Glucocorticoid-induced TNF receptor family-related protein functions as a costimulatory molecule for murine eosinophils.

Eosinophils are typical effector cells associated with type 2 immune responses and play key roles in the pathogenesis of allergic diseases. These cells are activated by various stimuli, such as cytokines, chemokines, and growth factors, but the regulatory mechanisms of eosinophil effector functions remain unclear. Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR), a transmembrane protein belonging to the tumor necrosis factor (TNF) receptor superfamily, is a well-known regulatory molecule for T cell activation. Here, we show that GITR is also constitutively expressed on eosinophils and functions as a costimulatory molecule for these cells. Although degranulation was unaffected by GITR engagement of murine bone marrow-derived eosinophils, secretion of inflammatory cytokines such as interleukin (IL)-4, IL-6, and IL-13 from IL-33-activated bone marrow-derived eosinophils was augmented by anti-mouse GITR agonistic antibody (DTA-1). In conclusion, our results provide a new regulatory pathway of cytokine secretion from eosinophils in which GITR functions as a costimulatory molecule.

Lipoteichoic Acid Inhibits Lipopolysaccharide-Induced TLR4 Signaling by Forming an Inactive TLR4/MD-2 Complex Dimer.

LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow-derived macrophages from TLR2-/- mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-ß without affecting cell surface expression of TLR4. LTA did not suppress IL-1ß-induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.

Minimal structure of IRAK-1 to induce degradation of TRAF6.

Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1-102), a serine/proline/threonine-rich ProST domain (amino acids 103-197), a central kinase domain with an activation loop (amino acids 198-522), and the C-terminal C1 and C2 domains (amino acids 523-712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585-591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants-including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.

Application of a Recombinant Three-Factor Chromogenic Reagent, PyroSmart, for Bacterial Endotoxins Test Filed in the Pharmacopeias.

Assays using lysate reagents prepared from horseshoe crab hemocyte extract (limulus amoebocyte lysate, LAL) are commonly and widely used to detect and measure endotoxin in parenteral drugs and medical devices. However, lysate reagents suffer from lot-to-lot variations leading to possible fluctuations in testing. Also, this continued usage of lysate reagents leads to the possible decline of the horseshoe crab population. Recently, a new recombinant chromogenic reagent, PyroSmart, consisting of three recombinant factors was introduced to the market. There are now three recombinant products; two with recombinant factor C reagents and PyroSmart with the complete recombinant LAL system. We evaluated the applicability of the reagent to the harmonized bacterial endotoxins test in the United States, European and Japanese pharmacopeias. The recombinant product showed equivalent potency of thirteen endotoxins from different bacterial strains to conventional chromogenic lysate reagents as long as their assay modes are identical. All analytical characteristics or assay parameters of the reagent satisfied the acceptance criteria which are set for the use for the bacterial endotoxins test filed in the pharmacopeias. All of 109 parenteral drugs tested can be measured with PyroSmart within respective maximum allowable dilutions. The lot-to-lot variation in recovery of endotoxin added in the parenteral drugs for PyroSmart was equal to or less than those of six limulus lysate reagents. In conclusion, the present study suggests that the recombinant reagent, PyroSmart, provide a good alternative to the LAL reagents with better lot-to-lot variation.

Activation through toll-like receptor 2 on group 2 innate lymphoid cells can induce asthmatic characteristics.

BACKGROUND: Type 2 innate lymphoid cells (ILC2s) are one of the sources of IL-5 and IL-13 in allergic airway inflammation. Innate immune receptors such as Toll-like receptors (TLRs) expressed on epithelial cells could contribute to ILC2 activation through IL-33 production, but a direct effect of TLRs on ILC2s remains to be elucidated. OBJECTIVES: We hypothesized that TLRs can directly activate lung ILC2s and participate in the pathogenesis of asthma. METHODS: After intranasal administration of IL-33 to wild-type (WT), TLR2KO and TLR4KO female mice, ILC2s were isolated from harvested lungs. ILC2s were incubated with IL-2 and TLR stimulants (pam3csk4 (PAM), house dust mite extract (HDM)). In some experiments, TLR2 or dectin-1 signalling inhibitors were used. As an in vivo model, the mice were treated with IL-33 and rested until lung recruitment of eosinophils regressed. Then they were treated intranasally with PAM + HDM or vehicle and analysed. RESULTS: In vitro stimulation of isolated ILC2s showed that PAM could induce IL-13 and IL-5 production, and HDM had a synergistic effect on this stimulation. Both effects were dependent on TLR2 and NF-κB signalling. PAM + HDM stimulation of WT mice led to increased ILC2s, airway hyperresponsiveness and increased levels of both neutrophils and eosinophils in bronchoalveolar lavage fluid. These observations were dependent on TLR2. CONCLUSIONS & CLINICAL RELEVANCE: TLR2 can directly activate lung ILC2s, an effect that is augmented by HDM. Asthmatic characteristics mediated through the TLR2 pathway were evident in the in vivo mice model. These data implicate a new pathway of ILC2 activation in the pathogenesis of asthma.

