Guanosine and its modified derivatives are endogenous ligands for TLR7

Abstract

Toll-like receptor (TLR) 7and 8 were considered to recognize single-strand RNA (ssRNA) from viruses. Although these receptors also respond to synthetic small chemical ligands, such as CL075 and R848, it remains to be determined whether these receptors sense natural small molecules or not. In the structure of human TLR8 (huTLR8) with ssRNA, there are two ligand-binding sites: one binds a uridine and the other binds an oligoribonucleotide (ORN). This finding demonstrates that huTLR8 recognizes degradation products of ssRNA, suggesting the presence of natural small ligands. We here show that TLR7 works as the sensor for guanosine (G)/2'-deoxyguanosine (dG) in the presence of ORN where ORN strengthens TLR7 interaction with G/dG. In addition, modified nucleosides such as 7-methylguanosine, 8-hydroxyguanosine (8-OHG) and 8-hydroxydeoxyguanosine (8-OHdG) activated TLR7 with ORNs. Importantly, 8-OHdG-a well-known oxidative DNA damage marker with unknown function-induced strong cytokine production comparable to G and dG both in mouse and human immune cells. Although 8-OHdG bound TLR7/ORN with lower affinity than dG did in isothermal titration calorimetry, administered 8-OHdG was metabolically more stable than dG in the serum, indicating that 8-OHdG acts on TLR7 as an endogenous ligand in vivo To address a role of G analogs in the disease state, we also examined macrophages from Unc93b1 (D34A/D34A) mice, which suffer from TLR7-dependent systemic inflammation, and found that Unc93b1 (D34A/D34A) macrophages showed significantly enhanced response to G alone or 8-OHdG with ORN. In conclusion, our results provide evidence that G, dG, 8-OHG and 8-OHdG are novel endogenous ligands for TLR7.

Deoxyguanosine is a TLR7 agonist

“…In the future, it will therefore be interesting to determine the structure of TLR7 in complex with dG. In contrast to our observations, earlier studies reported that dG does not activate TLR7 in WT cells . We speculate that these differences may be due to assay sensitivity and the type and/or activation status of the cells used; indeed, Shibata et al.…”

“…We speculate that these differences may be due to assay sensitivity and the type and/or activation status of the cells used; indeed, Shibata et al. found that dG treatment induces cytokines in hyperresponsive Unc93b1 mutated cells .…”

Nucleic acid-sensing TLRs: trafficking and regulation

“…In addition to guanosine, TLR7 can also bind and respond to deoxyguanosine, a breakdown product of DNA [62]. This raises the possibility that, in addition to RNA, TLR7 can also respond to DNA degradation products in the presence of oligoribonucleotides.…”

Conservation and Divergence of Ligand Recognition and Signal Transduction Mechanisms in Toll-Like Receptors

“…60) Guanosine and its derivatives activate TLR7 in combination with ssRNA. 61) Accordingly, the activated dimer form of TLR7 is induced by simultaneous binding to ssRNA and guanosine, similar to TLR8. In addition, chemical ligands alone, such as R848, can also induce TLR7 dimerization.…”

“…63) Similar to the enhanced activation of TLR8 by uridine in the presence of ssRNA, 62) guanosine, when used alone, cannot activate or induce dimerization of TLR7 effectively, according to the results of an nuclear factorkappaB (NF-κB) reporter assay and biochemical assay using recombinant proteins, but can induce the dimerization of TLR7 and activate TLR7 when used with ssRNA. 60,61) …”