Single-cell RNA-seq provides insight into the underdeveloped immune system of germ-free mice.

Germ-free mice exhibit profound immunological immaturity. Despite recent studies emphasizing the role of specific bacterium-derived metabolites in immune cell development and differentiation, the mechanisms linking microbiota absence to systemic immune deficits remain incompletely defined. Here, droplet-based single-cell RNA sequencing of bone marrow and peripheral blood from both germ-free and specific pathogen-free mice was performed, identifying 25 transcriptionally distinct cell types. Neutrophil apoptosis was elevated in germ-free mice, potentially due to the absence of niacin dehydrogenase, a metabolite primarily produced by Pseudomonas. In addition, germ-free mice exhibited increased excretion of 5'-methylthioadenosine, enhanced ERK activation driven by reactive oxygen species, and disruption of bone marrow stromal antigen 2 signaling. Monocytes and CD8 ⁺ T cells from germ-free mice showed diminished responses to interferon-β and interferon-γ, consistent with heightened viral susceptibility. These findings establish a microbiota-dependent regulatory pathway linking immunodeficiency to microbial absence in germ-free mice, confirmed through complementary validation techniques.