Single-Stranded Oligonucleotides Can Inhibit Cytokine Production Induced by Human Toll-Like Receptor 3

Abstract

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.

Agonist and Antagonist Recognition by RIG-I, a Cytoplasmic Innate Immunity Receptor

“…1B except css27, consistent with reports that ssRNA require a 5Ј-triphosphate to act as an RIG-I agonist (25,26). Double-stranded RNAs dsR27, pIC25, pIC115, and poly(I:C) were able to activate RIG-I signaling in the absence of the 5Ј-triphosphates from 6-to 13-fold (Fig.…”

“…Modified ODNs with phosphorothioate backbones can bind to the ectodomain of TLR3 and inhibit signaling (26). We wanted to assess whether ODNs such as 2006 can affect RIG-I activity in the cellbased assay (Fig.…”

“…A similar assay was previously used to map template recognition by the hepatitis C virus polymerase (29) and also substrate recognition by the severe acute respiratory syndrome coronavirus endoribonuclease (30). Briefly, 2006 was synthesized with a 3Ј amine to allow cross-linking to NHS-succimidyl ester-modified resin (26). R-HR cross-linked to 2006 on the NHS resin was digested with trypsin, and the resin was extensively washed in an attempt to remove peptides that are not covalently linked.…”

Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1

“…B, shown is the effects of siRNA to Unc93b1 on TLR4-dependent signal transduction. Cells were transfected to express TLR4 and its co-activators and induced with 1 g/ml lipopolysaccharide as described previously (47). The firefly luciferase reporter is driven by the NF-B promoter and normalized to the Renilla luciferase driven by the thymidine kinase promoter.…”

Targeting DAMP Activation of Toll-Like Receptors: Novel Pathways to Treat Rheumatoid Arthritis?

“…Further evidence for a role for the endosomal TLRs in CIA has also come from studies with short DNA oligonucleotides (ODNs). These act as Immunoregulatory Sequences (IRS) and inhibit endosomal TLR activity (Barrat et al, 2005;Ranjith-Kumar et al, 2008;Lenert, 2010). Prophylactic administration of ODNs in CIA and CpG-induced arthritis has been shown to abrogate disease progression (Zeuner et al, 2002;Dong et al, 2004).…”