Single-cell RNA-seq provides insight into the underdeveloped immune system of germ-free mice
Germ-free mice feature a profoundly underdeveloped immune system. Despite recent studies that emphasize the role of specific bacteria-derived metabolites in immune cell development and differentiation, it remains unclear how the lack of microbiota leads to immune deficiencies. Here, we performed droplet-based single-cell RNA sequencing to analyze the bone marrow and peripheral blood of both germ-free and specific pathogen-free mice, identifying 25 distinct cell types. Our findings suggest that neutrophil apoptosis in germ-free mice may be associated with the absence of niacin dehydrogenase, which is derived primarily from Pseudomonas. In addition, germ-free mice exhibited elevated excretion of 5'-methylthioadenosine, increased ERK activation induced by reactive oxygen species, and cessation of the bone marrow stromal antigen 2 signaling pathway in germ-free mice. The responses of monocytes and CD8+ T cells to interferon ß and interferon ? were reduced in germ-free mice, which explains their increased susceptibility to viruses. Together, we identified a regulatory mechanism that connects immunodeficiency with the absence of microbiota in germ-free mice and validated these findings using multiple techniques. Overall design: Groups: Compare two conditions (e.g., germ-free (GF) vs. specific-pathogen-free (SPF) mice) Tissues: Collect multiple relevant tissues (e.g., peripheral blood, bone marrow, spleen)