**4. Disabling neutrophils and other immune cells in CF airways**

Excess neutrophil recruitment to the lungs results in the discharge of their destructive weapons not only directed to kill pathogens (see Section 2) but also to damage the lung and airway tissue. A large number of mediators produced by neutrophils, mainly oxidants and proteases, escape from neutrophils during cell death and phagocytosis. NE, a serine protease capable of digesting several substrates including structural proteins, is a direct mediator degrading elastin, which drives towards bronchiectasis and bronchomalacia [18]. Importantly, NE is associated with lung function decline [107]. In the lung, the main protease inhibitors, the prototypical α1‐antitrypsin (α1‐AT) secreted by hepatocytes and secretory leukoprotease inhibitor (SLPI) produced by the respiratory epithelium in bronchi and bronchioles, are designed to oppose free proteases and prevent their deleterious effects. These protease inhibitors are eventually overwhelmed by the protease burden in the lung and degraded by bacterial and human NE. It has been documented that despite normal antigenic concentrations of α1‐ AT and SLPI in children with CF, the majority of α1‐AT and SLPI were complexed and/or degraded [108]. In addition, CF airways are exposed to ROS (O2 , H2 O2 , HOCl), derived mainly from the host's immune response. This oxidative stress exacerbates pulmonary deterioration and advances bronchiectasis in patients with CF [109]. Similar to the protease/antiprotease balance, antioxidants produced by airway epithelial cells (reduced glutathione [GSH] and thiocyanate [SCN− ]) are overwhelmed by the burden of oxidants in the CF airways. Activated neutrophils are also capable of oxidizing glutathione by HOCl [110], contributing to GSH deficiency in CF airways. Hypochlorous acid is also able to oxidize calprotectin thereby inhibiting its ability to sequester manganese and zinc ions and consequently to limiting the growth of *S. aureus* and *P. aeruginosa* [111]. Moreover, it has been documented that ROS suppresses CFTR function [112] and that NE degrades CFTR [113], further worsening the CF pathophysiologic vicious cycle.

wild‐type mice reconstituted with wild‐type neutrophils [98]. Altogether, these data strongly indicate that the lack of functional CFTR could result in excessive NF‐κB activation in neutro-

CF neutrophils have a reduced phagocytic activity [19, 99] and defects in the respiratory burst, attributed to disrupted chloride transport to the phagolysosome [65, 100–102]. While wild‐type CFTR is transported to neutrophil phagosomes, the F508del protein is not targeted efficiently to these organelles [64], explaining why a correct chlorination of phagosomes in CF does not occur and hence the bactericidal defect. A still debated question is, however, the CFTR expression in neutrophils. Morris and colleagues, although found a defect in iC3b‐ mediated phagocytosis, did not detect CFTR in circulating and airway neutrophils by either immuno‐labelling or a Western blot [99]. Others found that CFTR expression was limited or undetectable in neutrophils by flow cytometry and also that no role for CFTR in neutrophil‐ mediated phagocytosis was observed [103]. On the other hand, Zhou and colleagues found CFTR at the phagosome level, although a lentiviral‐expressing system was used to achieve high protein levels. It might be that CFTR, expressed in hematopoietic stem/progenitor cells [104, 105], is down‐regulated to low levels during neutrophil maturation, which is nevertheless sufficient for neutrophil phagocytic and killing activities. The lack/dysfunction of CFTR in the bone marrow may lead to an irreversible functional defect. In this context, it is worth mentioning that knocking out CFTR in the myeloid compartment of mice resulted in poor survival, increased inflammation with recruitment of neutrophils, elevated cytokine production, and inability to resolve infection upon challenge with *P. aeruginosa*‐loaded agarose beads

phils and therefore propagate a hyper‐inflammatory response.

128 Role of Neutrophils in Disease Pathogenesis

to mimic a chronic pulmonary infection [106].

**4. Disabling neutrophils and other immune cells in CF airways**

degraded [108]. In addition, CF airways are exposed to ROS (O2

thiocyanate [SCN−

Excess neutrophil recruitment to the lungs results in the discharge of their destructive weapons not only directed to kill pathogens (see Section 2) but also to damage the lung and airway tissue. A large number of mediators produced by neutrophils, mainly oxidants and proteases, escape from neutrophils during cell death and phagocytosis. NE, a serine protease capable of digesting several substrates including structural proteins, is a direct mediator degrading elastin, which drives towards bronchiectasis and bronchomalacia [18]. Importantly, NE is associated with lung function decline [107]. In the lung, the main protease inhibitors, the prototypical α1‐antitrypsin (α1‐AT) secreted by hepatocytes and secretory leukoprotease inhibitor (SLPI) produced by the respiratory epithelium in bronchi and bronchioles, are designed to oppose free proteases and prevent their deleterious effects. These protease inhibitors are eventually overwhelmed by the protease burden in the lung and degraded by bacterial and human NE. It has been documented that despite normal antigenic concentrations of α1‐ AT and SLPI in children with CF, the majority of α1‐AT and SLPI were complexed and/or

