Common Interaction Surfaces of the Toll-Like Receptor 4 Cytoplasmic Domain Stimulate Multiple Nuclear Targets

ABSTRACT Toll-like receptor 4 (TLR4) mediates the host response to lipopolysaccharide (LPS) by promoting the activation of pro- and anti-inflammatory cytokine genes. To activate each gene, numerous signal transduction pathways are required. The adaptor proteins MyD88 and TIRAP contribute to the activation of several and possibly all pathways via direct interactions with TLR4's Toll/interleukin-1 receptor (IL-1R) (TIR) domain. However, additional adaptors that are required for the activation of specific subsets of pathways may exist, which could contribute to the differential regulation of target genes. Furthermore, it remains unknown whether direct interactions that have been reported between TIR domains and other proteins are required for TLR4 signaling. To address these issues, we systematically mutated the TLR4 TIR domain in the context of a CD4/TLR4 fusion protein. Several exposed residues defining at least two structural surfaces were required in macrophages for activation of the proinflammatory IL-12 p40 and anti-inflammatory IL-10 promoters, as well as promoters dependent on individual transcription factors. Interestingly, the same residues were required by all promoters tested, suggesting that the signaling pathways diverge downstream of the adaptors. The mutant phenotypes provide a framework for future studies of TLR4 signaling, as the interaction supported by each critical surface residue will need to be defined.

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Oxymatrine inhibits lipopolysaccharide-induced inflammation by down-regulating Toll-like receptor 4/nuclear factor-kappa B in macrophages

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2014-0362 ◽

2015 ◽

Vol 93 (4) ◽

pp. 253-260 ◽

Cited By ~ 22

Author(s):

Yu Zhang ◽

Ruhong Yan ◽

Yae Hu

Keyword(s):

Nitric Oxide ◽

Interleukin 1 ◽

Chinese Herb ◽

Mrna Levels ◽

Nuclear Factor ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Anti Inflammatory

Oxymatrine (OMT) is the quinolizidine alkaloid extracted from the Chinese herb Sophora flavescens Ait. that has many pharmacological effects and is used for the treatment of some inflammatory diseases. In this study, RAW264.7 cells and THP-1 differentiated macrophages were pretreated with various concentrations of OMT at 2 h prior to treatment with lipopolysaccharide (LPS) (1.0 μg/mL) for different durations. We detected the anti-inflammatory effect of OMT in LPS-stimulated macrophages and investigated the molecular mechanism. We showed that OMT pretreatment significantly inhibited the LPS-induced secretion of nitric oxide (NO), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) in supernatant, attenuated the mRNA levels of inducible nitric oxide synthase (iNOS), IL-1β, TNF-α, and Toll-like receptor 4 (TLR4), increased TLR4 and phosphorylation of inhibitor of kappa B-alpha (p-IBα) in cytosol, and decreased the nuclear level of nuclear factor-κB (NF-κB) p65 in macrophages. In conclusion, OMT exerts anti-inflammatory properties in LPS-stimulated macrophages by down-regulating the TLR4/NF-κB pathway.

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The Small GTPase Arf6 Is Essential for the Tram/Trif Pathway in TLR4 Signaling

Journal of Biological Chemistry ◽

10.1074/jbc.m113.499194 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1364-1376 ◽

Cited By ~ 19

Author(s):

Tim Van Acker ◽

Sven Eyckerman ◽

Lieselotte Vande Walle ◽

Sarah Gerlo ◽

Marc Goethals ◽

...

Keyword(s):

Plasma Membrane ◽

Critical Role ◽

Adaptor Proteins ◽

Small Gtpase ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Mouse Macrophages ◽

Adp Ribosylation Factor ◽

Irf3 Activation

Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal−/− mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.

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Identification of Interaction Sites for Dimerization and Adapter Recruitment in Toll/Interleukin-1 Receptor (TIR) Domain of Toll-like Receptor 4

Journal of Biological Chemistry ◽

10.1074/jbc.m111.282350 ◽

2011 ◽

Vol 287 (6) ◽

pp. 4088-4098 ◽

Cited By ~ 46

Author(s):

Celia Bovijn ◽

Peter Ulrichts ◽

Anne-Sophie De Smet ◽

Dominiek Catteeuw ◽

Rudi Beyaert ◽

...