IRF1 supports DNA binding of STAT1 by promoting its phosphorylation.

The signal transducer and activator of transcription 1 (STAT1), a pivotal transcription factor in Janus kinase (JAK)-STAT signaling, regulates the expression of a wide range of immune-related genes, including interferon (IFN) regulatory factor 1 (IRF1). In this study, we found that IRF1 could induce STAT1 phosphorylation and in turn STAT1 activation. When IRF1 was transiently expressed in HEK293 cells, STAT1 phosphorylated at Y701, dimerized and bound to an oligonucleotide containing a gamma-activated sequence (GAS) derived from the IRF1 promoter. IRF1 expression also induced GAS-dependent promoter reporter activity, and phosphorylation of JAK1, a kinase upstream of STAT1. Although no direct interaction between IRF1 and STAT1 was observed, the transactivation domain of IRF1 was required for IRF1-mediated STAT1 activation, indicating the involvement of gene product(s) regulated by IRF1. Moreover, supernatants from cells expressing IRF1 induced phosphorylation of STAT1 and JAK1, and subsequent GAS binding by STAT1 that could not be blocked by treatment with antibodies against IFN-ß or IFN-γ. IFN-γ-induced STAT1 phosphorylation persisted for up to 30 h following stimulation of HEK293, but declined in IRF1-deficient HEK293 cells. IRF1-promoter activity induced by IFN-γ was also reduced in IRF1-deficient HEK293 cells, which could be rescued by complementation with IRF1. Together these results indicate that IRF1 promotes DNA binding of STAT1, which can in turn participate in a positive feedback loop of JAK-STAT signaling.

AKT1 distinctively suppresses MyD88-depenedent and TRIF-dependent Toll-like receptor signaling in a kinase activity-independent manner.

We found that AKT1, a primary effector molecule of PI3K-AKT signaling, distinctively suppressed Toll-like receptor (TLR)-mediated MyD88-dependent and Toll/IL-1R domain-containing adaptor inducing IFN-ß (TRIF)-dependent signaling by inhibiting NF-κB activation and IRF3 activity independently of its kinase activity. In AKT1 knockout RAW264.7 cells, lipopolysaccharide (LPS)-induced transcription and protein production of cytokines including IL-1ß and TNF-α (regulated by the MyD88-dependent pathway), as well as IFN-ß and RANTES (C-C motif chemokine ligand 5: CCL-5; regulated by the TRIF-dependent pathways) was enhanced compared to wild type cells. In response to LPS stimulation, AKT1 knockout cells also exhibited enhanced NF-κB and IFN-ß promoter activities, which were reduced to a level comparable to that in wild type cells by complementation with either AKT1 or its kinase-dead mutant (AKT1-KD). Expression of AKT1 or AKT1-KD similarly suppressed NF-κB and IFN-ß promoter activities induced by LPS and other TLR ligands in wild type cells. Analysis of NF-κB activation caused by transient expression of proteins involved in the MyD88-dependent pathway in TLR signaling revealed that AKT1 suppressed signaling that occurs between activation of IKKß and that of NF-κB. In contrast, AKT1 appeared to suppress the IFN-ß promoter through inhibition of IRF3 activity itself. These results demonstrate a novel, non-kinase function of AKT1 that inhibits TLR signaling, and suggest the multifunctional nature of AKT1.

NF-κB activation via MyD88-dependent Toll-like receptor signaling is inhibited by trichothecene mycotoxin deoxynivalenol.

