Intestinal Epithelial Cell Line HT-29 Research Articles

Protective Responses of the Intestinal Epithelial Cell Line HT-29 Cells Exposed to Dephosphorylated Salmonella Flagellin [RESEARCH NOTE

This study aimed to describe the effects of Salmonella Typhimurium flagellin (SFL) dephosphorylated by sweet potato purple acid phosphatase (PAP) on the protective responses of the intestinal epithelial cell line HT-29 cells. The enzyme was reported to display a broad substrate specificity for various organic phosphorylated conjugates and phosphoproteins. Dephosphorylation of SFL by sweet potato PAP decreased to 35% in the presence of 0.05 mM vanadate as compared with the negative control (p < 0.05). Intact SFL and the SFL treated with sweet potato PAP did not remarkably induce the activation of caspase-3 in HT-29 cells at all the tested levels of the substrate. Intact SFL maximally induced the release of IL (interleukin)-8 in HT-29 cells at 1000 ng/mL (p < 0.05). However, the SFL treated with the enzyme inhibited the release of IL-8 at over 100 ng/mL of the substrate as compared with intact SFL, resulting in an approximately 8-fold decrease even at 1000 ng/mL (p < 0.05). The SFL treated with the enzyme decreased the activation of the total ERK1/2 in the cells to 1.9 and 1.7–fold at 10 and 1000 ng/mL of the substrate, respectively, as compared with intact SFL (p < 0.05). In conclusion, sweet potato PAP could be a promising tool for controlling excessive inflammation during Salmonella infection in animal husbandry, and the enzyme could be a safe alternative that can overcome the drawbacks of chemotherapy.

Synergistic Inhibitory Effect of Lactobacillus Cell Lysates and Butyrate on Poly I:C-Induced IL-8 Production in Human Intestinal Epithelial Cells.

Postbiotics include cell lysates (CLs), enzymes, cell wall fragments, and heat-killed bacteria derived from probiotics. Although postbiotics are increasingly being considered for their potential health-promoting properties, the effects of postbiotics on virus-mediated inflammatory responses in the intestine have not been elucidated. Hence, the present study aimed to examine whether CLs of Lactipantibacillus plantarum (LP CL) and Lacticaseibacillus rhamnosus GG (LR CL) could inhibit virus-mediated inflammatory responses in the human intestinal epithelial cell line HT-29 in vitro. Pretreatment with LP CL and LR CL significantly inhibited interleukin (IL)-8 production, which was induced by poly I:C, a synthetic analog of double-stranded RNA (dsRNA) viruses, at the mRNA and protein levels in HT-29 cells. However, peptidoglycans and heat-killed L. plantarum and L. rhamnosus GG did not effectively inhibit IL-8 production. LP CL and LR CL attenuated the poly I:C-induced phosphorylation of ERK and JNK and the activation of NF-κB, suggesting that these CLs could inhibit poly I:C-induced IL-8 production by regulating intracellular signaling pathways in HT-29 cells. Furthermore, among the short-chain fatty acids, butyrate enhanced the inhibitory effect of CLs on poly I:C-induced IL-8 production at the mRNA and protein levels in HT-29 cells, while acetate and propionate did not. Taken together, these results suggest that both LP CL and LR CL could act as potent effector molecules that can inhibit virus-mediated inflammatory responses and confer synergistic inhibitory effects with butyrate in human intestinal epithelial cells.

P047 Osteopontin in colitis-associated carcinoma (CAC)

