Elsevier

Biochemical Pharmacology

Volume 77, Issue 8, 15 April 2009, Pages 1391-1400
Biochemical Pharmacology

Suppression of the TRIF-dependent signaling pathway of Toll-like receptors by luteolin

https://doi.org/10.1016/j.bcp.2009.01.009Get rights and content

Abstract

Toll-like receptors (TLRs) play important roles in induction of innate immune responses for both host defense against invading pathogens and wound healing after tissue injury. Since dysregulation of TLR-mediated immune responses is closely linked to many chronic diseases, modulation of TLR activation by small molecules may have therapeutic potential against such diseases. Expression of the majority of lipopolysaccharide-induced TLR4 target genes is mediated through a MyD88-independent (TRIF-dependent) signaling pathway. In order to evaluate the therapeutic potential of the flavonoid luteolin we examined its effect on TLR-stimulated signal transduction via the TRIF-dependent pathway. Luteolin suppressed activation of Interferon regulatory factor 3 (IRF3) and NFκB induced by TLR3 and TLR4 agonists resulting in the decreased expression of target genes such as TNF-α, IL-6, IL-12, IP-10, IFNβ, CXCL9, and IL-27 in macrophages. Luteolin attenuated ligand-independent activation of IRF3 or NFκB induced by TLR4, TRIF, or TBK1, while it did not inhibit TLR oligomerization. Luteolin inhibited TBK1-kinase activity and IRF3 dimerization and phosphorylation, leading to the reduction of TBK1-dependent gene expression. Structural analogs of luteolin such as quercetin, chrysin, and eriodictyol also inhibited TBK1-kinase activity and TBK1-target gene expression. These results demonstrate that TBK1 is a novel target of anti-inflammatory flavonoids resulting in the down-regulation of the TRIF-dependent signaling pathway. These results suggest that the beneficial activities of these flavonoids against inflammatory diseases may be attributed to the modulation of TLR-mediated inflammatory responses.

Introduction

Toll-like receptors (TLRs) play a critical role in host defense by sensing invading microbial pathogens and initiating innate and adaptive immune responses. Activation of the immune system by bacterial or viral infection stimulates inflammatory responses that are necessary for host defense against invading pathogens. However, dysregulation of TLR-mediated cellular responses can lead to chronic inflammation, which in turn contributes to the development and progress of many inflammatory diseases. Accumulating evidence shows the close relationship between TLRs and various inflammatory diseases including septic shock, atherosclerosis, rheumatoid arthritis, diabetes and cancer [1], [2]. Understanding how TLR activation can be modulated may thus provide new opportunities to develop effective therapeutics for chronic inflammatory diseases [3].

Broadly, TLRs can activate two branches of downstream signaling pathways, MyD88- and TRIF-dependent pathways culminating in the expression of inflammatory gene products including cytokines and chemokines. MyD88 is a common downstream adaptor leading to the activation of the IKKβ complex and the NFκB transcription factor [4]. TRIF, another adaptor molecule of TLRs, is mainly responsible for regulating MyD88-independent pathways [5]. TRIF activates the downstream kinases, TBK1 and IKKɛ, leading to the phosphorylation and activation of IRF3 and the consequent expression of type I IFNs and IFN-inducible genes [6]. IFNβ and IFN-inducible genes such as IP-10 and iNOS are critical inducers of endotoxic shock following LPS exposure. The survival rate of IFNβ- and iNOS-deficient mice was significantly greater than that of controls following induction of experimental sepsis [7], [8], [9]. Mice deficient in type I IFN receptors were also highly resistant to infection by the gram-positive bacterium, Listeria monocytogenes[10]. Since the expression of IFNβ is mainly dependent on activation of the TRIF-signaling pathway, these data suggest an important role for the TRIF-dependent signaling pathway in mediating inflammatory responses.

Flavonoids abundant in plants are widely known to exert various biological activities including anti-inflammatory and anti-cancer effects [11]. Luteolin, a flavonoid compound found in many herbal extracts including celery, green pepper, and chamomile, is known to have anti-inflammatory activity. Oral administration of luteolin to mice suppressed the inflammatory responses in acute and chronic inflammation animal models such as carrageenan-induced paw edema, air pouch models and the cotton pellet granuloma test [12]. Treatment of mice with luteolin alleviated inflammatory responses and decreased bacterial load in pulmonary infection with Chlamydia pneumoniae[13]. Intraperitoneal injection of luteolin greatly increased the survival rate of mice in a sepsis model induced by LPS challenge, and also reduced the level of inflammatory markers such as serum levels of TNF-α and IL-6, ICAM-1 expression in liver, and leukocyte infiltration in the lung and liver [14]. However, the molecular target of luteolin in TLR signaling pathways has not been fully identified.

Luteolin inhibits the activation of IKKβ[15]. This finding suggests a role for luteolin in MyD88-dependent signaling pathway since IKKβ is one of the major kinases downstreams of MyD88. However, it is not known if luteolin can regulate TRIF-dependent (MyD88-independent) signaling following TLR stimulation. Since the TRIF-dependent pathway is responsible for the expression of more than 70% of LPS-induced genes [16] and TRIF-dependent genes significantly contribute to endotoxin lethality [7], the modulation of the TRIF-dependent pathway of TLRs might be a useful and novel anti-inflammatory strategy. Therefore, we investigated whether flavonoids such as luteolin can modulate the TRIF-dependent signaling pathway of TLRs, and we identified their anti-inflammatory targets in this pathway. Our study will provide insight to understand how the dietary modulation of TLR activation can be an attractive strategy for reducing the risk of the inflammatory diseases.

Section snippets

Cell culture

RAW264.7 cells and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) heat inactivated fetal bovine serum (FBS, Invitrogen), 100 units/ml Penicillin, and 100 μg/ml Streptomycin (Invitrogen). Ba/F3 cells, a murine pro-B cell line, expressing TLR4 (Flag- or GFP-tagged), CD14, and MD2 were cultured in RPMI1640 medium as described previously [17]. Bone marrow cells isolated from wild type (C57BL/6) mice (Japan SLC, Inc., Japan) were cultured in DMEM containing

Luteolin suppresses the activation of TLR3 and TLR4

To investigate if luteolin modulates TRIF-dependent signaling pathway of TLRs, we first determined whether luteolin affects the activation of TLR3 and TLR4 which have TRIF as an adaptor molecule. Luteolin inhibited IRF3-reporter gene expression induced by LPS (TLR4 agonist) or poly(I:C) (TLR3 agonist) in mouse macrophage cell line, RAW264.7 (Fig. 1A and B). In addition, NFκB-reporter gene expression induced by LPS or poly(I:C) was prevented by luteolin (Fig. 1C and D). Furthermore, luteolin

Discussion

Accumulating evidence now suggests the importance of TRIF-dependent signaling pathway of TLRs in inflammatory responses and development of certain chronic diseases. It was reported that the expression of more than 70% of LPS-induced genes was modulated in a TRIF-dependent manner suggesting the significant contribution of TRIF-signaling to TLR4-mediated immune responses [16]. Especially, the expression of IFNβ and IFN-inducible genes that are critical mediators of endotoxic shock [7] was

Acknowledgements

We thank Dr. Charles B. Stephensen in USDA Western Human Nutrition Research Center, Davis, CA, USA for critical readings and scientific editing. This work was supported by the grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A080830) (to J.Y.L.) and by grants DK064007-01A2, from the National Institutes of Health (to D.H.H).

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