Abstract
Current paradigms of chronic obstructive pulmonary disease (COPD) treatment suggest stratifying patients by their symptoms, utilising three main drug classes, but it is unclear if this approach will substantially alter the progression of the disease in the long term. More treatment options are needed which target the underlying pathology of the condition. Whilst many inflammatory cells are implicated in COPD, the neutrophil is by far the most abundant and has been extensively associated with disease pathogenesis. Neutrophil products are thought to be key mediators of inflammatory changes in the airways of COPD patients, causing pathological features such as emphysema and hypersecretion of mucus. High rates of bacterial colonisation and recurrent infective exacerbations of COPD, as well as evidence of neutrophil-associated host damage suggest that neutrophil functions may be impaired in COPD. This concept is supported by studies demonstrating impaired migratory accuracy and increased degranulation and reactive oxygen species release, with some evidence of altered cellular signalling pathways which might be exploitable as therapeutic targets. This review discusses our evolving understanding of neutrophil function in both health and COPD and highlights the role of this cell in disease pathogenesis, to determine whether this key inflammatory mediator represents a viable therapeutic target to prevent disease progression.
Introduction
Chronic obstructive pulmonary disease (COPD) remains a significant global health challenge. It has not seen the same improvements in morbidity and mortality as many other chronic inflammatory diseases with only one novel drug class reaching the market in the past 20 years (Citation1). After 25 years of smoking approximately 30–40% of adults would have developed COPD (Citation2) but despite most patients having this shared risk factor, COPD is heterogeneous in presentation, the age of onset and speed of decline. Whilst the disease is defined by the presence of airflow obstruction, there are a number of recognised clinical phenotypes. These include a predominance of emphysema (Citation3), obstructive bronchiolitis (Citation3, Citation4), the presence or absence of chronic bronchitis, frequent exacerbations (Citation5) and a faster decline in lung function (Citation6), and patients exhibit a spectrum of these features. Clinical phenotypes are stable within individual patients (Citation6) and cluster within families (Citation7) and thus are likely to reflect genetic traits. Indeed, genome wide association studies in COPD have identified a large number of signals for genes associated with lung development, lung parenchyma formation, and repair and epigenetic regulation such as the inositol phosphate pathway (Citation8, Citation9). These studies suggest there may be a wide range of therapeutic targets in different subsets of COPD patients, providing hope for personalised medicine. However, genes linked to lung development may not be therapeutically targetable in an adult with lung disease, drug development is costly and slow and the burden of COPD is rising fastest in low income countries (Citation10). A more common and shared targetable mechanism across disease phenotypes could provide a treatment for a larger number of patients.
The complex inflammatory milieu in COPD
Most pro-inflammatory mediators and immune cells have been shown to be raised in patients with COPD (Citation11, Citation12) and this inflammation is heightened and self-sustaining in smokers who are susceptible to COPD in contrast to those smokers who are not (Citation13, Citation14). However, while many cells and mediators have been implicated in COPD pathogenesis at some level, few have reliably demonstrated their importance as therapeutic targets in human studies. For example, despite the promising resistance to COPD-like lung damage shown by TNFα receptor (Citation15) or IL-1 receptor 1 knock-out mice (Citation16) studies targeting these individual mediators in unselected cohorts of patients with COPD have been disappointing with TNFα and IL-1 receptor 1 inhibition showing no improvements in disease endpoints (Citation17, Citation18). This might suggest that, unlike murine models, only sub groups of patients will respond to individual mediator-based therapies. In support of this concept, there is variability in inflammatory patterns between patients, even when matched for age, smoking status and disease severity (Citation19) which might reflect specific genetic traits as shown in some studies of TNFα (Citation20) and IL-1β (Citation21) polymorphisms.
