3-(3-Hydroxyphenyl)propionic Acid-Induced Trained Immunity in Bone Marrow-Derived Macrophages Is Associated with Glutamate-Related Immunometabolic Reprogramming

Background/Objectives: Gut microbiota-associated metabolites may regulate trained immunity, but the underlying metabolites and mechanisms remain unclear. This study aimed to identify candidate inducers and characterize associated immunometabolic changes in bone marrow-derived macrophages (BMDMs). Methods: Among 59 gut microbial metabolites screened in BMDMs from female C57BL/6 mice, 3-(3-hydroxyphenyl)propionic acid (3HPP) was identified based on enhanced TNF-α and IL-6 production following lipopolysaccharide (LPS) restimulation after a 5-day rest. Subsequent analyses included concentration–response and CCK-8 assays, transcriptomic and qPCR analyses, and assessments of metabolic indices, mitochondrial reactive oxygen species (mtROS), and macrophage surface markers. Results: 3HPP was identified as a candidate inducer of trained immunity, as evidenced by the significantly higher levels of TNF-α and IL-6 following LPS restimulation compared with the control group. Concentration–response and CCK-8 assays supported 10 μM as the working concentration. Transcriptomic analysis and qPCR indicated altered expression of genes involved in amino acid metabolism (Gls2 and Cbs), glutathione metabolism (Slc7a11, Gclc, and Gpx1), glutamate-related signaling (Grm3), glycolysis and immunometabolic regulation (Mtor and Hk2), mitochondrial regulation (Ppargc1a), and epigenetic regulation (Kdm5a). Biochemical assays showed increased extracellular lactate and glutamate levels, decreased cellular GSH levels, combined GSH + GSSG pool, and GSH/GSSG ratio, and an increased mtROS-associated signal. Flow cytometry revealed reduced CD86 and CD206 expression, with a more pronounced decrease in CD206-positive cells. Conclusions: 3HPP induces a trained immune phenotype in BMDMs accompanied by glutamate-related immunometabolic changes, altered glutathione homeostasis, and changes in mitochondrial redox status and macrophage surface-marker expression. These findings provide new insight into the potential role of microbiota-associated metabolites in shaping innate immune memory.