Single-cell RNA sequencing reveals intrinsic and microbiota-associated sex differences in murine lung.

Sex-based differences in respiratory disease outcomes are well recognized. However, the underlying immunological mechanisms driving this dimorphism remain incompletely understood. While sex hormones influence immune cell development and function, the role of commensal microbes in shaping sex-specific lung immunity has not been explored. Here, we used single-cell RNA sequencing (scRNAseq) and flow cytometry to profile lung immune cells in male and female mice housed under specific pathogen-free (SPF) or germ-free (GF) conditions. Under SPF conditions, males exhibited a striking myeloid bias, with increased monocytes and macrophages, along with broad upregulation of inflammatory mediators, including S100a8, S100a9, and Il1b, across multiple cell types, and enrichment of TNF and interferon (IFN) signaling pathways. In contrast, females displayed lymphocyte-skewed profiles, with higher frequencies of T cells, B cells, and natural killer (NK) cells. Interestingly, these sex-based differences in immune composition and inflammatory programs were largely absent in GF mice, suggesting that microbial exposure influences baseline immunological dimorphism between males and females. Notably, select sex-associated immune differences, including female-biased NK cell enrichment, persisted irrespective of microbial status, suggesting intrinsic, microbiota-independent programming. Together, these findings indicate that commensal microbes may modulate sex-specific lung immunity, potentially amplifying pre-existing intrinsic differences, highlighting the intersection of extrinsic (microbial) and intrinsic (sex-linked) factors in shaping baseline mucosal immunity.