To complicate matters further, there is significant intra-patient variability in the concentration of plasma and sputum mediators and cells on a day-to-day basis. Some mediators increase while others decrease suggesting the variability not only reflects dilution but also fluctuations in specific components of the inflammatory load (Citation19, Citation22). This supports an alternative explanation to the negative trial results reported to date, where there is so much compensation and overlap within the complex inflammatory storm that is established COPD (Citation23) that end-cell effects can be driven by an alternative cytokine, should one be abrogated. For example, toll-like receptors, TNFα and IL-1 signalling to NF-κB all converge on a common IκB kinase complex that phosphorylates the NF-κB inhibitory protein IκBα, despite the upstream signalling components being to a large part receptor-specific (Citation24). Despite the effects of these mediators being synergistic when they converge on the same pathway, inhibiting one has not proved efficacious enough to impact robustly on cellular inflammation or COPD disease progression. Potentially targeting the functions of the end-cell and not the intermediatory cytokine might be more effective and there is a strong rationale for targeting the neutrophil in COPD.
Classical neutrophil functions in health
Neutrophils are the most abundant leukocyte, accounting for 70% of all circulating white blood cells. They are short-lived cells (with a half-life of around eight hours), with basal production of 1–2 × 1011 neutrophils/day in health; though this can increase to 1012 during infection (Citation25). Following myeloblastic differentiation in the bone marrow, the mobilisation of terminally differentiated neutrophils into the circulation is tightly controlled by bone marrow signals and circulating growth factors. Interactions between neutrophil CXCR4 and bone marrow stromal cell CXCL12 cause cell retention or cell return to the bone marrow and increasing neutrophil CXCR2 results in neutrophil release into the circulation (Citation26, Citation27).
Neutrophils are characterised by the presence of a multi-lobed nucleus and granular cytoplasm, due to the presence of azurophillic (primary), specific (secondary) and gelatinase (tertiary) granules, as well as secretory vesicles. These granules and vesicles contain a complex and specialised arsenal of proteins which facilitate cell to cell communication and functional modification, neutrophil migration from the systemic circulation through the dense extracellular matrix to areas of inflammation, microbial killing and tissue remodelling, degradation and repair. Some of these proteins are listed in .
Table 1. The contents of human neutrophil granules and secretory vesicles.
In COPD, a significant physiological role of these granular contents is bacterial killing, achieved mainly when bacteria are ingested into phagosomes that fuse with lysosymes containing proteinases, bacteriocidal proteins such as perforin and granzyme B (the two molecules known as the cytotoxic entity of natural killer cells and of cytotoxic T lymphocytes) (Citation28) and reactive oxygen species following the formation of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase from subunits within the cytosol. However, before this can occur, circulating neutrophils must reach the site of infection, and they do so following chemotactic signals in a gradient-dependent manner (Citation29). ATP released from chemoattractant-stimulated neutrophils amplifies the external migratory signal, controlling gradient sensing and migrational speed in an autocrine and paracrine manner, recruiting more neutrophils to the site of inflammation (Citation30). Circulating neutrophils then cross the endothelium via the Leukocyte Adhesion Cascade (LAC). Here, neutrophils interact with endothelial cells at sites of high inflammatory signalling by initially rolling on the endothelial surface by reversibly binding selectins which are found on both the neutrophil and endothelial cells. This transitions to firm adhesion by the sequential activation of integrins, followed by transendothelial migration (Citation31).
Once within inflamed tissue, the neutrophil further utilizes chemotactic gradients created by host-derived inflammatory cytokines (e.g. Interleukin-8) and bacterial products (e.g., lipopolysaccharide [LPS] and N-formyl-methionyl-leucyl-phenylalanine [fMLP]) released from the site of infection to navigate towards invading pathogens (Citation29). Activated neutrophils have a clear “front” and “back” and use this polarised form to amplify external gradients internally within the cell, allowing accurate mobilisation of internal structures (generation of pseudopods for migration, granules for phagolysis) towards the inflammatory or infectious insult. Chemotactic signals such as Leukotriene B4 (LTB4), integrin binding and neutrophil ATP release promotes the formation of neutrophil “swarms” within these damaged tissues (Citation32) sealing off the sites of infection and maximising the clearance of bacteria and cell debris by phagocytosis and netosis (Citation33). During these episodes, neutrophils far outnumber any other immune cells in the inflamed tissue.
