zearalenone and Inflammation

In the past decade, mycotoxin zearalenone (ZEN)-induced gastrointestinal adverse effects have been increasingly attracting worldwide attention. This study aimed to determine the gastrointestinal adverse effects of ZEN in Drosophila melanogaster (D. melanogaster) and reveal possible mechanisms of action of ZEN in insects. Here, chronic exposure of D. melanogaster to ZEN killed flies in a dose-dependent manner (2-20 µM). ZEN (20 µM) decreased the survival rates and climbing ability of flies, and activated immune deficiency-mediated intestinal immunity in midgut, leading to the production of antimicrobial peptides. Meanwhile, ZEN exposure induced morphological alteration of adult midgut. Further study suggested that high levels of oxidative stress was observed in ZEN-treated midgut due to the imbalance between the production of reactive oxygen species and the expression and activities of cellular antioxidant enzyme, including superoxide dismutase and catalase. ZEN-induced oxidative stress then caused cell death, impaired gut barrier function and increased gut permeability, leading to oxidative injury in midgut. Subsequently, ZEN-induce midgut injury further disrupted intestinal stem cell (ISC) homeostasis, stimulating ISC proliferation and tissue regeneration, but did not alter cell fate specification of ISC. Additionally, activation of Jun N-terminal kinase pathway was involved in ZEN-induced oxidative injury and tissue regeneration in midgut. Antioxidant vitamin E alleviated ZEN-induced oxidative injury to midgut epithelium. Collectively, this study provided additional evidences for ZEN-induced gastrointestinal adverse effects from an invertebrate model, extended our understanding of the mechanisms mediating mycotoxin toxicity in organisms.

Topics: Animals; Antioxidants; Drosophila melanogaster; Inflammation; Mycotoxins; Oxidative Stress; Zearalenone

Zearalenone (ZEA) is a mycotoxin commonly found in cereals and feedstuffs, which can induce oxidative stress and inflammation to cause liver damage in humans and animals. Betulinic acid (BA) is extracted from pentacyclic triterpenoids of many natural plants and has anti-inflammatory, and anti-oxidation biological activities in many studies. However, the protective effect of BA on liver injury induced by ZEA has not been reported. Therefore, this study aims to explore the protective effect of BA on ZEA-induced liver injury and its possible mechanism. In the mice experiment, ZEA exposure increased the liver index and caused histopathological impairment, oxidative damage, hepatic inflammatory responses, and increased hepatocyte apoptosis. However, when combined with BA, it could inhibit the production of ROS, up-regulate the proteins expression of Nrf2 and HO-1 and down-regulate the expression of Keap1, and alleviate oxidative damage and inflammation in the liver of mice. In addition, BA could alleviate ZEA-induced apoptosis and liver injury in mice by inhibiting the endoplasmic reticulum stress (ERS) and MAPK signaling pathways. In conclusion, this study revealed the protective effect of BA on the hepatotoxicity of ZEA for the first time, providing a new perspective for the development of ZEA antidote and the application of BA.

Topics: Animals; Apoptosis; Betulinic Acid; Chemical and Drug Induced Liver Injury, Chronic; Endoplasmic Reticulum Stress; Humans; Inflammation; Kelch-Like ECH-Associated Protein 1; Mice; NF-E2-Related Factor 2; Oxidative Stress; Signal Transduction; Zearalenone

Hepatotoxic contaminants such as zearalenone (ZEA) are widely present in foods. Marine algae have a wide range of potential applications in pharmaceuticals, cosmetics, and food products. Research is ongoing to develop treatments and products based on the compounds found in algae. Fucoxanthin (FXN) is a brown-algae-derived dietary compound that is reported to prevent hepatotoxicity caused by ZEA. This compound has multiple biological functions, including anti-diabetic, anti-obesity, anti-microbial, and anti-cancer properties. Furthermore, FXN is a powerful antioxidant. In this study, we examined the effects of FXN on ZEA-induced stress and inflammation in HepG2 cells. MTT assays, ROS generation assays, Western blots, and apoptosis analysis were used to evaluate the effects of FXN on ZEA-induced HepG2 cell inflammation. Pre-incubation with FXN reduced the cytotoxicity of ZEA toward HepG2 cells. FXN inhibited the ZEA-induced production of pro-inflammatory cytokines, including IL-1 β, IL-6, and TNF-α. Moreover, FXN increased HO-1 expression in HepG2 by activating the PI3K/AKT/NRF2 signaling pathway. In conclusion, FXN inhibits ZEA-induced inflammation and oxidative stress in hepatocytes by targeting Nrf2 via activating PI3K/AKT signaling.

