interleukin-8 and deoxynivalenol

Deoxynivalenol (DON) is the most prevalent mycotoxin in human food and is ubiquitously detected in human bodyfluids. DON leads to intestinal barrier dysfunction, as observed from animal- and cell culture models with the known disadvantages. Here we present the effects of DON in a gut-on-a-chip model, as the first study incorporating the effects of intestinal flow. Using the OrganoPlate 3-lane, Caco-2 cells were seeded against an extracellular matrix (ECM) and formed leak tight tubules. DON was then applied in different concentrations (3 μM to 300 μM) via the apical or the basolateral channel. Permeability was assessed using continuous TEER and barrier integrity assays (BIA). Zonulin-1, toxicity (LDH) and proinflammatory status (IL-8) was analyzed. DON exposure led to a dose dependent decrease in para-and transcellular barrier integrity, which was more sensitive to basal than apical application (route). Timelaps/Continuous TEER measurements however revealed bidirectional effects, with even TEER-inducing effects of lower concentrations (until 10 μM). IL-8 secretion into luminal supernatants was only induced by apical DON. Attributed to the flow, the barrier-disintegrating effects of DON start at higher concentrations than in other culture models. The barrier was more sensitive to basolateral DON, even though DON had to pass the ECM; and IL-8 secretion was independent of TEER-alterations. Thus, the gut-on-a chip model might be a good alternative to further characterize the bidirectional effects of DON with reasonable throughput incorporating flow.

Topics: Animals; Caco-2 Cells; Humans; Interleukin-8; Intestinal Mucosa; Lab-On-A-Chip Devices; Mycotoxins

Deoxynivalenol (DON), one of the most abundant mycotoxins in cereal products, was recently detected with other mycotoxins and the emetic bacterial toxin cereulide (CER) in maize porridge. Within a cereal-based diet, co-exposure to these toxins is likely, hence raising the question of combinatory toxicological effects. While the toxicological evaluation of DON has quite progressed, consequences of chronic, low-dose CER exposure are still insufficiently explored. Information about the combinatory toxicological effects of these toxins is lacking. In the present study, we investigated how CER (0.1-100 ng/mL) and DON (0.01-10 µg/mL) alone and in a constant ratio of 1:100 (CER:DON) affect the cytotoxicity and immune response of differentiated human intestinal Caco-2 cells. While DON alone reduced cell viability only in the highest concentration (10 µg/mL), CER caused severe cytotoxicity upon prolonged incubation (starting from 10 ng/mL after 24 h and 48 h, 2.5 ng/mL and higher after 72 h). After 72 h, synergistic effects were observed at 2.5 ng/mL CER and 0.25 µg/mL DON. Different endpoints of inflammation were investigated in interleukin-1β-stimulated Caco-2 cells. Notably, DON-induced interleukin-8 transcription and secretion were diminished by the presence of 10 and 25 ng/mL CER after short-term (5 h) incubation, indicating immunosuppressive properties. We hypothesise that habitual consumption of cereal-based foods co-contaminated with CER and DON may cause synergistic cytotoxic effects and an altered immune response in the human intestine. Therefore, further research concerning effects of co-occurring bacterial toxins and mycotoxins on the impairment of intestinal barrier integrity, intestinal inflammation and the promotion of malnutrition is needed.

Topics: Caco-2 Cells; Cell Survival; Depsipeptides; Diet; Emetics; Food Contamination; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Intestinal Mucosa; Intestines; Mycotoxins; Trichothecenes

Extensive research has revealed the association of continued oxidative stress with chronic inflammation, which could subsequently affect many different chronic diseases. The mycotoxin deoxynivalenol (DON) frequently contaminates cereals crops worldwide, and are a public health concern since DON ingestion may result in persistent intestinal inflammation. There has also been considerable attention over the potential of DON to provoke oxidative stress. In this study, the cytoprotective effect of Schisandrin A (Sch A), one of the most abundant active dibenzocyclooctadiene lignans in the fruit of Schisandra chinensis (Turcz.) Baill (also known as Chinese magnolia-vine), was investigated in HT-29 cells against DON-induced cytotoxicity, oxidative stress and inflammation. Sch A appeared to protect against DON-induced cytotoxicity in HT-29 cells, and significantly lessened the DON-stimulated intracellular reactive oxygen species and nitrogen oxidative species production. Furthermore, Sch A lowered DON-induced catalase, superoxide dismutase and glutathione peroxidase antioxidant enzyme activities but maintains glutathione S transferase activity and glutathione levels. Mechanistic studies suggest that Sch A reduced DON-induced oxidative stress by down-regulating heme oxygenase-1 expression via nuclear factor (erythroid-derived 2)-like 2 signalling pathway. In addition, Sch A decreased the DON-induced cyclooxygenase-2 expression and prostaglandin E2 production and pro-inflammatory cytokine interleukin 8 expression and secretion. This may be mediated by preventing DON-induced translocation of nuclear factor-κB, as well as activation of mitogen-activated protein kinases pathways. In the light of these findings, we concluded that Sch A exerted a cytoprotective role in DON-induced toxicity in vitro, and it would be valuable to examine in vivo effects.