Phagocytosis is an active, receptor-dependent process through which a phagocyte internalises material into membrane-bound vacuoles (Citation34). Phagocytosis of opsonised particles (by IgG, for example) occurs in seconds (Citation35), faster than the minutes required for macrophage phagocytosis of bacteria (Citation36). The most extensively studied opsonin receptors of neutrophils, the Fc receptors (e.g. FcγRIIA [CD32], FcγIIIb [CD16]) bind IgG-bound particles triggering a signalling cascade involving the tyrosine kinase Syk and phosphatidylinositol 3-kinase (PI3K). Complement molecules also opsonise pathogens by interacting with neutrophil complement receptors (e.g. CR1 [CD35], CR3 [CD11b/CD18]) (Citation37–40). Once bacteria are enveloped, bacterial killing occurs through the delivery of neutrophil granules and the NADPH oxidase machinery into the vacuole by membrane trafficking (Citation29, Citation41, Citation42). These processes are depicted in .
Figure 1. Phagosome maturation.
The neutrophil NADPH oxidase machinery, activated by delivery of its membrane-bound components to the phagosome, pumps electrons into the phagosomal space to generate toxic ROS. Membrane trafficking allows delivery of primary and secondary granules to the phagosomal membrane, which release a variety of microbicidal proteins into the phagosomal space. MPO released from primary granules reacts with ROS to further produce highly toxic substances. Adapted from (Citation161).
In contrast with the highly acidic pH of macrophage phagolysosomes, the neutrophil phagosome appears to undergo a transient alkalisation, which is optimal for neutrophil protease activity (Citation43). Then, with maturation and acidification of the phagolysosome, Reactive Oxygen Species (ROS) are released into phagosomes with NADPH oxidase acting as a channel for electrons from the cytosol into phagosomal vacuoles, stimulating reduction of oxygen (O2) to the superoxide anion O2 - (Citation44). Superoxide can then dismutate, a process accelerated by the enzyme superoxide dismutase (SOD), to form the highly oxidative hydrogen peroxide (H2O2), which can react further (the MPO-halide-hydrogen peroxide antibacterial system) to form strongly bactericidal hypohalous acids (e.g., HOCl) (Citation45–50). The role of ROS and NADPH oxidase in elimination of pathogens was long-believed to be a dominant killing mechanism in neutrophils, however, the exact role of these processes in neutrophil bacterial killing has come under recent scrutiny with some research supporting their role as facilitatory rather than obligatory (Citation48).
Neutrophils are also able to produce extracellular traps (NETs), consisting of a backbone of uncondensed chromatin to which bactericidal products such as cathepsins, MPO and nuclear histones are bound (Citation51). Microscopic studies suggest NETs are responsible for the killing of a wide range of pathogens, including gram-negative (Citation51) and gram-positive (Citation51, Citation52) bacteria as well as fungi (Citation53). However, NETosis occurs later in neutrophil activation than other killing processes such as phagocytosis or generation of ROS (Citation54) and this appears to be a “final act” for most neutrophils, when faced with overwhelming infection.
The crucial role of neutrophils in clearing infection is amply evidenced by the devastating outcomes seen when neutrophil numbers are reduced or functions are inhibited including during neutropenic sepsis, chronic granulomatous diseases (most commonly associated with abnormalities in cytochrome b558, preventing NADPH oxidase formation (Citation55)); Chediak–Higashi Syndrome (characterised by giant lysosomal granules which lack neutrophil elastase and cathepsin G (Citation56)) and specific granule deficiency, where neutrophil migration is comprised due to failure to upregulate surface receptors upon activation (Citation57); this is more fully reviewed in (Citation58).
The capacity for neutrophil-induced host damage and its mitigation
The same cytotoxic proteins crucial in bacterial killing also have the potential for significant host harm, as exemplified by neutrophil elastase (NE). Each neutrophil contains on average 400 elastase-positive granules (Citation59) and each granule contains approximately 67,000 molecules of NE at a mean concentration of 5.33 mMol (Citation60). Free NE is inhibited by Alpha 1 anti-trypsin (AAT) but on a one-to-one molar basis and the average concentration of AAT in healthy individuals in 30 μMol, resulting in a localised area of uninhibited and thus active NE around the cell following degranulation. Free NE can cause significant damage to host by a number of mechanisms () and degranulation can occur during migration, frustrated phagocytosis or “sloppy eating” and netosis as well as during cellular necrosis. NE is not the only protein implicated in host damage and many other granular products have been implicated including proteinase 3 (Citation61), reactive oxygen species generation (Citation62) and MPO-mediated nitrosative stress (Citation63). In stable COPD, sputum samples can contain up to 10 × 106/ml neutrophils (Citation22); therefore, the potential for lung damage is high.
