Gut-derived small extracellular vesicles support trained innate immune tolerance in murine microglial cells.

INTRODUCTION: Microglia are the resident immune cells of the central nervous system (CNS) that maintain tissue homeostasis and contribute to the pathogenesis of neuroinflammatory disorders. As innate immune cells, microglia can acquire memory-like states that exert long-term effects on CNS function and disease susceptibility. Increasing evidence highlights a dynamic interaction between the gut microbiota and the CNS, shaping microglial maturation and responsiveness throughout life. In addition to soluble microbial metabolites, gut-derived extracellular vesicles (EVs), including vesicles of microbial origin, have emerged as important mediators of microbiota-host communication capable of modulating brain homeostasis and inflammatory signaling; however, their role in programming microglial immune memory remains unclear. METHODS: Here, we examined whether gut-derived small EVs influence memory-like features of primary murine microglia in vitro. Microglia were primed with small EVs followed by a secondary lipopolysaccharide (LPS) challenge, and inflammatory signaling, metabolic activity, epigenetic markers, and effector functions (migration and phagocytosis) were assessed. RESULTS: Small EV priming followed by secondary LPS challenge induced a trained innate immune tolerance phenotype characterized by reduced pro-inflammatory mediator release and attenuated TLR2/4-MyD88-p38 MAPK signaling. This tolerant state was accompanied by suppressed glycolytic activity and decreased levels of activating histone H3 marks, indicating coordinated metabolic and epigenetic reprogramming. Notably, despite diminished inflammatory signaling, small EV-primed microglia displayed enhanced migratory and phagocytic capacities associated with increased ERK1/2 activation. DISCUSSION: Together, these findings indicate that gut-derived small EVs can imprint memory-like programs in microglia that restrain inflammatory activation while preserving essential effector functions in vitro, suggesting a mechanism by which microbiota-brain communication may shape neuroinflammatory responses.