Macrophages induce the innate immunity by recognizing pathogens through Toll-like receptors (TLRs), which sense pathogen-associated molecular patterns. Myeloid differentiation factor 88 (MyD88), which is an essential adaptor molecule for most TLRs, mediates the induction of inflammatory cytokines through nuclear factor κB (NF-κB). Trichothecene mycotoxin deoxynivalenol (DON) shows immunotoxic effects by interrupting inflammatory mediators produced by activated macrophages. The present study investigates the effect of DON on NF-κB in activated macrophages through MyD88-dependent pathways. DON inhibited NF-κB-dependent reporter activity induced by MyD88-dependent TLR agonists. In addition, lipopolysaccharide-induced phosphorylation of interleukin-1 receptor-associated kinase 1 and inhibitor κBα were attenuated by DON. Furthermore, DON downregulated the expression level of MyD88. These results suggest that DON inhibits NF-κB activation in macrophages stimulated with TLR ligands via MyD88-dependent TLR signals. Therefore exposure to DON may lead to the inhibition of MyD88-dependent pathway of TLR signaling.

Zinc- and oxidative property-dependent degradation of pro-caspase-1 and NLRP3 by ziram in mouse macrophages.

The NLRP3 inflammasome, composed of caspase-1, NLRP3 and ASC, plays a critical role in the clearance of microbial pathogens. Here, we found that the treatment of mouse macrophages with the zinc-containing dithiocarbamate ziram, a widely used fungicide in agriculture, caused a decrease in pro-caspase-1 and NLRP3 levels while not affecting ASC level. Ziram did not affect levels of pro-caspase-1 and NLRP3 mRNA, and no cleavage products of pro-caspase-1 including p10 subunit, which is an autocleavage product of pro-caspase-1, were detected, indicating that the decrease was associated with degradation of these proteins. The decrease was inhibited by SH-type antioxidants, N-acetyl cysteine, dithiothreitol and 2-mercaptoethanol, or a metal chelator EDTA but not by inhibitors of proteasome, lysosomes, autophagy and matrix metalloproteases. Thiram, a comparator for ziram that does not contain zinc, showed a weaker decrease in protein levels. Furthermore, the zinc-containing dithiocarbamate, zinc diethyldithiocarbamate, efficiently decreased the levels of pro-caspase-1 and NLRP3, whereas dithiocarbamates, dimethyldithiocarbamate and diethyldithiocarbamate without zinc, were less active. The organic zinc compound [3,4-toluenedithiolato(2-)]zinc hydrate did not induce a decrease in protein levels. Ziram also inhibited IL-1ß production by macrophages in response to lipopolysaccharide and bacterial clearance during Salmonella infection of macrophage cells. These results indicate that ziram causes degradation of pro-caspase-1 and NLRP3 in a zinc- and oxidative property-dependent manner and suggest that exposure to ziram may compromise the clearance of microbial pathogens.

Toll-like receptors required for dermatophagoides farinae to activate NF-κB.

Body and excrement extracts from Dermatophagoides farinae were used to study stimulation of Toll-like receptors (TLRs). The excrement extract stimulated nuclear factor (NF)-κB-dependent reporter activity to an extent similar to lipopolysaccharide (LPS) in a mouse macrophage cell line, J774A.1, but the activity of the body extract was negligible. The excrement extract also activated NF-κB in HEK293 cells expressing TLR1/TLR2, TLR2/TLR6 and CD14/TLR4/MD-2, whereas no activation was observed in cells expressing TLR3, TLR5, TLR7, TLR8 or TLR9. Although the excrement extract required co-expression of CD14, TLR4 and MD-2 in HEK293 cells to activate NF-κB, efficient activation was still observed in I-13.35 cells, a bone-marrow macrophage cell line established from LPS-hypo-responsive C3H/HeJ mice. The excrement extract activated NF-κB in HEK293 cells expressing TLR2 alone, but the activation was significantly increased by co-expression of CD14. Polymyxin B inhibited CD14/TLR4/MD-2- and CD14/TLR2-mediated activation of NF-κB but not the activation in I-13.35 cells. These results indicate that CD14/TLR4/MD-2-dependent and CD14/TLR2-dependent mechanisms are involved in the activation of NF-κB by the excrement extract of D. farinae and suggest that the extract also contains substances that activate NF-κB through non-TLR-mediated mechanisms.

