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Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production
Published on: March 2, 2014
Cell-specific regulation of nucleic acid sensor cascades: a controlling interest in the antiviral response
Saskia C Stein1, Eric Lam, Erik Falck-Pedersen
1Weill Medical College of Cornell University, Department of Microbiology and Immunology, Molecular Biology Graduate Program, New York, New York, USA.
Abstract:
In this study, we examined the capacities of non-antigen-presenting cell types to propagate antiviral signals following infection with recombinant adenovirus or by direct nucleic acid transfection. Three murine cell lines (RAW264.7 macrophages as a positive control, FL83B hepatocytes, and MS1 endothelial cells) were assessed following exposure to adenovirus, DNA, or RNA ligands. Based on primary (interferon response factor 3 [IRF3] phosphorylation) and secondary (STAT1/2 phosphorylation) response markers, we found each cell line presented a unique response profile: RAW cells were highly responsive, MS1 cells were modified in their response, and FL83B cells were essentially nonresponsive. Comparative reverse transcription-quantitative PCR (RT-qPCR) of nucleic acid sensing components revealed major differences between the three cell types. A prominent difference was at the level of adaptor molecules; TRIF, MyD88, MAVS, and STING. TRIF was absent in MS1 and FL83B cells, whereas MyD88 levels were diminished in FL83B hepatocytes. These differences resulted in compromised TLR-mediated activation. While the cytosolic adaptor MAVS was well represented in all cell lines, the DNA adaptor STING was deficient in FL83B hepatocytes (down by nearly 3 log units). The absence of STING provides an explanation for the lack of DNA responsiveness in these cells. This hypothesis was confirmed by acquisition of IRF3 activation in Flag-STING FL83B cells following DNA transfection. To consolidate the central role of adaptors in MS1 endothelial cells, short hairpin RNA (shRNA) knockdown of STING and MAVS resulted in a ligand-specific loss of IRF3 responsiveness. In contrast to the requirement for specific adaptor proteins, a requirement for a specific DNA sensor (AIM2, DDx41, or p204) in the IRF3 activation response was not detected by shRNA knockdown in MS1 cells. The data reveal that cell-specific regulation of nucleic acid sensing cascade components influences antiviral recognition responses, that controlling levels of adaptor molecules is a recurring strategy in regulating antiviral recognition response functions, and that comparative RT-qPCR has predictive value for antiviral/innate response functions in these cells.
Insights
Cell type significantly impacts antiviral responses by altering nucleic acid sensing pathways. Key adaptor molecules like STING are crucial for DNA sensing, and their absence or low levels impair antiviral signal propagation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Non-antigen-presenting cells play a role in antiviral immunity.
- Nucleic acid sensing pathways are critical for detecting viral infections.
- Cell-specific differences in these pathways can affect antiviral responses.
Purpose of the Study:
- To investigate the antiviral signaling capacities of non-antigen-presenting cells.
- To determine how cell type influences responses to viral nucleic acids.
- To identify molecular differences, particularly adaptor molecules, that explain these responses.
Main Methods:
- Exposure of murine cell lines (macrophages, hepatocytes, endothelial cells) to adenovirus or nucleic acid transfections.
- Assessment of interferon response factor 3 (IRF3) and STAT1/2 phosphorylation as markers of antiviral signaling.
- Comparative reverse transcription-quantitative PCR (RT-qPCR) to quantify nucleic acid sensing components, including adaptor molecules.
- Validation using STING-complemented cells and short hairpin RNA (shRNA) knockdown experiments.
Main Results:
- Murine cell lines exhibited distinct antiviral response profiles: RAW264.7 macrophages were highly responsive, MS1 endothelial cells showed modified responses, and FL83B hepatocytes were largely unresponsive.
- Differences in adaptor molecule expression (TRIF, MyD88, MAVS, STING) correlated with response levels; notably, STING deficiency in FL83B hepatocytes explained their lack of DNA responsiveness.
- shRNA knockdown of STING and MAVS in MS1 cells confirmed their essential role in IRF3 activation, while specific DNA sensors were not found to be rate-limiting.
Conclusions:
- Cell-specific regulation of nucleic acid sensing components dictates antiviral recognition.
- Modulating adaptor molecule levels is a key strategy for controlling innate antiviral responses.
- Comparative RT-qPCR is a valuable tool for predicting cellular antiviral functions.
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