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Abstract: Aims: The purpose of this study was to evaluate the therapeutic potential of the helenalin in Toll-like receptor (TLR) signaling pathways. Main methods: RAW264.7 cells were transfected with a NF-κB, IFNβ PRDIII-I, or IP-10 luciferase plasmid and then luciferase enzyme activities were determined by luciferase assay. The expression of iNOS, COX-2, and IP-10 and phosphorylation of IRF3 were determined by Western blotting. The levels of IP-10 were determined with culture medium by using IP-10 ELISA kit. TBK1 kinase activity was determined by MBP assay kit. Key findings: Helenalin inhibited transcription factor NF-κB and IRF3 activation, which was induced by TLR agonists as well as its target genes, such as COX-2, iNOS, and IP-10. Helenalin attenuated ligand-independent activation of NF-κB induced by MyD88, IKKβ, and p65, and IRF3 induced by TRIF, TBK1, or IRF3. Furthermore, helenalin inhibited TBK1 kinase activity in vitro. Significance: TLRs are primary sensors that detect a wide variety of microbial components and play an important role in the induction of innate immune. To evaluate the therapeutic potential of helenalin, we examined its effect on signal transduction via the TLR signaling pathways. Our results suggest that beneficial effects of helenalin on chronic inflammatory diseases are mediated through modulation of TLR signaling pathways by targeting TBK1. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) play important inductive roles in innate immune responses for host defense against invading microbial pathogens. Activation of TLR4 by lipopolysaccharide (LPS) induces dimerization of TLR4 and, subsequently, activation of downstream signaling pathways including nuclear factor-kappa B and interferon regulatory factor 3. TLR4 dimerization may be an early regulatory event in activating signaling pathways induced by LPS. Here, biochemical evidence is reported that isoliquiritigenin, one of the major ingredients derived from licorice root, inhibits LPS-induced TLR4 dimerization resulting in inhibition of nuclear factor-kappa B and interferon regulatory factor 3 activation, and cyclooxygenase-2 and inducible nitric oxide synthase expression. These results suggest that isoliquiritigenin modulates TLR-mediated signaling pathways at the receptor level. Furthermore, these results suggest that TLRs themselves may be important targets for the prevention of chronic inflammatory diseases. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) play an important role in recognition of microbial components and induce innate immune responses by recognizing invading microbial pathogens leading to the activation of the adaptive immune responses. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and TRIF-dependent pathways leading to the expression of pro-inflammatory cytokines and type I interferons (IFNs). The MyD88- and TRIF-dependent pathways lead to the activation of NF-κB and IRF3 through the activation of IKK-β and TBK1, respectively. Selenium is an essential trace element nutrient possessing anticarcinogenic properties. Here, we attempted to identify the molecular targets of selenium in TLR signaling pathways. Selenium inhibited NF-κB activation induced by poly[I:C] (TLR3 agonist), LPS (TLR4 agonist) or overexpression of MyD88 or IKK-β which is the key kinase of MyD88-dependent signaling pathway. Selenium inhibited IRF3 activation induced by poly[I:C], LPS or the overexpression of TRIF or TBK1. Selenium also suppressed the expression of COX-2 and iNOS and the endogenous IFNβ mRNA induced by poly[I:C] or LPS. Therefore, our results suggest that selenium can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs leading to decreased inflammatory gene expression. [Copyright &y& Elsevier]

Toll-like receptors (TLRs) play significant roles in recognizing the pathogen-associated molecular patterns that induce innate immunity, and subsequently, acquired immunity. In general, TLRs have two downstream signaling pathways, the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways, which lead to the activation of nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). 1-[5-methoxy-2-(2-nitrovinyl)phenyl]pyrrolidine (MNP) has been previously synthesized in our laboratory. To evaluate the therapeutic potential of MNP, its effect on signal transduction via the TLR signaling pathways was examined. MNP was shown to inhibit the activation of NF-κB and IRF3 induced by TLR agonists, as well as to inhibit the expression of cyclooxygenase-2, inducible nitric oxide synthase, and interferon inducible protein-10. MNP also inhibited the activation of NF-κB and IRF3 induced by the overexpression of downstream signaling components of the MyD88- or TRIF-dependent signaling pathways. These results suggest that MNP can modulate MyD88- and TRIF-dependent signaling pathways of TLRs, leading to decreased inflammatory gene expression. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptors (TLRs) recognize many pathogen-associated molecular patterns and induce innate immunity. TLR signaling pathways induce the activation of various transcription factors, such as nuclear factor-κB (NF-κB), leading to the induction of pro-inflammatory gene products, such as inducible nitric oxide synthase (iNOS). Here, we investigated the effect of an (E)-1-(2-(2-nitrovinyl)phenyl)pyrrolidine (NVPP), previously synthesized in our laboratory, on inflammation by modulating NF-κB activation and iNOS expression induced by TLR agonists in murine macrophages. NVPP suppressed NF-κB activation and iNOS expression induced by lipopolysaccharide (TLR4 agonist), polyriboinosinic polyribocytidylic acid (TLR3 agonist), and macrophage-activating lipopeptide 2kDa (TLR2 and TLR6 agonist). All the results suggest that NVPP is suitable for development as a new anti-inflammatory drug. [Copyright &y& Elsevier]

Abstract: Aims: The aim of this study was to evaluate the therapeutic potential of the phenethyl isothiocyanate (PEITC) in Toll-like receptor (TLR) signaling pathways. Main methods: To evaluate the cytotoxic nature of PEITC in RAW 264.7 cells, cytotoxicity was determined using the MTS cell viability assay. RAW264.7 cells were transfected with a nuclear factor-κB (NF-κB), interferon β (IFNβ) PRDIII-I, or interferon inducible protein-10 (IP-10) luciferase plasmid and then luciferase enzyme activities were determined by luciferase assay. The expression of inducible nitric oxide synthase (iNOS) and phosphorylation of interferon regulatory factor 3 (IRF3) were determined by Western blotting. The levels of IP-10 were determined with culture medium by using an IP-10 enzyme-linked immunosorbent assay (ELISA) kit. Key findings: PEITC suppressed the activation of IRF3 and the expression of IP-10 induced by lipopolysaccharide (LPS) or polyinosinic–polycytidylic acid (poly[I:C]). Significance: TLRs play an important role in the induction of innate immune responses for host defense against invading microbial pathogens. PEITC found in cruciferous vegetables has an effect on treatment of many chronic diseases. Our results suggest that beneficial effects of PEITC on chronic inflammatory diseases are mediated through modulation of Toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent signaling pathway of TLRs. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) recognize molecular structures derived from microbes and initiate innate immunity. The stimulation of TLR4 by lipopolysaccharide (LPS) triggers the activation of the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent major downstream signaling pathways. Previously, we synthesized a fumaryl oxazolidinone derivative, 4-oxo-4-(2-oxo-oxazolidin-3-yl)-but-2-enoic acid ethyl ester (OSL07) and demonstrated that it inhibits activation of nuclear factor kappa B (NF-κB) by inhibiting the MyD88-dependent pathway of TLRs. TLR4 and the downstream signaling components are good therapeutic targets for many chronic inflammatory diseases. Here, it is investigated whether OSL07 modulates TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by OSL07. OSL07 inhibited LPS-induced NF-κB and interferon regulatory factor 3 activation by targeting TLR4 dimerization. These results suggest that OSL07 can modulate TLR4 signaling pathway leading to decreased inflammatory gene expression. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptors (TLRs) recognize molecular structures derived from microbes including bacteria, viruses, yeast, and fungi, and regulate the activation of innate immunity. All TLR signaling pathways culminate in the activation of nuclear factor-κB (NF-κB) transcription factor leading to the induction of inflammatory gene products including cytokines and cyclooxygenase-2 (COX-2). In the present report, we demonstrate biochemical evidence that the fumaryl oxazolidinone derivative 4-Oxo-4-(2-oxo-oxazolidin-3-yl)-but-2-enoic acid ethyl ester (OSL07), which was previously synthesized in our laboratory, inhibits the NF-κB activation induced by TLR agonists and overexpression of downstream signaling components of TLRs, MyD88 and IKKβ. OSL07 also inhibits TLR agonist-induced COX-2 expression. These results indicate that the anti-inflammatory effects of OSL07 are caused by the modulation of the immune responses regulated by TLR signaling pathways. [Copyright &y& Elsevier]