Role of toll-like receptor 4 in mediating multiorgan dysfunction in mice with acetaminophen induced acute liver failure.

Strategies for the prevention of multiorgan dysfunction (MOD) in acetaminophen (APAP)-induced acute liver failure (ALF) are an unmet need. Our study tested the hypothesis that sterile inflammation induced by APAP in a mouse model would activate toll-like receptor 4 (TLR4) in the liver and extrahepatic organs and lead to the progression of ALF and MOD and that the administration of the novel TLR4 antagonist STM28 (a peptide formed of 17 amino-acids) would prevent liver injury and associated MOD. ALF and, subsequently, MOD were induced in TLR4-knockout (KO) mice (B6.B10ScN-Tlr4 (lpsdel) /JthJ) and wild-type (WT) mice (C57BL/6) with APAP (500 mg/kg). A second set of experiments was conducted to evaluate the effects of a pretreatment with a novel TLR4 antagonist, STM28, on APAP-induced MOD in CD1 mice. Animals were sacrificed at the coma stage, and plasma, peripheral blood cells, liver, kidneys, and brain were collected. Biochemistry values and cytokines were measured. Liver and kidneys were studied histologically and were stained for TLR4 and activated Kupffer cells, and the expression of nuclear factor kappa B-p65 was quantified with western blotting. Brain water was measured in the frontal cortex. After APAP administration, TLR4-KO (NFkBp65) mice were relatively protected from liver necrosis and end-organ dysfunction and had significantly better survival than WT controls (P < 0.01). STM28 attenuated liver injury and necrosis, reduced creatinine levels, and delayed the time to a coma significantly. The increases in cytokines in the plasma and liver, including TLR4 expression and the activation of Kupffer cells, after APAP administration were reduced significantly in the STM28-treated animals. The increased number of circulating myeloid cells was reduced significantly after STM28 treatment. In conclusion, these data provide evidence for an important role of the TLR4 antagonist in the prevention of the progression of APAP-induced ALF and MOD.

Lipid IVa incompletely activates MyD88-independent Toll-like receptor 4 signaling in mouse macrophage cell lines.

We investigated the difference in the effect of synthetic lipid A compounds on MyD88-dependent and -independent Toll-like receptor 4 (TLR4) signaling in mouse macrophage cells. At higher concentrations, Escherichia coli-type hexa-acylated lipid A 506, Salmonella-type hepta-acylated lipid A 516, the lipid A precursor lipid IVa and monophosphoryl lipid A induced similar levels of production of the MyD88-dependent cytokine IL-1ß although their potencies varied, whereas the maximum production of the MyD88-independent cytokine RANTES induced by lipid IVa was less than 50% that of other lipid A compounds. A maximum level of NF-κB activation, which is involved in IL-1ß gene transcription, was also induced to a similar level by these four lipid A compounds, while the maximum level of IFN-ß promoter activity induced during MyD88-independent signaling was also less than 50% for lipid IVa stimulation compared with other lipid A compounds. Early IκBα phosphorylation activated by MyD88-dependent signaling was similarly induced by 506 and lipid IVa, whereas lipid IVa barely stimulated the phosphorylation of IRF3, a MyD88-independent transcription factor, although efficient phosphorylation was observed with 506 stimulation. These results indicate that lipid IVa has limited activity toward MyD88-independent signaling of TLR4, in macrophage cell lines, despite having efficient activity in the MyD88-dependent pathway.

Application of matrix-assisted laser desorption ionization-time of flight mass spectrometry for discrimination of laboratory-derived antibiotic-resistant bacteria.

The ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to discriminate laboratory-derived antibiotic-resistant bacterial strains of known genetic origin was examined. A computer-based cluster analysis of spectral data successfully discriminated the majority of single- as well as multiple-antibiotic-resistant Escherichia coli strains examined. Cluster analysis of Staphylococcus aureus strains with different levels of novobiocin resistance showed that as the degree of resistance increased similarity to the wild-type strain decreased. These results demonstrate that MALDI-TOF MS is capable of discriminating antibiotic-resistant bacterial strains and may have potential for differentiating bacterial strains with varying degrees of antibiotic-resistance.

IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6.

