Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE

Figure 2

Identification of RIG-I as the key sensor in type I IFN response in RPE. (a, b) RNA but not DNA could be sensed by RPE to induce type I IFN response, suggesting the lack of DNA sensors in unstimulated RPE. THP-1 and THP-1 cGAS KO cells were used as controls (a). Cells were treated with indicated inducers at 0.25 μg/mL, with or without the Lipofectamine transfection reagent, and intracellular ISG15 was measured by ELISA 24 h after stimulation. Each bar represents biological replicates () and is indicated as . Note that ARPE-19 without transfection did not respond to most tested inducers except for poly(I:C). compared with corresponded vehicle-treated groups. compared with same induces in parental cells. (c) Type I IFN response was induced via the RIG-I–MAVS–IRF3 axis in ARPE-19 cells. ARPE-19 cells were cultured 10 d after reaching confluence for screening purposes. Different key nodes for the activation of the IFN pathway were knocked down using siRNAs (at least 3 different siRNAs for each gene). Cells were then treated with indicated nucleic acids, and the release of secreted IFN-β was measured by ELISA 24 h after stimulation. Similar results were observed in 3 independent experiments with different inducers (indicated in different lines) or different siRNAs (shown as different column); a single representative experiment is shown as an example. A heatmap was used to better visualize the percentage (%) change in the results. The efficiency of the mRNA knockdown was validated by qPCR and shown at the top in the form of heatmap. About ~70% inhibition was observed in most tested siRNAs.

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