Topics: Catalase; Cell Cycle Checkpoints; Cell Death; Cell Nucleus; Cell Survival; Cyclooctanes; Cyclooxygenase 2; Cytoprotection; Dinoprostone; Enterocytes; Gene Expression Regulation; Glutathione Peroxidase; Heme Oxygenase-1; HT29 Cells; Humans; Inflammation; Inflammation Mediators; Interleukin-8; Lignans; Lipid Peroxidation; MAP Kinase Signaling System; Models, Biological; NF-E2-Related Factor 2; NF-kappa B; Nitrites; Oxidative Stress; Polycyclic Compounds; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase; Trichothecenes

Adlay is a cereal crop that has long been used as traditional herbal medicine and as a highly nourishing food. However, deoxynivalenol (DON), the most prevalent trichothecene mycotoxin worldwide, frequently spoils grains, including adlay,

Topics: Animals; Cell Line; Cell Proliferation; Chemokines; Diet; ELAV-Like Protein 1; Enterocytes; HT29 Cells; Humans; Inflammation; Interleukin-8; Plant Extracts; Poaceae; Protein Kinase C; rhoA GTP-Binding Protein; Signal Transduction; Swine; Trichothecenes

Topics: Cell Line; Cell Survival; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Respiratory System; Trichothecenes

Caco-2 monolayers were pretreated (24 h) with or without specific oligosaccharides or DP fractions of VGOS (DP2 to DP6) before being exposed for 12 or 24 h to the fungal toxin deoxynivalenol (DON). Transepithelial electrical resistance and lucifer yellow permeability were measured to investigate barrier integrity. A calcium switch assay was used to study the reassembly of tight junction proteins. Release of CXCL8, a typical marker for inflammation, was quantified by ELISA.. In comparison with PGOS, FOS and inulin, VGOS showed the most pronounced protective effect on the DON-induced impairment of the monolayer integrity, acceleration of the tight junction reassembly and the subsequent CXCL8 release. DP2 and DP3 in concentrations occurring in VGOS prevented the DON-induced epithelial barrier disruption, which could be related to their high prevalence in VGOS. However, no effects of the separate DP GOS fractions were observed on CXCL8 release.. This comparative study demonstrates the direct, microbiota-independent effects of oligosaccharides on the intestinal barrier function and shows the differences between individual galacto- and fructo-oligosaccharides. This microbiota-independent effect of oligosaccharides depends on the oligosaccharide structure, DP length and concentration.

Topics: Caco-2 Cells; Epithelial Cells; Gastrointestinal Microbiome; Humans; Interleukin-8; Intestines; Inulin; Oligosaccharides; Structure-Activity Relationship; Trichothecenes

Deoxynivalenol (DON), the most prevalent mycotoxin worldwide, leads to economic losses for animal food production. Swine is a most sensitive domestic animal to DON due to rapid absorption and low detoxification by gut microbiota. Specifically, DON can severely damage pig intestinal tissue by disrupting the intestinal barrier and inducing inflammatory responses. We evaluated the effects of several mycotoxin detoxifiers including bentonites, yeast cell wall components, and mixture-typed detoxifier composed of mineral, microorganisms, and phytogenic substances on DON-insulted intestinal barrier and pro-inflammatory responses using in vitro porcine enterocyte culture model. DON-induced disruption of the in vitro gut barrier was attenuated by all three mycotoxin detoxifiers in dose-dependent manners. These mycotoxin detoxifiers also suppressed DON-induced pro-inflammatory chemokine expression to different degrees, which was mediated by downregulation of mitogen-activated kinases and early growth response-1. Of note, the mixture-typed detoxifier was the most prominent mitigating agent at the cellular levels whereas the high dose of bentonite clay also had suppressive action against DON-induced pro-inflammatory insult. The in vitro porcine enterocyte-based assessment of intestinal barrier integrity and inflammatory signals provides sensitive and simplified alternative bioassay of feed additives such as detoxifiers against enteropathogenic mycotoxins with comprehensive mechanistic confirmation.

Topics: Animal Feed; Animals; Bentonite; Cell Line; Cell Wall; Chemokine CCL2; Diatomaceous Earth; Enterocytes; Food Contamination; Interleukin-8; Swine; Trichothecenes; Yeasts

Considering the diverse toxic effects of the

Topics: Caco-2 Cells; Cell Survival; Epithelial Cells; Humans; Interleukin-8; L-Lactate Dehydrogenase; Oligosaccharides; Trichothecenes

