(380) Neutrophil Extracellular Traps Disrupt Bronchial Epithelial Barrier Function: Alpha-1 Antitrypsin to the Rescue

Background: Bronchial epithelia serve an important barrier function to limit entry of pathogens and injurious agents into the submucosa and bloodstream. Epithelial cells form an effective barrier through a complex junctional protein network that maintains cell-cell contact and limits paracellular passage. E-cadherin is a crucial adherens junctional protein that is widely expressed in human epithelia and is a key component of the intact human airway mucosa. Decreased epithelial barrier function has been reported in CF. Respiratory infections, such as with Pseudomonas aeruginosa, are associated with loss of E-cadherin. We sought to understand the impact of Neutrophil Extracellular Traps (NETs) on barrier function in human bronchial epithelia.

Methods: We exposed non-CF primary human bronchial epithelial cells (HBE) and a human airway epithelial cell line (wtCFBE41o- cells) to isolated human NETs in their apical compartment for 2-18 hours. We assessed epithelial barrier function by transepithelial electrical resistance (TEER), transepithelial flux of FITC-labeled dextran, immunofluorescence by confocal microscopy and protein expression by Western blot. To demonstrate direct binding of alpha-1 antitrypsin (A1AT) to NET neutrophil elastase (NE), we performed Western blots on supernatants of HBE exposed to NETs with and without A1AT pretreatment and, separately, a co-immunoprecipitation for A1AT and NE.

Results: We demonstrate that NETs disrupt airway epithelial barrier function by decreasing TEER in NHBE (49.9% +/- 24%; p< 0.0001) and wtCFBE (48.7% +/- 14.7%; p< 0.0001) at 18 hours. NET exposure increased paracellular flux in wtCFBE beginning at 8 hours (p=0.0044) through 24 hours (p < 0.0001). Intact epithelia exposed to NETs demonstrated visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein (52.9% +/- 7.8%; p=0.0006) and the appearance of cleaved E-cadherin peptides in a time-dependent manner. DNase did not alter NET-driven changes in HBE TEER or E-cadherin. We showed that A1AT significantly decreased NET protease activity of NE by 72.8%, proteinase 3 (PR3) by 90.6% and cathepsin G (CG) by 99.0%. A1AT co-localized with NET NE and NET proteinase 3, but not consistently with cathepsin G in HBE supernatants exposed to NETs with and without pretreatment of A1AT. To prove A1AT directly binds NET proteases, we performed co-immunoprecipitation on HBE supernatants and detected NET NE:A1AT complex formation.

Conclusions: Human NETs contribute to loss of airway epithelial barrier function by degrading E-cadherin. Exogenous A1AT inhibited NET serine protease activity and limited NET-driven cleavage of E-cadherin by directly binding NET NE and proteinase 3. Pretreatment of NETs with alpha-1 antitrypsin preserved human bronchial epithelial integrity and resistance.

Acknowledgements: Acknowledgments: Funding by: NIH NHLBI 1K08HL124191, CFF K Boost HUDOCK20, NIH NHLBI K08HL124191-04S1, RDP CCHMC pilot Cystic Fibrosis Foundation, NAREN19R0, Parker B Francis.

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