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.
Highlights
The innate immune system employs pathogen-recognition receptors (PRRs) such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs) to sense pathogen-associated molecular patterns (PAMPs) [1]
An unrelated peptide (URP) derived from LRR14 of human TLR5 was used as a control, according to our previous results [3]
The results demonstrated that synthetic peptide (SP) could decrease CpG ODN-induced TNF-α and IL-6 release from RAW264.7 cells dose-dependently
Summary
The innate immune system employs pathogen-recognition receptors (PRRs) such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs) to sense pathogen-associated molecular patterns (PAMPs) [1]. Internalized CpG ODN within the endosome initiates TLR9-mediated signaling via sequential recruitment of MyD88, interleukin receptor associated kinase (IRAK) and TNF-α receptor associated factor 6 (TRAF6), which in turn activate important downstream transcription factors, such as NF-κB and AP-1, culminating in the induction of proinflammatory cytokines such as TNF-α and IL-6 [3]. In autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, self-DNA/RNA-protein complexes activate TLR9 [6]. CpG ODN/TLR9 signaling could be considered a potential target for treating these diseases. Various TLRs and their adapter proteins have many universal molecules; MyD88-target inhibitors can destroy the immune system [8]. We investigated an inhibitory CpG ODN designed as a TLR9 antagonist to block immunostimulatory CpG ODN to treat sepsis and lupus nephritis [2]. It is important to identify new inhibitors that target TLR9 or CpG ODN and safely. In this study, a synthetic LRR11 peptide (SP) was synthesized, and its influence on TLR9 signaling was investigated
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