Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides

Int J Mol Sci. 2016 Feb 22;17(2):242. doi: 10.3390/ijms17020242.

Abstract

Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

Keywords: CpG oligodeoxynucleotides; TLR9; Toll-like receptor 9; internalization; nuclear factor; phosphorylation; proinflammatory cytokine; synthetic peptide.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dose-Response Relationship, Drug
  • Gene Expression Regulation / drug effects
  • Humans
  • Interleukin-6 / metabolism*
  • Lipopeptides / pharmacology
  • Lipopolysaccharides / pharmacology
  • Mice
  • Oligodeoxyribonucleotides / antagonists & inhibitors*
  • Peptides / pharmacology*
  • RAW 264.7 Cells
  • Signal Transduction / drug effects
  • Toll-Like Receptor 9 / metabolism*
  • Tumor Necrosis Factor-alpha / metabolism*

Substances

  • CPG-oligonucleotide
  • Interleukin-6
  • Lipopeptides
  • Lipopolysaccharides
  • Oligodeoxyribonucleotides
  • Pam(3)CSK(4) peptide
  • Peptides
  • TLR9 protein, human
  • Toll-Like Receptor 9
  • Tumor Necrosis Factor-alpha