Table 2. The actions of neutrophil elastase.
To protect the host, neutrophils have evolved a number of safe-guarding mechanisms. Neutrophils exist in three potential states of activation; quiescent, primed or activated, akin to a tri-coloured warning system and primed cells both enable a faster response to full activation but also permit a return to the quiescent state prior to degranulation if full activation is not needed (Citation71). Cellular cross-talk controls aspects of neutrophil activity. For example, during inflammatory events, activated platelets adhere to circulating blood neutrophils causing neutrophil-derived and arachidonic acid-containing extracellular vesicles to be taken up into the platelet in a Mac1-dependent fashion. Within the platelet, arachidonic acid is used to synthesise thromboxane A2 which increases ICAM-1 on endothelial cells, facilitating the movement of neutrophils from the blood stream into the tissues (Citation72). This co-operation between three cell types (platelet, neutrophil and endothelial cell) is thought to prevent unwarranted neutrophil migration into tissues and subsequent tissue damage. Degranulation is not an “all or nothing” response and neutrophils release different vesicles or granules depending on the environmental triggers encountered. Different granule sub-types are mobilised and released dependent on cytosolic-free calcium levels, which allows coordination of granule release to match cellular requirement, e.g. mobilisation of azurophilic granules occurs only once the neutrophil comes into contact with a significant inflammatory stimuli while mobilisation of secretory vesicles occurs during the initial stages of cell polarisation (Citation73).
Changing perceptions of the neutrophil
The classical view of neutrophils is that they are a uniform population of short lived cells and have a narrow range of responses, in contrast to the ever-expanding sub-populations of other cell types. However, this perception is being challenged and there is evidence that neutrophils may be more diverse than first thought. Neutrophils are more transcriptionally active than previously appreciated (Citation74) and their lifespan appears to change depending on activation status and environmental circumstances (Citation75). Furthermore, experimental models have described an increasing number of neutrophil “phenotypes” which have different functional characteristics and appear more distinct than simply being “young” (or freshly released from the bone marrow) or “old” (prior to returning to the marginated pools and apoptosis) cells. Not all neutrophils stay in tissues for clearance by efferocytosis, and a subset have been shown to “reverse transmigrate” back into the systemic circulation (Citation76). Studies have described anti-inflammatory neutrophils that are involved in tumour clearance (Citation77) and there are descriptions of neutrophils which appear to modulate T-cell function via Mac 1 signalling, leading to dampening of the immune response (Citation78), so called “anti-inflammatory” neutrophils. In murine models of ventilator-associated acute lung injury, there appear to be two distinct waves of neutrophil in the airways; the first is the classical neutrophil which has been associated with significant tissue damage (Citation79). The second appears to be a pro-angiogenic neutrophil (Citation80), characterised by increased MMP-9 release (Citation81, Citation82) with a distinctive pattern of surface markers and an association with improved clinical outcomes (Citation83). It remains unclear what the relevance of these cellular phenotypes are in clinical practice, including in COPD.
Neutrophils in COPD
Neutrophils are considered central to the pathogenesis of COPD. Airway neutrophilia is a feature of COPD regardless of the clinical phenotype, severity of disease, rapidity of decline or age of onset. Indeed, even in the subset of patients with eosinophilic COPD, neutrophils remain the predominant cell (Citation84). Their numbers and products in sputum and airway lavage fluid correlate with disease severity, as determined by the degree of airway obstruction, decline in FEV1 or severity of emphysema present (Citation85–88). Histological studies have not always reported the presence of neutrophils within the airway wall (Citation89, Citation90), however, neutrophils are not tissue resident but transitory visitors, migrating through to the airways.
