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Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain.

ABSTRACT: Toll/IL-1R resistance (TIR) domain-containing adapter-inducing IFN-? (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NF?B. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation in wild-type cells. TF5 failed to inhibit LPS-induced cytokine and kinase activation in TRIF-deficient immortalized bone-marrow-derived macrophage, but was fully inhibitory in MyD88 knockout cells. TF5 does not block macrophage activation induced by TLR2, TLR3, TLR9, or retinoic acid-inducible gene 1/melanoma differentiation-associated protein 5 agonists. Immunoprecipitation assays demonstrated that TF4 binds to TLR4 but not TRIF-related adaptor molecule (TRAM), whereas TF5 binds to TRAM strongly and TLR4 to a lesser extent. Although TF5 prevented coimmunoprecipitation of TRIF with both TRAM and TLR4, site-directed mutagenesis of the TRIF B helix residues affected TRIF-TRAM coimmunoprecipitation selectively, as these mutations did not block TRIF-TLR4 association. These results suggest that the folded TRIF TIR domain associates with TRAM through the TRIF B helix region, but uses a different region for TRIF-TLR4 association. The B helix peptide TF5, however, can associate with either TRAM or TLR4. In a mouse model of TLR4-driven inflammation, TF5 decreased plasma cytokine levels and protected mice from a lethal LPS challenge. Our data identify TRIF sites that are important for interaction with TLR4 and TRAM, and demonstrate that TF5 is a potent TLR4 inhibitor with significant potential as a candidate therapeutic for human sepsis.

SUBMITTER: Piao W

PROVIDER: S-EPMC3839745 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain.

Piao Wenji W Ru Lisa W LW Piepenbrink Kurt H KH Sundberg Eric J EJ Vogel Stefanie N SN Toshchakov Vladimir Y VY

Proceedings of the National Academy of Sciences of the United States of America 20131105 47

Toll/IL-1R resistance (TIR) domain-containing adapter-inducing IFN-β (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NFκB. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation ...[more]

PMID: 24194546

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Project description:BACKGROUND: TIR-domain-containing adapter-inducing interferon-? (TRIF) is the sole downstream adaptor of Toll-like receptor (TLR)3, which is one of the major signaling pathways in immune cells leading to neuroinflammation in the central nervous system. Overexpression of TRIF may lead to activation of inflammatory responses, and contribute to pathophysiological progression in both acute and chronic neurodegenerative retinal diseases. In the present study, was aimed to elucidate the contributions of TRIF to optic nerve (ON) regeneration and retinal ganglion cell (RGC) survival following injury to the ON, a widely studied model of central nervous system injury and of degenerative diseases such as glaucoma. METHODS: We used retrograde labeling with a fluorochrome, hydroxystilbamidine (Fluorogold) to evaluate RGC survival, and immunostaining with growth-associated protein-43 to evaluate axon regeneration in an ON crush model. Changes in microglial cytokines following RGC injury was examined with ELISA and real-time PCR. In vivo studies were carried out in wild-type and trif-/- mice. A Transwell co-culture system and migration test were used to mimic the crosstalk between microglia and RGCs. TRIF-associated downstream adaptors were determined by western blotting. RESULTS: Compared with wild-type (WT) mice, TRIF knockout (KO) mice displayed a robust ability to regenerate axons 3 or 7 days after nerve injury. In addition, RGC survival was considerably higher in trif-/- than in WT mice. ON lesion induced less microglial activation in trif-/- than in WT mice. and more WT microglia distorted and migrated toward the foramen opticum. In the transwell system, few trif-/- microglia migrated through the membrane when stimulated by the performed lesion on RGC axons in a transwell system. Inactivation of microglial cells in trif-/- mice was associated with reduced production of inflammatory cytokines, as detected with real-time RT-PCR and ELISA. Furthermore western blot analysis showed that activation of known downstream effectors of TRIF, including TBK1, IKK? and NF-?B, were significantly inhibited by TRIF deficiency. CONCLUSION: Our results indicate that TRIF deficiency promotes ON axon regeneration by attenuating microglial activation and consequently reducing the release of harmful cytokines via NF-?B inactivation.

Dataset Information

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain.

Publications

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain.

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