The integrity of the epithelial layer in the gastrointestinal tract protects organisms from exposure to luminal antigens, which are considered the primary cause of chronic intestinal inflammation and allergic responses. The common wheat-associated fungal toxin deoxynivalenol acts as a specific disruptor of the intestinal tight junction network and hence might contribute to the pathogenesis of inflammatory bowel diseases.. The aim of the current study was to assess whether defined galacto-oligosaccharides (GOSs) can prevent deoxynivalenol-induced epithelial dysfunction.. Human epithelial intestinal Caco-2 cells, pretreated with different concentrations of GOSs (0.5%, 1%, and 2%) for 24 h, were stimulated with 4.2-μM deoxynivalenol (24 h), and 6/7-wk-old male B6C3F1 mice were fed a diet supplemented with 1% GOSs for 2 wk before being orally exposed to deoxynivalenol (25 mg/kg body weight, 6 h). Barrier integrity was determined by measuring transepithelial electrical resistance (TEER) and intestinal permeability to marker molecules. A calcium switch assay was conducted to study the assembly of epithelial tight junction proteins. Alterations in tight junction and cytokine expression were assessed by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, or ELISA, and their localization was visualized by immunofluorescence microscopy. Sections of the proximal and distal small intestine were stained with hematoxylin/eosin for histomorphometric analysis.. The in vitro data showed that medium supplemented with 2% GOSs improved tight junction assembly reaching an acceleration of 85% after 6 h (P < 0.05). In turn, GOSs prevented the deoxynivalenol-induced loss of epithelial barrier function as measured by TEER (114% of control), and paracellular flux of Lucifer yellow (82.7% of prechallenge values, P < 0.05). Moreover, GOSs stabilized the expression and cellular distribution of claudin3 and suppressed by >50% the deoxynivalenol-induced synthesis and release of interleukin-8 [IL8/chemokine CXC motif ligand (CXCL8)] (P < 0.05). In mice, GOSs prevented the deoxynivalenol-induced mRNA overexpression of claudin3 (P = 0.022) and CXCL8 homolog keratinocyte hemoattractant (Kc) (Cxcl1) (P = 0.06) as well as the deoxynivalenol-induced morphologic defects.. The results demonstrate that GOSs stimulate the tight junction assembly and in turn mitigate the deleterious effects of deoxynivalenol on the intestinal barrier of Caco-2 cells and on villus architecture of B6C3F1 mice.

Topics: Animals; Caco-2 Cells; Claudin-3; Epithelial Cells; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Male; Mice; Oligosaccharides; Permeability; Tight Junctions; Trichothecenes

The presence of deoxynivalenol (DON), a mycotoxin produced by Fusarium species, has been reported worldwide in food and feedstuffs. Even though oral intake is the main route of exposure, DON inhalation is also of concern in workers and exposed population. Particulate matter (PM) is one of the most important causes of air quality detriment and it induces several adverse health effects. Therefore it is of primary importance to understand possible combined effects of DON and PM. The alveolar type II, A549, and the bronchial epithelial, BEAS-2B, cell lines were exposed for 24 h to different concentrations of DON (10-1000 ng/ml), PM10 (5 μg/cm(2), sampled in summer or winter season), and a combination of these pollutants. Cell death, interleukins release and cell cycle alteration were analysed; protein array technique was also applied to evaluate proteins activation related to MAP-kinases cascade. Our results demonstrate that low doses of PM and DON used alone have scarce toxic effects, while induce cytotoxicity and inflammation when used in combination. This observation outlines the importance of investigation on the combined effects of air pollutants for their possible outcomes on human health.

Topics: Air Pollutants; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Lung; Particulate Matter; Seasons; Trichothecenes

The porcine intestinal epithelium is a primary target for mycotoxin deoxynivalenol (DON) and lipopolysaccharides (LPS). Although epithelial cells are exposed to these toxins mainly from the luminal-chyme compartment an exposure from the blood side resulting from systemic absorption cannot be excluded. Thus, we investigated the effect of DON and LPS, alone or combined, on porcine intestinal epithelial cells IPEC-J2 on a transcriptional, translational and functional level when administered either from apical or basolateral. IPEC-J2 cells were cultured on 12-well inserts in complete medium at 5% CO2 and 39°C and subjected to following treatments: control (CON), 2000 ng/mL DON, 1 μg/mL LPS or DON+LPS for 72 h, either from apical or basolateral. Transepithelial electrical resistance (TEER), protein and IL-8 content were measured and microarray analysis, qRT-PCR (IL-8, zonula occludens-1 ZO-1, β-actin), Western Blot (ZO-1, β-actin) and immunofluorescence (ZO-1) were performed. Data of at least three independent experiments were analysed with ANOVA and Dunnett's post hoc test. Basolateral DON resulted in significantly lower cell counts (p<0.05) with larger cells (p<0.01), whereas apical DON reduced total (p<0.001) and specific protein content (IL-8 content CON vs. DON: 2378 pg/3 mL vs. 991 pg/3 mL; p<0.001). Transcripts of ß-actin and ZO-1 were significantly upregulated in response to DON, irrespective of direction, whereas IL-8 mRNA remained unaffected. However, ZO-1 spatial distribution in the tight junction and its function (TEER) were detrimentally affected by basolateral DON only. In conclusion, direction of DON exposure affected IPEC-J2 differently on a translational and functional level, but was mainly inconsequential on a transcriptional level.

Topics: Actins; Animals; Cell Count; Cells, Cultured; Epithelial Cells; Escherichia coli; Interleukin-8; Intestines; Lipopolysaccharides; Swine; Tight Junctions; Trichothecenes; Up-Regulation; Zonula Occludens-1 Protein

