-positive bacteria, some Propionibacterium freudenreichii strains exhibit anti-inflammatory activity, notably via surface proteins such as the surface-layer protein B (SlpB). We have hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host. In order to demonstrate their production in this species, EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, and their physicochemical characterization, using transmission electron microscopy and nanoparticle tracking analysis (NTA), revealed shapes and sizes typical of EVs. Proteomic characterization showed that EVs contain a broad range of proteins, including immunomodulatory proteins such as SlpB. In silico protein-protein interaction predictions indicated that EV proteins could interact with host proteins, including the immunomodulatory transcription factor NF-κB. This potential interaction has a functional significance because EVs modulate inflammatory responses, as shown by IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells. Indeed, EVs displayed an anti-inflammatory effect by modulating the NF-κB pathway; this was dependent on their concentration and on the proinflammatory inducer (LPS-specific). Moreover, while this anti-inflammatory effect partly depended on SlpB, it was not abolished by EV surface proteolysis, suggesting possible intracellular sites of action for EVs. This is the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. This study has enhanced our understanding of the mechanisms associated with the probiotic activity of P. freudenreichii and identified opportunities to employ bacterial-derived EVs for the development of bioactive products with therapeutic effects.

Intestinal Epithelial Cells Express Immunomodulatory ISG15 During Active Ulcerative Colitis and Crohn's Disease.

Background and AimsIntestinal epithelial cells [IECs] secrete cytokines that recruit immune cells to the mucosa and regulate immune responses that drive inflammation in inflammatory bowel disease [IBD]. However, experiments in patient-derived IEC models are still scarce. Here, we aimed to investigate how innate immunity and IEC-specific pattern recognition receptor [PRR] signalling can be involved in an enhanced type I interferon [IFN] gene signature observed in colon epithelium of patients with active IBD, with a special focus on secreted ubiquitin-like protein ISG15.MethodsGene and protein expression in whole mucosa biopsies and in microdissected human colonic epithelial lining, in HT29 human intestinal epithelial cells and primary 3D colonoids treated with PRR-ligands and cytokines, were detected by transcriptomics, in situ hybridisation, immunohistochemistry, western blots, and enzyme-linked immunosorbent assay [ELISA]. Effects of IEC-secreted cytokines were examined in human peripheral blood mononuclear cells [PBMCs] by multiplex chemokine profiling and ELISA.ResultsThe type I IFN gene signature in human mucosal biopsies was mimicked in Toll-like receptor TLR3 and to some extent tumour necrosis factor [TNF]-treated human IECs. In intestinal biopsies, ISG15 expression correlated with expression of the newly identified receptor for extracellular ISG15, LFA-1 integrin. ISG15 was expressed and secreted from HT29 cells and primary 3D colonoids through both JAK1-pSTAT-IRF9-dependent and independent pathways. In experiments using PBMCs, we show that ISG15 releases IBD-relevant proinflammatory cytokines such as CXCL1, CXCL5, CXCL8, CCL20, IL1, IL6, TNF, and IFNγ.ConclusionsISG15 is secreted from primary IECs upon extracellular stimulation, and mucosal ISG15 emerges as an intriguing candidate for immunotherapy in IBD.

An altered gene expression profile in tyramine-exposed intestinal cell cultures supports the genotoxicity of this biogenic amine at dietary concentrations

Tyramine, histamine and putrescine are the most commonly detected and most abundant biogenic amines (BA) in food. The consumption of food with high concentrations of these BA is discouraged by the main food safety agencies, but legal limits have only been set for histamine. The present work reports a transcriptomic investigation of the oncogenic potential of the above-mentioned BA, as assessed in the HT29 human intestinal epithelial cell line. Tyramine had a greater effect on the expression of genes involved in tumorigenesis than did histamine or putrescine. Since some of the genes that showed altered expression in tyramine-exposed cells are involved in DNA damage and repair, the effect of this BA on the expression of other genes involved in the DNA damage response was investigated. The results suggest that tyramine might be genotoxic for intestinal cells at concentrations easily found in BA-rich food. Moreover, a role in promoting intestinal cancer cannot be excluded.