from the host's immune response. This oxidative stress exacerbates pulmonary deterioration and advances bronchiectasis in patients with CF [109]. Similar to the protease/antiprotease balance, antioxidants produced by airway epithelial cells (reduced glutathione [GSH] and

]) are overwhelmed by the burden of oxidants in the CF airways. Activated

, H2 O2

, HOCl), derived mainly

An important role in the degradation of structural proteins in CF airways is played synergistically by serine proteases, such as NE, proteinase 3, and cathepsin G [114]. In cystic fibrosis, neutrophil activation and degranulation result in the excessive release of proteinase 3, cathepsin G, and NE into the extracellular medium as active enzymes. Part of these serine proteases are exposed at the cell surface of immune cells and are important as modulators of the inflammatory response. Proteinase 3 has been shown to convert IL‐8 to more potent, amino‐terminally truncated forms [115], indicating that neutrophil proteases released in the inflamed lung convert IL‐8 to enhance its chemotactic activity. Besides serine proteases, neutrophil‐derived metalloproteinases, including MMP‐8 and MMP‐9, have also been involved in CF lung disease and chronic neutrophilic inflammation [116]. NE contributes to MMP‐9 activation early in CF disease as the ratio of active/pro‐enzyme MMP‐9 was found to be higher in the presence of free neutrophil elastase activity, but not infection, and active MMP‐9 was associated with progression of bronchiectasis [117]. In the context of CF, it is important to recall that neutrophil proteases increase mucin secretion in the airways and reduce ciliary beat frequency, contributing to the impairment in mucociliary clearance [118, 119], induce airway epithelial cells to produce neutrophil chemoattractants [120], and activate the apical epithelial sodium channel ENaC [121].

Unopposed serine proteases and metalloproteinases are responsible for degradation of soluble pattern recognition receptors (PRRs). NE proteolytic activity present in the CF sputum has been shown to degrade the prototypic long pentraxin PTX3, explaining the low levels of this PRR in CF airway secretions [122]. Released cathepsin G upon neutrophil activation degrades both components of the extracellular matrix and the surfactant protein A, a peptide that facilitates bacterial clearance by alveolar macrophages [123]. MMP‐9 cleaves the pulmonary collectin surfactant protein D (SP‐D) more efficiently than NE; this cleavage causes SP‐D to no longer be able to agglutinate bacteria and affects SP‐D's innate immune functions, as bacteria are no longer efficiently phagocytosed by alveolar macrophages in vitro [124].

High levels of neutrophil proteases further worsen the immune response by disabling immune cell functions. NE has several potential roles in disabling neutrophils including cleavage of opsonophagocytosis proteins, such as iC3b, complement receptor 1 (CR1) and C5a receptor [125–127], the chemokine receptor CXR1 [128], and TIM3 receptor leading to decreased galectin‐9/TIM3 interactions [129]. Overall, the loss of these proteins is responsible for suboptimal local neutrophil priming and bacterial clearance. PMN‐derived cathepsin G also thwarts efficient phagocytosis by macrophages, resulting in the cleavage of receptors and causing inefficient opsonization and impaired bacterial killing [18]. Cathepsin G cleavage of serum amyloid P component (SAP) renders it anti‐opsonic, as evidenced by the increased binding of SAP to *P. aeruginosa* LPS and inhibition of phagocytosis in vitro [130], thus sequestering bacteria within the lung and potentially contributing to persistent infections in CF. Cathepsin G also interferes with removal of neutrophilic apoptotic bodies, since it mediates the degradation of the macrophage phosphatidylserine receptors with failure to resolve inflammation because of the lack of efferocytosis [62, 131]. Also, NK cells and lymphocytes are disabled by neutrophil serine proteases. Cathepsin G determines a proteolytic cleavage of NKp46, a crucial activating receptor expressed on NK cells, an effect also determined by the CF sputum [132]. NE cleaves T‐cell receptors CD2, CD4, CD8, and CD14, impairing monocyte activation and also blocking dendritic cell maturation and antigen presentation [133, 134].