Macrocyclic trichothecenes have been frequently detected in fungi in water-damaged buildings and exhibited higher toxicity than the well-studied trichothecenes; however, the mechanism underlying their toxicity has been poorly understood. In this study, transcriptional regulation of the cytokine interleukin (IL)-8 by a macrocyclic trichothecene, verrucarin A (VA), in human monocytic THP-1 cells is reported. Consistent with previous findings, VA was 100-fold more cytotoxic than deoxynivalenol (DON), while ochratoxin A (OA) was not cytotoxic. In cells transduced with the wild-type IL-8 promoter luciferase construct, VA induced a biphasic dose response composed of an upregulation of luciferase expression at low concentrations of 0.01-1 ng/ml and a downregulation at high levels of 10 ng/ml and higher. In contrast, DON induced a sigmoid-shaped dose response with the EC50 of 11.6 ng/ml, while OA did not markedly affect the IL-8 expression. When cells were transduced with IL-8 promoter with a mutation of transcription factor nuclear factor-κB (NF-κB)-binding site, VA (1 ng/ml), DON (1000 ng/ml), and tumor necrosis factor (TNF) α (20 ng/ml)-induced luciferase expression were impaired. In addition, the NF-κB inhibitor caffeic acid phenethyl ester inhibited VA-, DON-, and TNFα-induced luciferase expression. Mutation of the CCAAT/enhancer-binding protein (CEBP) β binding site of the IL-8 promoter affected only DON-, but not VA- and TNFα-induced luciferase expression. Taken together, these results suggested that VA activated IL-8 promoter via an NF-κB-dependent, but not CEBPβ-dependent, pathway in human monocytes.

Topics: CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Interleukin-8; Lentivirus; Luciferases; Monocytes; NF-kappa B; Ochratoxins; Promoter Regions, Genetic; Signal Transduction; Trichothecenes; Tumor Necrosis Factor-alpha; Up-Regulation

Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is toxic to many animal species, with pigs being the most sensitive species to the toxin. The aim of the present study was to determine the effects of DON on pig polymorphonuclear cells (PMNs), the first line of defense against infection.. PMNs isolated from pig blood samples were stimulated with LPS to mimic infection. DON (0.5-10 μM) altered three main functions of pig PMNs: LPS-induced secretion of IL-8, chemotaxis, and phagocytosis capability. This alteration of PMN properties was due to apoptotis induced by DON exposure. Using Western blot and flow cytometry, we demonstrated that this process included the permeabilization of the mitochondrial outer membrane and the activation of caspase-3. The effect of DON was mediated by the phosphorylation of the p38 mitogen-activated protein kinase within the first 30 min of exposure.. This study provides evidence that low concentrations of DON can alter the immune functions of porcine PMNs and suggests the involvement of p38 mitogen-activated protein kinase in the signal transduction pathway. These immunosuppressive effects of DON may have implications for humans and/or animals when eating contaminated food/feed.

Topics: Animals; Caspase 3; Cell Death; Chemotaxis; Enzyme Activation; Escherichia coli; Hydrogen Peroxide; Interleukin-8; Lipopolysaccharides; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinase 3; Mycotoxins; Neutrophils; p38 Mitogen-Activated Protein Kinases; Phagocytosis; Swine; Trichothecenes

The effects of the trichothecene mycotoxin deoxynivalenol (DON) and its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) on human intestinal cell Caco-2 were investigated by the studies of transepithelial transport, gene expression, and cytokine secretion. Permeability across a Caco-2 cell monolayer was evaluated by transport study. Transport rates were ranked as DON, 3ADON<15ADON in apical-basolateral direction. 15ADON showed the highest permeability, induced the highest decrease in transepithelial electrical resistance (TEER), and prompted significant Lucifer Yellow permeability. These results showed that 15ADON affect paracellular barrier function extremely. In addition, gene expressions induced by toxins were screened by DNA microarray for investigating cellular effect on Caco-2 cell. The most remarkable gene induced by DON and 15ADON was inflammatory chemokine IL-8 and thus mRNA expression and secretion of IL-8 were analyzed by PCR and ELISA. Both DON and acetylated DONs could induce mRNA expression and production of IL-8. In particular, ELISA assay showed that the ability to produce IL-8 was ranked as 3ADON

Topics: Biological Transport; Caco-2 Cells; Cell Survival; Gene Expression Profiling; Humans; Interleukin-8; Intestinal Absorption; Intestinal Mucosa; Oligonucleotide Array Sequence Analysis; Trichothecenes

The trichothecene deoxynivalenol (DON) binds to eukaryotic ribosomes and triggers p38-driven proinflammatory gene expression in the macrophage-a response that is dependent on both double-stranded RNA-activated protein kinase (PKR) and hematopoietic cell kinase (Hck). Here we elucidated critical linkages that exist among the ribosome and these kinases during the course of DON-induced ribotoxic stress in mononuclear phagocytes. Similar to PKR inhibitors, Hck inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2) suppressed p38 activation and p38-driven interleukin 8 (IL-8) expression in the U937 human monocyte cell line. U937 cells stably transfected with a PKR antisense vector (U9K-A1) displayed marked reduction of DON-induced p38 activation and IL-8 expression as compared to cells transfected with empty vector (U9K-C2), with both responses being completely ablated by PP2. Western analysis of sucrose density gradient fractions revealed that PKR and Hck interacted with the 40S ribosomal subunit in U9K-C2 but not U9K-A1 cells. Subsequent transfection and immunoprecipitation studies with HeLa cells indicated that Hck interacted with ribosomal protein S3. Consistent with U937 cells, DON induced p38 association with the ribosome and phosphorylation in peritoneal macrophages from wild-type but not PKR-deficient mice. DON-induced phosphorylation of ribosome-associated Hck in RAW 264.7 murine macrophages was also suppressed by 2-aminopurine (2-AP). Both 2-AP and PP2 inhibited DON-induced phosphorylation of p38 as well as two kinases, apoptosis signal-regulating kinase 1 and mitogen-activated protein kinase 3/6, known to be upstream of p38. Taken together, PKR and Hck were critical for DON-induced ribosomal recruitment of p38, its subsequent phosphorylation, and, ultimately, p38-driven proinflammatory cytokine expression.