Effect of Heat Treatment on the Antitumor Activity of Lactoferrin in Human Colon Tumor (HT29) Model.

To investigate the effect of heat treatment on the antitumor activity of lactoferrin in colon cancer cells and colon tumors, the HT-29 (human intestinal epithelial tumor cell) cell line was exposed to lactoferrin and various heat treatments. The impacts on cell proliferation, invasion, and migration were observed in vitro, and nude mice bearing HT29 tumors were administered lactoferrin and underwent various heat treatments in vivo. In the HT29 cell proliferation test using transwell and scratch analyses, lactoferrin (20 mg/mL) without or with heat treatment (50 and 70 °C) significantly inhibited cell proliferation, migration, and invasion (compared with the control, p < 0.05), while lactoferrin with heat treatment (100 °C) did not affect these parameters. In vivo, HT29 tumor weight was significantly reduced in the lactoferrin (without heat treatment and with 50 and 70 °C treatment) groups (1.59 ± 0.20, 1.67 ± 0.25, and 2.41 ± 0.42 g, compared with the control, p < 0.05), and there was no significant difference between the control (3.73 ± 0.33 g) and the 100 °C treatment group (3.58 ± 0.29 g). Moreover, 100 °C heat treatment reduced inhibition of the VEGFR2/VEGFA/PI3K/Akt/Erk1/2 angiogenesis pathway by lactoferrin. In summary, HT29 tumors were effectively suppressed by lactoferrin via inhibition of VEGFR2/VEGFA/PI3K/Akt/Erk1/2 pathway, and heat treatment affected the antitumor activity of lactoferrin in a temperature-dependent manner.

Ameliorative effect of Alnus japonica ethanol extract on colitis through the inhibition of inflammatory responses and attenuation of intestinal barrier disruption in vivo and in vitro.

Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract caused by high levels of pro-inflammatory cytokines and epithelial barrier dysfunction. Alnus japonica Steud. (Betulaceae) has been used in traditional Asian medicine. However, the potential of A. japonica for the treatment of intestinal inflammation has not been investigated. This study investigated the effects of ethanol extract from A. japonica bark (AJE) on colonic mucosa injury in mice with dextran sodium sulfate (DSS)-induced colitis. Treatment with AJE ameliorated pathological damage and the histopathologic features of DSS-induced colitis. The administration of AJE also inhibits DSS-induced pro-inflammatory cytokines expression, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2. Notably, AJE administration attenuated the reduction of tight junction proteins, zonula occludens (ZO)-1 and occludin, in DSS-induced colitis. In addition, AJE increased heme oxygenase (HO)-1 expression and prevented DSS-induced apoptosis in colonic epithelial cells. Furthermore, in vitro studies demonstrated that AJE inhibits TNF-α-induced IL-8, IL-1β, and COX-2 expression in human intestinal epithelial HT-29 cells and tert-butyl hydroperoxide-induced reduction of ZO-1 and occludin expression in human intestinal epithelial Caco-2 cells. AJE-induced HO-1 protein expression was also found in both HT-29 and Caco-2 cells. Taken together, our findings demonstrated that AJE inhibits intestinal inflammation and protects against intestinal barrier disruption in mice with DSS-induced colitis in vivo and human intestinal epithelial cells in vitro. These results suggest that AJE might have beneficial effects for the treatment of IBD.

Oregonin inhibits inflammation and protects against barrier disruption in intestinal epithelial cells

