Murine Toll-like receptor 8 is a nucleic acid multi-sensor detecting 2',3'-cyclic monophosphate guanosine as well as combinations of ribo-, deoxy-, cyclic nucleotides, and nucleosides

Toll-like receptor 8 (TLR8) in humans senses RNA degradation products and elicits an inflammatory immune response. In contrast, the ligand specificity and function of its murine counterpart mTLR8, long considered non-functional, remain poorly defined. Here, we established an agonist combination model of poly-deoxythymidine (poly-dT) DNA and TLR7/8 binding site 1 agonists such as uridine or the benzazepine compound TL8-506, which activates mTLR8, while suppressing mTLR7 signaling. Extensive agonist analysis based on this model revealed that 2,3-cyclic guanosine monophosphate (2,3-cGMP) serves as a natural ligand for mTLR8, suggesting functionality of its binding site 1 without engagement of site 2. In addition, 2,3-cyclic uridine monophosphate, bacterial single-stranded (ss) DNA, double-stranded (ds) DNA fragments, microRNAs, ssRNA derived from HIV1, SARS-CoV-2, or bacterial sources all potentiate mTLR8 sensing of site 1 agonists. All these stimuli induce distinct inflammatory responses from murine macrophages and microglia via TLR8. In vivo, intrathecal administration of TL8-506 and poly-dT led to microglial accumulation and neuronal injury in the murine cerebral cortex through TLR8, highlighting the potential neuropathological consequences of mTLR8 activation. Taken together, our study defines mTLR8 as a nucleic acid sensor detecting 2,3-cGMP as well as combinations of ssDNA, dsDNA, ssRNA fragments, 2,3-cyclic nucleotide monophosphates, and nucleosides, with implications for host defense and neuroinflammation.