TRAF6 plays a crucial role in signal transduction of the Toll-like receptor (TLR). It has been reported that TRAF6 catalyzes the formation of unique Lys63-linked polyubiquitin chains, which do not lead to proteasome-mediated degradation. Here we found that stimulation of J774.1 cells with various TLR ligands led to decreases in TRAF6 protein levels that occurred at a slower rate than IκBα degradation. The decrease in TRAF6 was inhibited by proteasome inhibitors MG-132, lactacystin and N-acetyl-leucyl-leucyl-norleucinal. Among intracellular TLR signaling molecules MyD88, IRAK-4, IRAK-1, TRAF6, and IKKß, only IRAK-1 expression downregulated TRAF6 in HEK293 cells. The amount of TRAF6 expressed either transiently or stably was also reduced by co-expression of IRAK-1 and no TRAF6 cleavage products were detected. The levels of either a TRAF6 N-terminal deletion mutant or a ubiquitin ligase-defective mutant were not affected by IRAK-1 expression. Downregulation of TRAF6 required the TRAF6-binding site (Glu544, Glu587, Glu706) of IRAK-1 but not its catalytic site (Asp340). Upon IRAK-1 transfection, no significant TRAF6 ubiquitination was detected. Instead, TRAF6-associated IRAK-1 was ubiquitinated with both Lys48- and Lys63-linked polyubiquitin chains. TRAF6 downregulation was inhibited by co-expression of the E3 ubiquitin ligase Pellino 3, whose Lys63-linked polyubiquitination on IRAK-1 is reported to compete with Lys48-linked IRAK-1 polyubiquitination. Expression of IRAK-1 inhibited IκBα phosphorylation in response to TLR2 stimulation. These results indicate that stimulation of TLRs induces proteasome-dependent downregulation of TRAF6. We conclude that TRAF6 associated with ubiquitinated IRAK-1 is degraded together by the proteasome and that IRAK-1 possesses a negative regulatory role on TLR signaling.

Application of matrix-assisted laser desorption ionization-time of flight mass spectrometry for discrimination of Escherichia strains possessing highly conserved ribosomal RNA gene sequences.

We evaluated the capability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to discriminate twelve Escherichia strains: E. blattae, E. fergusonii, E. hermanii and nine E. coli, whose ribosomal RNA (rRNA) gene sequence homologies are in the range of 96-100%. Similarities obtained by MALDI-TOF MS were found to be 78-92% among the E. coli strains, and 74% between E. coli and E. fergusonii. E. blattae and E. hermanii showed only 32% similarity when compared to the other species. Thus, MALDI-TOF MS provides capability of distinguishing bacterial species or even strains possessing highly conserved rRNA gene sequences.

Urban aerosols induce pro-inflammatory cytokine production in macrophages and cause airway inflammation in vivo.

Urban air pollution, especially in developing countries, is a crucial environmental problem. Urban aerosols may contain various kinds of substances and induce harmful effects such as allergic diseases. Therefore, it is critical to clarify the biological effects of urban aerosols on human health. In this study, we evaluated the induction of airway inflammation in vitro and in vivo due to exposure of urban aerosols. We investigated cytokine production and nuclear factor-kappaB (NF-kappaB) activation after stimulation of macrophage cells by exposure of urban aerosols. Urban aerosols were found to induce the production of interleukin (IL)-8, tumor necrosis factor-alpha and IL-1beta on macrophage cells. In addition, we showed that NF-kappaB pathway regulated the urban aerosols-induced inflammatory cytokine response. Moreover, the intranasal administration of urban aerosols resulted in increases in the total cell number in bronchoalveolar lavage and infiltration of eosinophils in lung tissue. These results indicate that urban aerosols induce respiratory inflammation and onset of inflammatory disease due to an activation of the immune system.

Inhibitory effects of soluble MD-2 and soluble CD14 on bacterial growth.

The effects of the soluble forms of the endotoxin receptor molecules sMD-2 and sCD14 on bacterial growth were studied. When Escherichia coli and Bacillus subtilis were incubated at 37 degrees C for 18 hr with either sMD-2 or sCD14, growth of these bacteria was significantly inhibited as evaluated by viable cell counts and NADPH/NADH activity. A mutant of sCD14 (sCD14d57-64) lacking a region essential for LPS binding did not inhibit the growth of E. coli, whereas this mutant did inhibit the growth of B. subtilis. Addition of excess PG to the bacterial culture reversed the inhibitory effect of sMD-2 on the growth of B. subtilis, but not on the growth of E. coli. Furthermore, when evaluated by ELISA, both sMD-2 and sCD14 bound specifically to PG. Taken together, these results indicate that sMD-2 and sCD14 inhibit the growth of both Gram-positive and Gram-negative bacteria and further suggest that binding to PG and LPS is involved in the inhibitory effect of sMD-2 on Gram-positive bacteria and of sCD14 on Gram-negative bacteria, respectively.

