Comparison of naturalization mouse model setups uncover distinct effects on intestinal mucosa depending on microbial experience

Introduction: Concerns regarding the translational value of preclinical mouse models have been addressed by introducing various approaches of ‘naturalizing’ research mice, which provide them with more diverse microbiomes and physiological immune responses. We have previously shown that ‘feralized’ mice, that is, inbred laboratory mice raised in a farmyard-like, microbe-rich environment exhibit a shifted gut microbiota, matured immunophenotype, and reduced severity of colorectal cancer. Similar studies occasionally involve co-housing with wild or pet-store-raised mice as microbial donors integrating species-specific commensals and pathogens. To what extent these different practices of microbial exposure are crucial for the resulting mouse phenotype remains unclear. Methods: Here, we present the first side-by-side comparison of different methods to naturalize laboratory mice: co-housing with wild-caught house mice, feralization in a farmyard-like habitat only, or a combination of the two, with conventional clean laboratory mice as a reference. Results: Independent of the method, the naturalized colon-mucosa microbiota, was colonized by several Helicobacter species, and the colonic intestinal epithelial cells of naturalized mice displayed elevated expression of genes encoding antimicrobial peptides, mucus components, and reactive-oxygen-species-producing enzymes. They further showed significantly increased resident memory T cells in the colonic lamina propria and effector memory T cells in the mesenteric lymph nodes. The most pronounced changes of these parameters occurred in mice co-housed with wild-caught mice, while feralized mice displayed phenotypes that were intermediate between laboratory and co-housed mice. Conclusion: These findings enhance our understanding of naturalization model setups and effects on the gut barrier and immune system, thereby aiding future decisions on the utilization of naturalized mouse models.