Novel insights related to CF neutrophils

In this issue of Blood, Pohl et al demonstrate that neutrophils from patients with mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) have impaired exocytosis of gelatinase and specific granules, altered ion homeostasis resulting in deactivation of the small GTPase Rab27a, and compromise bacterial killing abilities. These neutrophil defects were corrected by ivacaftor treatment, an ion channel potentiator, which has shown beneficial effects in pulmonary function in cystic fibrosis (CF) patients.¹ 

Cystic fibrosis is a disease associated with mutations in the CFTR gene, which results in enhanced susceptibility to pulmonary infections, with massive neutrophil recruitment to the lungs. The defect in CFTR function in the lung epithelium results in constitutive activation of a key transcription factor, nuclear factor κB, with concomitant production of proinflammatory cytokines such as interleukin-8 and tumor necrosis factor (TNF)-α.²  CF patients suffer from chronic bacterial infections, with large number of infiltrating neutrophils that are unable to eliminate the bacteria from the infected airways and may also contribute to tissue damage. Significant progress has been made in the understanding of the pathogenesis of CF and the interplay between the resident epithelial cells and macrophages and the recruited neutrophils. However, studies linking the dysfunction of the CFTR chloride channel with the regulation of the innate immune response, in particular with the modulation of neutrophil functions, are sparse. The study by Pohl et al sheds new insight into this gap of knowledge, demonstrating that TNF-α− or formyl-methionyl-leucyl phenylalanine-stimulated neutrophils from CF patients carrying the class II (ΔF508/ΔF508) or class III (ΔF508/G551D) mutations showed a significant decrease of lactoferrin and matrix metalloproteinase (MMP)-9 release and disturbed ion homeostasis compared with healthy controls or non-CF patients with bronchiectasis. Both the impaired neutrophil granule exocytosis and changes in cytosolic ion concentrations were attributed to dysfunction of the CFTR channel because similar results were observed when cells from healthy controls were treated with a CFTR pharmacologic inhibitor.¹ 

Sequential exocytosis of the 4 granule subsets is postulated to contribute to sequential neutrophil activation during an inflammatory response, through release of granule contents and incorporation of proteins into the plasma membrane. Furthermore, it has been reported that granule exocytosis prepares neutrophils for enhanced phagocytosis and killing of bacteria and that it contributes to TNF-α−mediated priming of neutrophil respiratory burst response.³  It has been shown that the small GTPase, Rab27a, is a fundamental player in the regulation of neutrophil granule exocytosis.^4,5  Neutrophils from Rab27a knockout mice or its effectors showed impaired extracellular reactive oxygen species production associated with deficient recruitment of granules to the plasma membrane.^4,5  Furthermore, Zhou et al showed that the CFTR channel is recruited to neutrophil phagosomes and is essential for the production of hypochlorous acid (HOCl) and an effective antimicrobial activity and that recruitment to the phagosome was impaired in the ΔF508-CFTR.⁶  Hence, the enhance susceptibility of CF patients to microbial infections might be related to the dysfunction of CFTR channel in neutrophils that results in deactivation of Rab27a impairing granule exocytosis and inefficient antimicrobial activity.¹ 

Another major contribution by Pohl et al relates to the correction observed in neutrophil granule exocytosis, Rab27a activation, and restored magnesium levels in CF patients who were heterozygous for the G551D mutation (G551D/ΔF508) that were receiving ivacaftor, a pharmacologic potentiator of CFTR channel, treatment. In a series of well-design studies, Pohl et al show that the defective extracellular release of granule components from CF neutrophils resulted in impaired microbial killing of Pseudomona aeruginosa, one of the key pathogens associated with CF. The percent of P aeruginosa survival when the bacteria were exposed to the supernatant collected from TNF-α−stimulated neutrophils from CF patients with the ΔF508/ΔF508 or G551D/ΔF508 genotypes was significantly higher than when the bacteria were exposed to the supernatants collected from stimulated cells from healthy controls. However, degranulated proteins present in the supernatant from TNF-α−stimulated neutrophils from CF patients that were under ivacaftor treatment showed similar effective antimicrobial activity as when the bacteria were exposed to supernatants collected from TNF-α−stimulated cells from healthy donors. These data highlight the importance of exocytosis, and in particular Rab27a function, in the process of extracellular bacterial killing by neutrophils.

Both the lung epithelium and the immune cells contribute to effective clearance of bacterial infection in CF patients. It has been shown that ivacaftor treatment improves lung function in CF patients compared with placebo.⁷  However, this study provides some critical novel evidence of how mutations of the CFTR channel affects neutrophils key functional responses compromising their antimicrobial capacity and the molecular mechanisms of how ivacaftor might correct the CFTR channel abnormalities and benefit patients with CF.