Saturday, September 7, 2024
9/7/2024
Project Summary Exposure to ionizing radiation (IR) is associated with inflammation due to an upregulation of inflammatory mediators and hyperactivity of the cells responsible for their production. The key immune cells involved in IR- induced inflammation, both as exacerbators and extinguishers, are macrophages (MΦ). Thus, deciphering of the signaling mechanisms responsible for IR-induced activation and polarization of these highly radioresistant cells is important for the identification of therapeutic targets to manage acute and counteract chronic IR- related inflammation. Along with damage-associated molecular patterns, released from irradiated tissues, radiation itself directly impacts MΦ through DNA damage and modulation of transcription that alters the MΦ phenotype. This effect is implemented through the activation of specific DNA and RNA sensors and downstream signaling pathways, resulting in differential expression of type I interferons (IFN-I) and interferon- stimulated genes (ISGs). It is well established that DNA fragments activate the STING/TBK1 pathway, thereby triggering IFN-I expression. However, we recently demonstrated that the cytoplasmic RNA sensors, RIG-I-like receptors (RLRs) and their adaptor protein MAVS, also play a significant role in MΦ activation after IR. We found that activation of the RLR family member MDA5 by double-stranded RNA, due IR-induced transcription of endogenous retroviruses (ERVs), leads to increased expression of IFN-I and inflammation- related genes. This finding generates two important questions that need to be resolved. First, whether the signaling from RLRs is critical for prolonged IR-induced activation of MΦ. Our data that ERV RNA-induced MDA5 signaling and IFN-I expression, in turn, upregulates retroviral transcription, suggests that there is a positive feedback loop for ERV expression that maintains recurrent activation of the RLR-MAVS pathway and thereby inflammatory activation of MΦ. Thus, we propose a time-course study of the involvement of RLR- MAVS signaling, IFN-I response and ERV transcription in the short- and long-term effects of gamma IR using MΦ with knocked out MAVS or IFN-I receptor and in vivo using the wild-type C57BL/6J mice and a MAVS- knockout strain. The second question is whether IR dose influences the impact of RNA sensors versus DNA sensors, with respect to the IR-induced DNA damage. We found that increases in IR dose led to a decline in RLR and MAVS levels, whereas the DNA sensing proteins were consistently upregulated. To test our hypothesis that high IR doses result in DNA sensing pathway activation and a STING-mediated mechanism of IFN-I expression that determines MΦ activation, we will investigate the dose-dependent disruption of RLR- RNA complexes with MAVS, DNA damage and accumulation of DNA fragments and RNA:DNA duplexes to assess their impact on RLR and STING-TBK1 signaling using MΦ cultures and the indicated above mouse strains, along with STING-knockout mice. We expect to identify nucleic acid-related signaling mechanism(s) responsible for the radiation-induced changes in MΦ polarity and their pathological inflammatory activation.
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