ABSTRACT
Toll-like receptors (TLRs) play a crucial role in danger recognition and induction of innate immune response against bacterial and viral infections. The TLR adaptor molecule, toll-interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF), facilitates TLR3 and TLR4 signaling, leading to the activation of the transcription factor, NF-κB and interferon regulatory factor 3 (IRF3). Andrographolide, the active component of Andrographis paniculata, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the role of andrographolide in TLR signaling pathways. Andrographolide suppressed NF-κB activation as well as COX-2 expression induced by TLR3 or TLR4 agonists. Andrographolide also suppressed the activation of IRF3 and the expression of interferon inducible protein-10 (IP-10) induced by TLR3 or TLR4 agonists. Andrographolide attenuated ligand-independent activation of IRF3 following overexpression of TRIF, TBK1, or IRF3. Furthermore, andrographolide inhibited TBK1 kinase activity in vitro. These results indicate that andrographolide modulates the TRIF-dependent pathway of TLRs by targeting TBK1 and represents a potential new anti-inflammatory candidate.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Diterpenes/therapeutic use , Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Andrographis/immunology , Animals , Chemokine CXCL10/metabolism , Interferon Regulatory Factor-3/metabolism , Mice , NF-kappa B/metabolism , RAW 264.7 Cells , Signal Transduction , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/metabolism , Transcriptional ActivationABSTRACT
Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) and triggers the activation of myeloid differention factor 88 (MyD88) and the Toll/interleukin-1 receptor domain-containing adapter, inducing interferon-ß (TRIF)-dependent major downstream signaling pathways. To evaluate the therapeutic potential of 1-[5-methoxy-2-(2-nitrovinyl)phenyl]pyrrolidine (MNP), previously synthesized in our laboratory, its effect on signal transduction via the TLR signaling pathways was examined. Here, we investigated whether MNP modulates the TLR4 signaling pathways and which anti-inflammatory target in TLR4 signaling is regulated by MNP. MNP inhibited the activation of nuclear factor-κB (NF-κB) induced by LPS (TLR4 agonist), and it also inhibited the expression of cyclooxygenase-2 and inducible nitric oxide synthase. MNP inhibited LPS-induced NF-κB activation by targeting TLR4 dimerization in addition to IKKß. These results suggest that MNP can modulate the TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.
Subject(s)
Nitro Compounds/pharmacology , Protein Multimerization/drug effects , Pyrrolidines/pharmacology , Toll-Like Receptor 4/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Cells, Cultured , Cyclooxygenase 2/biosynthesis , Dose-Response Relationship, Drug , I-kappa B Kinase/antagonists & inhibitors , Lipopolysaccharides , Mice , NF-kappa B/biosynthesis , Nitric Oxide Synthase Type II/biosynthesis , Signal Transduction/drug effectsABSTRACT
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-ß (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.
Subject(s)
Nitro Compounds/pharmacology , Pyrrolidines/pharmacology , Toll-Like Receptors/agonists , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Chemokine CXCL10/metabolism , Cyclooxygenase 2/metabolism , HEK293 Cells , Humans , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Mice , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Nitro Compounds/chemistry , Pyrrolidines/chemistry , RAW 264.7 Cells , Signal Transduction/drug effectsABSTRACT
Toll-like receptor 4 (TLR4) recognizes LPS and triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter, inducing interferon-ß (TRIF)-dependent major downstream signaling pathways. Previously, we presented biochemical evidence that 1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine (FPP), which was synthesized in our laboratory, inhibits NF-κB activation induced by LPS. Here, we investigated whether FPP modulates the TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by FPP. FPP inhibited LPS-induced NF-κB activation by targeting TLR4 dimerization. These results suggest that FPP can modulate the TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.