Background and aimsOregonin, a major diarylheptanoid derivative isolated from Alnus japonica, exerts anti-inflammatory effects; however, little is known about the effect of oregonin in intestinal inflammation. The current study investigated the potential of oregonin for clinical applications in the treatment of inflammatory bowel disease (IBD) and elucidated its underlying molecular mechanisms. MethodsThe anti-inflammatory effect of oregonin in tumor necrosis factor-α (TNF-α)-stimulated human intestinal epithelial HT-29 cells was investigated. In addition, the protective effect of oregonin was determined against disruption of the intestinal barrier in tert-butyl hydroperoxide (t-BH)-stimulated human intestinal epithelial Caco-2 cells. ResultsOregonin suppressed the expression of cyclooxygenase-2 (COX-2), intercellular adhesion molecule-1 (ICAM-1), IL-8, and IL-1β, and inhibited activation of nuclear factor κB (NF-κB) in HT-29 cells stimulated with TNF-α. Oregonin increased heme oxygenase-1 (HO-1) expression through the ERK1/2 and JNK-dependent signaling pathway, which contributed to the oregonin-mediated suppression of COX-2 expression in the HT-29 cells stimulated with TNF-α. Moreover, oregonin induced AMP-activated protein kinase (AMPK) activation. Knockdown of AMPK abolished the induction of HO-1 protein by oregonin and suppression of oregonin-mediated ICAM-1 and COX-2 expression in the HT-29 cells stimulated with TNF-α. Oregonin prevented the t-BH-induced increase in monolayer permeability through inhibition of the reduction in expression of zonula occludens-1 and occludin in Caco-2 cells. Targeting HO-1 by siRNA transfection attenuated the oregonin-mediated prevention of loss of tight junction proteins and increase in permeability. ConclusionThe findings of this study suggest that oregonin is a potential candidate for treatment of IBD by preventing mucosal inflammation and barrier disruption.

Upregulation of aquaporin 3 expression by diterpenoids in Euphorbia pekinensis is associated with activation of the NF-κB signaling pathway in the co-culture system of HT-29 and RAW 264.7 cells

This study was designed to evaluate the toxic effects of diterpenoids separated from the roots of Euphorbia pekinensis, a type of widely used traditional Chinese medicine. This herb has intestinal toxicity associated with its complex diterpenoids. In this study, the diterpenoids (pekinenin A, pekinenin C, pekinenin F, pekinenin G, yuexiandajisu A, (−)-(1S)-15-hydroxy-18-carboxycembrene) elevated the expression of interleukin 1 beta and tumor necrosis factor alpha in a dose-dependent manner at doses of 6.25, 12.5, and 25 μM in RAW264.7 monocultures. Pekinenin C increased the expression of phosphorylated IκB and phosphorylated p65 in RAW264.7 monocultures, indicating that it stimulated a substantial inflammatory response and activated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. A co-culture model of RAW 264.7 mouse macrophage cells and HT-29 human intestinal epithelial cells was established to study the correlation between inflammation and aquaporin (AQP) expression and to evaluate the toxicity of different diterpenoids from E. pekinensis. Pekinenin C (6.25, 12.5, and 25 μM) increased AQP3 mRNA and protein expression of HT-29 cells in the co-culture system in a dose-dependent manner but not in HT-29 monocultures. AQP3 mRNA and protein expression peaked at 2 and 3 h of HT-29 cells in the co-culture system, respectively. In contrast, their expression peaked more slowly in the monoculture system. After the specific NF-κB inhibitor BAY11-7082 (5, 10, and 20 μM) was added to the co-culture system, the release of cytokines and increased AQP3 expression caused by pekinenin C were inhibited. Comparisons of the representative monomeric compound pekinenin C, diterpenoid monomer mixtures, and total diterpenoids from E. pekinensis showed that the monomer mixtures had the most toxicity. In conclusion, this study demonstrated that E. pekinensis induces inflammation and increases the expression of AQP3, causing disorders of water metabolism, which may lead to gastrointestinal side effects such as diarrhea.

Isoliquiritigenin inhibits TNF-α-induced release of high-mobility group box 1 through activation of HDAC in human intestinal epithelial HT-29 cells

