Toll-like receptors (TLRs) play an important role in the innate immune response to pathogens1. TLRs detect PAMPs (pathogen-associated molecular patterns) and stimulate immune cells via the MyD88-dependent interleukin 1 receptor (IL-1R)–TLR signaling pathway, which leads to activation of the transcription factor NF-κB2. In humans, ten members of this family (TLR1 to TLR10) have been reported to date. TLR2, TLR4 and TLR5 are crucial for the recognition of peptidoglycan, lipopolysaccharide and flagellin, respectively3,4,5. TLR6 associates with TLR2 and recognizes lipoproteins from mycoplasma6. TLR9 detects bacterial DNA containing unmethylated CpG motifs and TLR3 activates immune cells in response to double-stranded RNA7,8,9. The natural ligands for TLR1, TLR7, TLR8 and TLR10 are not known, although a synthetic compound with antiviral activity has now been described as a ligand for TLR710.

Hemmi et al. reported in Nature Immunology that, in experiments that used gene-deficient mice, the antiviral imidazoquinoline resiquimod (R848) activates immune cells via the TLR7 MyD88-dependent signaling pathway10. They showed that macrophages from MyD88- and TLR7-deficient mice do not respond to R848 stimulation, whereas macrophages from wild-type mice strongly induce the transcription factor NF-κB and the secretion of proinflammatory cytokines, such as tumor necrosis factor-α, as well as the regulatory cytokine IL-12. In addition, Hemmi et al. showed, by genetic complementation in HEK293 cells, that human TLR7 confers responsiveness to R84810.

In the quest to identify ligands for TLRs, we also screened immunostimulatory synthetic compounds for their potential to activate HEK293 cells that were transiently transfected with TLR cDNAs and a NF-κB luciferase reporter plasmid. We found that R848 induced NF-κB activation in HEK293 cells transfected with human TLR8 (GenBank accession number AF245703) in a dose-dependent manner (Fig. 1). In accordance with the findings of Hemmi et al., human and murine TLR7 (GenBank accession numbers AF240467 and AY035889) also mediate R848 recognition (Fig. 1)10. TLR7 showed a higher sensitivity to R-848, but TLR8 was able to induce NF-κB more effectively than TLR7 when higher concentrations of R-848 were used (Fig. 1). In contrast, HEK293 cells transiently transfected with murine TLR8 (GenBank accession number AY035890) did not activate NF-κB after stimulation with R848 (data not shown), which suggests that TLR8 is nonfunctional in mice. This mute phenotype is in accordance with the observation that TLR7-deficient mice do not respond to R848, even though TLR8 is present10.

Figure 1: R-848 induces NF-κB activation via TLR8 or TLR7.
figure 1

HEK293 cells were transfected by electroporation with vectors expressing human TLR8, human TLR7 or murine TLR7 and a NF-kB luciferase reporter plasmid. Sixteen hours after transfection, cells were stimulated with R848 (commercially synthesized by GLSynthesis, Worcester, MA) at the indicated concentrations for a further 7 h. TLR2-, TLR3- and TLR4-transfected cells did not induce NF-kB activation after stimulation with R848; a negative result was also scored for murine TLR8 (data not shown10). Data are mean ± s.d. from one representative of three independent experiments.

These results show that both human TLR7 and TLR8 can independently mediate recognition of the same antiviral compound, imidazoquinoline R848, which suggests a possible redundancy among these receptors. The differential dose response observed between these receptors may allow fine-tuning of the immune response to high or low concentrations of this molecule as well as other ligands. Further studies will be necessary to identify the natural ligands for both receptors and clarify the status and function of murine TLR8.