Spatial metabolic and phenotypic characterization of the germ‐free mouse model

The gut microbiome has been strongly linked to health and disease, exerting its systemic effects through host and bacterial molecules that disseminate from the intestine. Understanding how these molecules may contribute to, exacerbate, or even improve specific health conditions is a key mechanistic challenge in microbiome research. Using the germ-free mouse model, we used a spatial biology approach to map the location of small molecules in intestinal and systemic tissues in addition to phenotyping cells in their vicinity. Significant differences were noted in molecular species across all tissues tested, including the ileum, colon, spleen, lung, liver, and kidney, with the greatest number of changes in the liver. Molecules putatively identified as phenol sulfate and 5-amino valeric acid betaine were noted to have significantly changed in abundance in the germ-free mouse intestine as well as systemically. Phenotypic characterization of germ-free mouse organs identified significant alterations in immune cell numbers indicative of an aberrant immune response, underlining the critical role of the microbiome in immune stimulation and priming, even at sites distal from the intestine. Our findings highlight the significant molecular and cellular changes that occur in the absence of a gut microbiota, identifying key microbiome-derived metabolites and host phenotypic signatures.