Abstract Background Inflammatory bowel disease (IBD) patients pose a lifelong risk of developing colitis-associated carcinoma (CAC). Current understanding and mechanisms of CAC development stem from murine CAC models while studies on human CAC carcinogenesis are scarce. Thus, we aimed to understand CAC carcinogenesis, using human samples from patients suffering from Crohn′s disease (CD), CD-associated CAC (CD-CAC), ulcerative colitis (UC), and UC-associated CAC (UC-CAC) and further validated the findings in vitro. Methods We obtained FFPE specimens from 40 patients that suffered from either one of the IBDs or CACs and furthermore inflammation-free healthy controls (10 patients per group). These samples were micro-dissected, followed by RNA isolation and Nanostring gene expression analysis. Our data revealed a strong upregulation of SPP1 (Secreted Phosphoprotein-1) gene encoding for osteopontin (OPN) upon comparing heathy controls to IBD and to CAC. We then used in vitro-models to elucidate the mechanism of OPN-induced carcinogenesis. We stimulated intestinal epithelial cell lines HT-29/B6 and T84 cells with rhOPN (recombinant human OPN), following bulk RNAseq. We validated the findings using Seahorse assay for analysis of mitochondrial respiration (determining oxygen consumption rate, OCR). Western phospho-blots were done to unravel signal transduction events. Results Nanostring gene expression data revealed the following: (i) SPP1 gene encoding for OPN and epithelial-to-mesenchymal (EMT) transcription factors fibronectin-1 (FN1) and ZEB1 were strongly upregulated in UC-CAC and CD-CAC patients when compared to UC and CD. (ii) IL17-pathway genes were significantly downregulated in UC-CAC patients. (iii) Tight junction and polarity genes were altered in both UC-CAC and CD-CAC patients. rhOPN stimulation in vitro to HT29/B6 and T84 cells excluded OPN as the trigger of EMT but uncovered upregulated mitochondrial respiratory chain genes as a potential trigger for tumour cell proliferation. Validation of RNAseq findings by Seahorse assay revealed that after sequential injections of oligomycin, FCCP, rotenone and antimycin A both, HT-29/B6 and T84 cells, had higher basal and maximal OCRs in response to rhOPN (Fig. 1). T84, but not HT-29/B6 cells also showed an upregulated ATP production in response to rhOPN. Conclusion SPP1 expression correlates with CAC carcinogenesis. Putative underlying pathways rather include modulation of mitochondrial respiratory chain activity by differential gene expression than induction of EMT.

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.

Functional Properties of Dunaliella salina and Its Positive Effect on Probiotics.

The unicellular green microalga Dunaliella is a potential source of a wide range of nutritionally important compounds applicable to the food industry. The aim of this study was to assess the effect of Dunaliella salina dried biomass on the growth and adherence of 10 strains of Lactobacillus, Lacticaseibacillus, and Bifidobacterium. The immunomodulatory, antioxidant, and cytotoxic effects of D. salina on human peripheral mononuclear cells and simulated intestinal epithelial cell lines Caco-2 and HT-29 were evaluated. Furthermore, the hypocholesterolemic effects of the microalgae on lipid metabolism in rats fed a high-fat diet were analyzed. The addition of D. salina biomass had a positive effect on the growth of nine out of 10 probiotics and promoted the adherence of three bifidobacteria strains to human cell lines. The antioxidant and immunomodulatory properties of D. salina were concentration-dependent. The inflammatory cytokines (TNF-α and IL-6) were significantly increased following Dunaliella stimulation at the lowest concentration (0.5% w/v). Eight week supplementation of D. salina to the diet of hypercholesteromic rats significantly decreased the serum concentrations of LDL-C, VLDL, IDL-B, and IDL-C. D. salina is not cytotoxic in intestinal cell models; it promotes adherence of selected bifidobacteria, it affords immunomodulatory and antioxidant effects, and its addition to diets may help decrease atherosclerosis risk factors.

EB1089 promotes the expression of vitamin D receptor in the intestinal epithelial cell line HT-29 and reduces lipopolysaccharide-induced inflammatory response.

BackgroundEB1089 is a vitamin D receptor (VDR) agonist that reduces the inflammatory response. The specific pathway through which the inflammatory response is reduced is unclear, and this study is intended to investigate its effect on the inflammatory response through an LPS (lipopolysaccharide)-induced inflammation model of the intestinal epithelial cell line HT-29.MethodsWe divided HT-29 cells into a control group, LPS group, and LPS + EB1089 group. Cell proliferation was detected with a Cell Counting Kit-8 (CCK-8) kit, and the apoptosis rate was determined through flow cytometry, the lactate dehydrogenase (LDH), interleukin-18 (IL-18), and interleukin-1β (IL-1β) contents were measured with enzyme-linked immunosorbent assay (ELISA), and the expression levels of ASC, Caspase-1, NLRP3, VDR, TLR4, MYD88, NF-κB and other proteins in the cells were detected by western blot.ResultsPretreatment with EB1089 pretreatment reduced the LPS-induced apoptosis of HT-29 cells. LDH content was evidently lower in the LPS + EB1089 group than in the LPS group. The LDH content in the LPS + EB1089 group was evidently lower than that in the LPS group. The protein expression levels of ASC, Caspase-1, NLRP3, TLR4, MyD88, and NF-κB in the LPS group were evidently increased compared with those in the control group; however, the protein expression of VDR evidently decreased. The protein expression levels of Caspase-1, NLRP3, TLR4, MyD88, and NF-κB in the LPS + EB1089 group were evidently lower than those in the LPS group; however, the VDR protein expression evidently increased in the LPS + EB1089 group.ConclusionsEB1089 promoted the VDR expression and reduced the LPS induced inflammation in HT-29 cells.