The association between COPD and neutrophils is further evidenced by Alpha-1 Antitrypsin Deficiency (AATD). AATD is the most extensively documented genetic risk factor for COPD, where the reduction of AAT predisposes to increased proteinase-mediated degradation of extracellular matrix and development of emphysema (Citation91). Emphysema occurs even in the absence of smoking in some individuals with AATD and rates of decline in lung function are faster in those who smoke. Furthermore, there is evidence that emphysema progression is slowed by augmentation with replacement Alpha-1 Anti-trypsin (Citation92) although the effect of augmentation on FEV1 decline is less clear (Citation93).
Neutrophil products have been shown to cause all of the pathological features of COPD in animal and cell-based models. As described in and using NE as an exemplar, serine proteases not only degrade all components of the extracellular matrix but also activate matrix metalloproteinases (MMPs) from their inactive pro-forms and inhibit the inhibitors of MMPs, increasing the proteinase burden (Citation94) implicated in COPD (Citation95). Proteinases are potent stimulants of mucus secretion in the airways and reduce mucociliary clearance by impeding ciliary function leading to symptoms of chronic bronchitis (Citation96–98). They also cleave immunoglobulins and components of the complement cascade, potentially causing an opsono-receptor mismatch (Citation64). There is evidence to link neutrophil degranulation with bacterial colonisation. In murine models, neutrophil recruitment to the mucosa has been associated with a neutrophil-elastase-dependent change in the composition of the intestinal microbiota, facilitating colonisation (Citation99). In cystic fibrosis, neutrophil recruitment to the airways is associated with prolonged neutrophil survival, activation and altered functions consistent with a phenotypic change which permits the co-habitation of neutrophils and microbes (Citation100). In COPD, and sputum NETosis correlates with the dominance of Haemophilus species in the lung microbiome (Citation101). In theory, in COPD, this might result in a vicious circle, where inflamed or damaged tissue support bacterial colonisation and subsequent infection, which leads to more inflammation and more neutrophil activation and recruitment to the airways.
In support of this, there is consistent evidence of heightened neutrophil activity in COPD. AαVAL360 is a neutrophil elastase-specific fibrinogen degradation product, thus a footprint of elastase activity, and plasma levels have been shown to be raised in stable COPD. This suggests either systemic activity of neutrophils in COPD or potentially a significant overspill from the lungs into the circulation (Citation102). COPD sputum also contains neutrophil products including human neutrophil lipocalin (HNL) and myeloperoxidase (MPO) (Citation103).
Further evidence of the importance of neutrophils in COPD comes from imaging studies. 18-fluorodeoxyglucose positron emission tomography in COPD patients show enhanced uptake caused by active neutrophils in the emphysematous regions of the lungs and this correlates with measures of disease severity (Citation104). The release of neutrophil proteinases into these damaged lungs will contribute to the degradation of elastin and type III collagen, leading to the destruction of alveolar tissue and consequently centrilobular emphysema, apparent in these imaging studies and in many cases of COPD (Citation105, Citation106). Elegant pathological studies by Hogg et al. (Citation107) suggest small airways dysfunction and destruction may precede the development of emphysema and may reflect the earliest pathological changes in the lungs of patients with COPD. Neutrophils have also been implicated in small airways disease, showing a relationship with neutrophilic infiltration and tomographic measures of air trapping (Citation108).
