Single-cell transcriptomics reveal alveolar macrophage-specific responses in single-hit ozone exposure model in mice

Alveolar macrophages (AMs), a highly plastic immune cell population, are among the first responders to inhaled ozone (O₃) and ozonated products in the lung airspaces. However, a comprehensive understanding of how AMs respond to O₃, particularly across different exposure concentrations, remains incomplete. To address this knowledge gap, we exposed adult male C57BL/6J mice to filtered air (FA), 1 ppm O₃, or 1.5 ppm O₃ for 3 h. Mice were euthanized 22-24 h after the end of exposure, and experiments were conducted to assess cellular recruitment, inflammatory mediator analyses, and gene expression alterations. Compared with FA-exposed mice, O₃-exposed mice exhibited increased recruitment of AMs and neutrophils into the lung airspaces, consistent with elevated levels of the macrophage- and neutrophil-specific chemokines, i.e., monocyte chemotactic protein 3 (MCP-3), MCP-5, and macrophage inflammatory protein 2. To delineate the transcriptomic landscape of AMs following O₃ exposure and determine how these alterations relate to AM heterogeneity and functional states, we subjected AMs to single-cell RNA sequencing analyses. Differentially expressed gene (DEG) analysis of the AM population revealed significant transcriptional changes in both the 1-ppm and 1.5-ppm O₃-exposed groups. Compared with AMs from the FA-exposed group, AMs from both O₃-exposed groups exhibited enrichment of pathways such as oxidative phosphorylation, eIF2 signaling, and noncanonical NF-κB signaling. Furthermore, AMs from 1-ppm O₃-exposed mice exhibited enrichment of the IL-10 signaling pathway, whereas AMs from 1.5-ppm O₃-exposed mice were uniquely enriched for DNA damage bypass and repair pathways. Interestingly, uniform manifold approximation and projection analysis of annotated AMs in three experimental groups identified five distinct subclusters. DEGs and ingenuity pathway analyses of these subclusters revealed O₃ concentration-dependent enrichment of pathways associated with protein translation, cholesterol biosynthesis, and mitochondrial biogenesis. Further analyses revealed that exposure to different concentrations of O₃ results in cluster-specific alterations in the expression of gene signatures associated with macrophage activation. In addition, AMs from 1.5-ppm O₃-exposed mice displayed increased expression of proliferation-associated gene signatures. Taken together, these findings identify O₃ concentration-dependent transcriptomic alterations in AMs and associated functional modulations at single-cell resolution.<b>NEW & NOTEWORTHY</b> This study addresses a critical knowledge gap regarding the response of alveolar macrophages to ozone, a potent inhaled toxicant. Specifically, we demonstrate that a single exposure to two experimentally relevant concentrations of ozone significantly alters functionally relevant gene expression in alveolar macrophages, as revealed by single-cell resolution analysis.