Topics: Animals; eIF-2 Kinase; Enzyme Activation; Enzyme Inhibitors; HeLa Cells; Humans; Interleukin-8; Macrophages, Peritoneal; Mice; Mononuclear Phagocyte System; p38 Mitogen-Activated Protein Kinases; Phagocytes; Proto-Oncogene Proteins c-hck; Pyrimidines; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Trichothecenes; U937 Cells

The intake of deoxynivalenol (DON), a mycotoxin contaminating cereal food items, causes gastro-intestinal illness in human and animal. This study investigated whether intracellular inflammatory cascades (MAPKs and NF-κB), cell maturity (proliferating vs. differentiated), cell state (control vs. inflamed) and exposure duration (chronic vs. acute) affect IL-8 secretion and PGE-2 synthesis in Caco-2 cells exposed to plausible intestinal concentrations (50, 500 and 5000 ng/ml) of DON. IL-8 secretion and PGE-2 synthesizing capacity were dose-dependently upregulated in differentiated Caco-2 cells exposed to DON during 24h, reaching an increase of ∼25 and 1.7-fold respectively, whereas transcript level of IL-8 and COX-2 were increased by ∼40 and 17-fold. Similar results were obtained with proliferating cells. The upregulation decreased upon simultaneous incubation with inhibitors of MAPKs ERK1/2 or p38 or of transcription factor NF-κB. IL-8 secretion and PGE-2 synthesizing capacity increased respectively by ∼15 and 2-fold after chronic 21 day incubation with DON (50 ng/ml). IL-8 production was exacerbated (∼510-fold versus negative control) upon simultaneous exposure to inflammatory stimuli. These results suggest activation of inflammatory pathways in intestinal epithelial cells exposed chronically or acutely to DON. The sensitivity to DON, whereas not affected by cell differentiation, is exacerbated by the presence of additional stimuli.

Topics: Caco-2 Cells; Cell Differentiation; Cell Proliferation; Dinoprostone; Dose-Response Relationship, Drug; Humans; Inflammation; Inflammation Mediators; Interleukin-8; Intestinal Mucosa; Time Factors; Trichothecenes

CCAAT/enhancer-binding protein homologous protein (CHOP) is a crucial stress-responsive factor in various mucosal injuries, including cellular translational stress conditions. In this study, chemical ribosome-inactivating stresses were assessed for their effects on stress-inducible CHOP expression and its association with epithelial inflammatory cytokine production. Several representative ribotoxic agents (deoxynivalenol, anisomycin, and 15-acetyldeoxynivalenol) enhanced CHOP expression and its nuclear translocation in human intestinal epithelial cells. Moreover, CHOP was a strong positive regulator of IL-8 production, but CHOP-mediated IL-8 production was inversely associated with expression of the mucosal regulatory factor peroxisome proliferator-activated receptor γ (PPARγ). Based on our recent report that PPARγ is a negative regulator of mRNA stability of IL-8, PPARγ was linked to a notable mRNA stabilizing protein, HuR, since ribotoxin-induced IL-8 mRNA is stabilized by HuR protein. Expression of exogenous PPARγ suppressed ribotoxin-triggered cytoplasmic translocation of HuR. In contrast, PPARγ-regulating CHOP was a positive modulator of HuR protein export from nuclei. Taken together, the results indicate that ribotoxin-induced CHOP protein is positively associated with production of proinflammatory cytokine IL-8, but it downregulates PPARγ action, subsequently allowing the cytosolic translocation of HuR protein and stabilization of IL-8 mRNA in gut epithelial cells. CHOP and PPARγ may represent critical mechanistic links between ribotoxic stress and proinflammatory cytokine production, and they may have a broader functional significance with regard to gastrointestinal stresses by toxic mucosal insults.

Topics: Anisomycin; Antigens, Surface; Blotting, Western; Cell Line; ELAV Proteins; ELAV-Like Protein 1; Enzyme-Linked Immunosorbent Assay; Gene Expression; Gene Expression Regulation; Humans; Immunity, Mucosal; Interleukin-8; Intestinal Mucosa; Microscopy, Confocal; PPAR gamma; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Transcription Factor CHOP; Transfection; Trichothecenes

HuR/Elav-like RNA binding protein 1 (ELAVL1) positively regulates mRNA stability of AU-rich elements (ARE)-containing transcript such as pro-inflammatory cytokines. Ribotoxic stresses can trigger the production of pro-inflammatory mediators by enhancing mRNA stability and the transcriptional activity. We investigated the effects of ribotoxin deoxynivalenol (DON) on HuR translocation and its involvement in the regulation of the pro-inflammatory interleukin-8 (IL-8) mRNA stability. Exposure to the muco-active DON induced nuclear export of both endogenous and exogenous HuR RNA binding protein in human intestinal epithelial cells. Moreover, the interference with HuR protein production suppressed ribotoxic DON-induced IL-8 secretion and its mRNA stability. Cytoplasmic HuR protein interacted with IL-8 mRNA and the complex stabilization was due to the presence of 3'-untranslated region of the transcript. Partly in terms of IL-8-modulating transcription factors, HuR protein was demonstrated to be positively and negatively associated with DON-induced early growth response gene 1 (EGR-1) and activating transcription factor 3 (ATF3), respectively. HuR was a critical mechanistic link between ribotoxic stress and the pro-inflammatory cytokine production, and may have a broader functional significance with regard to mucosal insults since ribotoxic stress responses are also produced upon interactions with the diverse environment of gut.

