Sunday, May 18, 2025
5/18/2025
The NET Impact on Lung Epithelia in Cystic Fibrosis and Mucoobstruction - Abstract Cystic fibrosis (CF) lung disease has fundamentally changed in the era of highly effective CFTR modulators, but the inflammation mediating progressive lung destruction and bacterial infections remain major clinical problems. Treatments for people with other mucoobstructive lung diseases are even more limited. Our proposal will address the fundamental gap in our understanding regarding the molecular mechanisms by which NETs contribute to airway damage and infection in mucoobstructive lung diseases. People with mucoobstructive disease have high neutrophil extracellular trap (NET) burdens in their lungs. NETs are web- like structures with a DNA backbone studded with immunomodulatory proteins (e.g., neutrophil elastase=NE). NETs trap and kill microorganisms, however, we have demonstrated that NETs drive epithelial inflammation and cause lung tissue injury. We postulate that NETs are critical mediators of inflammation and infection in mucoobstructive diseases, but their role is unclear. We propose that NETs act through targeting of E-cadherin, an epithelial junction protein that is bound to intracellular β-catenin, which can activate NF-κB signaling to increase cytokines. In preliminary work, we demonstrated that NET exposure separately leads to: cleavage of E-cadherin, activation of β-catenin and NF-κB, enhanced IL-1 signaling, leading to secretion of TNF-α and IL-8, apoptosis, release of free iron and increased intracellular Pseudomonal (PA) density in primary human bronchial epithelia. We have shown that alpha-1 antitrypsin (A1AT) mitigates many of these deleterious NET effects. We discovered that the βENaC-Tg mouse model of mucoobstructive lung disease—with increased lung NETs and delayed clearance of PA—have spontaneously increased: E-cadherin cleavage, TNF-α and IL- 8 homologues and apoptosis. In this proposal we will coalesce these individual findings into a cohesive molecular mechanism and test our central hypothesis that A1AT inhibition of NET-mediated cleavage of E- cadherin will limit inflammatory cytokine signaling, which will reduce epithelial apoptosis and lung PA density. In Aim1, we will test our hypothesis that NET-mediated cleavage of extracellular E-cadherin in bronchial epithelium activates β-catenin to translocate to the nucleus and activate NF-κB signaling that increases inflammatory cytokines (IL-1 & TNF-α). In Aim 2, we will test our hypothesis that NETs drive TNF-α-induced apoptosis of the bronchial epithelium, which releases iron to foster PA growth and infection. In Aim 3 we will use our novel gene delivery tool to test our hypothesis that transplanted pulmonary macrophages will produce continuous and local hA1AT that decreases NET-driven NE activity, E-cadherin cleavage, inflammatory cytokine secretion, apoptosis and decrease PA infection in mucus obstruction. The impact of our study will be to generate new insights into the fundamental biology of NET: epithelial interactions, which are critical to driving pathogenesis in mucoobstructive lung diseases and generalizable to other NET-mediated diseases.
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