Aromatic Microbial Metabolite Hippuric Acid Potentiates Pro-Inflammatory Responses in Macrophages through TLR-MyD88 Signaling and Lipid Remodeling

Abstract The gut microbiome generates a diverse array of metabolites that actively shape host immunity, yet the pro-inflammatory potential of microbial metabolites remains poorly understood. In this study, we identified hippuric acid, an aromatic gut microbe-derived metabolite, as a potent enhancer of pro-inflammatory responses using a murine bacterial infection model and a non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. Administering hippuric acid intraperitoneally in murine models of Escherichia coli infection or LPS-induced inflammation significantly heightened pro-inflammatory responses and innate immune cell activation. In vitro, hippuric acid selectively potentiated M1-like macrophage polarization (LPS + IFNγ) but had no effect on M2-like polarization (IL-4). Hippuric acid further enhanced responses to diverse MyD88-dependent TLR ligands, but not TRIF-dependent TLR3, implicating a possible mechanism of action via activation of TLR-MyD88 signaling. Genetic deletion of MyD88 abrogated the pro-inflammatory effects of hippuric acid both in vitro and in vivo, confirming its dependence on the MyD88 pathway. Transcriptomic and lipidomic analyses revealed that hippuric acid promoted cholesterol biosynthesis and lipid accumulation, linking microbial metabolism to lipid-driven immune activation. Notably, hippuric acid similarly enhanced pro-inflammatory responses in human macrophages, and its elevated levels correlated with increased sepsis mortality, highlighting its potential clinical relevance. These findings establish hippuric acid as a previously unrecognized microbial-derived inflammatory modulator, bridging gut microbial metabolism, lipid remodeling, and innate immune signaling, and offer new insights into its role in infection and inflammation. Competing Interest Statement The authors have declared no competing interest.