Elastase Exocytosis by Airway Neutrophils Is Associated with Early Lung Damage in Children with Cystic Fibrosis

Abstract

Rationale: Neutrophils are recruited to the airways of individuals with cystic fibrosis (CF). In adolescents and adults with CF, airway neutrophils actively exocytose the primary granule protease elastase (NE), whose extracellular activity correlates with lung damage. During childhood, free extracellular NE activity is measurable only in a subset of patients, and the exocytic function of airway neutrophils is unknown.Objectives: To measure NE exocytosis by airway neutrophils in relation to free extracellular NE activity and lung damage in children with CF.Methods: We measured lung damage using chest computed tomography coupled with the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis scoring system. Concomitantly, we phenotyped blood and BAL fluid leukocytes by flow and image cytometry, and measured free extracellular NE activity using spectrophotometric and Förster resonance energy transfer assays. Children with airway inflammation linked to aerodigestive disorder were enrolled as control subjects.Measurements and Main Results: Children with CF but not disease control children harbored BAL fluid neutrophils with high exocytosis of primary granules, before the detection of bronchiectasis. This measure of NE exocytosis correlated with lung damage (R = 0.55; P = 0.0008), whereas the molecular measure of free extracellular NE activity did not. This discrepancy may be caused by the inhibition of extracellular NE by BAL fluid antiproteases and its binding to leukocytes.Conclusions: NE exocytosis by airway neutrophils occurs in all children with CF, and its cellular measure correlates with early lung damage. These findings implicate live airway neutrophils in early CF pathogenesis, which should instruct biomarker development and antiinflammatory therapy in children with CF.

Neutrophil Adaptations upon Recruitment to the Lung: New Concepts and Implications for Homeostasis and Disease

“…Importantly, they have been shown to develop a unique inflammatory phenotype after recruitment into the CF airway lumen, where they maintain viability and exocytose their primary granules but have reduced ability to phagocytose bacteria in a distinct fate now called GRIM (granule releasing, immunomodulatory, and metabolically active) neutrophils [105,116,117]. Exocytosis of the primary granules, which are usually sequestered in the cytoplasm, results in release of effectors including NE, Cathepsin G, MPO and Arg1, which has been found to correlate with disease progression in mice with CF-like lung disease and young children and older patients with CF [118][119][120]. This pro-inflammatory phenotype with reduced ability to clear pathogens poses an intriguing paradox and is becoming a focal point in addressing CF lung disease.…”

Cystic Fibrosis: Pathophysiology of Lung Disease

“…70 Neutrophils present in CF airways as assessed by BAL have a phenotype typical of an increased exocytosis rate that correlates with lung damage. 75 Recent data indicate that structural lung damage in CF as determined by computed tomography scan more closely correlates with the frequency of detection of BAL HLE than it does with the detection of infection. 76 These results suggest that free HLE in the CF airway is driving much of the CF lung disease (►Table 1).…”

Progress in Model Systems of Cystic Fibrosis Mucosal Inflammation to Understand Aberrant Neutrophil Activity

“…In 2005, a pioneering study assessing bronchoalveolar lavage fluid (BALF) after CF newborn screening demonstrated that infection in the first year of life is linked to early airway inflammation (7). Subsequent surveillance studies have now established that for most children, inflammation, altered microbiome, active neutrophil proteolytic function, and lung damage all become evident within the first 2 years of life (8)(9)(10)(11), before children are old enough to be treated with CFTR modulators (12,13). Lung function declines can be evident in infants and continue into childhood (14-16).…”

“…Neutrophil elastase (NE) and other serine proteases are a central component of the neutrophil antimicrobial arsenal, stored in the primary granules that are the last granule to mobilize and are highly resistant to fusion with the outer membrane (23). Yet uninhibited NE activity can be detected in over 30% of BALF samples from young children with CF (11,24). Activity of NE is considered one of the most significant biomarkers in CF lung disease, as activity significantly correlates with lung damage and functional declines at all stages of life with CF (25-28).…”

“…In an age related cohort of non-CF children admitted for acute respiratory distress syndrome (ARDS), neutrophil exocytosis and reduced bacterial killing was observed in individuals co-infected with virus and bacteria but not viral infection alone, suggesting that neutrophil exocytosis may be linked to responses against polymicrobial infection (93). This relationship with infection is yet to be established in early CF disease, however neutrophil exocytosis markers correlate positively with disease severity more so than free NE activity (11). Therefore, changes in airway neutrophil functional markers may be more reliable indicators of disease progression in children with CF and should be a focus of early CF lung disease research.…”