**6. The effect of CFTR mutation targeted therapy on neutrophil function**

There can be no doubt that our grasp and understanding of the complex physiology of CFTR protein function has progressed majorly and permitted the development of exciting new treatments designed to target the basic defects of CF. Along with this, continuing improve‐ ments in clinical care have allowed improved outcomes for patients with CF including reduced morbidity and preserved lung function. As CFTR has been shown to be an integral membrane channel on neutrophils, the influence of new CFTR therapeutics on neutrophil function is an area of intense interest.

Class I mutations are thought to affect 5% of the CF population in Western society and in this class of *CFTR* mutations, there is complete absence of stable CFTR protein (due to non-sense mutations from premature stop codons) and thus replacement of the defective *CFTR* gene or changes in how the protein is made is required. However, the focus relating to the research surrounding gene addition therapy is its potential use in all class mutations in CF, with the ultimate aim of functional gene insertion and normalized *CFTR* expression. Studies on gene addition therapy have been developed to replace the mutant *CFTR* gene, and in this respect a phase II trial with non-viral lipid vector for DNA instillation has commenced. The study investigators have recommended monthly inhaled therapy for one-year duration (NCT 01621867). Further developments involve a lentiviral vector for gene therapy in this patient population [147] and the collective results of gene therapy on neutrophil function will be an exciting area of research in the future.

A second area of immense interest is the therapeutic use of aminoglycosides (e.g., gentamycin) and ataluren to cause read through of premature stop codons thereby allowing translation to continue to the end of transcription [148, 149]. Ataluren (PTC 124) is under investigation for its use and role in targeting premature stop codons. This compound has the potential to allow processing of premature stop codons, resulting in the production of normal length and functional CFTR, with insertion at the cell surface. Its beneficial effects have been proven with analysis of nasal chloride transport. The phase III trial in 238 patients with CF failed to achieve its primary end point (improvement in FEV1) at 48 weeks, except in a small subgroup of patients not on concomitant nebulised aminoglycoside treatment [150].

reactive oxygen species (ROS) is very specific to neutrophil apoptosis. The observation that neutrophils isolated from CGD patients which are known to be NADPH oxidase defective display a significant delayed spontaneous cell death relative to that of neutrophils from healthy donors is important [140]. This suggests that activation of the NADPH oxidase, with conse‐ quent production of ROS, is involved in spontaneous apoptosis and in regulating the pro‐

**Figure 5. Summary schematic of apoptosis pathways in the neutrophil.** [1] Intrinsic mitochondrial pathway (red): mito‐ chondria release cytochrome c (Cyt C) in response to cellular stress. Together with apoptotic protease activating factor-1 (Apaf-1) and procaspase-9, Cyt C will form the apoptosome complex. This results in the proteolytic activation of the pro‐ caspase. Mature caspase-9 can then proteolytically activate caspase-3. [2] Extrinsic/ligand death-receptor pathway (black): death factors such as tumour necrosis factor alpha (TNF-alpha) and Fas ligand (FasL) trigger apoptosis by binding on death receptors such as tumour necrosis factor receptor 1 (TNFR1) and Fas. The death receptors recruit procaspase-8 by means of an adaptor protein, TNF-R1-associated death domain (TRADD), or Fas associated death domain protein (FADD). After cleavage the mature caspase-8 can then directly activate caspase-3. [3] Endoplasmic reticulum (ER) stress pathway (yellow bolt): the ER can also induce apoptosis as a reaction to ER stress by releasing intracytosolic calcium (Ca2+) there‐ by activating effector caspases or through the expression of pro-apoptotic transcription factors such as CHOP. These dif‐ ferent initiation pathways converge further downstream into activation of caspase-3. The effector caspase-3 cleaves ICAD (inhibitor of CAD) and releases it from CAD (caspase-activated DNase). CAD translocates from the cytoplasm to the nu‐ cleus and can now act as active endonuclease and fragment DNA. Inhibitors of apoptosis such as myeloid leukaemia cell differentiation protein (Mcl-1) and X-linked inhibitor of apoptosis (XIAP) act upstream against pro-apoptotic Bcl-2 fami‐

grammed cell death of neutrophils during phagocytosis.

262 Cystic Fibrosis in the Light of New Research

ly members at the mitochondria and downstream, directly inhibit caspases, respectively.

Class II (e.g., *∆F508*) and III *CFTR* mutations (e.g., *G551D*) have seen the advent of CFTR corrector and potentiator therapies. Corrector agents facilitate appropriate protein folding and targeting to the cell membrane. The correctors lumacaftor (VX 809) or VX 661 are designed to promote increased quantities of ΔF508 CFTR at the cell membrane surface. VX 809 has shown good results *in vitro* but it is likely that adequate correction of ΔF508 CFTR will need multicompound drug therapy [147, 151]. The potentiator ivacaftor (VX 770) was originally devel‐ oped to augment the activity and efficiency of the abnormal CFTR protein (Figure 6).

**Figure 6.** CFTR function in human epithelia. Panel (A) illustrates defective CFTR Cl- transport due to the *G551D* muta‐ tion. Image (B) illustrates corrected function post VX-770 (ivacaftor) treatment.

Use of this therapy involved a well-designed randomized, double blind, placebo-controlled trial. The study subjects had at least one *G551D CFTR* mutation and were randomly assigned to ivacaftor 150 mg twice daily or placebo for 48 weeks. The treatment group showed a sustained improvement from baseline in FEV1 by 10% compared with the placebo group. They were also 55% less likely to suffer a pulmonary exacerbation compared with their placebo counter parts, had higher health scores, gained weight and normalization of their sweat chloride levels. These benefits were sustained for the duration of the trial and the frequency of adverse events in the two groups was equivocal [152]. A shortcoming of the use of ivacaftor is that only 2–3% of individuals with CF have the *G551D* mutation and research is currently underway to ascertain whether ivacaftor may be employed for other mutations. A multicentre, phase II trial on the use of the CFTR corrector lumacaftor together with the CFTR potentiator ivacaftor for the treatment of patients with CF with the *ΔF508 CFTR* mutation was performed. The primary outcome was change in sweat chloride concentration with combina‐ tion therapy in the *∆F508* subjects, however a minimal effect on sweat chloride level was observed [153]. It is postulated that the reason behind this was that the potentiator rendered the CFTR protein less stable and increased its removal from the cell membrane. However, currently there is a very motivated area of research into double or triple combination treatment for synergistic ΔF508 CFTR correction [154].

Of relevance to the circulating neutrophil and as *CFTR* mRNA transcripts have been reported in neutrophils at an expression level similar to those found in monocytes and alveolar macrophages [24, 33], the effect of CFTR corrector and potentiator therapies on neutrophil function is an anticipated area of research. In a study by Pohl *et al*. (2014), the mechanism leading to impaired degranulation by CF neutrophils was shown to involve altered ion homeostasis caused by defective CFTR function and significantly decreased levels of GTPbound Rab27a. Of major importance, treatment of *G551D* patients with ivacaftor, normalized neutrophil cytosolic ion levels, and activation of Rab27a thereby leading to increased degra‐ nulation and pseudomonal killing. These results confirm that intrinsic alterations of circulating neutrophils from patients with CF are corrected by ivacaftor thus illustrating additional clinical benefits for CFTR modulator therapy. In line with this concept, neutrophils of ivacaftor-treated subjects demonstrated decreased cell surface CD63, a marker of primary granule release [155].
