Ribonuclease activity undermines immune sensing of naked extracellular RNA.

Cell membranes are thought of as barriers to extracellular RNA (exRNA) uptake. While naked exRNAs can be spontaneously internalized by certain cells, functional cytosolic delivery has been rarely observed. Here, we show that extracellular ribonucleases (RNases)-primarily from cell culture supplements-have obscured the study of exRNA functionality. When ribonuclease inhibitor (RI) is added to cell cultures, naked exRNAs can trigger pro-inflammatory responses in dendritic cells and macrophages, largely via endosomal Toll-like receptors (TLRs). Moreover, naked exRNAs can escape endosomes, engaging cytosolic RNA sensors. In addition, naked extracellular mRNAs can be spontaneously internalized and translated by various cell types in an RI-dependent manner. In vivo, RI co-injection amplifies naked-RNA-induced activation of splenic lymphocytes and myeloid leukocytes. Furthermore, naked RNA is inherently pro-inflammatory in RNase-poor compartments like the peritoneal cavity. These findings demonstrate that naked RNA is bioactive without requiring vesicular encapsulation, making a case for nonvesicular-exRNA-mediated intercellular communication.