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the immune system can launch an appropriate response. This is achieved using a broad range of Toll-like receptors (TLRs) to sense pathogenic molecules associated with bacteria. For example, LPS is detected by TLR4, and flagellin by TLR5. In CF, dysfunction in these systems contributes to airway pathology via the combination of TLR hyper-responsiveness and an abundance of TLR agonists that are not

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competently removed by mucociliary clearance (15). The manner in which airway dysfunction leads to TLR overstimulation can be illustrated by the case of the protease neutrophil elastase (NE). This enzyme is released in large quantities by CF neutrophils (30),

activates TLR4 and causes exaggerated release of the cytokine interleukin-8 (IL-8) (31). IL-8 itself is a potent chemoattractant for NE- producing neutrophils, leading to an amplified immune response and positive feedback loop of immune system stimulation (13). Other components of the CF lung milieu have similar effects, for example haem, released into the lung from microbleeds resulting from tissue damage, also activates the TLR system in a similar manner (32). Sex also has a role in the immune response to infection, the prognoses for female CF patients are worse than that in males. The cause for the

discrepancy is thought to involve the female hormone 17β-estradiol.

Although its role in vivo is complex, this hormone may have a net pro- inflammatory effect in CF. Exogenous application of the hormone in

male CFTR-/-mice has been shown to exacerbate inflammation in P.

aeruginosa infection and lead to increased lung bacterial burden (33). The hormone also contributes to the sexual dichotomy of the disease by promoting the early conversion of P. aeruginosa to a mucoid phenotype. It has been observed that the median age of chronic infection with this phenotype is 1.7 years earlier in females and that the frequency of infective exacerbations is increased (34).

The constant activation of innate immune receptors by pathogens (and host molecules) leads to a dysregulated overproduction of cytokines

such as IL-6 and tumour necrosis factor-α (TNF-α). The resulting pro-

inflammatory cascade then recruits neutrophils from the bloodstream to combat the inflammatory insult. In the healthy lung these immune cells are vital for killing bacteria, using three mechanisms:

phagocytosis, granule release of agents such as NE, and neutrophil extra-cellular trap formation (13). This component of the immune response is also dysregulated in CF. Numerous studies have

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normal and CF neutrophils that significantly contribute to lung damage rather than ameliorate it. For example, phagocytosis by neutrophils has been shown to be impaired in paediatric CF patients compared to controls (35). CFTR is present in phagolysosomes of neutrophils, and

its defective activity leads to lower Cl- in the compartments, which is

required for their activity against P. aeruginosa (36). In a neutrophil of a healthy patient, a phagocytosed foreign particle triggers the

formation of O2- by the enzyme NADPH oxidase, which quickly forms

H2O2. Myeloperoxidase, another enzyme, can then catalyse

halogenation (and death) of the foreign particle by using H2O2, Cl- and

the correct pH conditions to form hypochlorite. This is called the

respiratory burst and can cause oxygen consumption by neutrophils to

increase 100-fold (37). Inappropriate O2- production by CF neutrophils

has also been linked to tissue damage. One study observed that after

an exacerbation, CF neutrophils release larger quantities of O2-

compared to healthycontrols. The spontaneous release of NE was

also elevated (38). In a similar manner, and despite the fact that the NE content of the granules of CF neutrophils is not increased, an elevated elastase response to bacterial stimulus compared to normal neutrophils has been noted with CF neutrophils after pre-incubation with bronchoalveolar lavage (BAL) fluid. This indicates that CF neutrophils intrinsically release more NE in response to bacterial stimulus (30).

Neutrophil chemotaxis (movement in response to a chemical stimulus) in CF infection has been demonstrated to differ in comparison to healthy controls, displaying a degree of hypo-responsiveness to IL-8. This may illustrate receptor desensitisation as a result of persistently high levels of IL-8 present in the lung. However, as noted in a review of CF neutrophil dysfunction, the high levels of IL-8 in CF ensures that recruitment is not reduced in vivo and in the absence of infection, CF neutrophils can out-migrate their non-CF counterparts (37).

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1.2.1 Sequelae of neutrophil dysfunction in CF

While they are essential to clearing infection, the dysfunctional action of neutrophils in CF leads to tissue damage and remodelling (13). The neutrophil-dominated response to P. aeruginosa leads to the release of large quantities of NE which, apart from its ability to stimulate IL-8 release as detailed above, causes extensive tissue damage (39, 40). Thus, the exaggerated inflammatory response leads to long-term reduction in lung function and is associated with premature death (17, 19, 20). Neutrophils represent approximately 70% of the airway

inflammatory cell population in CF, in contrast to 1% in healthy

patients (41). This is mostly related to elevated neutrophil chemokine levels in the lung as a result of the ineffective clearance of P.

aeruginosa (39). The lungs of the majority of CF patients appear normal at birth, however chronic neutrophilic inflammation begins early, with one study finding that neutrophils represented on average 31.5% of immune cells for a group of 1 to 5 year old children (42). High NE levels overwhelm epithelial antiprotease defences, which normally protect against proteolytic damage, and can inactivate other components of the immune response, such as complement and immunoglobulins (41). The enzyme may further contribute to impaired bacterial clearance by inhibiting the receptors that mediate pathogen recognition such as CD16 (37). It can also cleave flagellin,

compromising the host’s ability to recognise and react to the molecule (43).

Dysfunctional neutrophil death is a major contributor to the lung

disease of CF. The necrosis of the large number of neutrophils results in the release of extra-cellular DNA (eDNA), further increasing mucous viscosity (19). When neutrophils, which normally have a short lifespan, are inhibited in the normal process of controlled apoptosis, tissue damage can be increased by the uncontrolled release of pro-

inflammatory substances. Host and bacterial mediators such as LL-37 (44) and LPS (45) have been shown to delay apoptosis, an effect which may help clear infection in healthy lungs but has the potential to

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exacerbate lung disease in CF. To resolve inflammation properly, neutrophils must first apoptose and then be cleared to prevent the above effects. Many of the conditions of the CF lung confound this process. For example, pyocyanin, a metabolite produced by P. aeruginosa, has been shown to impair the engulfment of apoptotic neutrophils by macrophages, leading to a large increase in necrotic neutrophils in an in vivo mouse model (46).

In a healthy lung, a balance exists between proteases and anti-

proteases, such as secretory leukoprotease inhibitor (SLPI), α1-

antitrypsin (A1AT), and elafin. This facilitates tight regulation of protease activities and is required to avoid the lung damage and immune dysfunction detailed above (42). Despite their potential for damage in CF, proteases have an important protective role in a

healthy lung and this can be illustrated by the effects of their absence. S. aureus and C. albicans have both been shown to be markedly more virulent in mice lacking neutrophil-granule proteases (47). In another study, an NE-knockout mouse model was found to be more

susceptible to Klebsiella pneumoniae and Escherichia coli (E. coli) infection than wildtype mice, confirming that NE is crucial for the antibacterial action of neutrophils (48).

In CF the protease-antiprotease balance is dysregulated and proteases produced from the neutrophil-dominated epithelial inflammation can overwhelm the inhibitory defences (42). Unlike in many other lung diseases, the issue is not one of antiprotease deficiency but more of enzyme excess. High neutrophils numbers mean that large quantities of unopposed proteases are released into the ASL (41). This leads to lung matrix destruction and inflammation (49). There are several proteases from different sources, both host and bacterial, including serine, aspartyl, and metallo-proteases that are relevant to CF (32).

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