Topics: Apoptosis; Humans; Inflammation; NF-E2-Related Factor 2; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Zearalenone

This study aimed to evaluate endophytic fungi isolated from Tocoyena bullata and Humiria balsamifera plant species for their antimycobacterial and anti-inflammatory activities, focusing on severe pulmonary tuberculosis cases which are often associated with exacerbated inflammation.. Mycobacterium suspensions were incubated with the samples for 5 days. RAW 264.7 macrophages stimulated with LPS were also incubated with them for 24 h to assess the inhibition of inflammatory mediator production and cytotoxicity. C57BL/6 mice were infected with Mtb M299 and treated for 15 days with lasiodiplodin (Lasio).. Endophytic fungus Sordaria tamaensis, obtained from T. bullata, was the most promising. Its ethanolic extract impaired mycobacterial growth with MIC50 (µg/ml): 1.5 ± 0.6 (BCG), 66.8 ± 0.1 (H37Rv) and 80.0 ± 0.1 (M299). (R)-(+)-Lasio showed MIC50 92.2 ± 1.8 µg/ml (M299). In addition, Lasio was able to inhibit NO, IL-1β and TNF-α production and was not cytotoxic for macrophages. M. tuberculosis-infected C57BL/6 animals treated by Lasio reduced the number of acid-fast bacilli, lung pathology, leucocyte influx and proinflammatory cytokine production in the lungs. The class IIa fructose 1,6-bisphosphate aldolase was the predicted hypothetical target of Lasio.. (R)-(+)-Lasio stood out as a promising anti-TB compound, exhibiting anti-inflammatory and antimycobacterial effects, as well as low cytotoxicity.

Topics: Animals; Anti-Inflammatory Agents; Antitubercular Agents; Caco-2 Cells; Humans; Inflammation; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; RAW 264.7 Cells; Rubiaceae; Sordariales; Tuberculosis, Pulmonary; Zearalenone

Zearalenone (ZEN)-contaminated diets induce detrimental effects on the bovine reproduction. Recently, we reported that active sperm induce pro-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. This study aimed to investigate the impact of presence of ZEN on the sperm-uterine crosstalk in vitro. BEECs monolayers were stimulated by ZEN (10, 100, and 1000 ng/mL) for 0, 3, 6, 12, or 24 h and gene expressions were analyzed by real-time PCR. Moreover, BEECs were pre-exposed to ZEN (10, 100, and 1000 ng/mL) for 24 h then, co-incubated with sperm for 6 h. Conditioned media (CM) from a sperm-BEECs co-culture, after pre-exposure to ZEN, were harvested and exploited to challenge either polymorphonuclear cells (PMNs) or sperm. Both PMNs phagocytic activity toward sperm and sperm motility parameters were then assessed. Results showed that ZEN alone induced pro-inflammatory responses in BEECs through the induction of mRNA expressions of pro-inflammatory cytokines (TNFA and IL1B) and PGES1 at different time points. Pre-exposure of BEECs to ZEN, amplified the sperm-triggered upregulation of pro-inflammatory cytokines (TNFA and IL1B) and chemokine IL8 mRNA abundance in BEECs. Sperm-BEECs conditioned media, primed by ZEN, stimulated the PMNs phagocytosis for sperm whereas suppressed sperm motility parameters. Taken together, these findings indicate that the presence of ZEN augments the pro-inflammatory cascade triggered by sperm in BEECs, provokes PMNs phagocytosis for sperm, and reduces sperm motility parameters. Such immunological reactions may create a hostile environment for sperm competence and survival in the bovine uterus, thus impair fertility.

