Blocking TIR Domain Interactions in TLR9 Signaling

Area of Science:

• Immunology
• Molecular Biology
• Drug Discovery

Background:

• Toll-like receptor 9 (TLR9) activation by ligands triggers NF-κB signaling and pro-inflammatory cytokine production.
• Dysregulated TLR9 activation is implicated in the pathogenesis of various inflammatory diseases.
• Targeting TLR9 signaling offers a potential therapeutic avenue for managing inflammatory conditions.

Purpose of the Study:

• To investigate the potential of cell-permeating decoy peptides (CPDPs) derived from the TLR9 Toll/IL-1R resistance (TIR) domain as inhibitors of TLR9 signaling.
• To identify specific CPDPs with potent and selective inhibition of TLR9-mediated inflammatory responses.
• To elucidate the mechanism of action and in vivo efficacy of the most potent TLR9-specific CPDP.

Main Methods:

• Design and synthesis of CPDPs based on the TLR9 TIR domain.
• Assessment of CPDP inhibitory activity on TLR9-induced cytokine production in cell-based assays.
• Evaluation of CPDP specificity against other Toll-like receptors (TLR2/1, TLR4, TLR7).
• Analysis of CPDP binding to TLR9 and adapter protein (TIRAP, MyD88) TIR domains using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging.
• In vivo assessment of CPDP efficacy in a mouse model of oligonucleotide-induced inflammation and lethality.

Main Results:

• CPDP 9R34, incorporating AB loop, β-strand B, and N-terminal BB loop residues, demonstrated the most potent inhibition of TLR9 signaling.
• Other CPDPs derived from α-helices C, D, and E showed less potent inhibition.
• 9R34 exhibited TLR9 specificity, without inhibiting TLR2/1, TLR4, or TLR7 signaling.
• The N-terminally truncated 9R34-ΔN retained potent TLR9 inhibitory activity.
• 9R34-ΔN specifically bound to TLR9 TIR domains and, to a lesser extent, TIRAP TIR, but not MyD88 TIR.
• Intraperitoneal administration of 9R34-ΔN effectively suppressed systemic cytokine production and lethality in mice challenged with oligonucleotides.

Conclusions:

• A potent and TLR9-specific CPDP, 9R34-ΔN, was identified, targeting both receptor dimerization and adapter recruitment.
• The findings suggest that TLR TIR domains interact via homologous surfaces, forming filamentous structures.
• This study provides a promising therapeutic candidate for TLR9-mediated inflammatory diseases.