Abstract Rationale Asthma is a chronic, heterogeneous respiratory disease affecting over 25 million Americans, including 7 million children. It is characterized by airway inflammation, remodeling, and metabolic dysregulation of the airway epithelium. Despite current anti-inflammatory and bronchodilator therapies, asthma exacerbations remain a major cause of morbidity and mortality. Recent evidence suggests that bronchial epithelial cells from asthmatic patients exhibit altered metabolic signatures characterized by increased inflammation, reactive oxygen species (ROS) production, and oxidative stress- hallmarks of mitochondrial dysfunction. However, the mechanistic link between bronchoconstriction and epithelial metabolic alterations remains unclear. Club cell secretory protein (CC16), a highly abundant anti-inflammatory and antioxidant protein in the lung, protects against airway remodeling, but its levels are reduced in asthma. This project investigates how bronchoconstriction affects CC16 production and how loss of CC16 contributes to epithelial metabolic dysregulation. Methods Mouse tracheal epithelial cells (MTECs) were isolated, cultured to confluence, and differentiated into pseudostratified epithelium at an air-liquid interface. Wild-type (WT) and CC16-deficient (Cc16-/-) MTECs were treated with recombinant CC16 (rCC16) or vehicle for 48 hours, and apical secretions were collected for mass spectrometry, or live cells for Seahorse XF Cell Mito Stress Test. Proteomic analyses were performed using HPLC-ESI-MS/MS on an Orbitrap Fusion Lumos mass spectrometer, followed by label-free quantification with Progenesis QI software. A transmembrane compression system simulated bronchoconstriction by applying 0.43 psi (30 cmH2O) pressure for 3 hours at 37 °C. Apical secretions were collected for mass spectrometry. Results Proteomic analysis revealed that rCC16 treatment upregulated proteins involved in glycolysis, NADPH regeneration, and oxidative stress responses in WT MTECs. qRT-PCR confirmed increased expression of glycolytic enzymes ALDOA and ALDOC in human bronchial epithelial cells. CC16-deficient MTECs exhibited reduced maximal OCR, indicating impaired mitochondrial respiration, while rCC16 treatment restored OCR to WT levels. Under mechanical compression, WT MTECs displayed 53 downregulated and 118 upregulated proteins. Notably, CC16 (uteroglobin) was significantly reduced at both protein and mRNA levels, indicating that mechanical stress suppresses CC16 expression. Pressurized cells showed enrichment of pathways related to fatty acid oxidation and ATP generation, consistent with a metabolic shift following CC16 loss. Conclusions CC16 supports epithelial metabolic homeostasis by enhancing glycolytic and mitochondrial activity. Its suppression during bronchoconstriction likely disrupts energy balance, contributing to epithelial dysfunction in asthma. Restoring CC16 may protect airway metabolism during bronchoconstrictive stress. Future studies will assess whether rCC16 pretreatment prevents metabolic and mitochondrial defects induced by mechanical compression. This abstract is funded by: R01HL142769 (Ledford), T32 HL007249-44/45 (Iannuzo), R21AI178782 (Ledford), T32 HL007249-48 (Berthiaume Fox), ARCS Scholarship Phoenix Chapter (Berthiaume)
Fox et al. (Fri,) studied this question.