Deoxynivalenol and nivalenol inhibit lipopolysaccharide-induced nitric oxide production by mouse macrophage cells.

Deoxynivalenol (DON) and nivalenol (NIV), trichothecene mycotoxins, are secondary metabolites produced by Fusarium fungi. Trichothecene mycotoxins cause immune dysfunction, thus leading to diverse responses to infection. The present study evaluated the effect of DON and NIV on nitric oxide (NO) production by RAW264 cells stimulated with lipopolysaccharide (LPS). LPS-induced NO production was reduced in the presence of these toxins. The transcriptional activation and expression of inducible NO synthase (iNOS) by LPS were also repressed by these toxins. DON or NIV inhibited LPS-induced expression of interferon-beta (IFN-beta), which plays an indispensable role in LPS-induced iNOS expression. These results indicate that DON and NIV inhibit the LPS-induced NO and IFN-beta production, which both play an important role for host protection against invading pathogens, and suggests that the inhibition of these factors may be involved in the immunotoxic effects of these mycotoxins.

Caspase-3 is activated and rapidly released from human umbilical vein endothelial cells in response to lipopolysaccharide.

Endothelial cell injury/dysfunction is considered to play a critical role in the pathogenesis of severe sepsis and septic shock. Although it is considered that endothelial cell apoptosis is involved in endothelial injury/dysfunction, physiological involvement remains ambiguous since the induction of apoptosis requires the inhibition of endogenous apoptosis inhibitors. Here we show that caspase-3 activation, a biological indicator of apoptosis, is observed in response to lipopolysaccharide (LPS) stimulation even under the influence of endogenous apoptosis inhibitors, and that activated caspase-3 is rapidly released from human umbilical vein endothelial cells (HUVEC). In the presence of cycloheximide (CHX), an increase in intracellular caspase-3/7 activity in response to LPS was not detected in HUVEC up to 24 h following stimulation even in the presence of LPS-binding protein (LBP), soluble CD14 and soluble MD-2, whereas the decrease in cell viability and increase in release of the cellular enzyme lactate dehydrogenase (LDH) were observed in a soluble CD14/LBP-dependent manner. On the other hand, even in the absence of CHX, a significant increase in caspase-3/7 activity and a cleaved caspase-3 fragment with a slight increase in LDH release was observed in culture supernatants in response to LPS. This increase in caspase-3/7 activity was observed even when LDH release was undetected. These results indicate that caspase-3 is activated by LPS under physiological conditions and suggest that HUVEC escape from cell death by rapidly releasing activated caspase-3 into extracellular space. Failure of this escape mechanism may result in endothelial injury/dysfunction.

A novel TLR4-binding peptide that inhibits LPS-induced activation of NF-kappaB and in vivo toxicity.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. It is a ligand for Toll-like receptor 4 (TLR4), which plays an essential role in innate immunity. Macrophages and dendritic cells exposed to LPS overproduce proinflammatory mediators, leading to septic shock. In this study, we screened for peptides that associate with TLR4 with a yeast two-hybrid screen using the human TLR4 extracellular domain as bait. A peptide (STM28) isolated from the screen inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in human and mouse macrophage cells and interacted with TLR4 in yeast and mammalian cells. STM28 showed no inhibitory effects against NF-kappaB activation induced by TLR1/2, TLR3 and TLR9 ligands in a mouse macrophage cell line, RAW 264. In addition, STM28 suppressed LPS-induced tumor necrosis factor-alpha production by differentiated THP-1 cells. Moreover, LPS-induced lethality in d-galactosamine-sensitized mice was significantly repressed by STM28 in a dose-dependent manner. These results demonstrate that STM28 selectivity inhibits TLR4-induced macrophage activation, and suggest that STM28 may have utility as a novel therapeutic agent for Gram-negative bacterial sepsis.