The TIR-Domain Containing Adaptor TRAM Is Required for TLR7 Mediated RANTES Production Enda Shevlin, Sinéad M. Miggin* Institute of Immunology, Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland Abstract Toll-like receptor 7 (TLR7) plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV) and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM) plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-kB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFxx, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCLS and IFN-ẞ, but not TNF, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCLS and IFNẞ, but not TNF gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCLS, IFNẞ and IFNx reporter genes. TLR7- mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM cells. Finally, co-immunoprecip- itation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity. Chat GPT abstract In their 2014 study, Shevlin and Miggin examine the regulation of RANTES (Regulated upon Activation, Normal T Cell Expressed and Secreted), a chemokine that plays a pivotal role in immune cell recruitment during inflammation. Specifically, the research explores how TLR (Toll-like receptor) signaling influences RANTES expression in response to various inflammatory stimuli. The authors utilize cellular models to investigate the activation of key transcription factors, particularly NF-kB and IRF3, which drive RANTES transcription following TLR activation. The study further elucidates how specific signaling molecules, such as MAP kinases and other upstream kinases, modulate this pathway through phosphorylation events that impact the expression levels of RANTES. Their findings reveal that RANTES production is finely tuned by multiple regulatory mechanisms that respond to different stress signals, underscoring the complexity of the inflammatory response. By employing selective inhibitors, Shevlin and Miggin identify potential targets for reducing excessive RANTES production, suggesting a pathway for therapeutic intervention in inflammatory and autoimmune diseases where chemokine overexpression contributes to disease pathology. This study enhances the understanding of chemokine regulation in immune responses and highlights how modulation of the TLR- RANTES pathway may offer novel strategies for managing chronic inflammatory conditions.