Toll-Like Receptor 8 Stability is Regulated by Ring Finger 216 in response to circulating MicroRNAs.

Toll-Like Receptor 8 (TLR8) is an intracellular pattern recognition receptor that senses RNA in endosomes to initiate innate immune signaling through NF-κB, and mechanisms regulating TLR8 protein abundance are not completely understood. Protein degradation is a cellular process controlling protein levels, accomplished largely through ubiquitin transfer directed by E3-ligase proteins to substrates. Here were show that TLR8 has a short half-life in THP-1 monocytes (~1 h), and that TLR8 is ubiquitinated and degraded in the proteasome. Treatment with the TLR8 agonist R848 causes rapid depletion of TLR8 levels at short time points, an effect blocked by proteasomal inhibition. We show a novel role for Ring Finger Protein 216 (RNF216), and E3-ligase which targets TLR8 for ubiquitination and degradation. RNF216 over-expression reduces TLR8 levels, while RNF216 knockdown stabilizes TLR8. In humans with Acute Respiratory Distress Syndrome (ARDS), we describe a potential role for TLR8 activation by circulating RNA ligands, as plasma and extracted RNA fractions from ARDS subjects activated TLR8 in vitro. MicroRNA (miRNA) expression profiling revealed several circulating miRNA's from ARDS subjects. MicroRNA-mimics promoted TLR8 proteasomal degradation in THP-1 cells. These data show that TLR8 proteasomal disposal through RNF216 in response RNA ligands regulate TLR8 cellular concentrations and may have implications innate immune signaling. Additionally, TLR8 activation by circulating RNA ligands may be a previously under-recognized stimulus contributing to excessive innate immune signaling characteristic in ARDS.