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Previously, we developed a murine model of dust mite, ragweed, and Aspergillus (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.<\/jats:p>","DOI":"10.1165\/rcmb.2024-0358oc","type":"journal-article","created":{"date-parts":[[2025,3,19]],"date-time":"2025-03-19T16:48:32Z","timestamp":1742402912000},"page":"586-599","update-policy":"https:\/\/doi.org\/10.1164\/crossmarkpolicies","source":"Crossref","is-referenced-by-count":4,"title":["Single-Cell RNA Sequencing Identifies a Unique Macrophage Population in a Mouse Model of Ozone-induced Asthma Exacerbation"],"prefix":"10.1093","volume":"73","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5387-9026","authenticated-orcid":false,"given":"Jess L.","family":"Ray","sequence":"first","affiliation":[{"name":"Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Dorothy M. 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