Aromatic microbial metabolite hippuric acid enhances inflammatory responses in macrophages via TLR-MyD88 signaling and lipid remodeling

The gut microbiome produces diverse metabolites shaping immunity, yet their pro-inflammatory potential remains unclear. Using untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics, we identified hippuric acid-an aromatic, microbe-derived metabolite-as a potent enhancer of inflammatory responses during Escherichia coli infection. Hippuric acid administration heightened inflammation, activated innate immune cells, and reduced survival in infected mice. In vitro, hippuric acid selectively potentiated M1-like (lipopolysaccharide [LPS] or LPS+interferon gamma [IFNγ]) macrophage pro-inflammatory responses but had no effect on M2-like (interleukin [IL]-4) polarization. It enhanced responses to myeloid differentiation primary response 88 (MyD88)-dependent Toll-like receptor (TLR) ligands but not TRIF-, STING-, or NOD2-mediated stimuli. Genetic deletion of MyD88 abolished hippuric-acid-induced pro-inflammatory responses. Transcriptomic and lipidomic analyses revealed increased cholesterol biosynthesis and lipid accumulation, while reducing cellular cholesterol blunted the pro-inflammatory effects of hippuric acid. Notably, hippuric acid also enhanced pro-inflammatory responses in human macrophages, and its elevated levels correlated with sepsis mortality, linking microbial metabolism, lipid remodeling, and innate immunity.