Progression of Cystic Fibrosis Lung Disease from Childhood to Adulthood: Neutrophils, Neutrophil Extracellular Trap (NET) Formation, and NET Degradation

The review addresses how CFTR gene mutations drive the progression of cystic fibrosis lung disease from early life to adulthood, focusing on neutrophil biology, neutrophil extracellular trap (NET) formation, and NET clearance. It highlights that neutrophilic inflammation can precede detectable infection, escalates through childhood, and, together with infections, leads to mucus dehydration, increased viscosity, and airway obstruction. Neutrophils in CF airways contribute to tissue damage via cytotoxic granule proteins, extracellular DNA, and NETs, which exacerbate mucus plugging. The paper aims to synthesize mechanisms by which CF airway conditions (pH, salt, cytokine milieu, altered neutrophil phenotypes) modulate NETosis, and to discuss therapeutic strategies that reduce detrimental NET-related pathology while preserving antimicrobial function.

The review synthesizes evidence on: (1) CFTR structure, regulation (PKA-phosphorylated R-domain), and mutation classes (I–VI) with clinical relevance (e.g., common F508del Class II; gating defects like G551D responsive to ivacaftor). (2) Pathophysiology of mucus dehydration: CFTR-ENaC dysregulation increases intracellular Na+/Cl−, dehydrates airway surface liquid, reduces mucociliary clearance; reduced HCO3− secretion and oxidative crosslinking thicken mucus; neutrophil-derived DNA and actin further increase viscosity; Th2/Th17 cytokines contribute to goblet hyperplasia and neutrophil recruitment. (3) Infection and inflammation: early airway inflammation may precede infection; elevated IL-8, IL-1β, TNF-α, elastase in CF airways; structural lung disease seen early by CT; chronic infections with Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia; additive inflammatory effects of co-infections; lung transplantation outcomes and complications. (4) Neutrophil biology: development and trafficking (CXCR4/CXCR2), high pulmonary marginated pool; CF neutrophil phenotypes show impaired HOCl-mediated killing, delayed apoptosis, altered ROS depending on compartment, and increased survival; CF neutrophils express CFTR important for phagolysosomal chlorination. (5) NETosis mechanisms: NOX-dependent pathway (RAF-MEK-ERK signaling, ROS, histone modification, autophagy) and NOX-independent pathway (calcium influx, SK3 activation, mitochondrial ROS, PAD4-mediated citrullination). CF neutrophils exhibit mitochondrial dysfunction and elevated intracellular calcium, potentially biasing toward NETosis, including NOX-independent routes. Pathogens common in CF (P. aeruginosa, S. aureus, Aspergillus fumigatus) induce NETs; bacterial motility and LPS/pyocyanin implicated. (6) Vicious cycle: NETs elevate sputum DNA, increase viscosity, scaffold proteases (elastase, MPO) that cause tissue damage; NET DNA may promote bacterial biofilm and pathoadaptation; clinical P. aeruginosa strains can become resistant to NET-mediated killing. (7) NET clearance: DNase (dornase alfa) reduces viscosity but actin inhibits DNase I and DNase can release active proteases from NET scaffolds; actin-resistant DNase variants (e.g., alidornase alfa) and DNase1L3 noted; macrophage/monocyte roles in NET clearance are impaired in CF due to adhesion/transmigration defects. (8) Therapeutic avenues: optimize DNase (actin-resistant), combine with antiproteases, consider anti-histone strategies (with immunogenicity caveats), target biofilm dispersion, and identify small molecules that suppress excessive NETosis without impairing host defense. Modulators of pH, ROS, kinase pathways, and ion channels are highlighted; CF airway L-arginine deficiency and neutrophil arginase-mediated T cell suppression may influence viral exacerbations.

