Single-Cell RNA Sequencing Identifies a Unique Macrophage Population in a Mouse Model of Ozone-induced Asthma Exacerbation

Ozone (O₃) inhalation triggers asthmatic airway hyperresponsiveness (AHR), but the mechanisms are unknown. Previously, we developed a murine model of dust mite, ragweed, and <i>Aspergillus</i> (DRA)-induced allergic lung inflammation followed by O₃ exposure for mechanistic investigation. The present study used single-cell RNA sequencing for unbiased profiling of cells within the lungs of mice exposed to DRA, O₃, or DRA + O₃ to identify components of the immune cell niche that contribute to AHR. Alveolar macrophages (AMs) had the greatest number of differentially expressed genes after DRA + O₃, most of which were unique to the two-hit exposure. After DRA + O₃, AMs activated transcriptional pathways related to cholesterol biosynthesis, degradation of the extracellular matrix, endosomal Toll-like receptor processing, and various cytokine signals. We also identified AM and monocyte subset populations that were unique to the DRA + O₃ group. These unique AMs activated gene pathways related to inflammation, sphingolipid metabolism, and bronchial constriction. The unique monocyte population had a gene signature that suggested phospholipase activation and increased degradation of the extracellular matrix. Flow cytometric analysis of BAL immune cells showed recruited monocyte-derived AMs after DRA and DRA + O₃, but not after O₃ exposure alone. O₃ alone increased BAL neutrophils, but this response was attenuated in DRA + O₃ mice. DRA-induced changes in the airspace immune cell profile were reflected in elevated BAL cytokine/chemokine levels after DRA + O₃ compared with O₃ alone. The present work highlights the role of monocytes and AMs in the response to O₃ and suggests that the presence of distinct subpopulations after allergic inflammation may contribute to O₃-induced AHR.