Abstract
In the established model of classical trained immunity, metabolic and epigenetic hubs serve as central integrators of innate memory. While typically associated with proinflammatory reprogramming, the regulation of autophagy and cellular proteostasis remains essential for guiding macrophage differentiation and ensuring efficient pathogen clearance without excessive inflammation. In this study, we demonstrate that sodium butyrate (SB), a short-chain fatty acid, induces a functional profile that diverges from the canonical pathways observed in classical innate immune training. The induction of an innate reprogrammed state in chicken macrophages by SB is strictly dependent on the cellular developmental stage, occurring only during the early stages of differentiation from chicken bone marrow-derived macrophages but not in fully differentiated cells. This suggests that SB primarily facilitates an innate immune reprogramming with a specific temporal window of sensitivity. Our results show that SB-reprogrammed chicken macrophages exhibit enhanced reactive oxygen species generation, altered cytokine expression, and an increased capacity to kill a diverse range of bacteria. Treatment with chemical inhibitors further demonstrated that these heightened antibacterial effects are directly attributed to increased reactive oxygen species production and autophagy. In summary, these findings indicate that SB induces functional outcomes distinct from classical trained immunity and can elicit innate immune memory through alternative regulatory axes. Our data suggest that distinct innate reprogramming states give rise to alternative activation programs and that innate immune memory exists along a spectrum of phenotypes rather than as a single, uniform state.