Toll-like receptor 8 (TLR8) is a single-stranded RNA sensing receptor and is localized in the cellular compartments, where it encounters foreign or self-nucleic acids and activates innate and adaptive immune responses. However, the mechanism controlling intracellular localization TLR8 is not completely resolved. We previously revealed the intracellular localization of TLR8 ectodomain (ECD), and in this study, we investigated the mechanism of the intracellular localization. Here we found that TLR8 ECDs from different species as well as ECDs from different TLRs are all intracellularly localized, similarly to the full-length porcine TLR8. Furthermore, porcine, bovine, and human TLR8 ECDs are all localized in cell endosomes, reflecting the cellular localization of TLR8. Intriguingly, none of post-translational modifications at single sites, including glycosylation, phosphorylation, ubiquitination, acetylation, and palmitoylation alter porcine TLR8-ECD endosomal localization. Nevertheless, the signal peptide of porcine TLR8-ECD determines its endosomal localization. On the other hand, signaling regulator UNC93B1 also decides the endosomal localization of porcine, bovine, and human TLR8 ECDs. The results from this study shed light on the mechanisms of not only TLR8 intracellular localization but also the TLR immune signaling.

To compare porcine TLR8 (pTLR8) and human TLR8 (hTLR8) signaling pathways and downstream genes, we activated the pTLR8 and hTLR8 reporter cells with TLR8 agonist R848, and subjected the stimulated cells together with non-stimulated control cells for transcriptome analysis. There are 1157 differentially expression genes (DEGs) in R848 activated hTLR8 cells, whereas 502 DEGs in R848 activated pTLR8 cells. Among these DEGs, 804 genes are hTLR8 specific, 149 genes are pTLR8 specific, and 353 genes are hTLR8 and pTLR8 common. Related Results were published in reference [Ao, 2019].