Optimal oligonucleotide sequences for TLR9 inhibitory activity in human cells: lack of correlation with TLR9 binding

Robert F Ashman; J Adam Goeken; Eicke Latz; Petar Lenert

doi:10.1093/intimm/dxq473

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Optimal oligonucleotide sequences for TLR9 inhibitory activity in human cells: lack of correlation with TLR9 binding

Journal article

Open access

Peer reviewed

Optimal oligonucleotide sequences for TLR9 inhibitory activity in human cells: lack of correlation with TLR9 binding

Robert F Ashman, J Adam Goeken, Eicke Latz and Petar Lenert

International immunology, Vol.23(3), pp.203-214

03/2011

DOI: 10.1093/intimm/dxq473

PMCID: PMC3053407

PMID: 21393636

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Abstract

Toll-like receptor (TLR)9 performs our innate response to bacterial DNA, warning us of the presence of infection. Inhibitory oligodeoxyribonucleotides (INH-ODN) have been developed that selectively block activation of mouse TLR9. Their inhibitory motif consisting of CCx(not-C)(not-C)xxGGG (x = any base) also reduces anti-DNA antibodies in lupus mice. The current study demonstrates that this motif also provides the sequences required to block TLR9 in human B cells and human embryonic kidney (HEK) cells transfected with human TLR9. However, extending the sequence by four to five bases at the 5' end enhanced activity and this enhancement was greater when a phosphorothioate (pS) backbone replaced the native phosphodiester (pO) backbone. A series of pO-backbone INH-ODN representing a 500-fold range of activity in biologic assays was shown to cover less than a 2.5-fold range of avidity for binding human TLR9-Ig fusion protein, eliminating TLR9 ectodomain binding as the explanation for sequence-specific differences in biologic activity. With few exceptions, the relative activity of INH-ODN in Namalwa cells and HEK/human TLR9 cells was similar to that seen in mouse B cells. INH-ODN activity in human peripheral blood B cells correlated significantly with the cell line data. These results favor the conclusion that although the backbone determines strength of TLR9 binding, critical recognition of the INH-ODN sequence necessary for biologic activity is performed by a molecule that is not TLR9. These studies also identify the strongest INH-ODN for human B cells, helping to guide the selection of INH-ODN sequences for therapeutics in any situation where inflammation is enhanced by TLR9.

Flow Cytometry

Cell Line

Toll-Like Receptor 9 - antagonists & inhibitors

Anti-Inflammatory Agents - pharmacology

Humans

Oligodeoxyribonucleotides - chemistry

Molecular Sequence Data

Toll-Like Receptor 9 - immunology

Animals

B-Lymphocytes - immunology

Oligodeoxyribonucleotides - genetics

Base Sequence

Phosphorothioate Oligonucleotides - pharmacology

Protein Binding - drug effects

Mice

Phosphorothioate Oligonucleotides - chemistry

Oligodeoxyribonucleotides - pharmacology

DNA-Binding Proteins

Details

Title: Subtitle: Optimal oligonucleotide sequences for TLR9 inhibitory activity in human cells: lack of correlation with TLR9 binding
Creators: Robert F Ashman - Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
J Adam Goeken
Eicke Latz
Petar Lenert
Resource Type: Journal article
Publication Details: International immunology, Vol.23(3), pp.203-214
DOI: 10.1093/intimm/dxq473
PMID: 21393636
PMCID: PMC3053407
ISSN: 0953-8178
eISSN: 1460-2377
Grant note: R01 AI083713 / NIAID NIH HHS R01 HL093262 / NHLBI NIH HHS R01AI0437374 / NIAID NIH HHS
Language: English
Date published: 03/2011
Academic Unit: Pathology; Immunology; Internal Medicine
Record Identifier: 9984094596402771

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