- Early neutrophilic inflammation occurs in CF airways even before overt infection and escalates with age, contributing to structural lung damage and decline in function. - NETs and extracellular DNA are abundant in CF sputum/BAL and correlate with disease severity; NETs increase mucus viscosity and carry cytotoxic proteases (elastase, MPO) that injure tissue. - CF airway milieu (reduced pH, altered ion concentrations, cytokines) and CF neutrophil intrinsic changes (mitochondrial dysfunction, elevated intracellular Ca2+, delayed apoptosis) enhance susceptibility to NETosis via both NOX-dependent and NOX-independent pathways. - Common CF pathogens (P. aeruginosa, S. aureus, A. fumigatus) effectively induce NETs; P. aeruginosa clinical strains may develop resistance to NET-mediated killing, and NET DNA can promote biofilm formation. - DNase therapy (dornase alfa) reduces sputum viscosity but may increase protease activity by releasing proteases from NET scaffolds; actin in sputum inhibits DNase I, motivating interest in actin-resistant DNases (e.g., DNase1L3, alidornase alfa) that showed lung function improvement in early trials. - Monocyte/macrophage dysfunction in CF impairs NET clearance from airways, contributing to extracellular DNA accumulation; CFTR correctors can partially restore monocyte integrin function in vitro. - CFTR modulators (e.g., ivacaftor for G551D) can reduce neutrophil activation and may decrease NETotic propensity, suggesting genotype-specific modulation of innate immune dysfunction. - Potential therapeutic targets include pathways regulating NETosis (RAF-MEK-ERK, JNK, Akt), ion channels (SK3), ROS sources (NOX/mitochondria), PAD4-mediated histone citrullination, airway pH/ionic milieu, and biofilm dispersal strategies.

The reviewed evidence supports a central role for neutrophils and NETs in CF lung disease progression. NET formation, driven by CF airway conditions and persistent infection, contributes to a feed-forward cycle of mucus obstruction, protease-mediated tissue damage, and impaired bacterial clearance, thereby addressing the question of how neutrophil biology links to worsening CF pathology. Understanding distinct NETosis pathways (NOX-dependent versus NOX-independent) in CF provides mechanistic entry points for selective intervention aimed at reducing excessive NET burden without compromising essential antimicrobial functions. Therapeutic implications include refining DNase approaches (to overcome actin inhibition and mitigate protease release), combining DNase with antiproteases, modulating airway pH and ion transport, deploying CFTR modulators to correct immune cell phenotypes, and targeting biofilms to shift pathogens to more vulnerable states. The interplay between neutrophils and adaptive immunity (e.g., arginase-mediated T cell suppression and implications for viral exacerbations) further underscores the systemic impact of neutrophil dysfunction and NETs in CF, highlighting avenues for reducing exacerbation frequency and preserving lung function.

Neutrophils are pivotal to innate defense but, in CF, defective CFTR in epithelial and immune cells contributes to persistent inflammation, chronic infection, and excessive NET formation. NET-derived DNA and proteases exacerbate mucus obstruction and tissue damage, amplifying disease progression. While DNase and antiprotease therapies offer partial benefit, their limitations and potential to increase proteolytic activity necessitate improved strategies. Advancing mechanistic insight into NETosis pathways in CF, optimizing NET clearance (e.g., actin-resistant DNases, macrophage support), targeting biofilms, modulating airway pH/ions, and employing CFTR modulators to normalize immune cell function represent key future directions. Integrative therapies that preserve neutrophil antimicrobial efficacy while curbing harmful NET-related effects are essential to improving outcomes in CF lung disease.

As a narrative review, the article synthesizes existing literature without presenting new primary experimental data; thus, conclusions depend on the quality and heterogeneity of cited studies. Mechanistic details of NETosis in CF in vivo (temporal triggers, relative contributions of NOX-dependent vs NOX-independent pathways) remain incompletely defined. Clinical evidence for next-generation DNases (e.g., actin-resistant variants) and anti-NET strategies in CF is preliminary, and potential adverse effects (e.g., increased free protease activity, immunogenicity of anti-histone approaches) require rigorous evaluation. The complexity of CF airway microbiota and host immunity limits generalizability across ages, genotypes, and stages of disease.