Topics: Animals; Cattle; Cells, Cultured; Coculture Techniques; Cytokines; Epithelial Cells; Estrogens, Non-Steroidal; Female; Inflammation; Male; Neutrophils; Phagocytosis; Sperm Motility; Spermatozoa; Uterus; Zearalenone

The complex microbial community in food environment is a major problem of human or animal health and safety. Mycotoxins and food-borne bacteria can both induce inflammation in the body and cause a series of changes in biological functions. In this study, mice were gavaged with low doses of ZEA, DON, or ZEA + DON, and then infected with L. monocytogenes. A cytokine microarray, including 40 inflammation-related serum cytokines, and proteomics were used to verify the effects of ZEA, DON, and ZEA + DON on the host inflammation and biological function after L. monocytogenes infection. The results showed that mononucleosis after bacterial infection was inhibited by ZEA, DON, and ZEA + DON, while the balance of macrophage differentiation was shifted toward M2-type. ZEA, DON, and ZEA + DON decreased the levels of serum proinflammatory cytokines IL-1β and IL-12 after infection. In addition, the signal of the NF-κB pathway was inhibited. Proteomic results showed that ZEA, DON, and ZEA + DON led to biological dysfunction in ribosomal and metabolic cells, primarily leading to abnormal ribosomal hyperfunction. This study showed that ZEA, DON, and ZEA + DON can aggravate disease progression by inhibiting the inflammatory response following foodborne bacterial infection. These metabolites may also disrupt normal biological functions, which may lead to ribosomal hyperfunction, making bacterial clearance more difficult.

Topics: Animals; Cytokines; Inflammation; Mice; Proteomics; Trichothecenes; Zearalenone

Topics: Aflatoxin B1; Caspase 3; Cell Line; Claudin-1; Cyclooxygenase 2; Epithelial Cells; Humans; Inflammation; Interleukin-8; Mycotoxins; NF-kappa B; Occludin; Probiotics; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Trichothecenes; Tumor Necrosis Factor-alpha; Zearalenone

Topics: Animals; Dietary Supplements; Fusarium; Inflammation; Lactobacillales; Mice; Oxidative Stress; Zearalenone

TGFβ-activated kinase 1 (TAK1) is a master regulator that drives multiple cell death and proinflammatory signaling pathways, making it a promising therapeutic target to treat ischemic stroke. However, whether targeting TAK1 could improve stroke outcomes has never been tested in female subjects, hindering its potential translation into clinical use. Here we examined the therapeutic effect of 5Z-7-Oxozeaenol (OZ), a selective TAK1 inhibitor, in ovariectomized female mice after middle cerebral artery occlusion (MCAO). OZ significantly reduced neuronal cell death and axonal injury at the acute stage and mitigated neuroinflammation at the subacute stage after MCAO in ovariectomized female mice. Consistent with RNA sequencing analysis that TAK1 activation contributed to microglia/macrophage-mediated inflammatory responses in the post-stroke brain, inhibition of TAK1 with OZ caused phenotypic shift of microglia/macrophages toward an inflammation-resolving state. Furthermore, microglia/macrophage-specific TAK1 knockout (TAK1 mKO) reproduced OZ's effects, causally confirming the role of TAK1 in determining proinflammatory microglial/macrophage responses in post-stroke females. Post-stroke treatment with OZ for 5 days effectively promoted long-term neurological recovery and the integrity of both gray matter and white matter in female mice. Together, the TAK1 inhibitor OZ elicits long-lasting improvement of stroke outcomes in female mice, at least partially through enhancing beneficial microglial/macrophage responses and inflammation resolution. Given its therapeutic efficacy on both male and female rodents, TAK1 inhibitor is worth further investigation as a valid treatment to ischemic stroke.

