A18-08 Osteopontin Contributes to Ozone-induced Pulmonary Responses via Monocyte-derived Dendritic Cells

Abstract Rationale Ozone exposure induces airway hyperresponsiveness (AHR) and neutrophilic airway inflammation, a distinct non-type 2 asthma phenotype resistant to corticosteroids; however, the underlying mechanisms remain unclear. Osteopontin, a multifunctional cytokine encoded by Spp1, has been implicated in inflammatory and remodeling processes of asthma. This study aimed to clarify the role of osteopontin and its producing cells in ozone-induced airway responses. Methods Male BALB/c mice were exposed to ozone (2 ppm) or ambient air for 3 hours. AHR, bronchoalveolar lavage fluid (BALF) cell counts, and cytokine and chemokine levels in lung tissue were analyzed. Osteopontin was quantified by ELISA. Single-cell RNA sequencing with cell-cell communication (CellChat) analysis was performed to identify Spp1-expressing cell clusters and downstream signaling. Flow cytometry and airway measurements were conducted in mice treated with clodronate liposomes, to impair monocyte-derived dendritic cells (moDCs), or with an osteopontin inhibitor. RNA sequencing of bone marrow-derived neutrophils stimulated with recombinant osteopontin was also performed. Results Ozone exposure markedly enhanced AHR and increased neutrophils in BALF, along with elevated osteopontin in lung tissue. Cytokines such as granulocyte colony-stimulating factor (G-CSF), IL-6, and leukemia inhibitory factor (LIF) were significantly upregulated. Single-cell RNA sequencing revealed that Spp1 expression was predominantly increased in DCs, especially moDCs, with predicted intercellular signaling toward neutrophils, macrophages, fibroblasts, pericytes, and smooth muscle cells. Clodronate liposome treatment reduced lung moDCs and osteopontin levels, significantly attenuating ozone-induced neutrophilic inflammation and AHR. Neutralization of osteopontin similarly decreased BALF neutrophils and tended to reduce AHR. In vitro, osteopontin stimulation of neutrophils activated oxidative phosphorylation-related pathways and upregulated NDUFA1, a mitochondrial complex I gene associated with ATP synthesis, suggesting direct metabolic activation of neutrophils. Conclusions Osteopontin plays a pivotal role in ozone-induced pulmonary responses by mediating neutrophilic airway inflammation and AHR through moDC-derived Spp1 signaling. Mechanistically, osteopontin not only amplifies DC-neutrophil and macrophage crosstalk but also directly enhances neutrophil mitochondrial activity via NDUFA1 induction. These findings highlight osteopontin as a potential therapeutic target for neutrophilic, steroid-resistant asthma triggered by environmental pollutants. This abstract is funded by: AstraZeneca