Topics: Antigens, Surface; Cell Line; ELAV Proteins; ELAV-Like Protein 1; Humans; Interleukin-8; Intestinal Mucosa; Protein Transport; Ribosomes; RNA Stability; RNA-Binding Proteins; Trichothecenes

Translational inhibitors such as the trichothecene mycotoxin deoxynivalenol (DON) and ribosomal inhibitory proteins (RIPs) induce mitogen-activated protein kinase (MAPK)-driven chemokine and cytokine production by a mechanism known as the ribotoxic stress response (RSR). Double-stranded RNA-activated protein kinase (PKR) associates with the ribosome making it uniquely positioned to sense 28S ribosomal RNA damage and initiate the RSR. We have previously shown that PKR mediates DON-induced MAPK phosphorylation in macrophages and monocytes. The purpose of this study was to test the hypothesis that PKR is essential for induction of interleukin (IL)-8 expression in monocytes by DON and two prototypical RIPs, ricin, and Shiga toxin 1 (Stx1). Preincubation of human monocytic U937 cells with the PKR inhibitors C16 and 2-aminopurine (2-AP) blocked DON-induced expression of IL-8 protein and mRNA. Induction of IL-8 expression was similarly impaired in U937 cells stably transfected with a dominant negative PKR plasmid (UK9M) as compared with cells transfected with control plasmid (UK9C). Nuclear factor-kappa B binding, which has been previously shown to be a requisite for DON-induced IL-8 transcription, was markedly reduced in UK9M cells as compared with UK9C cells. As observed for DON, ricin-, and Stx1-induced IL-8 expression was suppressed by the PKR inhibitors C16 and 2-AP as well as impaired in UK9M cells. Taken together, these data indicate that PKR plays a common role in IL-8 induction by DON and the two RIPs, suggesting that this kinase might be a critical factor in RSR.

Topics: Dose-Response Relationship, Drug; eIF-2 Kinase; Humans; Interleukin-8; Monocytes; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Protein Synthesis Inhibitors; Ricin; RNA, Messenger; Shiga Toxin 1; Signal Transduction; Transcription Factor RelA; Transfection; Trichothecenes; U937 Cells; Up-Regulation

Mycotoxins are fungal secondary metabolites responsible of food-mediated intoxication in animals and humans. Deoxynivalenol, ochratoxin A and patulin are the best known enteropathogenic mycotoxins able to alter intestinal functions resulting in malnutrition, diarrhea, vomiting and intestinal inflammation in vivo. Although their effects on intestinal barrier and transport activities have been extensively characterized, the mechanisms responsible for their pro-inflammatory effect are still poorly understood. Here we investigated if mycotoxin-induced intestinal inflammation results from a direct and/or indirect pro-inflammatory activity of these mycotoxins on human intestinal epithelial cells, using differentiated Caco-2 cells as model and interleukin 8 (IL-8) as an indicator of intestinal inflammation. Deoxynivalenol was the only mycotoxin able to directly increase IL-8 secretion (10- to 15-fold increase). We also investigated if these mycotoxins could indirectly stimulate IL-8 secretion through: (i) a modulation of the action of pro-inflammatory molecules such as the interleukin-1beta (IL-1beta), and/or (ii) an increase in the transepithelial passage of non-invasive commensal Escherichia coli. We found that deoxynivalenol, ochratoxin A and patulin all potentiated the effect of IL-1beta on IL-8 secretion (ranging from 35% to 138% increase) and increased the transepithelial passage of commensal bacteria (ranging from 12- to 1544-fold increase). In addition to potentially exacerbate established intestinal inflammation, these mycotoxins may thus participate in the induction of sepsis and intestinal inflammation in vivo. Taken together, our results suggest that the pro-inflammatory activity of enteropathogenic mycotoxins is mediated by both direct and indirect effects.

Topics: Bacteria; Caco-2 Cells; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Intestinal Mucosa; Mycotoxins; NF-kappa B; Ochratoxins; p38 Mitogen-Activated Protein Kinases; Patulin; Permeability; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Trichothecenes

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. In human intestinal Caco-2 cells, DON activates the mitogen-activated protein kinases (MAPKs). We hypothesized a link between DON ingestion and intestinal inflammation, and used Caco-2 cells to assess the effects of DON, at plausible intestinal concentrations (250-10,000 ng/ml), on inflammatory mediators acting downstream the MAPKs cascade i.e. activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 (IL-8) secretion. In addition, Caco-2 cells were co-exposed to pro-inflammatory stimuli in order to mimic an inflamed intestinal epithelium. Dose-dependent increases in NF-kappaB activity and IL-8 secretion were observed, reaching 1.4- and 7.6-fold, respectively using DON at 10 microg/ml. Phosphorylation of inhibitor-kappaB (IkappaB) increased (1.6-fold) at DON levels <0.5 microg/ml. Exposure of Caco-2 cells to pro-inflammatory agents, i.e. 25 ng/ml interleukin-1beta, 100 ng/ml tumor necrosis factor-alpha or 10 microg/ml lipopolysaccharides, activated NF-kappaB and increased IL-8 secretion. Synergistic interactions between these stimuli and DON were observed. These data show that DON induces NF-kappaB activation and IL-8 secretion dose-dependently in Caco-2 cells, and this effect was accentuated upon pro-inflammatory stimulation, suggesting DON exposure could cause or exacerbate intestinal inflammation.

