The study demonstrates that mRNA, both in vitro transcribed and endogenous, can activate Toll-like receptor 3 (TLR3) and induce immune responses in human dendritic cells (DCs). Using a luciferase reporter assay in HEK293 cells expressing TLR3, the authors show that in vitro transcribed mRNA, including natural and 2′-fluoro-substituted mRNA, activates NF-κB and induces interleukin-8 secretion. This activation is dose-dependent and involves tyrosine phosphorylation. In DCs, mRNA treatment upregulates the expression of TLR3, interferon regulatory factor-1, tumor necrosis factor-α, and interleukin-1 receptor-associated kinase-M mRNA, leading to increased secretion of interferon-α and other cytokines. Endogenous RNA released from necrotic cells also stimulates DCs, and this effect can be inhibited by pretreatment of the necrotic cells with RNase. These findings suggest that RNA, likely through its secondary structure, is a potent host-derived activator of TLR3, which has potential physiological relevance in modulating immune responses to damaged tissues.The study demonstrates that mRNA, both in vitro transcribed and endogenous, can activate Toll-like receptor 3 (TLR3) and induce immune responses in human dendritic cells (DCs). Using a luciferase reporter assay in HEK293 cells expressing TLR3, the authors show that in vitro transcribed mRNA, including natural and 2′-fluoro-substituted mRNA, activates NF-κB and induces interleukin-8 secretion. This activation is dose-dependent and involves tyrosine phosphorylation. In DCs, mRNA treatment upregulates the expression of TLR3, interferon regulatory factor-1, tumor necrosis factor-α, and interleukin-1 receptor-associated kinase-M mRNA, leading to increased secretion of interferon-α and other cytokines. Endogenous RNA released from necrotic cells also stimulates DCs, and this effect can be inhibited by pretreatment of the necrotic cells with RNase. These findings suggest that RNA, likely through its secondary structure, is a potent host-derived activator of TLR3, which has potential physiological relevance in modulating immune responses to damaged tissues.