**2. The debate on CFTR expression by neutrophils**

the pancreas, liver, and intestinal tract, but the most severe complications often occur in the lung. CF related symptoms, although variable among patients, are typically present early in life and increase in severity with age despite aggressive therapeutic intervention. CFTR absence or malfunction causes defective ion transport across the epithelium, reduction in pericillary liquid volume, and persistent mucus hypersecretion. As a consequence, mucus accumulates on the airway surface and leads to chronic bacterial infection, exacerbated airway inflammation, and lung injury. Pulmonary inflammation in the CF setting has been observed as early as infancy and there is evidence of structural lung disease present in children with CF as young as 10 weeks old, with 50%–70% of children presenting with bronchiectasis by 3–5 years [2-6]. Additional studies have shown neutrophil-dominated airway inflammation in children with CF [7, 8], and with increasing age elevated levels of proinflammatory mediators including interleukin (IL)-8 [9], tumour necrosis factor-alpha (TNF-alpha) [10], and leukotriene B4 (LTB4) [11] serve to escalate the chronic neutrophil presence in the pulmonary circulation. Indeed, key studies have demonstrated that neutrophil-released granule proteins, particularly neutrophil elastase (NE), play a crucial pathological role (Figure 1) [12, 13]. In the healthy lung, neutrophils represent approximately 1% of the inflammatory cells; however, they account for 60–70% of the total cell count in CF bronchial lavage fluid [14, 15] and ~25% of CF children

250 Cystic Fibrosis in the Light of New Research

were positive for free NE activity in airway samples at 3 months old [16].

**Figure 1.** The potential effects of active NE in CF. Neutrophil elastase (NE) has the potential to cleave structural mole‐ cules and antiproteases causing an anti-protease imbalance and also activate enzymes and receptors that play a crucial pathological role in CF. Abbreviations used: NE, neutrophil elastase; SLPI, secretory leukocyte protease inhibitor; TIMPs, tissue inhibitors of metalloproteinases; MMP, metalloproteinase; TACE, tumour necrosis factor-alpha-convert‐ ing enzyme; TLRs, toll-like receptors; EGFR, epidermal growth factor receptor; PAR, protease activated receptors.

The year 2015 marks the 26th anniversary of the identification and isolation of the *CFTR* gene from epithelial cells [23]. This initial discovery was vital in the development of CFTR-targeted pharmacotherapy and resulted in an explosion of research aimed at identifying the expression of *CFTR* mRNA transcripts and protein levels in epithelial cells and non-epithelial cells alike. Although CF is a pleiotropic disease that affects multiple organs, disease progression is most pronounced in the airways and therefore researchers initially studied the expression of *CFTR* in epithelial cells found within the lungs. In this regard, Yoshimura and colleagues (1991) demonstrated the expression of *CFTR* mRNA transcripts in human bronchial epithelial cells [24], and Trapnall *et al*. demonstrated equal expression of *CFTR* mRNA transcripts from isolated respiratory tract epithelial cells from healthy controls and homozygous and hetero‐ zygous *∆F508* patients with CF [25]. The level of research at this stage indicated that the expression of the *CFTR* was epithelial cell specific, yet the conclusions drawn from a further study by Yoshimura suggested that the *CFTR*-gene had characteristics of a house-keeping gene, suggesting that it may be present in other cell types [26]. This latter manuscript prompted researchers to examine *CFTR* mRNA transcripts in other cell types and in late 1991, Yoshimura demonstrated the expression of low levels of *CFTR* mRNA transcripts in non-epithelial cells, including T-lymphocytes, neutrophils, monocytes, and alveolar macrophages, all of human origin [26]. This was the first record of *CFTR* gene expression in a multitude of immune cell types, and the authors suggested an important role for CFTR Cl- transport. In agreement, it was later demonstrated that alveolar macrophages from *CFTR* deficient mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria due to impaired acidification of the phagosome. Of interest, in this later study CFTR protein was not detected in either murine or human neutrophils [27] and further studies have shown CFTR independent phagosomal acidification in macrophages [28, 29]. Nevertheless, the expression of *CFTR* mRNA transcripts in human macrophages was con‐ firmed by Del Porto, who published that the bactericidal capabilities of macrophages was CFTR dependent, indicating an important functional role for the CFTR protein in these immune cells [30].

Up to this point, no connection between abnormal neutrophil function and the expression of CFTR protein in human neutrophils had been made, and whether or not neutrophils express functional CFTR was still the topic of great debate among leading scientists and clinicians [5, 8, 13, 14], with relevance for the development of CFTR-targeting pharmacotherapy. This is emanating from worldwide laboratories equally committed to the pursuit of knowledge on the cause for impaired neutrophil activity in CF and also the potential consequence of the loss of functional CFTR Cl- channels. Indeed, there is still great controversy as to the true nature of dysregulated neutrophil activity in CF and, for example, in 2010, McKeon and colleagues could only detect low levels of *CFTR* mRNA transcripts in human neutrophils by either reverse transcriptase polymerase chain reaction (RT-PCR) or real time PCR methods of amplification [31]. The authors could not detect CFTR protein expression in membrane or cytosolic fractions, or cell lysates from human neutrophils by Western blot analysis, suggesting that human neutrophils do not express CFTR protein and that the dysregulated neutrophil function in CF was due to the inflammatory status of the individual [31]. Similarly, a study by Morris *et al.,* (2005) who investigated altered phagocytosis of neutrophils in CF due to cell priming, did not detect CFTR protein in human neutrophils by Western blot analysis [32].

Over the years, a number of reasons for not detecting CFTR protein in neutrophil cell fractions by Western blot analysis have been put forward and include the susceptibility of CFTR protein to degradation, a lack of reliable anti-CFTR antibodies, and also boiling of cell fractions prior to electrophoresis. As these issues were addressed researchers have detected CFTR protein in human neutrophils and have established functional roles for membrane associated CFTR. In line with this concept, in 2006, Painter and colleagues demonstrated the presence of CFTR protein in human neutrophil membrane phagolysosomes by confocal microscopy, and also verified the expression of *CFTR* at a mRNA level, and at a protein level by Western blot analysis [33]. Interestingly, in this same study results demonstrated the expression of CFTR protein by Western blot analysis in a human myeloid cell line (HL-60 cells) differentiated into neutrophillike cells [33]. This is in contrast to the study by Yoshimura *et al*., who could not detect *CFTR* mRNA transcripts in HL-60 cells [24]. Further studies have identified *CFTR* mRNA transcripts in differentiated HL-60 cells and demonstrated CFTR protein localisation to the phagocytic vacuole, strengthening the similarities between HL-60 cells and human neutrophils [34]. Furthermore, expression of CFTR protein was confirmed by Pohl *et al*. (2014) in healthy control neutrophils and levels were severely impaired in neutrophils isolated from people with CF carrying the *∆F508* or *G551D* mutation. By flow cytometry, this latter study also demonstrated that the percentage of CFTR positive cells significantly increased post phorbol ester stimulation [35], indicating upregulation of CFTR to the plasma membrane post activation. Interestingly, bacterial lipopolysaccharide (LPS)-stimulated murine neutrophils also demonstrated in‐ creased CFTR protein expression when compared to unstimulated cells, suggesting a role for CFTR in bacterial mediated neutrophil cell activation [36].