Description of a Newly Isolated Blautia faecis Strain and Its Benefit in Mouse Models of Post-Influenza Secondary Enteric and Pulmonary Infections.

The expanding knowledge on the systemic influence of the human microbiome suggests that fecal samples are underexploited sources of new beneficial strains for extra-intestinal health. We have recently shown that acetate, a main circulating microbiota-derived molecule, reduces the deleterious effects of pulmonary Streptococcus pneumoniae and enteric Salmonella enterica serovar Typhimurium bacterial post-influenza superinfections. Considering the beneficial and broad effects of acetate, we intended to isolate a commensal strain, producing acetate and potentially exploitable in the context of respiratory infections. We designed successive steps to select intestinal commensals that are extremely oxygen-sensitive, cultivable after a freezing process, without a proinflammatory effect on IL-8 induction, and producing acetate. We have identified the Blautia faecis DSM33383 strain, which decreased the TNFα-induced production of IL-8 by the intestinal epithelial cell line HT-29. The beneficial effect of this bacterial strain was further studied in two preclinical models of post-influenza Streptococcus pneumoniae (S.p) and Salmonella enterica serovar Typhimurium (S.t) superinfection. The intragastrical administration of Blautia faecis DSM33383 led to protection in influenza-infected mice suffering from an S.p. and, to a lesser extent, from an S.t secondary infection. Altogether, this study showed that Blautia faecis DSM33383 could be a promising candidate for preventive management of respiratory infectious diseases.

LncRNA NEAT1 mediates intestinal inflammation by regulating TNFRSF1B.

BackgroundInflammatory bowel disease (IBD) is a chronic nonspecific intestinal disease. Our previous work showed that long non-coding RNA (LncRNA) nuclear enriched abundant transcript 1 (NEAT1) plays an important role in IBD. In the current study, we aimed to explore the underlying mechanism by which NEAT1 participates in the development of the disease.MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression of NEAT1 and tumor necrosis factor superfamily member 1B (TNFRSF1B) in clinical specimens and dextran sulfate sodium (DSS) colitis mice. Inflammatory cell models were established by stimulating human normal intestinal epithelial cell line NCM460 and human colon cancer cell line HT-29 with tumor necrosis factor alpha (TNF-α). Expressions of inflammatory cytokines such as interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were detected by enzyme-linked immunosorbent assay (ELISA) or RT-qPCR, TNFRSF1B, NF-κB p65 and p-NF-κB p65 followed by the knockdown or overexpression of NEAT1 and TNFRSF1B were analyzed by western blotting, and the regulatory effects of NEAT1 on TNFRSF1B were detected by RNA pull-down experiments and RNA-decay assay. The translocation of NF-κB p65 to the nucleus was detected by immunofluorescence.ResultsIn patients’ specimens and DSS colitis mouse models, NEAT1 and TNFRSF1B expression were up-regulated compared with the control group. TNF-α stimulation increased NEAT1 and TNFRSF1B expression and activated NF-κB signaling pathway by increasing the translocation of NF-κB p65 to the nucleus. In the presence of TNF-α stimulation, NEAT1 knockdown reduces the expression of TNFRSF1B and the translocation of NF-κB p65, thereby relieves cell inflammation. These effects can be reversed by the overexpression of TNFRSF1B.In addition, NEAT1 is involved in inflammatory response by up-regulating the mRNA levels of TNFRSF1B, and knocking down NEAT1 can alleviate inflammation by down-regulating TNFRSF1B. Moreover, NEAT1 co-precipitates TNFRSF1B mRNA in RNA-pulldown assay, and the presence of NEAT1 stabilizes the mRNA of TNFRSF1B.ConclusionsOur results showed that LncRNA NEAT1 promotes NF-κB p65 translocation and mediates intestinal inflammation by regulating TNFRSF1B.

