Multi-omics of controlled microbial naturalization uncovers sex-dimorphic immune maturation 2254508

Abstract Introduction Mouse modeling is the cornerstone of mechanistic immunology, yet widely used laboratory mice maintained in overly hygienic environments harbor predominantly naïve immune systems that poorly emulate human immune status. “Naturalized” or “dirty” models with diverse microbial exposures promote immune maturation, but current methods lack reproducibility and broad applicability, including poor compatibility with sex-based comparisons, preventing widespread adoption. Methods We develop a controlled and reproducible naturalization method for diverse mechanistic applications, integrated with longitudinal deep multi-omics across tissues, including proteomics, scRNA/VDJ-seq, shotgun metagenomics, and spectral flow cytometry. Results The method leverages natural cecal microbiota from large cohorts of well-defined male/female pet-store mice, viably preserved for consistent administration by oral gavage to laboratory mice–with compatibility across experimental variables. The model induces broad immune activation, with sustained maturation and functional adaptation across innate and adaptive immune cells in circulation and tissues, including durable alterations to bone marrow populations. We further resolve maturation trajectories at the single-cell level, revealing unified heightened interferon signatures, expansion of age-associated atypical subsets, as well as convergent terminal differentiation of CD4+ and CD8+ T cells resembling human TEMRAs. Leveraging this model, we probe immune sex dimorphism and uncover a female bias in interferon, extrafollicular B cell, and Ly49+ cytotoxic T cell responses, in contrast to a male bias in IL-15-induced T cell bystander activation–with broad implications for human inflammation and autoimmunity. Conclusion We present a widely applicable method for mouse microbial naturalization and comprehensive multi-omics resource of controlled immune maturation–providing a natural platform for translational mechanistic discovery, including immune sex dimorphism. Funding Source PD Soros Fellowship, NIH (F30AI188889 & T32GM136651), and Yale University Topic Categories Mucosal and Regional Immunology (MUC)