Cellular dysfunction as a mediator of COPD
Neutrophil responses should be tightly controlled to prevent host damage and the high number of neutrophils present in airway secretions in COPD should mitigate against bacterial colonisation. Despite this, there is tissue damage and recurrent infections are common. There is evidence to suggest that neutrophils from patients with COPD are impaired in function which might explain these injurious effects. COPD neutrophils demonstrate migratory inaccuracy, able to migrate towards chemotactic signals with greater speed, but decreased accuracy than neutrophils from age-matched healthy controls (Citation109, Citation110). This defect was not present in smokers without COPD and has been described in mild to chronic disease, which suggests it is an early development in COPD and might theoretically precede the disease, although this has not been tested. In vitro modelling suggests the migratory defect results in a longer and more convoluted migratory paths, increasing the secretion of damaging enzymes and delaying bacterial killing (Citation109). There is limited data about the phagocytic functions of COPD neutrophils. In some studies, ingestion of opsonised species have been found to be reduced in COPD neutrophils compared to age-matched healthy controls (Citation111, Citation112). However, in other studies, there are no differences between the phagocytic abilities of COPD neutrophils and controls (Citation113, Citation114). This suggests any defect may be stimulant, environment (tissue or blood neutrophil) and assay dependent or only seen in a subset of patients. Flow cytometric studies demonstrate enhanced respiratory burst in neutrophils from patients with COPD as compared to healthy smokers (Citation115) and increased markers of oxidative activity both in COPD airways and systemically suggest increased ROS-producing capacity in stable disease (Citation116). Neutrophils from patients with COPD are also able to degranulate more readily to stimuli and increased quantities of NETs and NET-producing neutrophils are observed in the sputum of both stable and exacerbating COPD patients (Citation117, Citation118). All of these functions favour tissue damage.
It is unclear why neutrophil functions may be altered in COPD. Some other chronic inflammatory conditions report a profound change in neutrophil gene expression profile which might support a phenotypic shift in these cells (Citation83, Citation119) and there is some evidence to support a similar concept in COPD. A recent study identified different neutrophil phenotypes in COPD based on the expression of regulated proteins and hierarchical clustering without clear differences in clinical phenotype (Citation120) but this was cross-sectional and could not assess when neutrophil phenotype was altered. It might reflect differences in genetic traits between patients but also might reflect epigenetic changes within the neutrophil following long-term exposure to inflammation. DNA damage has been seen in COPD due to oxidative stress (Citation121) and in other conditions transcriptional changes have been described in neutrophils and are associated with poor neutrophil migratory accuracy (Citation122, Citation123). Epigenetic changes are long-lived, and the systemic inflammation present in COPD could theoretically affect neutrophil progenitor cells, leading to sustained aberrant functions and subsequent inflammation. In support of this concept, epigenetic changes have been described in bone marrow macrophage progenitor cells in type 2 diabetes (Citation124), which is also a pro-inflammatory disease.
Neutrophil functions during exacerbations of COPD
COPD exacerbations are heterogeneous in their cause (viral, bacterial, environmental, associated with co-morbidities), severity and duration (Citation125) and the majority of studies of inflammation during exacerbations have included small number of patients. However, they have confirmed the presence of neutrophils during these episodes (Citation126). Increased serum levels of damage-associated molecular patterns (DAMPs) have been described during COPD exacerbations and gene expression of DAMP receptors (including toll-like receptor (TLR2 and TLR4) in circulating neutrophils have been shown to be significantly increased during these events supporting neutrophil activation (Citation127). Furthermore, there is evidence of increased neutrophil elastase activity during COPD exacerbations compared with the stable state (Citation128) and spontaneous ROS production in sputum neutrophils is up to 45% higher during exacerbations of COPD compared with stable disease (Citation129). In summary, it appears that the potential for neutrophil-associated tissue damage seen in stable COPD is heightened in exacerbations, with more neutrophils, more activation and more proteinase and ROS release. It is unknown if the functional differences noted in stable patients with COPD are also present or even amplified during exacerbations.
Neutrophils and cellular cross talk in COPD
Neutrophils do not act in isolation, and many cells have altered functions in COPD, including macrophages (Citation130), T cells (Citation131), B cells (Citation131, Citation132), eosinophils (Citation133) and platelets (Citation134), which have all been implicated in tissue damage. The close cross talk between cells which allows an efficient response to infection but then limits host damage appears to be lost in COPD and it might be the accumulation of cell dysfunction which leads to such a damaging neutrophil response. For example, alveolar macrophages are less efficient at phagocytosing bacteria in COPD (Citation135). When activated, these cells release inflammatory mediators and macrophage-specific and scavenged proteinases, recruiting more neutrophils to the airways, which in turn promote monocyte recruitment. Activated CD8+ T cells in COPD demonstrate more aggressive, pro-inflammatory responses than seen in smokers without COPD in response to microbial TLR ligands (Citation136), leading to more neutrophil recruitment. Neutrophil clearance by macrophage efferocytosis is reduced in COPD (Citation137), increasing the likelihood that these cells will undergo necrosis, increasing the proteinase burden in lung tissue and thus leading to a vicious cycle of inflammatory damage. Also, fibroblasts in COPD have a reduced capability for tissue repair (Citation138) and endothelial and epithelial dysfunction have been widely described (Citation139). Perhaps a combination of diverse genetic mutations or transcriptional changes in patients, when associated with appropriate environmental inflammatory exposures (such as smoking) can cooperatively cause variations in immune, airway and endothelial cells which are sustained, lead to damaging neutrophil recruitment and activation and ultimately to COPD ().