Intracellular Transport of Ribosome-Inactivating Proteins Depends on Annexin 13.

In the present study, we assessed the role of annexin 13 membrane-binding protein (ANXA13) in the intracellular transport of vesicles containing type II ribosome-inactivating proteins (RIP-IIs). A modified human intestinal epithelial cell line HT29 was used, in which the expression of ANXA13 was significantly reduced. The cytotoxic effect of ricin and viscumin was evaluated by modification of 28S ribosome RNA. The observed differences in the activity of toxins on the parental and modified HT29 lines indicate that ANXA13 plays a different role in the intracellular transport of vesicles containing the RIP-IIs.

Effects of Aluminum on the Integrity of the Intestinal Epithelium: An in Vitro and in Vivo Study.

Background:Aluminum (Al) is the most abundant and ubiquitous metal in the environment. The main route of human exposure to Al is through food and water intake. Although human exposure to Al is common, the influence of Al on the gastrointestinal tract remains poorly understood.Objectives:We aimed to further understand the toxic effect of Al and to elucidate the underlying cellular mechanisms in the intestinal barrier.Methods:The human intestinal epithelial cell line HT-29 and C57BL6 mice were exposed to at () and body weight (13 weeks), respectively. In cell culture experiments, intracellular oxidative stress, inflammatory protein and gene expression, and intestinal epithelial permeability were measured. In animal studies, histological examination, gene expression, and myeloperoxidase (MPO) activity assays were conducted.Results:Cellular oxidative stress level (superoxide production) in -treated cells (, ) was approximately 38-fold higher than that of the control. Both protein and mRNA expression of tight junction (TJ) components (occludin and claudin-1) in -treated cells (, ) was significantly lower than that of the control. Transepithelial electrical resistance (TEER) decreased up to 67% in -treated cells (, ) compared with that of the control, which decreased approximately 7%. Al activated extracellular signal-regulated kinase 1/2 and nuclear factor-kappa B (), resulting in mRNA expression of matrix metalloproteinase-9, myosin light-chain kinase, and inflammatory cytokines [tumor necrosis factor alpha (), (), and IL-6] in HT-29 cells. Moreover, oral administration of to mice induced pathological alteration, MPO activation, and inflammatory cytokine (, , and IL-6) production in the colon.Conclusion:Al induced epithelial barrier dysfunction and inflammation via generation of oxidative stress, down-regulation of the TJ proteins, and production of inflammatory cytokines in HT-29 cells. In addition, Al induced toxicity in the colon by increasing the levels of inflammatory cytokines and MPO activity and induced histological damage in a mouse model. Our data suggest that Al may be a potential risk factor for human intestinal diseases. https://doi.org/10.1289/EHP5701

Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.

Listeria monocytogenes cells under nutrient deprivation showed reduced ability to infect the human intestinal cell line HT-29.

This study aimed to evaluate the effect of two types of stress, cold and nutritional, on the viability and the in vitro virulence of the foodborne pathogenic bacteria Listeria monocytogenes. Ten diverse isolates were kept in phosphate-buffered saline (PBS) at optimal (37 °C) or at refrigeration temperature (7 °C), for 1 and 7 days. The viability of the cells [log colony-forming units (c.f.u.)/ml] and their in vitro virulence, before and after storage in these conditions, were investigated. In vitro virulence (log PFA) was evaluated using the human intestinal epithelial cell line HT-29 in plaque-forming assays (PFAs).Results/Key findings. In general, when compared with the conditions at 37 °C, the exposure at 7 °C for 7 days seemed to increase the resistance of the isolates to nutritional stress. Nutritional stress per se acted significantly to decrease the in vitro virulence of the isolates. After 7 days of nutrient deprivation, whether at optimal or at refrigeration temperature, the majority of the isolates assumed a low-virulence phenotype. Our results suggest that when L. monocytogenes are in refrigerated post-processing environments that are unable to support their growth they may increase their resistance to nutritional stress and may decrease their virulence. This should be considered when performing risk assessments for refrigerated ready-to-eat (RTE) foods.

