Elucidating the Function of Redox-Signaling Mechanisms Underlying Trained Immunity in Alveolar Macrophages 2303944

Abstract Introduction Trained immunity represents a functional state of the innate immune system characterized by long-term epigenetic and metabolic reprogramming, enabling enhanced and rapid responses to future pathogenic challenges. While nitric oxide (NO) and reactive oxygen species (ROS) production are recognized as part of the innate immune response to pathogens, their role in establishing innate immune memory remains poorly understood. This study examines the role of redox signaling in establishing trained immunity in alveolar macrophages (AMs). Methods To investigate the function of redox signaling, AMs were treated with flagellin as the first stimulus, and redox inhibitors were used to block ROS/NO production during flagellin treatment. After 5 days of resting, cells were rechallenged with LPS. To analyze redox-dependent training, we used a combination of techniques, such as qPCR, ELISA, NO quantification, flow cytometry, mass spectrometry, and bulk RNA sequencing. Further histone modifications and metabolic changes were confirmed by Western blot and Seahorse analysis. Results Flagellin-induced trained immunity resulted in enhanced pro-inflammatory gene expression and cytokine production upon LPS restimulation. Additionally, flagellin increased the production of ROS/NO, and blocking the accumulation of these redox molecules significantly reduced the training effect. Interestingly, the treatment with inhibitors also induced training. Bulk RNA-Seq analysis revealed the upregulation of oxidative phosphorylation genes in trained cells. Mechanistically, flagellin-induced trained cells rewired AMs metabolism, increased glycolytic capacity, and histone acetylation and methylation levels. Furthermore, identification of redox-modified proteins revealed novel regulatory mechanisms in trained immunity. Conclusion Redox signaling is essential for establishing training in AMs, providing new insights for developing target-based immunotherapies in trained immunity. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)