Topics: Caco-2 Cells; Dose-Response Relationship, Drug; Enteritis; Humans; I-kappa B Kinase; Interleukin-1beta; Interleukin-8; NF-kappa B; Phosphorylation; Trichothecenes; Tumor Necrosis Factor-alpha

Mucosal epithelium senses external toxic insults and transmits the danger signals into the epithelial cells in order to activate a broad range of inflammatory responses. However, pre-exposure to the commensal endotoxins can induce inflammatory tolerance and maintain the homeostasis without excessive immune responses. We recently reported that ribotoxin deoxynivalenol (DON) and its derivatives elicited the pro-inflammatory response as the mucosal insults in human epithelial cells. Taking the knowledge into consideration, we tested the hypothesis that endotoxin pre-exposure can attenuate ribotoxin-induced epithelial interleukin-8 (IL-8) production via a tolerance mechanism. Pre-exposure to endotoxin repressed IL-8 release and its gene expression. However, inflammatory tolerance was not mediated by the attenuated NF-kappaB activation which has been generally recognized as the major mediator of LPS-mediated toll-like receptor (TLR) signaling pathway. Instead, pre-exposure to endotoxin was observed to trigger the delayed induction of peroxisome proliferator-activated receptor gamma (PPAR-gamma) which contributed to the diminished IL-8 production in the human epithelial cells. Moreover, endogenous PPAR-gamma agonist suppressed toxicant-mediated interleukin-8 production and IL-8 mRNA stability. Taken together, endotoxin induced hypo-production of pro-inflammatory cytokine IL-8 in the human epithelial cells, which was associated with the delayed activation of PPAR-gamma expression by pre-existing endotoxin.

Topics: Blotting, Western; Cell Line; Cell Survival; Early Growth Response Protein 1; Endotoxins; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Indicators and Reagents; Interleukin-8; Intestinal Mucosa; Luciferases; NF-kappa B; Plasmids; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Toll-Like Receptor 4; Transfection; Trichothecenes

The trichothecene mycotoxin deoxynivalenol (DON), commonly present in contaminated grains worldwide, induces expression of the chemokine interleukin (IL)-8 in human monocytes. The purpose of this study was to test the hypothesis that DON modulates transcriptional and posttranscriptional regulation of IL-8 expression in the U937 human monocyte model. When U937 cells were transfected with a wild-type IL-8 promoter luciferase construct (-162/+44 IL-8 LUC) and incubated with DON (1 mug/ml) or the positive control, lipopolysaccharide (LPS) (1 mug/ml), there was a significant increase in luciferase expression. Mutation of the nuclear factor-kappaB (NF-kappaB) binding site significantly impaired both DON- and LPS-induced luciferase expression. In contrast, mutating the activator protein-1 binding site resulted in significantly increased DON- and LPS-induced luciferase expression. CCAAT/enhancer-binding protein beta, octamer-1, or NF-kappaB repressing factor binding site mutations did not affect DON-induced luciferase activity. Consistent with reporter studies, the NF-kappaB inhibitor caffeic acid phenethyl ester completely ablated both DON-induced IL-8 mRNA and protein expression. When NF-kappaB subunit binding to a specific IL-8 promoter probe was evaluated by enzyme-linked immunosorbent assay (ELISA), DON was observed to increase p65 binding by 21-fold, have no effect on p50 binding and decrease p52 binding. DON was not found to stabilize IL-8 mRNA in U937 cells. Taken together, these data suggest that DON-induced IL-8 expression is likely to be mediated at the transcriptional level by NF-kappaB, specifically p65, but does not appear to involve mRNA stabilization.

Topics: CCAAT-Enhancer-Binding Protein-beta; Cells, Cultured; Gene Expression Regulation; Humans; Interleukin-8; Monocytes; NF-kappa B; p38 Mitogen-Activated Protein Kinases; RNA Stability; Transcription Factor RelA; Transcription, Genetic; Trichothecenes

Epithelium senses external toxic insults transmit the sentinel signals into the cells to reprogram a broad range of mucosal responses like epithelial inflammatory diseases. The purpose of this study was to test the hypothesis that ribotoxin DON evokes the epithelial sentinel signals which contributes to the pro-inflammatory cytokine interleukin-8 in human epithelial cells. Treatment with DON elevated interleukin-8 production in the human epithelial intestine 407 cells. Particularly, ribotoxin DON markedly elevated the phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK) which mediated DON-induced interleukin-8 (IL-8) production. DON-activated ERK1/2 also mediated the production of early growth response gene 1 (EGR-1) in the epithelial cell line and EGR-1 had the positive regulatory effect on the interleukin-8 production in the human epithelial cells. Taken all, DON-activated MAPK sentinel signals of the epithelial cells which promoted interleukin-8 production and its positive modulator EGR-1. These findings will provide insight into the possible mechanism associated with the early epithelial inflammation by ribotoxic insults.