Homology modeling and in silico prediction of Ulcerative colitis associated polymorphisms of NOD1

Cytosolic pattern recognition receptors play key roles in innate immune response. Nucleotide binding and oligomerisation domain containing protein 1 (NOD1) belonging to the Nod-like receptor C (NLRC) sub-family of Nod-like receptors (NLRs) is important for detection and clearance of intra-cellular Gram negative bacteria. NOD1 is involved in activation of pro-inflammatory pathways. Limited structural data is available for NOD1. Using different templates for each domain of NOD1, we determined the full-length homology model of NOD1. ADP binding amino acids within the nucleotide binding domain (NBD) of NOD1 were also predicted. Key residues in inter-domain interaction were identified by sequence comparison with Oryctolagus cuniculus NOD2, a related protein. Interactions between NBD and winged helix domain (WHD) were found to be conserved in NOD1. Functional and structural effect of single nucleotide polymorphisms within the NOD1 NBD domain associated with susceptibility risk to Ulcerative colitis (UC), an inflammatory disorder of the colon was evaluated by in silico studies. Mutations W219R and L349P were predicted to be damaging and disease associated by prediction programs SIFT, PolyPhen2, PANTHER, SNP&GO, PhD SNP and SNAP2. We further validated the effect of W219R and L349P mutation on NOD1 function in vitro. Elevated mRNA expression of pro-inflammatory cytokines IL8 and IL-1β was seen as compared to the wild type NOD1 in intestinal epithelial cell line HT29 when stimulated with NOD1 ligand. Thus, these mutations may indeed have a bearing on pathogenesis of inflammation during UC.

Chromosome region maintenance-1 (CRM1) regulates apoptosis of intestinal epithelial cells via p27kip1 in Crohn's disease

To investigate the role of chromosome region maintenance-1 (CRM1) in Crohn's disease (CD) and its potential pathological mechanisms. The expression and distribution of CRM1 in mucosal biopsies from patients with active CD and normal controls were detected by immunohistochemistry (IHC). We established a murine model of acute colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Western blot was performed to investigate the expression levels of CRM1, apoptotic markers (active caspase-3 and cleaved PARP), p27kip1 and p-p27ser10. IHC was performed to evaluate the distribution of CRM1, and double immunofluorescence (IF) was performed to evaluate the co-localization of CRM1 and active capase-3. Cells of the human intestinal epithelial cell line HT-29 were incubated with tumor necrosis factor-α (TNF-α) to establish an apoptotic in vitro model. Western blot was performed to determine the expression levels of CRM1, active caspase-3, cleaved PARP and p-p27ser10. Cytoplasmic and nuclear extracts were assessed to examine the translocation of CRM1. The interaction between CRM1 and p27kip1 was assessed by co-immunoprecipitation (co-IP) assays. Furthermore, we used small interfering RNA (siRNA) to knock down the protein expression of CRM1 in HT-29 cells and then measured the expression of active caspase-3, cleaved PARP and p-p27ser10. Flow cytometry was used to determine the effect of CRM1 on intestinal epithelial cell (IEC) apoptosis. We observed up-regulation of CRM1 accompanied by elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and p-p27ser10 in IECs of patients with active CD and in TNBS-induced colitis model cells. However, the expression of p27kip1 was negatively correlated with the expression patterns of CRM1, p-p27ser10 and apoptotic biochemical markers. Co-localization of CRM1 and active caspase-3 in IECs of the TNBS group further indicated the possible involvement of CRM1 in IEC apoptosis. By employing TNF-α-treated HT-29 cells as an in vitro IEC apoptosis model, we found that the expression levels of CRM1 and p-p27ser10 were in accordance with active caspase-3 and cleaved PARP. In addition, immunoprecipitation confirmed the physical interaction between CRM1 and p27kip1. siRNA knockdown of CRM1 significantly inhibited the phosphorylation of p27kip1 and the expression of active caspase-3 and cleaved PARP. In addition, flow cytometry analysis also showed that silencing CRM1 by siRNA inhibited TNF-α-induced cellular apoptosis in HT-29 cells. Up-regulated CRM1 may facilitate IEC apoptosis possibly through p27kip1 in CD, indicating an important role of CRM1 in the pathophysiology of CD.