Topics: Animals; Enzyme Inhibitors; Female; Infarction, Middle Cerebral Artery; Inflammation; Macrophages; MAP Kinase Kinase Kinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Ovariectomy; Recovery of Function; Zearalenone

Mast cells drive the inappropriate immune response characteristic of allergic inflammatory disorders via release of pro-inflammatory mediators in response to environmental cues detected by the IgE-FcεRI complex. The role of TGF-β-activated kinase 1 (TAK1), a participant in related signaling in other contexts, remains unknown in allergy. We detect novel activation of TAK1 at Ser412 in response to IgE-mediated activation under SCF-c-kit potentiation in a mast cell-driven response characteristic of allergic inflammation, which is potently blocked by TAK1 inhibitor 5Z-7-oxozeaenol (OZ). We, therefore, interrogated the role of TAK1 in a series of mast cell-mediated responses using IgE-sensitized murine bone marrow-derived mast cells, stimulated with allergen under several TAK1 inhibition strategies. TAK1 inhibition by OZ resulted in significant impairment in the phosphorylation of MAPKs p38, ERK, and JNK; and mediation of the NF-κB pathway via IκBα. Impaired gene expression and near abrogation in release of pro-inflammatory cytokines TNF, IL-6, IL-13, and chemokines CCL1, and CCL2 was detected. Finally, a significant inhibition of mast cell degranulation, accompanied by an impairment in calcium mobilization, was observed in TAK1-inhibited cells. These results suggest that TAK1 acts as a signaling node, not only linking the MAPK and NF-κB pathways in driving the late-phase response, but also initiation of the degranulation mechanism of the mast cell early-phase response following allergen recognition and may warrant consideration in future therapeutic development.

Topics: Animals; Bone Marrow Cells; Calcium; Cell Degranulation; Cytokines; Female; Gene Expression Regulation; Hypersensitivity; Immunoglobulin E; Inflammation; Inflammation Mediators; MAP Kinase Kinase Kinases; Mast Cells; Mice, Inbred C57BL; Models, Biological; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Phosphoserine; Proto-Oncogene Proteins c-kit; Receptors, IgE; RNA, Messenger; Signal Transduction; Zearalenone

The intestinal epithelium is the first barrier against food contaminants and is highly sensitive to Fusarium toxins, especially deoxynivalenol (DON) and zearalenone (ZEA). Here, we explored the effects of low doses of DON and/or ZEA in naturally moldy diets on intestinal functions in piglets, including inflammatory responses, epithelial barrier, and microbial composition. Piglets were treated with a control diet (CON), DON diet (1000.6 μg/kg), ZEA diet (269.1 μg/kg), and DON + ZEA diet (1007.5 + 265.4 μg/kg), respectively, for 3 weeks and then switched to the same CON diet for another 2 weeks. In the first period, even the selected low doses of DON or ZEA in the diet resulted in intestinal inflammation, diminish protein expression (claudin-4) and altered gut microbiota populations. Whereas upon switching to the CON diet for another 2 weeks, the deleterious effect of ZEA and DON on IL-1β and Bifidobacterium population could not be recovered. Additionally, combined DON and ZEA negatively affected body weight gain and feed consumption of piglets, as well as shown synergistic effects on evoking pro-inflammatory cytokines contents (TNF-α, IL-1β, and IL-6) and perturbing the cecum microbiota profile (E. coli, Lactobacillus, and Bifidobacterium). Collectively, chronic consumption of DON and ZEA contaminated feed or food, even at low doses, can induce intestinal damage and may have consequences for animal and human health.

