Impact Of RNA Structure On Induction Of Human Cellular Innate Immunity And Characterization Of Related Signaling Pathways

The innate immunity system defends the host from pathogen infection by triggering non-specific antiviral responses. Detection of foreign intruders is mediated by pathogen-associated molecular patterns (PAMP) that are recognized by pattern recognition receptors (PRRs). Retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and TLR3 are the major RNA sensors, located in the cytoplasm and endosomes, respectively, that elicit antiviral responses through interferon regulatory factor (IRF) 3. Although previous studies suggest that RIG-I mainly recognizes short 5'triphosphorylated dsRNA and ssRNA whereas MDA5 preferentially detects long dsRNA, systematic comparison of the ability of different types of RNAs to induce innate immune responses in human cells has still been limited. In this study, we generated dsRNA bacteriophageφ6 and influenza A virus-specific dsRNA and ssRNA molecules ranging from 58 to 2956 nts. Utilizing the RNA-dependent RNA polymerase of bacteriophageφ6, which is capable of primer-independent RNA synthesis from the very 3'end of the template, we replicated the ssRNA into perfectly duplexed dsRNA instead of bearing overhangs generated by annealing sense and antisense ssRNA together. We also systematically designed and biologically synthesized a set of blunt-end dsRNA and ssRNA molecules of varying lengths without 5'end phosphorylization by dephosphorylating dsRNA and ssRNA with Calf intestinal alkaline phosphatase (CLAP). In human monocyte-derived dendritic cells (moDCs), short dsRNAs efficiently induced the expression of interferon (IFN-a, IFN-βand IFN-λ-1) and proinflammatory (TNF-a, IL-6, IL-12, and CXCL10) cytokine genes. These genes were also induced by ssRNA molecules, but size-specific differences were not as pronounced as in dsRNA induced responses. However, long dsRNA induced higher expression of IL-1β. Moreover, we found that dephosphorylation of short ssRNA and dsRNA molecules led to a dramatic reduction in their ability to induce innate immune responses. Such a difference was not seen for larger ssRNAs. RNA induced cytokine responses correlated well with IFN regulatory factor (IRF) 3 phosphorylation suggesting that IRF3 plays a major role in both ssRNA and dsRNA induced responses in human moDCs. We also found that IFN activation was efficiently induced following recognition of short dsRNA by RIG-I and TLR3 in HEK cells, whereas ssRNA stimulation was less length dependent. So it can be conjectured that dsRNA stimulates innate immune responses mainly through RIG-I and TLR3 signaling pathways in human cells. Our data suggests that human DCs are extremely sensitive in recognizing foreign RNA and the responses depend on RNA size, form (ssRNA vs. dsRNA) and the level of 5'-phosphorylation.