Figure 2. Cellular interactions in COPD pathogenesis.
Inhaled irritants such as cigarette smoke stimulate bronchial epithelial cells and alveolar macrophages to secrete factors (CXCL8, LTB4, GM-CSF) that promote both neutrophil production in bone marrow and neutrophil migration towards inflamed airways, a process coordinated by activated platelets, endothelial cells and the neutrophil. Neutrophils infiltrate the airways in large numbers and produce serine proteases and elastolytic enzymes (e.g. NE, MMP-8,9, proteinase-3). These neutrophilic enzymes act alongside other activated immune cells (dotted line) to degrade alveolar tissue leading to emphysema, over-stimulate Goblet cells leading to hypersecretion of mucus, and cause fibrosis of the small airways, all of which are hallmark features of COPD. CXCL8, Chemokine (CXC motif) ligand 8; LTB4, Leukotriene B4; GM-CSF, Granulocyte-Macrophage Colony-Stimulating Factor; NE, Neutrophil Elastase; MMP, Matrix Metalloproteases.
Targeting the neutrophil in COPD
Despite a number of cells being implicated in COPD, arguably the cell with the most potential for damage is the neutrophil, due to cell numbers and the injurious potential of each cell. However, there is understandable caution in abrogating the function of this cell as this would place the host at risk of overwhelming infection. Instead, strategies have considered if numbers or functions (including pro-inflammatory responses) could be “normalised” in COPD.
Glucocorticosteroids are one of the most commonly prescribed anti-inflammatory medicines in obstructive lung diseases which mediate their effects in part through trans-repression of pro-inflammatory transcription factors such as nuclear factor-κB (NF-κB). There are a number of clinical and in vitro studies which describe reduced steroid responsiveness in COPD (including from COPD neutrophils due in part to reduced NF-κB activation) (Citation140) and large, late phase clinical trials of inhaled steroids do not suggest they prevent disease progression (Citation141). In light of this, steroids are not considered to be effective at targeting COPD-related neutrophilic inflammation.
Phoshodiesterase type-4 inhibitors (PDE4) such as Roflumilast have been shown to improve lung function, reduce exacerbation frequency and reduce inflammation in frequently exacerbating COPD (Citation142, Citation143). PDE4 is involved in the degradation of cyclic adenosine monophosphate (cAMP) and raised levels of cAMP promote airway smooth muscle relaxation and modulate inflammatory cell activity, reducing pro-inflammatory mediator release. In COPD, PDE4 inhibitors reduce neutrophil LTB4, protease and ROS release and reduce overall neutrophil recruitment to the airways (Citation144), suggesting potent and COPD-relevant anti-inflammatory actions.
Leukotriene B4 (LTB4) is a potent neutrophil chemoattractant, and it was hypothesised that blocking this chemokine might reduce neutrophil recruitment to the lungs. However, an open label study of an LTB4 antagonist in 17 COPD patients did not show a reduction in the chemotactic potential of sputum (Citation145), suggesting other mediators were able to compensate for the reduction in LTB4 activity. A six-month trial of a CXCR2 inhibitor (which is the receptor for a number of neutrophil chemoattractants including IL-8, Growth related oncogene-α and Epithelial-neutrophil activating peptide-78, all raised in COPD) reduced neutrophil counts, MPO and MMP-9 in sputum and improved FEV1 compared to placebo in over 600 patients with COPD but 18% of patients had to withdraw from the active arm due to neutropenia, suggesting perhaps this treatment was too potent in its effect (Citation146).
