Bronchoconstriction Modulates CC16 Levels and Subsequent Dysregulation of Epithelial Cell Metabolism in Asthma.

Introduction: : Asthma is a chronic, heterogenous respiratory disease affecting approximately 25 million people in the United States, including 7 million children. Asthma is characterized by airway inflammation, remodeling, and metabolic dysregulation in the airway epithelium. Despite the availability of bronchodilators and inhaled anti-inflammatory agents, current therapies do not ameliorate asthma exacerbations, or bronchoconstrictive events, contributing to significant morbidity and mortality. Recent evidence suggests that bronchial epithelial cells from asthmatic patients exhibit altered metabolic signatures. This metabolic shift of energetically demanding cells leads to increased inflammation, excessive reactive oxygen species production (ROS), and oxidative stress—hallmarks of mitochondrial dysfunction. While mitochondrial dysfunction has been implicated in asthmatic epithelial cells, the mechanistic link between bronchoconstriction and these metabolic alterations remains poorly defined. Club cell secretory protein (CC16) is the most abundant protein found in the lung and exerts key anti-inflammatory and antioxidant functions, culminating in protection against airway remodeling. Decreased levels of CC16 are characteristic of asthma and worsening respiratory disease. Thus, this project aims to use a transmembrane compression system to investigate how bronchoconstriction impacts production of CC16 in the airway, and how loss of CC16 may lead to metabolic dysregulation contributing to airway remodeling.

Materials and

Methods: : Mouse tracheal epithelial cell (MTEC) isolation and culture: Briefly, mouse tracheas were isolated and tracheal epithelium was collected and seeded in T75 culture flasks at 375k cells and grown at 37C, at 5% CO2 until 90% confluent (~1 week). MTECs were then transferred to Transwells (12-well plates) at a density of 89k cells/well and apically submerged for ~1 week. Air-lift was performed at ~80% confluence. Differentiated pseudostratified epithelium developed after 2 weeks at air.
rCC16 treatment: WT or Cc16-/- MTECs were treated with commercially purchased (R&D) recombinant CC16 (rCC16) or media (vehicle) for 48-hours.
Mass Spectroscopy: HPLC-ESI-MS/MS was performed in positive ion mode on a ThermoScientific Orbitrap Fusion Lumos Tribrid mass spectrometer with an EASY-Spray Source.
Label-free Quantitative Proteomics: Progenesis QI for proteomics software (version 2.4, Nonlinear Dynamics Ltd. Newcastle upon Tyne, UK) was used to perform ion-density based label-free quantification.
Transmembrane Compression Model System: The system includes pressurized mixed Ultra Zero Air, (20.9% Oxygen, 79% Nitrogen and < 0.1 ppm total hydrocarbons), humidifier, silicone tubing, and rubber stoppers sealing the apical transwell compartment. A manometer monitors real-time pressure. Pressure is set to 0.43 psi (30 cmH2O) and applied for 3 hours at 37C. Control wells were sealed with silicone plugs but not connected to pressure.
Oxygen consumption rate (OCR): Measured using the Extracellular Flux Analyzer using the MitoStress Assay kit. Cells were seeded at 65k/well in XFe 96 well plate. Oligomycin (1.5uM), FCCP (1uM), and Rotenone/AA (0.5uM) were added over the course of the assay for mitochondrial uncoupling.

Results, Conclusions, and Discussions:: Our findings highlight a key role for CC16 in regulating respiratory epithelial cell metabolism, particularly under mechanical stress conditions. Using mass spectrometry and quantitative proteomics, we found that recombinant CC16 (rCC16) treatment significantly upregulates proteins associated with Gene Ontology (GO) tags such as glycolysis, NADPH regeneration, and oxidative stress response in wild-type (WT) mouse tracheal epithelial cells (MTECs) (Figure 1). To increase the rigor of our findings, we verified this increase in expression of glycolytic enzyme (ALDOA, B, and C) transcripts by qRT-PCR in human bronchial epithelial cells (HBECs). ALDOA and ALDOC were significantly increased with rCC16 treatment in HBECs, suggesting this finding is translatable to the human airway epithelium.
CC16 also modulates mitochondrial respiration. Seahorse XF Mito Stress Tests showed that CC16-deficient (Cc16-/-) MTECs exhibited reduced maximal oxygen consumption rate (OCR), indicating impaired mitochondrial function. rCC16 treatment rescued this defect, restoring OCR to WT levels (Figure 2). This suggests CC16 directly supports mitochondrial respiratory capacity, likely enhancing energy production during metabolic stress.
We examined the impact of mechanical compression, an in vitro model of bronchoconstriction, on CC16 expression. Following 3 hours of pressure at 0.43 psi, WT MTECs had differentially expressed proteins in the apical compartment compared to controls. 53 proteins were significantly downregulated, and 118 proteins were significantly upregulated in compressed WT MTECs. Interestingly, CC16 (Uteroglobin) was among the 53 significantly downregulated proteins. Furthermore, compressed WT MTECs had significantly reduced CC16 (Scgb1a1= gene) mRNA expression, suggesting mechanical stress suppresses CC16 production (Figure 3). Concomitantly, we observed enrichment of GO tags related to mitochondrial metabolism, including fatty acid oxidation and ATP production immediately after pressure, and sustained for 48 hours measured by quantitative proteomics (not shown).
Taken together, these data support a model in which CC16 protects epithelial metabolic homeostasis by promoting glycolytic flux and mitochondrial respiration. Its suppression during bronchoconstriction may impair these functions, contributing to epithelial dysfunction observed in asthma. Future studies will focus on whether rCC16 pretreatment can prevent metabolic shifts and mitochondrial dysfunction caused by mechanical compression, and whether treating with rCC16 will protect epithelial metabolism during bronchoconstrictive episodes that occur during asthma.

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