Topics: Blotting, Western; Cell Line; Dose-Response Relationship, Drug; Early Growth Response Protein 1; Epithelial Cells; Humans; Interleukin-8; Intestinal Mucosa; Intestines; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trichothecenes

The effects of the ribotoxic trichothecene deoxynivalenol (DON) on mitogen-activated protein kinase (MAPK)-mediated IL-8 expression were investigated in cloned human monocytes and peripheral blood mononuclear cells (PBMC). DON (250 to 1000 ng/ml) induced both IL-8 mRNA and IL-8 heteronuclear RNA (hnRNA), an indicator of IL-8 transcription, in the human U937 monocytic cell line in a concentration-dependent manner. Expression of IL-8 hnRNA, mRNA and protein correlated with p38 phosphorylation and was completely abrogated by the p38 MAPK inhibitor SB203580. DON at 500 ng/ml similarly induced p38-dependent IL-8 protein and mRNA expression in PBMC cultures from healthy volunteers. Significantly increased IL-6 and IL-1beta intracellular protein and mRNA expression was also observed in PBMC treated with DON (500 ng/ml) which were also partially p38-dependent. Flow cytometry of PBMC revealed that DON-induced p38 phosphorylation varied among individuals relative to both threshold toxin concentrations (25-100 ng/ml) and relative increases in percentages of phospho-p38(+) cells. DON-induced p38 activation occurred exclusively in the CD14(+) monocyte population. DON was devoid of agonist activity for human Toll-like receptors 2, 3, 4, 5, 7, 8 and 9. However, two other ribotoxins, emetine and anisomycin, induced p38 phosphorylation in PBMC similarly to DON. Taken together, these data suggest that (1) p38 activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and (2) DON induced p38 activation in human monocytes via the ribotoxic stress response.

Topics: Anisomycin; Emetine; Gene Expression Regulation; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Monocytes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Synthesis Inhibitors; RNA, Messenger; Trichothecenes; U937 Cells

We examined whether the common crop mycotoxin deoxynivalenol (DON) from Fusarium species is toxic to human colonic (Caco-2), lung (A549) and monocytic (U937) cell lines. Moreover, since DON reportedly induces increased levels of Th2 cytokines and total IgE, and we have observed that mould extracts adjuvated allergy development in mice, possible adjuvant effect of DON on allergy was studied in a mouse model. For all the cells, exposure to DON for 24 h reduced cellular protein synthesis, proliferation and survival rate dose-dependently. In addition, production of IL-8 in the U937 cell line increased up to eight-fold at levels of DON just lower than the most toxic one, suggesting that IL-8 can be used as an additional index for cytotoxicity in mononuclear phagocytes. However, DON did not increase levels of allergen-specific IgE or IgG1 in the mouse model for allergy. These results suggest that DON, when inhaled or ingested, may have toxic effect on human alveolar macrophages and epithelial cells in lungs and colon, but does not increase the allergic response to allergens.

Topics: Adjuvants, Immunologic; Animals; Caco-2 Cells; Cell Division; Cell Line, Tumor; Cell Survival; Colon; Female; Humans; Hypersensitivity; Immunoglobulin E; Interleukin-8; Lung; Lymph Nodes; Mice; Mice, Inbred BALB C; Monocytes; Protein Biosynthesis; Trichothecenes; U937 Cells

The effects of deoxynivalenol (DON or vomitoxin) and four closely related 8-ketotrichothecenes on proinflammatory cytokine and chemokine production were evaluated in a clonal human macrophage model. U-937 cells, which represent a human monocytelike histocytic lymphoma, were differentiated into macrophages by preincubation with phorbol 12-myristate 13-acetate (PMA). Differentiated macrophages were incubated with DON in the absence or presence of lipopolysaccharide (LPS), and supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA) for the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), and for the chemokine interleukin-8 (IL-8). In the absence of LPS, DON at 500 or 1,000 ng/ml upregulated TNF-alpha production as early as 3 h and up to 6 h, whereas 100 to 1,000 ng/ml of DON significantly increased production of IL-6 from 3 to 24 h and IL-8 from 6 to 48 h. In cells costimulated with 0.2 microg/ml LPS, DON at 500 or 1000 ng/ml markedly superinduced TNF-alpha and IL-8 production. Although 100 ng/ml of DON also potentiated LPS-induced IL-6 production, 500 or 1,000 ng/ ml of the toxin suppressed the LPS-induced IL-6 response. Four other 8-ketotrichothecenes, fusarenon X, nivalenol, 3-acetyl DON, and 15-acetyl DON, were also capable of upregulating or suppressing TNF-alpha, IL-6, and IL-8 production at concentrations similar to that of DON. In total, the results suggest that DON and other 8-ketotrichothecenes have the potential to both directly induce and superinduce proinflammatory cytokine and chemokine expression in human macrophages, even at toxin concentrations that are cytotoxic.

Topics: Cells, Cultured; Cytokines; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-6; Interleukin-8; Macrophages; Models, Biological; Mycotoxins; Trichothecenes; Tumor Necrosis Factor-alpha; Up-Regulation