Topics: Animal Feed; Animals; Body Weight; Cecum; Cytokines; Diet; Dose-Response Relationship, Drug; Drug Synergism; Female; Food Contamination; Fusarium; Gene Expression; Hordeum; Immunity, Mucosal; Inflammation; Intestinal Mucosa; Jejunum; Swine; Tight Junction Proteins; Trichothecenes; Zea mays; Zearalenone

Zearalenone (ZEA), a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain. ZEA derivatives (α-zearalenol (α-ZOL), β-zearalenol (β-ZOL)) can also be produced by Fusarium spp. in corn stems infected by fungi in the field. Also, following oral exposure, zearalenone is metabolized in various tissues, particularly in the liver, the major metabolites being α-ZOL and β-ZOL. The co-exposure of cells to mixture of a combination of mycotoxins may cause an increase of toxicity produced by these mycotoxins. In this in vitro study, we investigated the combined effects of ZEA, α-ZOL, β-ZOL in binary mixtures on the viability and inflammatory response of human liver cancer cell line (HepG2). Cell viability was assessed after 72 h using a neutral red assay. Effect of the toxins and their binary combinations on the expression of genes involved in inflammation (IL-1β, TNF-α, and IL-8) were assessed through qPCR. Our viability data showed that irrespective of the toxin combinations, the toxins have synergistic effect. ZEA + α-ZOL and ZEA + β-ZOL mixtures have induced a slight to high antagonistic response on inflammatory cytokines at low concentrations that have turned into strong synergism for high concentrations. α-ZOL + β-ZOL showed antagonistic effects on inflammation for IL-1β and TNF-α, but act synergic for IL-8 at high toxin concentrations. This study clearly shows that co-contamination of food and feed with ZEA metabolites should be taken into consideration, as the co-exposure to mycotoxins might result in stronger adverse effect than resulted from the exposure to individual toxin.

Topics: Cell Survival; Cytokines; Drug Interactions; Gene Expression Regulation; Hep G2 Cells; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Mycotoxins; Tumor Necrosis Factor-alpha; Zearalenone; Zeranol

To ascertain whether zearalenone (ZEA) could induce intestinal inflammation and investigate its possible mechanism, we investigated inflammatory cytokine release and the activation of the NLRP3 inflammasome after ZEA treatment both in vitro or in vivo. First, intestinal porcine enterocyte cell line (IPEC-J2) cells and mouse peritoneal macrophages were treated with ZEA to detect NLRP3 inflammasome activation, and the role of reactive oxygen species (ROS) in ZEA-induced inflammation was investigated. Then, Balb/c mice were fed a gavage of ZEA, and the disease activity indices (DAIs) and histological analysis were used to assess intestinal inflammation. Our study showed that the mRNA expression of NLRP3 inflammasome, pro-interleukin-1β (pro-IL-1β), and pro-interleukin-18 (pro-IL-18) was up-regulated 0.5- to 1-fold and that the release of IL-1β and IL-18 increased from 48 pg mL

Topics: Animals; Cell Line; Estrogens, Non-Steroidal; Inflammasomes; Inflammation; Interleukin-18; Interleukin-1beta; Intestines; Macrophages; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species; RNA, Messenger; Swine; Zearalenone

Advanced glycation end products (AGEs), inflammatory-activated macrophages are essential in the initiation and progression of diabetic nephropathy (DN). TGF-β-activated kinase 1 (TAK1) plays a vital role in innate immune responses and inflammation. However, little information has been available about the effects of AGEs on the regulation of TAK1 expression and underlying mechanisms in AGEs-stimulated macrophage activation. We hypothesized TAK1 signal pathway in AGEs conditions could be a vital factor contributing to macrophage activation and inflammation. Thus, in the present study, we used bone marrow-derived macrophages (BMMs) to explore the functional role and potential mechanisms of TAK1 pathway under AGEs conditions. Results indicated that TAK1 played important roles in AGEs-induced mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B protein (NF-κB) activation, which regulated the production of monocyte chemo-attractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) in AGEs-stimulated macrophages. The results also suggested that TAK1 inhibitor (5Z-7-oxozeaenol) could inhibit AGEs-induced macrophage activation to down-regulate inflammatory cytokine production via MAPKs and NF-κB pathways, indicating that 5Z-7-oxozeaenol might be an immunoregulatory agent against AGEs-stimulated inflammatory response in DN.

