Abstract
Ozone (O (3) ) inhalation triggers asthmatic airway hyperresponsiveness (AHR), but the mechanisms by which this occurs are unknown. Previously, we developed a murine model of dust mite, ragweed, and aspergillus (DRA)-induced allergic lung inflammation followed by O (3) exposure for mechanistic investigation. The present study used single cell RNA-sequencing for unbiased profiling of immune cells within the lungs of mice exposed to DRA, O (3) , or DRA+O (3) , to identify the components of the immune cell niche that contribute to AHR. Alveolar macrophages (AMs) had the greatest number of differentially expressed genes following DRA+O (3) , most of which were unique to the 2-hit exposure. Following DRA+O (3) , AMs activated transcriptional pathways related to cholesterol biosynthesis, degradation of the extracellular matrix, endosomal TLR processing, and various cytokine signals. We also identified AM and monocyte subset populations that were unique to the DRA+O (3) 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 cytometry analysis of BAL immune cells showed recruited monocyte-derived AMs after DRA and DRA+O (3) , but not after O (3) exposure alone. O (3) alone increased BAL neutrophils but this response was attenuated in DRA+O (3) mice. DRA-induced changes in the airspace immune cell profile were reflected in elevated BAL cytokine/chemokine levels following DRA+O (3) compared to O (3) alone. The present work highlights the role of monocytes and AMs in the response to O (3) and suggests that the presence of distinct subpopulations following allergic inflammation may contribute to O (3) -induced AHR.