title: Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides.
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.
authors.affiliation: Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing 400038, China. xichunpan@163.com.
authors.forename: Xichun
authors.initials: X
authors.lastname: Pan
authors.affiliation: Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing 400038, China. libin6033@sina.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing 400038, China. km0640602@126.com.
authors.forename: Mei
authors.initials: M
authors.lastname: Kuang
authors.affiliation: Medical Research Center, Southwestern Hospital, the Third Military Medical University, Chongqing 400038, China. triplestars@163.com.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing 400038, China. cenyanyan@163.com.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Cen
authors.affiliation: Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing 400038, China. michel_0415@163.com.
authors.forename: Rongxin
authors.initials: R
authors.lastname: Qin
authors.affiliation: Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing 400038, China. dingguofu@126.com.
authors.forename: Guofu
authors.initials: G
authors.lastname: Ding
authors.affiliation: Medical Research Center, Southwestern Hospital, the Third Military Medical University, Chongqing 400038, China. zhengj99219@163.com.
authors.forename: Jiang
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing 400038, China. zhouh64@163.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhou
mesh_terms: D000818:Animals; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D055666:Lipopeptides / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000494:pharmacology*; D000067996:RAW 264.7 Cells; D015398:Signal Transduction / Q000187:drug effects; D051217:Toll-Like Receptor 9 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: C408982:CPG-oligonucleotide; D015850:Interleukin-6; D055666:Lipopeptides; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; C508075:Pam(3)CSK(4) peptide; D010455:Peptides; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha
keywords: CpG oligodeoxynucleotides; TLR9; Toll-like receptor 9; internalization; nuclear factor; phosphorylation; proinflammatory cytokine; synthetic peptide