Clostridium butyricum and butyrate reprogram macrophages to alleviate obesity by disrupting the LPS-EGR1 axis and its associated PI3K-Akt signaling

Obesity is closely associated with gut microbial dysbiosis and chronic low-grade inflammation, yet how microbial signals orchestrate systemic immune-metabolic dysfunction remains unclear. In adiposity-prone Ningxiang pigs (NF), we observed an expansion of Gram-negative bacteria, elevated circulating lipopolysaccharide (LPS), and reduced abundance of Clostridium butyricum and its metabolite butyrate. Fecal microbiota transplantation (FMT) from NF donors induced adiposity in lean Duroc-Landrace-Yorkshire (CF) recipients, accompanied by a significant decrease in C. butyricum colonization. In high-fat diet (HFD)-fed mice, supplementation with C. butyricum and butyrate effectively restored gut microbial homeostasis and ameliorated obesity. Integrated multi-organ transcriptomic analyses across pigs and long-tailed macaques revealed that early growth response 1 ( EGR1 ) is a central regulator of obesity-associated inflammation, with consistent enrichment of PI3K-Akt signaling. Single-cell RNA sequencing further demonstrated that EGR1 -high macrophages adopt a pro-inflammatory M1 phenotype, whereas EGR1 -low macrophages exhibit an anti-inflammatory M2 profile. Leveraging EGR1 macrophage-specific differentially expressed genes, both in silico knockout of EGR1 and pharmacological inhibition (EGR1i; IT25) confirmed the functional involvement of PI3K-Akt signaling downstream of EGR1 . Integrating metagenomic and single-cell data, we propose a conserved LPS- EGR1 axis that governs macrophage polarization in obesity. To experimentally validate this model, we established an LPS-potentiated HFD-induced obesity model in mice and an LPS-induced M1 polarization model in RAW264.7 macrophages. Treatment with C. butyricum , butyrate, arginine, or EGR1i significantly attenuated weight gain, downregulated EGR1 expression, and effectively suppressed M1 macrophage polarization, thereby restoring the M1/M2 balance. Notably, this process is accompanied by significant alterations in genes related to the PI3K-Akt signaling pathway, suggesting that this pathway may act in concert with EGR1 within this regulatory network. In summary, these cross-species experiments elucidate a fundamental mechanism whereby gut-derived LPS regulates macrophage polarization via EGR1 , thereby linking microbial dysbiosis to metabolic inflammation in mammals. Video Abstract CB and BA break the LPS-EGR1-M1 loop to combat obesity. CB: Clostridium butyricum. BA: Butyric acid. LPS: Lipopolysaccharide. GNB: Gram-negative bacteria. EGR1: Early Growth Response 1.