Although in its infancy, more recent studies have shifted paradigms in COPD treatment; focusing on correcting cell function rather than decreasing recruitment signals (Citation147). p38 mitogen-activated protein kinase activation is involved in neutrophil directional sensing (Citation148) and p38 signalling has been shown to be raised in COPD (Citation149) but Losmapimod (a p38 inhibitor) had no effect on endothelial function (Citation150), lung function, exercise tolerance (Citation151) or sputum neutrophils in COPD although inflammatory mediators were reduced (Citation152). Targeting matrix metalloproteinases in phase 2 trials has also provided no clear evidence of efficacy although a potential signal was seen in urinary desmosine (Citation153). Aberrant neutrophil functions have been linked to increased activity of the PI3K pathway within the neutrophil (Citation154) and more recent studies have focused on this as a potential target (Citation155), with on-going trials in acute exacerbations of COPD. Of note, the PI3K pathway includes a number of therapeutic targets, some of which can be modified using repurposed medications such as HMG Co-A reductase inhibitors (commonly called “statins”). In vitro, high concentration statins have been shown to reproduce the same positive effect on neutrophil migration seen using a PI3K inhibitor (Citation156) including in COPD (Citation157). Of interest, metformin, acting through AMP-activating kinase, may also impact on these cellular functions through the same signalling cascade (Citation158) and metformin has been associated with better clinical outcomes in some studies of COPD (Citation159).
The future
Current COPD treatment stratification using the most recent GOLD document suggests symptoms may help inform treatment decisions, with bronchodilation for breathlessness and inhaled steroids (with bronchodilators) for exacerbations, especially if there are eosinophils (Citation160), and recommends three main drug classes (beta2 agonists, muscurinic antagonists and corticosteroids). This approach to COPD management does not reflect the phenotype and endotype approach utilised in complex asthma or in stratified lung cancer trials, or the array of treatment options available for the treatment of cardiovascular disease.
It is likely that we will not modify the course of COPD if we do not treat the cellular causes of the pathology. Neutrophils and more recently neutrophil dysfunction has been implicated in many of the pathological features of COPD (from chronic bronchitis to emphysema). A potential analogy may be with type 2 diabetes. In this diverse condition there are likely to be many susceptibility factors, both genetic and environmental, but almost irrespective of the actual cause, controlling blood glucose to within normal levels can help prevent end organ damage. Neutrophil dysfunction may be a common cause or indeed a common outcome of the underlying defect in COPD which then contributes substantially to host damage. If these were true, maintaining the function of these cells to within normal parameters might control aspects of tissue damage for many patients with COPD. Neutrophil dysfunction may have many different causes in COPD (PI3K signalling in some, p38 in others) which could be targeted incrementally through a number of putative therapeutics. Alternatively, this defect might be associated with only a proportion of COPD patients. However, whatever the incidence of neutrophil dysfunction in COPD, clinical phenotypes are unlikely to identify the particular cellular cause of the disease in an individual (like breathlessness will not identify the cellular basis of lung cancer nor the exact genetic mutation present) and we will probably need a deeper approach to treatment stratification to advance our ability to halt disease progression.
Conclusions
Neutrophils are innate immune cells that have been widely implicated in the pathogenesis of COPD. They are a feature of all disease phenotypes and their ability to damage lung parenchyma as part of the inflammatory pathology of COPD has been well-described. There is mounting evidence of neutrophil dysfunction in COPD, which might explain the continued inflammatory response. All these data suggest the neutrophil may represent a unifying therapeutic target in COPD, but the necessary functions of these cells make this target a challenging one.
Normalising their activity whilst maintaining their ability to participate in host defence may be a crucial step in preventing COPD progression. However, it is unclear whether changing the function of one cell will have a positive or detrimental effect on other crucial cell functions; whether normalising neutrophils will have a positive effect on COPD clinically, and whether targeting a potential “end-cell” mechanism of COPD (such as aggressive and indiscriminate neutrophils) will help a wider range of patients than seen in studies of a particular inflammatory mediator. Understanding neutrophil signalling and how these cells interact with other cells in COPD may unlock new strategies for COPD treatment.
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References
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