Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

Abstract

i RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-␣) or alpha interferon (IFN-␣) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short doublestranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-␣ production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-␣, independent of 5=-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.

Sequence-dependent off-target inhibition of TLR7/8 sensing by synthetic microRNA inhibitors

“…As shown in Figure 5 , miR-224-5p mut2 AMO is more stable than the native miR-224-5p and miR-224-5p mut1 AMOs, and displayed the strongest TLR7 inhibitory activity. Given that 2′OMe RNAs have a strong affinity to TLR7 ( 13 ), it is reasonable to assume that the rules regulating their TLR7 activity are similar to that of immunostimulatory ssRNA, and that structure has an impact ( 20 – 21 , 44 ). We have previously shown that immunostimulatory motifs embedded in RNA with double-stranded regions were more potent activators of TLR8 ( 20 , 21 ).…”

It’s the Little Things (in Viral RNA)

“…It has been suggested that the changes in RNA secondary structures associated with inosine are detected through TLR7 and TLR8. These changes also lead to an increase in TNF-α production, which would mean that inosine serves as a molecular pattern to be recognized in the phagocytosed RNA, as these receptors are mainly expressed by antigen-presenting dendritic cells ( 78 ).…”

The Role of Nucleic Acid Sensing in Controlling Microbial and Autoimmune Disorders

“…More recently, researchers also determined that the RNA editing phenomenon of adenosine-to-inosine conversion enhances TLR7/8 activation. In this experiment, TLR7 sensing of ssRNA was enhanced in inosine-modified viral RNA (Sarvestani et al, 2014). The cooperative role between TLR7 and TLR8 is also an important part of the mechanism that results in an IFN response to ssRNA.…”