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

The gut microbiome generates a diverse array of metabolites that actively shape host immunity, yet the pro-inflammatory potential of microbial metabolites remains incompletely understood. Using a non-targeted, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics, we identified hippuric acid, an aromatic gut microbe-derived metabolite, as a potent enhancer of pro-inflammatory responses in Escherichia coli infection model. Intraperitoneal administration of hippuric acid significantly heightened pro-inflammatory responses, promoted innate immune cell activation, and reduced survival in infected mice. Similar pro-inflammatory effects were observed in an LPS-induced inflammation model. 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 augmented responses to multiple myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor (TLR) ligands, but not to TRIF-dependent TLR3, or to cytosolic innate immune stimuli such as STING and NOD2 agonists, implicating TLR-MyD88 signaling as a likely mechanism of action. 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 upregulated cholesterol biosynthesis and induced lipid accumulation. Pharmacological reduction of cellular cholesterol using fluvastatin or 25-hydroxycholesterol attenuated its pro-inflammatory effects. Notably, hippuric acid also enhanced pro-inflammatory responses in human macrophages, and its elevated levels correlated with increased sepsis mortality, underscoring its clinical relevance. Together, these findings identify hippuric acid as a previously unrecognized microbial-derived pro-inflammatory modulator that links gut microbial metabolism, lipid remodeling, and innate immune signaling, and offer new insights into its role in infection and inflammation.