Integrated transcriptomic and functional analyses reveal that NOX2 inhibition rewires the inflammatory landscape of macrophages.

Background: Macrophages are dynamic immune cells whose phenotype and function are shaped by environmental cues, including inflammatory stimuli and oxidative stress. A major source of macrophage-derived reactive oxygen species (ROS) is NADPH Oxidase 2 (NOX2), which is critical for microbial defense but also contributes to redox signaling and inflammatory responses. This increase in NOX2-based ROS can be both beneficial and detrimental, leading to the desire to modulate this key inflammatory pathway pharmacologically. However, while NOX2-driven ROS are well studied in host defense, the underlying macrophage transcriptional reprogramming that leads to inflammatory phenotypes, and the changes that occur to this programming under pharmacological inhibition, remain unclear. Methods: To address this gap, we used the selective small-molecule inhibitor GSK2795039 (GSK) to acutely block NOX2 activity in primary bone marrow-derived macrophages (BMDMs) under basal and lipopolysaccharide (LPS)-stimulated conditions. RNA sequencing and functional assays were performed to uncover the role of inflammation mediation due to NOX2 on transcriptional changes in macrophages. Results: RNA sequencing revealed that GSK alone induced modest transcriptional changes in resting macrophages, largely restricted to metabolic and stress-associated pathways. In contrast, co-treatment with LPS and GSK markedly reprogrammed the macrophage transcriptome, attenuating classical pro-inflammatory responses while enriching pathways associated with anti-inflammatory activation, tissue repair, extracellular matrix remodeling, and oxidative phosphorylation. Functional assays validated these transcriptomic findings. NOX2 inhibition under LPS activation reduced both intracellular and extracellular ROS, suppressed pro-inflammatory cytokine secretion (TNF-α, IL-6, IL-1β), and enhanced anti-inflammatory cytokines (IL-4, IL-10). Conclusion: Together, these results demonstrate that NOX2 inhibition does not broadly reprogram macrophages in the resting state but reshapes the inflammatory landscape of LPS-stimulated pro-inflammatory macrophages, shifting them toward a reparative, anti-inflammatory state even in the presence of strong activating stimuli. Our findings provide mechanistic insight into the immunomodulatory potential of NOX2 inhibition in inflammatory models.