Topics: Animals; Cells, Cultured; Chemokine CCL2; Diabetic Nephropathies; Down-Regulation; Glycation End Products, Advanced; Inflammation; Macrophage Activation; Macrophages; MAP Kinase Kinase Kinases; Mice; NF-kappa B; Signal Transduction; Tumor Necrosis Factor-alpha; Zearalenone

This work investigated the effect of Escherichia coli K88 and zearalenone contamination on pro-inflammatory gene expression (Toll like receptors, cytokines) and signalling molecules and the protective activity of a mixture of Lactobacilli sp. (Lactobacillus plantarum, Lactobacillus acidofilus and Lactobacillus paracasei) in porcine intestinal epithelial cells as part of the local immune system. IPEC-1 cell monolayer was exposed for 1 h to the individual or combined action of E. coli, zearalenone and lactobacilli mixture. Our results showed that TLRs (1-10) and cytokine (IL-1,-6,-8,-10, TNF-α, IFN-γ) genes expressed early (after 1 h of culture) in IPEC-1 cells. E. coli alone increased the TLRs mRNA expression, especially TLR4 and the inflammatory cytokines while ZEA alone showed either no effect or a marginally effect on TLRs, cytokines, and signalling genes when compared to untreated cells. The combined actions of the two contaminants lead to a synergistically up-regulation of key cytokines (IFN-γ, IL-10 and TNF-α) and TLRs (-2,-3,-4,-6, and -10). The live lactobacilli mixture was able to attenuate the pathogen and mycotoxin-induced response by downregulated the majority of inflammatory related genes suggesting that this mixture has an immunomodulatory potential and may be used to lower the inflammatory response.

Topics: Analysis of Variance; Animals; Cell Line; Cell Survival; Cytokines; DNA Primers; Enterocytes; Enterotoxigenic Escherichia coli; Gene Expression Regulation; Inflammation; Lactobacillus; Real-Time Polymerase Chain Reaction; RNA, Messenger; Swine; Toll-Like Receptors; Zearalenone

Male mice received lycopene for 10 days before a single oral administration of zearalenone (ZEA). After 48 h testes and blood were collected. Mice treated with lycopene/ZEA exhibited amelioration of the hematological changes. Lycopene prevented the reduction in the number and motility of spermatozoa and testosterone levels, indicating a protective effect in the testicular damage induced by ZEA. Lycopene was also effective in protecting against the decrease in glutathione-S-transferase, glutathione peroxidase, glutathione reductase and δ-aminolevulinic acid dehydratase activities caused by ZEA in the testes. Exposure of animals to ZEA induced modification of antioxidant and inflammatory status with increase of reduced glutathione (GSH) levels and increase of the oxidized glutathione, interleukins 1β, 2, 6, 10, tumor necrosis factor-α and bilirubin levels. Lycopene prevented ZEA-induced changes in GSH levels and inhibited the processes of inflammation, reducing the damage induced by ZEA. Altogether, our results indicate that lycopene was able to prevent ZEA-induced damage in the mice.

Topics: Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Bilirubin; Carotenoids; Glutathione Peroxidase; Glutathione Transferase; Inflammation; Interleukins; Lycopene; Male; Mice; Oxidative Stress; Porphobilinogen Synthase; Protective Agents; Testis; Testosterone; Tumor Necrosis Factor-alpha; Zearalenone

The toxicity of zearalenone (ZEA) was evaluated in swine spleen, a key organ for the innate and adaptative immune response. Weaned pigs were fed for 18 days with a control or a ZEA contaminated diet. The effect of ZEA was assessed on wide genome expression, pro- (TNF-α, IL-8, IL-6, IL-1β, IFN-γ) and anti-inflammatory (IL-10, IL-4) cytokines, other molecules involved in inflammatory processes (MMPs/TIMPs), as well as signaling molecules, (p38/JNK1/JNK2-MAPKs) and nuclear receptors (PPARγ/NFkB/AP-1/STAT3/c-JUN). Microarray analysis showed that 46% of total number of differentially expressed genes was involved in cellular signaling pathway, 13% in cytokine network and 10% in the inflammatory response. ZEA increased expression and synthesis of pro- inflammatory (TNF-α, IL-8, IL-6, IL-1β) and had no effect on IFN-γ, IL-4 and IL-10 cytokines in spleen. The inflammatory stimulation might be a consequence of JNK pathway activation rather than of p-38MAPK and NF-kB involvement whose gene and protein expression were suppressed by ZEA action. In summary, our findings indicated the role of ZEA as an immune disruptor at spleen level.

