Microbiota humanization drives human‐like metabolic and immune transcriptomic shifts in pigs

Abstract Pigs are increasingly recognized as promising candidates for clinical xenotransplantation and as large‐animal models for biomedical research; however, interspecies differences in gut microbiota, immune function, and metabolism remain major barriers. To address this, we established gut microbiota‐humanized (GMH) pigs by transplanting human fecal microbiota into antibiotic‐treated pigs. We systemically evaluated alterations in microbiota composition, serum metabolites, and immune cell profiles using integrated metagenomic, quasi‐targeted metabolomic and single‐cell transcriptomic (scRNA‐seq) analyses. Metagenomic profiling revealed a shift in the intestinal microbiota of GMH pigs toward a human‐like composition, characterized by enrichment of Bacteroidia and depletion of Bacilli . Metabolomic analysis showed that GMH pigs exhibited serum metabolite profiles more closely resembling those of humans. Among 423 detected serum metabolites, 136 that were lower in control pigs than in humans were upregulated in GMH pigs, whereas 79 that were elevated in control pigs decreased post‐transplantation. Notably, pathways related to tryptophan metabolism, bile acid biosynthesis, and fatty acid metabolism were enhanced in GMT pigs, while carbon‐related and glycolytic pathways were attenuated, indicating partial convergence toward human metabolic phenotype. Integration of microbial and metabolite data identified 20 and 33 metabolites associated with Bacteroidia and Bacilli , respectively. scRNA‐seq profiling of peripheral blood mononuclear cells demonstrated transcriptional and compositional remodeling of T cells, monocytes, and B cell subsets in GMH pigs. These findings demonstrated that human fecal microbiota can reshape both systemic metabolic and immune artitecture in pigs, offering a robust large‐animal platform for studying host‐microbiota interactions and advancing translational application in xenotransplantation and microbiome‐based therapeutics.