RESUMO
Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFkappaB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFkappaB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFkappaB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNbeta in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFkappaB or IRF3 induced by MyD88-dependent (MyD88, IKKbeta) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFkappaB activation. Further, cinnamaldehyde inhibited ligand-independent NFkappaB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.
Assuntos
Acroleína/análogos & derivados , Anti-Inflamatórios/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , Acroleína/farmacologia , Animais , Linhagem Celular , Humanos , Fator Regulador 3 de Interferon/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , NF-kappa B/antagonistas & inibidores , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/fisiologiaRESUMO
Toll-like receptors (TLRs), which are activated by invading microorganisms or endogenous molecules, evoke immune and inflammatory responses. TLR activation is closely linked to the development of many chronic inflammatory diseases including rheumatoid arthritis. Auranofin, an Au(I) compound, is a well-known and long-used anti-rheumatic drug. However, the mechanism as to how auranofin relieves the symptom of rheumatoid arthritis has not been fully clarified. Our results demonstrated that auranofin suppressed TLR4-mediated activation of transcription factors, NF-kappaB and IRF3, and expression of COX-2, a pro-inflammatory enzyme. This suppression was well correlated with the inhibitory effect of auranofin on the homodimerization of TLR4 induced by an agonist. Furthermore, auranofin inhibited NF-kappaB activation induced by MyD88-dependent downstream signaling components of TLR4, MyD88, IKKbeta, and p65. IRF3 activation induced by MyD88-independent signaling components, TRIF and TBK1, was also downregulated by auranofin. Our results first demonstrate that auranofin suppresses the multiple steps in TLR4 signaling, especially the homodimerization of TLR4. The results suggest that the suppression of TLR4 activity by auranofin may be the molecular mechanism through which auranofin exerts anti-rheumatic activity.
Assuntos
Auranofina/administração & dosagem , Linfócitos B/metabolismo , Lipopolissacarídeos/administração & dosagem , Receptor 4 Toll-Like/metabolismo , Animais , Antirreumáticos/administração & dosagem , Linfócitos B/efeitos dos fármacos , Linhagem Celular , Dimerização , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Ouro/administração & dosagem , CamundongosRESUMO
Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-kappaB. (-)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-kappaB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKbeta which is the key kinase in the canonical pathway for NF-kappaB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Catequina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/fisiologia , Animais , Catequina/química , Catequina/farmacologia , Linhagem Celular , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Expressão Gênica/genética , Humanos , Quinase I-kappa B/metabolismo , Interferon gama/genética , Luciferases/genética , Luciferases/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide , NF-kappa B/genética , NF-kappa B/metabolismo , Fenóis/química , Fenóis/farmacologia , Polifenóis , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Chá/química , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , Transfecção , Quinase Induzida por NF-kappaBRESUMO
Toll-like receptors play a key role in sensing microbial components and inducing innate immune responses. Ligand-induced dimerization of TLR4 is required for the activation of downstream signaling pathways. Thus, the receptor dimerization may be one of the first lines of regulation in activating TLR-mediated signaling pathways and induction of subsequent immune responses. LPS induces the activation of NF-kappaB and IRF3 through MyD88- or TRIF-dependent pathways. Curcumin, a polyphenol found in the plant Curcuma longa, has been shown to suppress the activation of NF-kappaB induced by various pro-inflammatory stimuli by inhibiting IKKbeta kinase activity in MyD88-dependent pathway. Curcumin also inhibited LPS-induced IRF3 activation. These results imply that curcumin inhibits both MyD88- and TRIF-dependent pathways in LPS-induced TLR4 signaling. However, in TRIF-dependent pathway, curcumin did not inhibit IRF3 activation induced by overexpression of TRIF in 293T cells. These results suggest that TLR4 receptor complex is the molecular target of curcumin in addition to IKKbeta. Here, we report biochemical evidence that phytochemicals (curcumin and sesquiterpene lactone) inhibit both ligand-induced and ligand-independent dimerization of TLR4. Furthermore, these results demonstrate that small molecules with non-microbial origin can directly inhibit TLRs-mediated signaling pathways at the receptor level. These results imply that the activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain dietary phytochemicals we consume daily.