Toll-like receptors (TLR) 7 and 8 are closely related members of the TLR family of pathogen-associated molecular pattern recognition receptors and have an important function in activation of innate immune responses upon viral infection. TLR7 can be activated selectively by the guanosine analogue loxoribine, whereas the imidazoquinoline derivative Resiquimod (R-848) activates both TLR7 and TLR8. We demonstrate that co-incubation of R-848 with thymidine homopolymer oligodeoxynucleotides (ODN) significantly increased activity of R-848 on TLR8-expressing HEK 293 cells, but abolished TLR7-mediated signaling. Similarly, the combination of loxoribine and thymidine ODN redirected the stimulatory effect of loxoribine away from TLR7, and toward TLR8. This alteration in ligand specificity was demonstrated both in TLRtransfected HEK cells, and also in human PBMC, with a corresponding change in cytokine production away from IFN-a secretion by TLR7-expressing plasmacytoid DC and toward IL-12, TNF-a and IFN-c secretion by TLR8-expressing monocytes and NK cells. These results demonstrate an unexpected plasticity in the ligand specificities of TLR7 and TLR8, and suggest a novel sequence-selective interaction between these receptors and synthetic phosphorothioate ODN.
IntroductionComprehension of the immune systems responses to pathogens has advanced dramatically since the discovery of the family of Toll-like receptors (TLR). TLR belong to the IL-1 receptor (IL-1R) superfamily and recognize so-called pathogen-associated molecular patterns (PAMP) thereby activating innate immune responses to bacterial, viral or fungal threats. In mammals, 11 TLR have been described so far. Natural pathogenderived ligands have been identified for many TLR: lipopolysaccharide (TLR4), triacyl lipopeptides (TLR1), lipopeptides and glycolipids (TLR2), flagellin (TLR5) and zymosan (TLR2 and TLR6) are some of these bacterial and fungal components. Non-methylated, CpG-containing DNA of bacterial or viral origin has been found to activate TLR9, whereas double-stranded viral RNA was demonstrated to target TLR3 (for review see [1]). Most recently, several groups identified singlestranded RNA as well as natural ligands for TLR7 and TLR8 [2][3][4]. In addition, certain short interfering RNA (siRNA) sequences have been identified as potential TLR7 ligands [5].TLR are type I membrane receptors with TLR 1, 2, 4, 5, and 6 located on the cell surface and TLR 7, 8, and 9 located within the cell [6][7][8][9]. TLR3 was found to be expressed intracellularly and at the cell surface in certain cell types, e.g., fibroblasts [10] or epithelial cells [11,12]. For TLR9, localization in the endoplasmic reticulum and recruitment to the endosomal compart- As activation of TLR is important for stimulating immune responses, the TLR family has been a target for pharmaceutical drug development. Synthetic ligands for several TLR have been developed and some are being tested in clinical trials. CpG-containing oligodeoxynucleotides (ODN), e.g., PF-3512676 (formerly known as CPG 7909),...