Topics: Animals; Gene Expression; Immunologic Factors; Inflammation; Interferon-gamma; Interleukins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mycotoxins; NF-kappa B; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Spleen; Swine; Zearalenone

The MAP3 kinase, TAK1, is known to act upstream of IKK and MAPK cascades in several cell types, and is typically activated in response to cytokines (e.g., TNF, IL-1) and TLR ligands. In this article, we report that in human neutrophils, TAK1 can also be activated by different classes of inflammatory stimuli, namely, chemoattractants and growth factors. After stimulation with such agents, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the inducible phosphorylation of ERK occurring in response to these stimuli but had little or no effect on that of p38 MAPK or PI3K. Inhibition of TAK1 also impaired MEKK3 (but not MEKK1) activation by fMLF. Moreover, both TAK1 and the MEK/ERK module were found to influence inflammatory cytokine expression and release in fMLF- and GM-CSF-activated neutrophils, whereas the PI3K pathway influenced this response independently of TAK1. Besides cytokine production, other responses were found to be under TAK1 control in neutrophils stimulated with chemoattractants and/or GM-CSF, namely, delayed apoptosis and leukotriene biosynthesis. Our data further emphasize the central role of TAK1 in controlling signaling cascades and functional responses in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions.

Topics: Apoptosis; Cells, Cultured; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Leukotrienes; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinase 3; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Zearalenone

Macrophages (MΦ) are functionally classified into two types, anti-inflammatory M2 and pro-inflammatory M1. Importantly, we recently revealed that soluble HIV-1 proteins, particularly the pathogenetic protein Nef, preferentially activate M2-MΦ and drive them towards an M1-like MΦ, which might explain the sustained immune activation seen in HIV-1-infected patients. Here, we show that the preferential effect of Nef on M2-MΦ is mediated by TAK1 (TGF-β-activated kinase 1) and macropinocytosis. As with MAP kinases and NF-κB pathway, Nef markedly activated TAK1 in M-CSF-derived M2-MΦ but not in GM-CSF-derived M1-MΦ. Two Nef mutants, which were unable to activate MAP kinases and NF-κB pathway, failed to activate TAK1. Indeed, the TAK1 inhibitor 5Z-7-oxozeaenol as well as the ectopic expression of a dominant-negative mutant of TAK1 or TRAF2, an upstream molecule of TAK1, inhibited Nef-induced signaling activation and M1-like phenotypic differentiation of M2-MΦ. Meanwhile, the preferential effect of Nef on M2-MΦ correlated with the fact the Nef entered M2-MΦ more efficiently than M1-MΦ. Importantly, the macropinosome formation inhibitor EIPA completely blocked the internalization of Nef into M2-MΦ. Because the macropinocytosis activity of M2-MΦ was higher than that of M1-MΦ, our findings indicate that Nef enters M2-MΦ efficiently by exploiting their higher macropinocytosis activity and drives them towards M1-like MΦ by activating TAK1.

Topics: Animals; Cell Differentiation; Cell Line; HIV-1; Humans; Inflammation; Macrophages; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; nef Gene Products, Human Immunodeficiency Virus; Pinocytosis; Zearalenone