P102 Hypoxia reduces inflammation through the downregulation of NLRP3/mTOR signaling and the activation of autophagy

Background: The impact of hypoxia on inflammatory bowel disease (IBD) is controversial with studies showing detrimental and protective effects. Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation that have been associated to the development of IBD. We investigated the functional impact of hypoxia on the development of colitis with special emphasis on autophagy and NLRP3 regulation. Methods: Healthy volunteers and patients with IBD, as well as wild-type, interleukin (IL)-10−/−, NLRP3−/− and IL-10−/− NLRP3−/− double knockout mice were subjected to hypoxia and changes in inflammatory signaling and gene expression were analyzed in colon biopsies using RT-qPCR and Western blotting. The effects of hypoxia on autophagy, NLRP3 regulation and inflammation were further assessed in vitro using the intestinal epithelial cell line HT-29. Results: Hypoxia significantly reduced tumor necrosis factor α, IL-6 and NLRP3 expression and increased autophagy gene expression in colon biopsies of patients with Crohn's disease. In normoxia, IL-10−/−, but not IL-10−/− Nlrp3−/− mice presented an accumulation of autophagy proteins and an increase in NF-κB activation and inflammatory cytokine expression, which was significantly reduced under hypoxia. In vitro, hypoxia-induced autophagy downregulated NF-κB signaling. Hypoxia also reduced NLRP3 expression, and silencing of NLRP3 activated autophagy following the dephosphorylation of mammalian target of rapamycin (mTOR). Co-immunoprecipitation experiments identified NLRP3 as a novel binding partner of mTOR. Conclusions: Our results show that hypoxia counteracts inflammation through a novel mechanism involving the downregulation of the binding partner of mTOR NLRP3 and subsequent activation of autophagy.

Effects of lactoferrin on the production of interferon-λ by the human intestinal epithelial cell line HT-29.

We examined the in-vitro effects of bovine lactoferrin (LF) on the production of interferon-λ (IFN-λ), an antiviral cytokine important for the defense of enterocytes, using the human intestinal epithelial cell line HT-29. HT-29 cell cultures were treated with LF for 1 h, and the cultures were stimulated with polyinosinic-polycytidylic acid (poly I:C). LF increased the concentration of IFN-λ in the culture supernatant after stimulation in a dose-dependent manner. A similar increase in the concentration of IFN-λ was observed in the supernatant of cells washed between treatment with LF and stimulation with poly I:C. At 6 and 24 h after stimulation with poly I:C (early and late phases, respectively) treated cultures contained significantly higher concentrations of IFN-λ1 in the culture supernatant, and significantly higher IFN-λ1 and IFN-λ2 mRNA levels, than controls. These results suggest that LF activates the innate cellular immunity of the enterocytes to double-stranded RNA and increases the production of IFN-λ.

Screening of Bifidobacteria and Lactobacilli Able to Antagonize the Cytotoxic Effect of Clostridium difficile upon Intestinal Epithelial HT29 Monolayer.

Clostridium difficile is an opportunistic pathogen inhabiting the human gut, often being the aetiological agent of infections after a microbiota dysbiosis following, for example, an antibiotic treatment. C. difficile infections (CDI) constitute a growing health problem with increasing rates of morbidity and mortality at groups of risk, such as elderly and hospitalized patients, but also in populations traditionally considered low-risk. This could be related to the occurrence of virulent strains which, among other factors, have high-level of resistance to fluoroquinolones, more efficient sporulation and markedly high toxin production. Several novel intervention strategies against CDI are currently under study, such as the use of probiotics to counteract the growth and/or toxigenic activity of C. difficile. In this work, we have analyzed the capability of twenty Bifidobacterium and Lactobacillus strains, from human intestinal origin, to counteract the toxic effect of C. difficile LMG21717 upon the human intestinal epithelial cell line HT29. For this purpose, we incubated the bacteria together with toxigenic supernatants obtained from C. difficile. After this co-incubation new supernatants were collected in order to quantify the remnant A and B toxins, as well as to determine their residual toxic effect upon HT29 monolayers. To this end, the real time cell analyser (RTCA) model, recently developed in our group to monitor C. difficile toxic effect, was used. Results obtained showed that strains of Bifidobacterium longum and B. breve were able to reduce the toxic effect of the pathogen upon HT29, the RTCA normalized cell-index values being inversely correlated with the amount of remnant toxin in the supernatant. The strain B. longum IPLA20022 showed the highest ability to counteract the cytotoxic effect of C. difficile acting directly against the toxin, also having the highest capability for removing the toxins from the clostridial toxigenic supernatant. Image analysis showed that this strain prevents HT29 cell rounding; this was achieved by preserving the F-actin microstructure and tight-junctions between adjacent cells, thus keeping the typical epithelium-like morphology. Besides, preliminary evidence showed that the viability of B. longum IPLA20022 is needed to exert the protective effect and that secreted factors seems to have anti-toxin activity.

