RESUMEN
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.
RESUMEN
Asthma is a chronic inflammatory airway disease characterized by acute exacerbations triggered by inhaled allergens, respiratory infections, or air pollution. Ozone (O3), a major component of air pollution, can damage the lung epithelium in healthy individuals. Despite this association, little is known about the effects of O3 and its impact on chronic lung disease. Epidemiological data have demonstrated that elevations in ambient O3 are associated with increased asthma exacerbations. To identify mechanisms by which O3 exposure leads to asthma exacerbations, we developed a two-hit mouse model where mice were sensitized and challenged with three common allergens (dust mite, ragweed and Aspergillus fumigates, DRA) to induce allergic inflammation prior to exposure to O3 (DRAO3). Changes in lung physiology, inflammatory cells, and inflammation were measured. Exposure to O3 following DRA significantly increased airway hyperreactivity (AHR), which was independent of TLR4. DRA exposure resulted in increased BAL eosinophilia while O3 exposure resulted in neutrophilia. Additionally, O3 exposure following DRA blunted anti-inflammatory and antioxidant responses. Finally, there were significantly less monocytes and innate lymphoid type 2 cells (ILC2s) in the dual challenged DRA-O3 group suggesting that the lack of these immune cells may influence O3-induced AHR in the setting of allergic inflammation. In summary, we developed a mouse model that mirrors some aspects of the clinical course of asthma exacerbations due to air pollution and identified that O3 exposure in the asthmatic lung leads to impaired endogenous anti-inflammatory and antioxidant responses and alterations inflammatory cell populations.