Keyword(s):

Interleukin 1 ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tir Domain ◽

Interaction Sites

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Bisdemethoxycurcumin and Its Cyclized Pyrazole Analogue Differentially Disrupt Lipopolysaccharide Signalling in Human Monocyte-Derived Macrophages

Mediators of Inflammation ◽

10.1155/2018/2868702 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 3

Author(s):

Serena Tedesco ◽

Morena Zusso ◽

Laura Facci ◽

Annalisa Trenti ◽

Carlotta Boscaro ◽

...

Keyword(s):

Human Blood ◽

Inflammatory Responses ◽

Interleukin 1 ◽

Human Monocyte ◽

Signalling Pathways ◽

Toll Like Receptor ◽

Blood Monocytes ◽

Toll Like Receptor 4 ◽

Anti Inflammatory ◽

Macrophage Subset

Several studies suggest that curcumin and related compounds possess antioxidant and anti-inflammatory properties including modulation of lipopolysaccharide- (LPS-) mediated signalling in macrophage cell models. We here investigated the effects of curcumin and the two structurally unrelated analogues GG6 and GG9 in primary human blood-derived macrophages as well as the signalling pathways involved. Macrophages differentiated from peripheral blood monocytes for 7 days were activated with LPS or selective Toll-like receptor agonists for 24 h. The effects of test compounds on cytokine production and immunophenotypes evaluated as CD80+/CCR2+ and CD206+/CD163+ subsets were examined by ELISA and flow cytometry. Signalling pathways were probed by Western blot. Curcumin (2.5–10 μM) failed to suppress LPS-induced inflammatory responses. While GG6 reduced LPS-induced IκB-α degradation and showed a trend towards reduced interleukin-1β release, GG9 prevented the increase in proinflammatory CD80+ macrophage subset, downregulation of the anti-inflammatory CD206+/CD163+ subset, increase in p38 phosphorylation, and increase in cell-bound and secreted interleukin-1β stimulated by LPS, at least in part through signalling pathways not involving Toll-like receptor 4 and nuclear factor-κB. Thus, the curcumin analogue GG9 attenuated the LPS-induced inflammatory response in human blood-derived macrophages and may therefore represent an attractive chemical template for macrophage pharmacological targeting.

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Toll or Interleukin-1 Receptor (TIR) Domain-containing Adaptor Inducing Interferon-β (TRIF)-mediated Caspase-11 Protease Production Integrates Toll-like Receptor 4 (TLR4) Protein- and Nlrp3 Inflammasome-mediated Host Defense against Enteropathogens

Journal of Biological Chemistry ◽

10.1074/jbc.m112.401406 ◽

2012 ◽

Vol 287 (41) ◽

pp. 34474-34483 ◽

Cited By ~ 157

Author(s):

Prajwal Gurung ◽

R. K. Subbarao Malireddi ◽

Paras K. Anand ◽

Dieter Demon ◽

Lieselotte Vande Walle ◽

...

Keyword(s):

Host Defense ◽

Nlrp3 Inflammasome ◽

Interleukin 1 ◽

Protease Production ◽

Toll Like Receptor ◽

Interferon Β ◽

Toll Like Receptor 4 ◽

Tir Domain

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Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011633 ◽

2020 ◽

Vol 295 (14) ◽

pp. 4661-4672 ◽

Cited By ~ 1

Author(s):

Ardeshir Ariana ◽

Norah A. Alturki ◽

Stephanie Hajjar ◽

Deborah J. Stumpo ◽

Christopher Tiedje ◽

...

Keyword(s):

Cell Death ◽

Rna Binding ◽

Tumor Necrosis Factor Receptor ◽

Adaptor Proteins ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Murine Macrophages ◽

Highly Sensitive ◽

Mitogen Activated Protein

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88−/−, Trif−/−, MyD88−/−Trif−/−, MK2−/−, and Zfp36−/− mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)–induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

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