ABSTRACT: Ozone (O3) is known to cause lung injury, which can influence the health and function of resident immune and non-immune cells of the respiratory tract. Alveolar macrophages (AMs), a highly plastic lung resident cell population, are among the first responders to the inhaled O3 and ozonized products. However, the complete understanding of how AMs respond to O3, particularly to different concentrations, remains elusive. Through single cell RNA sequencing (scRNA-seq), we aimed to profile AM transcriptome from O3-exposed mice and understand the relevance of these transcriptomic changes to the population and functional heterogeneity. Accordingly, we exposed C57BL/6J male mice to filtered air (FA), 1 ppm O3, or 1.5 ppm O3 for 3 hours. After 24h of exposure, bronchoalveolar lavage (BAL) was performed to harvest immune cells, which were subjected to ScRNA-seq. As compared to FA-exposed mice, O3-exposed mice exhibited increased alveolar macrophages and neutrophils in the lung airspaces, consistent with the elevated levels of macrophage and neutrophil chemokines, i.e., MIP-2, MCP-3, MCP-5. The ScRNA-Seq data analyses were performed to identify distinct and annotate distinct cellular clusters. AM clusters were analyzed for the alterations in gene expression and associated enrichment of biological pathways. Although the O3 exposure did not result in the appearance of a distinct cellular cluster, there was a significant difference in the number of differentially expressed genes (DEGs) between AMs from mice exposed to 1 ppm and 1.5 ppm O3, relative to those exposed to FA. As compared to AMs from FA-exposed group, AMs from 1 ppm and 1.5 ppm O3-exposed groups displayed enrichment of pathways including oxidative phosphorylation, EIF2 signaling, and non-canonical NF-kB signaling. Furthermore, AMs from 1 ppm O3-exposed mice were uniquely activated in IL-10 signaling pathway. On the other hand, AMs from 1.5 ppm O3-exposed mice were uniquely enriched in pathways including DNA damage bypass and repair pathways. Interestingly, UMAP analyses on annotated AMs resulted in five distinct subclusters. DEGs and IP analyses for each subcluster revealed O3 concentration-dependent enrichment of pathways relevant to protein translation, cholesterol biosynthesis and mitochondrial biogenesis. Further analyses revealed that O3 exposure results in cluster-specific alterations to the expression of gene signatures associated with macrophage activation. Finally, AMs from 1.5 ppm O3-exposed mice displayed elevated expression of proliferation associated gene signatures. Taken together, this study identified O3 concentration-dependent alterations in AMs transcriptomes and associated functional modulations at single cell resolution.