The suppression of pro-inflammatory cytokine-induced inflammation responses is an attractive pharmacological target for the development of therapeutic strategies for inflammatory bowel disease (IBD). In the present study, we evaluated the anti-inflammatory properties of flavonoid isoliquiritigenin (ISL) in intestinal epithelial cells and determined its mechanism of action. ISL suppressed the expression of inflammatory molecules, including IL-8, IL-1β and COX-2, in TNF-α-stimulated HT-29 cells. Moreover, ISL induced activation of Nrf2 and expression of its target genes, such as HO-1 and NQO1. ISL also inhibited the TNF-α-induced NF-κB activation in HT-29 cells. High-mobility group box 1 (HMGB1), which is one of the critical mediators of inflammation, is actively secreted from inflammatory cytokine-stimulated immune or non-immune cells. ISL inhibited HMGB1 secretion by preventing TNF-α-stimulated HMGB1 relocation, whereas the RNA and protein expression levels of cellular HMGB1 did not change in response to TNF-α or ISL. Moreover, we found that HMGB1 acetylation was associated with HMGB1 translocation to the cytoplasm and the extracellular release in TNF-α-stimulated HT-29 cells; however, ISL significantly decreased the amount of acetylated HMGB1 in both the cytoplasm and extracellular space of HT-29 cells. Histone deacetylase (HDAC) inhibition by Scriptaid abrogated ISL-induced HDAC activity and reversed the ISL-mediated decrease in acetylated HMGB1 release in TNF-α-stimulated HT-29 cells, suggesting that, at least in TNF-α-stimulated HT-29 cells, ISL suppresses acetylated HMGB1 release via the induction of HDAC activity. Together, the current results suggest that inhibition of HMGB1 release via the induction of HDAC activity using ISL may be a promising therapeutic intervention for IBD.

Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells.

Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene.

Bioactive molecules in the &amp;lt;10kDa fraction of the Lactobacillus helveticus R0389 and Lactobacillus rhamnosus R0011 secretomes down-regulate IL-8 production by HT-29 human intestinal epithelial cells induced by pro-inflammatory stimuli (MUC9P.752)

Abstract The human gut microbiome has recently been associated with maintaining intestinal epithelial cell (IEC) homeostasis at the gastrointestinal interface by modulating host mucosal immune responses through varied mechanisms. The aim of this study was to compare the immunomodulatory activities of the L. helveticus R0389 and L. rhamosus R0011 secretomes on IEC. The human IEC line HT-29 was activated with a range of innate immune stimulants, to allow delineation of differential effects of the secretomes on Interleukin-8 (IL-8) production induced by varied stimuli. HT-29 IEC were co-incubated with either L. helveticus R0389 or L. rhamnosus R0011 secretomes and one of the following pro-inflammatory stimuli: interleukin 1-β (IL-1β), tumor necrosis factor-α (TNF-α); the toll-like receptor (TLR) agonists poly (I:C) (TLR3) or lipopolysaccharide (LPS) (TLR4), and alterations in IL-8 production were measured via enzyme-linked immunosorbant assay (ELISA). The L. helveticus R0389 and L. rhamnosus R0011 secretomes were also subjected to size fractionation to determine the size of the bioactive constituent of the secretome. Down-regulation of IL-8 was observed in HT-29 IEC co-incubated with the &amp;lt;10kDa fraction of the L. helveticus R0389 secretome or the L. rhamnosus R0011 secretome, for all of the innate immune stimulants tested. These results suggest that both Lactobacilli may secrete a low molecular weight bioactive molecule that modulates host immune activity by IEC.

The prophylactic effect of probiotic Bacillus polyfermenticus KU3 against cancer cells

Bacillus polyfermenticus KU3 was isolated from kimchi, a Korean dish made from fermented vegetables and its potential probiotic characteristics were investigated. The spore cell of B. polyfermenticus KU3 was highly resistant to artificial gastric juice and survived for 24 h in artificial bile acid. B. polyfermenticus KU3 did not generate the carcinogenic enzymes, β-glucosidase, N-acetyl-β-glucosaminidase, and β-glucuronidase, and adhered strongly to HT-29 human intestinal epithelial cell lines. Using the [3–4,5-dimethylthiazol-2-yl]-2,5-diphenyletrazolium bromide assay, we found that B. polyfermenticus KU3 strongly inhibited the proliferation of cancer cells such as HeLa, LoVo, HT-29, AGS, and MCF-7 cells. The supernatant of B. polyfermenticus KU3 had an anticancer effect against HeLa and LoVo cells. Conversely, the proliferation of normal MRC-5 cells was not inhibited. We also demonstrated the anti-inflammatory activity of B. polyfermenticus KU3 under inflammatory conditions, as shown by the reduction in nitric oxide and proinflammatory cytokines (TNF-α, IL-10, TGF-β2, and COX-2). These results demonstrate the probiotic characteristics of B. polyfermenticus KU3 and provide evidence for the effect of this bacterium against various cancer cells.

