Microbiota-dependent transcriptional priming of lung innate immune cells in a mouse model of LPS-induced sepsis-associated lung injury.

Abstract

OBJECTIVES: While commensal microbiota are known to play essential roles in functional maturation of the innate immune system, the mechanisms by which microbial signals shape pulmonary immunity remain unclear. We performed single-cell RNA sequencing of lung immune cells from germ-free (GF) and conventional (CV) mice under normal physiological and LPS-induced septic conditions. METHODS: Lung immune cells were isolated from GF and CV mice exposed to normal or septic conditions. Single-cell RNA sequencing data were analyzed using standard pipelines with cell-type annotation and pathway profiling based on enrichment analyses. RESULTS: In GF mice, innate immune cell populations, including neutrophils, macrophages, and natural killer cells, exhibited an altered baseline transcriptional state characterized by reduced inflammatory readiness and a shift toward metabolic and stress-associated programs relative to these cell populations in CV mice. Neutrophils from GF mice exhibited a disrupted maturation trajectory with loss of transitional states and immature cell accumulation, suggesting that microbiota-derived cues are necessary to complete peripheral maturation and support a conserved systemic mechanism of microbiota-dependent innate immune differentiation. The lipopolysaccharide-responsive sub-cluster of macrophages exhibited high CCAAT enhancer-binding protein beta (Cebpb) expression in CV mice, which was linked to preferential engagement of inflammatory rather than homeostatic programs, whereas these macrophages in GF mice failed to induce Cebpb. During LPS-induced sepsis, lack of microbial priming results in blunted inflammatory responses and inadequate transcriptional network activation. CONCLUSIONS: Commensal microbiota influence transcriptional activity and maturation of pulmonary innate immune cells under experimental conditions, thereby influencing susceptibility to LPS-induced lung injury. Targeting microbiota-guided immune training pathways may allow modulation of pulmonary host defenses.