Data Sheet 1_Osteopontin from monocyte-derived dendritic cells mediates ozone-induced pulmonary responses in mice.docx

IntroductionAsthma is a heterogeneous disease characterized by airway inflammation and hyperresponsiveness (AHR). Ozone, a common air pollutant, induces a neutrophilic phenotype of asthma that is resistant to corticosteroids. Osteopontin has been implicated in airway inflammation, but its role in ozone-induced pulmonary responses remains unclear. We hypothesized that osteopontin contributes to neutrophilic airway inflammation and AHR through dendritic cell (DC)–neutrophil interactions.
MethodsMale BALB/c mice were exposed to ozone (2 ppm, 3 hours) or ambient air. AHR and bronchoalveolar lavage fluid (BALF) cell counts were assessed, and osteopontin levels were measured. Single-cell RNA sequencing of lung tissue was performed to identify osteopontin-producing cells and infer cell–cell communication using CellChat analysis. Functional validation included administration of clodronate liposomes to affect monocyte-derived DCs and an anti-osteopontin neutralizing antibody. In addition, RNA sequencing of bone marrow-derived neutrophils stimulated with osteopontin in vitro was conducted.
ResultsOzone exposure significantly increased neutrophils in BALF, enhanced AHR, and elevated osteopontin levels in lung tissue. Single-cell RNA sequencing showed DCs, particularly monocyte-derived DCs, as the main source of osteopontin, with downstream signaling to macrophages, fibroblasts, pericytes, smooth muscle cells, and neutrophils. Clodronate liposome treatment reduced osteopontin expression and attenuated ozone-induced neutrophilic inflammation and AHR. Neutralization of osteopontin suppressed neutrophilic airway inflammation but did not significantly improve AHR. In vitro, osteopontin stimulation of neutrophils upregulated NDUFA1, a mitochondrial complex I gene.
ConclusionsThe present findings indicate that ozone exposure induces osteopontin production primarily from monocyte-derived DCs, which promotes neutrophilic airway inflammation through DC–neutrophil interactions, while having a limited impact on airway hyperresponsiveness.