Salmonella enterica Serovar Enteritidis Modulates Intestinal Epithelial miR-128 Levels to Decrease Macrophage Recruitment via Macrophage Colony-Stimulating Factor

The mechanism underlying the ability of virulent Salmonella organisms to escape clearance by macrophages is incompletely understood. Here, we report a novel mechanism by which Salmonella escapes macrophages. Microarray and quantitative real-time polymerase chain reaction analyses were used to screen key microRNAs regulating Salmonella-host cell interactions. Target gene was tested using luciferase reporter and Western blot assays. The role of microRNA 128 (miR-128) was assayed using intestinal epithelial cells and a mouse infection model. The miR-128 level in human intestinal epithelial HT29 cells was strongly increased by infection with strain SE2472, and the elevation in miR-128 levels in mouse intestine and colon tissues correlated with the level of Salmonella infection in mice. Macrophage colony-stimulating factor (M-CSF) was identified as a target of miR-128, and increased miR-128 levels in epithelial cells due to infection with strain SE2472 significantly decreased the level of cell-secreted M-CSF, leading to impaired M-CSF-mediated macrophage recruitment. The secreted proteins from Salmonella were identified as possible effectors to induce miR-128 expression via the p53 signaling pathway. Moreover, intragastric delivery of anti-miR-128 antagomir into mice significantly increased M-CSF-mediated macrophage recruitment and suppressed Salmonella infection. Salmonella can upregulate intestinal epithelial miR-128 expression, which, in turn, decreases levels of epithelial cell-secreted M-CSF and M-CSF-induced macrophage recruitment.

Potential probiotic properties of phytase-producing Lactobacillus salivarius FC113

Probiotics have known efficacy as dietary supplements. Here, Lactobacillus strain F113 was characterized for probiotic use. Strain FC113 was selected as having the highest phytase activity (403.6 U) among tested strains showing acid tolerance and nitrite production. FC113 was tentatively identified as Lactobacillus salivarius based on an API 50 CHL assay and 16S rRNA gene analysis. The production of interleukin (IL)-1α and tumor necrosis factor (TNF)-α was measured in in vitro culture experiments. Cytokine production by L. salivarius FC113 at 1 × 107 CFU/ml was approximately 175.5 ± 36.40 pg/mL IL-1α and 353.5 ± 61.79 pg/mL TNF-α. L. salivarius FC113 was profoundly resistant to artificial gastric juice (pH 2.5, 1 % pepsin), and persisted for 24 h in artificial bile acid. According to results obtained with an API ZYM kit, L. salivarius FC113 did not generate carcinogenic enzymes. L. salivarius F113 had an inhibitory effect on food-borne pathogens, and adhered strongly to HT-29 human intestinal epithelial cell lines. These results show that L. salivarius FC113 has probiotic characteristics, and exhibits high feed bioavailability in the host animal, in addition to an immune-stimulating effect.

Human milk oligosaccharides reduceEntamoeba histolyticaattachment and cytotoxicityin vitro

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infant's intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasite Entamoeba histolytica, as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) and N-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site of E. histolytica infection. The objective of the present study was to investigate whether HMO reduce E. histolytica attachment and cytotoxicity. Our in vitro results show that physiological concentrations of isolated, pooled HMO detach E. histolytica by more than 80 %. In addition, HMO rescue E. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80 % of the HT-29 cells, while HMO with fucose α1-2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolished E. histolytica attachment and cytotoxicity at 8 mg/ml. Although our results need to be confirmed in vivo, they may provide one explanation for why breast-fed infants are at lower risk of E. histolytica infections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents.