DNA-PKcs regulates type I interferon responses in macrophages during bacterial infection 2334790

Abstract Introduction Macrophages, first responders to bacterial infection, integrate pattern recognition receptor (PRR) and interferon signaling to induce the production of reactive oxygen and nitrogen intermediates, which destroy intracellular bacteria. We have previously demonstrated that these genotoxic intermediates also damage macrophage genomic DNA, activating a DNA damage response (DDR) that is coordinated by kinases ATM and DNA-PKcs. This DDR modulates macrophage transcriptional responses during the innate immune response to infection. Though ATM has an established role in regulating cell-type-specific immune responses downstream of DNA damage, a potential role for DNA-PKcs in this regard has not been well-characterized. Thus, we asked whether or not DNA-PKcs regulates macrophage transcriptional responses during bacterial infection. Methods We compared the transcriptomes of wildtype and DNA-PKcs-deficient macrophages, after exposure to bacterial stimuli, using bulk RNA-sequencing. We also compared the transcriptomes of activated macrophages that were treated with a small molecule inhibitor of DNA-PKcs, AZD7648, relative to vehicle-only controls. Among the aforementioned experimental conditions, we assessed expression of a cohort of pro-inflammatory mediators using quantitative real-time PCR and ELISA. We also addressed relative activation of PRR- and interferon-regulated signaling cascades, among our experimental conditions, using immunoblotting. Results We find that DNA-PKcs signaling modulates the macrophage transcriptome of activated macrophages. Additionally, DNA-PKcs kinase activity is required for optimal STING-dependent and -independent type I interferon production in activated macrophages. Of note, we find reduced expression of some, but not all, interferon-stimulated genes (ISGs) in macrophages in which DNA-PKcs kinase activity is abrogated. Conclusion Taken together, these data suggest a potential role for DNA-PKcs in modulating ISG expression beyond its regulation of type I interferon production. Funding Source National Institutes of Health (1R16GM159931-01) Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)