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

Abstract

Alveolar macrophages (AMs), a highly plastic immune cell population, are among the first responders to the inhaled ozone (O3) and ozonized products in the lung airspaces. However, the complete understanding of how AMs respond to O3, particularly to different concentrations, remains elusive. Accordingly, we exposed C57BL/6J male adult mice to filtered air (FA), 1 ppm O3, or 1.5 ppm O3 for 3 hours. As compared to FA-exposed mice, O3-exposed mice exhibited increased recruitment of alveolar macrophages and neutrophils in the lung airspaces, which was consistent with the elevated levels of macrophage- and neutrophil-specific chemokines, i.e., MIP-2, MCP-3, and MCP-5. Next, to profile AM transcriptome from O3-exposed mice and understand the relevance of these transcriptomic changes in relation to their population heterogeneity and functionality, we performed single-cell RNA sequencing (scRNA-seq) analyses. The differentially expressed genes (DEGs) analyses on AM population identified significant changes in 1 ppm-exposed and 1.5 ppm O3-exposed mice. As compared to AMs from FA-exposed group, AMs from 1 ppm O3- 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 showed enrichment of IL-10 signaling pathway. On the other hand, AMs from 1.5 ppm O3-exposed mice were uniquely enriched in DNA damage bypass and repair pathways. Interestingly, UMAP analyses on annotated AMs resulted in five distinct subclusters. DEGs and ingenuity pathways (IP) analyses on 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 identifies O3 concentration-dependent alterations in AMs transcriptome and associated functional modulations at a single-cell resolution.