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Biomechanics

Biomechanics - Poster Session E

Poster P20 - Optimizing Mechanical Stress System to Investigate the Role of CC16 in Airway Remodeling and Cell Metabolism in Asthma

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Club cell secretory protein, also known as CCSP/CC16/CC10/Uteroglobin, has gained significant attention as a potential biomarker of lung epithelial cell injury. Recent studies by our group have highlighted the predictive value of low circulating CC16 levels in childhood lung function and increased risk for developing asthma or chronic obstructive pulmonary disease (COPD) in adult life (Guerra, 2023). Interestingly, CC16 is detectible in the serum, making it an attractive and accessible biomarker for airway disease. In fact, numerous studies indicate a consistent decrease in serum CC16 among those with asthma and COPD and decreases in lung function in the general population (Oh, 2018). Our previous studies have determined that CC16 is an important regulator of pulmonary epithelial-driven responses during infection. Because of CC16's role in host defense, we sought to characterize the response that rCC16 elicits in WT Mouse Tracheal Epithelial Cells (MTECS). Here, we use mass spectroscopy and quantitative proteomics to elucidate a strong metabolic signature in cells treated with rCC16. We also sought to characterize WT MTEC's metabolic response and impact on CC16 levels to transmembrane pressure by optimizing a previously established system for modeling asthma and airway remodeling (Drazen, 2001).

Materials and

Methods:  Mouse tracheal epithelial cell isolation and culture: MTEC isolation and harvest as previously described (Iannuzo, 2022). In short, mouse tracheas were isolated and tracheal epithelium was collected and seeded in T75 tissue culture-treated flasks at 375k and grown at 37C, 5% CO2 until 90% confluent. MTECs were then transfer to Transwell (12-well plates) at a density of 89k cells/well. These cells were "air-lifted" 1 week after plating, or at 80% confluence. Differentiated epithelium was accomplished after 2 weeks at air.

rCC16 treatment for MTECs: WT MTECs were treated apically with rCC16 or vehicle media for 48-hours.

Mass Spectroscopy: HPLC-ESI-MS/MS was performed as previously described in positive ion mode on a Thermo Scientific Orbitrap Fusion Lumos tribrid mass spectrometer fitted with an EASY-Spray Source (Iannuzo, 2023) (Kruse, 2017).

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 as previously described (Iannuzo, 2023), (Parker, 2019).

Application of apical compression on MTECs: As previously described (Drazen, 2001). Silicon plunger was sealed around apical transmembrane. 2 mL of DMEM/F12 was added to the basolateral media. 10 minutes of 30 cmH20 was applied intermittently for a total of 30 minutes.

Results, Conclusions, and Discussions: In WT MTECs, treatment with rCC16 led to the upregulation of proteins associated with cellular metabolism. From GO-Biological Process pathway enrichment analysis, 16 proteins were significantly associated with "Glycolytic Process", 5 proteins were significantly associated with "Pentose-Phosphate Shunt", and 14 proteins were significantly associated with "Response to Oxidative Stress". KEGG pathway enrichment analysis revealed similar pathways upregulated in rCC16 treatment, including "Glycolysis/Gluconeogenesis", and "Pentose Phosphate Pathway". These findings suggest that CC16 may play an important role in cell metabolism, including pathways like glycolysis, gluconeogenesis, and the pentose-phosphate pathway.
Given these findings, we anticipate that introducing transmembrane compression to WT MTECs may reduce their metabolic activity and that treatment with rCC16 may rescue this signature.
We are continuing to validate and optimize our transmembrane compression model in our WT MTECs. To avoid cell death by drying out the membrane, we increased our basolateral compartment volume from 1mL to 2 mL of media during periods of compression. Optimizing compression time at a pressure of 30cmH20 remains ongoing.

Acknowledgements (Optional):