K+ Channel Inhibition Differentially Regulates Migration of Intestinal Epithelial Cells in Inflamed vs. Non-Inflamed Conditions in a PI3K/Akt-Mediated Manner.

BackgroundPotassium channels have been shown to determine wound healing in different tissues, but their role in intestinal epithelial restitution–the rapid closure of superficial wounds by intestinal epithelial cells (IEC)–remains unclear.MethodsIn this study, the regulation of IEC migration by potassium channel modulation was explored with and without additional epidermal growth factor (EGF) under baseline and interferon-γ (IFN-γ)-pretreated conditions in scratch assays and Boyden chamber assays using the intestinal epithelial cell lines IEC-18 and HT-29. To identify possibly involved subcellular pathways, Western Blot (WB)-analysis of ERK and Akt phosphorylation was conducted and PI3K and ERK inhibitors were used in scratch assays. Furthermore, mRNA-levels of the potassium channel KCNN4 were determined in IEC from patients suffering from inflammatory bowel diseases (IBD).ResultsInhibition of Ca2+-dependent potassium channels significantly increased intestinal epithelial restitution, which could not be further promoted by additional EGF. In contrast, inhibition of KCNN4 after pretreatment with IFN-γ led to decreased or unaffected migration. This effect was abolished by EGF. Changes in Akt, but not in ERK phosphorylation strongly correlated with these findings and PI3K but not ERK inhibition abrogated the effect of KCNN4 inhibition. Levels of KCNN4 mRNA were higher in samples from IBD patients compared with controls.ConclusionsTaken together, we demonstrate that inhibition of KCNN4 differentially regulates IEC migration in IFN-γ-pretreated vs. non pretreated conditions. Moreover, our data propose that the PI3K signaling cascade is responsible for this differential regulation. Therefore, we present a cellular model that contributes new aspects to epithelial barrier dysfunction in chronic intestinal inflammation, resulting in propagation of inflammation and symptoms like ulcers or diarrhea.

Allyl methyl disulfide inhibits IL-8 and IP-10 secretion in intestinal epithelial cells via the NF-кB signaling pathway.

Garlic and its active constituents have shown versatile medicinal activities in the prevention and treatment of various disorders. Allyl methyl disulfide (AMDS) was identified as one of the major bioactive components in an effective inhalation fork remedy using fresh garlic paste in our previous study. In this work, we investigated the immunological properties of AMDS to elucidate the underlying mechanisms of the fork inhalation treatment using fresh garlic. The inhibition effect of AMDS on TNF-α-induced IL-8 and IP-10 production in intestinal epithelial cell lines HT-29 and Caco-2 was first evaluated. Pretreatment of the cells with AMDS attenuated IL-8 and IP-10 secretion induced by TNF-α in a dose-dependent manner in the non-cytotoxic concentration range of 20 to 150 μM. Mechanistic studies revealed that AMDS suppressed the accumulation of IL-8 mRNA and inhibited IкBα degradation and NF-кB p65 translocation into the nucleus at both the transcriptional and translational levels, suggesting that the attenuation effort of AMDS on cytokine IL-8 secretion might at least be partially related to the NF-κB signaling pathway. These results suggest that AMDS may be a promising phytochemical agent in the treatment of immunological disorders, such as ulcerative colitis, Crohn's disease, intestinal inflammatory diseases and others. In addition, the mechanistic study data indicated that immune modulation could be one of the therapeutic mechanisms of the effective fork treatment containing AMDS as one of the major components.

Bioactive molecules in the <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 <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.