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of Fusarium genera. Piglets were fed for 18 days with a control or a ZEN (316 ppb) contaminated diet. At the end of the experiment tissue samples were taken for assessment of: lymphocyte proliferation, monocytes and granulocytes respiratory burst, inflammatory cytokine synthesis in blood and liver, expression of genes involved in oxidative stress or in inflammation, plasma biochemical parameters, total antioxidant status and nitric oxide synthesis. In blood, ZEN increases the respiratory burst of monocytes and the inflammatory cytokine (TNF alpha, IL-1 beta, IFN gamma) synthesis, while in liver, ZEN decreases the synthesis of all inflammatory cytokines investigated. In liver and spleen, different effect on the expression of genes involved in oxidative stress and inflammation was observed. While in liver, ZEN decrease the expression of cyclooxigenase gene, but increase the expression of glutathione peroxydase and catalase genes; in spleen, ZEN induces a decrease of the superoxide dismutase gene expression together with an increase of the cyclooxigenase. In conclusion, our results showed that liver, spleen and blood may also be target tissues in weanling piglets fed ZEN contaminated diet, with different effects on oxidative stress and inflammation.

Topics: Animals; Base Sequence; Cell Proliferation; Cytokines; DNA Primers; Estrogens, Non-Steroidal; Gene Expression Profiling; Inflammation; Organ Size; Oxidative Stress; Real-Time Polymerase Chain Reaction; Swine; Zearalenone

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Implant particles may induce inflammatory response by activating the nuclear factor kappaB (NFkappaB) and mitogen activated protein kinases (MAPK). Previous studies have shown that these signaling pathways are involved in the transforming growth factor-beta activated kinase 1 (TAK1) in signaling cascades induced by the receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha) as well as interleukin-1beta (IL-1beta). In this study, the roles of the TAK1 pathway in the production of the pro-inflammatory cytokine TNF-alpha in RAW 264.7 cells exposed to titanium alloy particles were investigated. Endogenous TAK1 was phosphorylated upon simulating RAW 264.7 cells by titanium alloy particles. The critical role for TAK1 in p38MAPK and NFkappaB activation was as well confirmed by the inhibition of p38MAPK and NFkappaB activity following 5Z-7-oxozeaenol, a selective inhibitor of TAK1. Furthermore, it was found that TNF-alpha was completely blocked by 5Z-7-oxozeaenol in RAW 264.7 cells. These results suggest that the TAK1-MAPK-NFkappaB signaling pathways may be a potential pharmacological target that can prevent instability for arthroplasty prosthesis.

Topics: Alloys; Animals; Arthroplasty; Cell Line; Inflammation; Lipopolysaccharides; Macrophages; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Phosphorylation; Signal Transduction; Titanium; Tumor Necrosis Factor-alpha; Zearalenone

TAK1, a member of the mitogen-activated kinase kinase kinase (MAPKKK) family, participates in proinflammatory cellular signaling pathways by activating JNK/p38 MAPKs and NF-kappaB. To identify drugs that prevent inflammation, we screened inhibitors of TAK1 catalytic activity. We identified a natural resorcylic lactone of fungal origin, 5Z-7-oxozeaenol, as a highly potent inhibitor of TAK1. This compound did not effectively inhibit the catalytic activities of the MEKK1 or ASK1 MAPKKKs, suggesting that 5Z-7-oxozeaenol is a selective inhibitor of TAK1. In cell culture, 5Z-7-oxozeaenol blocked interleukin-1-induced activation of TAK1, JNK/p38 MAPK, IkappaB kinases, and NF-kappaB, resulting in inhibition of cyclooxgenase-2 production. Furthermore, in vivo 5Z-7-oxozeaenol was able to inhibit picryl chloride-induced ear swelling. Thus, 5Z-7-oxozeaenol blocks proinflammatory signaling by selectively inhibiting TAK1 MAPKKK.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; Cells, Cultured; Cyclooxygenase 2; Dose-Response Relationship, Drug; Enzyme Activation; Female; Genes, Reporter; Genetic Vectors; Humans; Immunoblotting; Inflammation; Inhibitory Concentration 50; Interleukin-1; Isoenzymes; Lactones; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Models, Chemical; p38 Mitogen-Activated Protein Kinases; Precipitin Tests; Prostaglandin-Endoperoxide Synthases; Protein Binding; Signal Transduction; Time Factors; Transfection; Zearalenone