Our evolving view of neutrophils in defining the pathology of chronic lung disease

Kyle T Mincham; Nicoletta Bruno; Aran Singanayagam; Robert J Snelgrove

doi:10.1111/imm.13419

. 2021 Oct 4;164(4):701–721. doi: 10.1111/imm.13419

Our evolving view of neutrophils in defining the pathology of chronic lung disease

Kyle T Mincham ¹, Nicoletta Bruno ¹, Aran Singanayagam ^1,², Robert J Snelgrove ^1,^✉

PMCID: PMC8561104 PMID: 34547115

Abstract

Neutrophils are critical components of the body's immune response to infection, being loaded with a potent arsenal of toxic mediators and displaying immense destructive capacity. Given the potential of neutrophils to impart extensive tissue damage, it is perhaps not surprising that when augmented these cells are also implicated in the pathology of inflammatory diseases. Prominent neutrophilic inflammation is a hallmark feature of patients with chronic lung diseases such as chronic obstructive pulmonary disease, severe asthma, bronchiectasis and cystic fibrosis, with their numbers frequently associating with worse prognosis. Accordingly, it is anticipated that neutrophils are central to the pathology of these diseases and represent an attractive therapeutic target. However, in many instances, evidence directly linking neutrophils to the pathology of disease has remained somewhat circumstantial and strategies that have looked to reduce neutrophilic inflammation in the clinic have proved largely disappointing. We have classically viewed neutrophils as somewhat crude, terminally differentiated, insular and homogeneous protagonists of pathology. However, it is now clear that this does not do the neutrophil justice, and we now recognize that these cells exhibit heterogeneity, a pronounced awareness of the localized environment and a remarkable capacity to interact with and modulate the behaviour of a multitude of cells, even exhibiting anti‐inflammatory, pro‐resolving and pro‐repair functions. In this review, we discuss evidence for the role of neutrophils in chronic lung disease and how our evolving view of these cells may impact upon our perceived assessment of their contribution to disease pathology and efforts to target them therapeutically.

Keywords: asthma, COPD, lung, neutrophil

Prominent neutrophilic inflammation is a hallmark feature of patients with chronic lung diseases such as chronic obstructive pulmonary disease, severe asthma, bronchiectasis and cystic fibrosis, with their numbers frequently associating with worse prognosis. We have classically viewed neutrophils as somewhat crude, terminally differentiated, insular and homogeneous protagonists of pathology, and thus, they have been viewed as attractive therapeutic targets in chronic lung diseases. However, given our evolving view of neutrophils, it is critical that we re‐evaluate their perceived contribution to disease pathology and reconsider strategies to target them therapeutically.

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OUR EVOLVING VIEW OF NEUTROPHILS

Neutrophils have classically been considered to be a short‐lived, terminally differentiated, phenotypically homogeneous population of cells whose main task is pathogen killing. Accordingly, they are equipped with a vast arsenal of toxic mediators to facilitate rapid clearance of invading microorganisms. However, owing to the capacity of their payload to also impart significant bystander host tissue damage, neutrophilic inflammation needs to be tightly regulated. Overzealous or persistent neutrophilic inflammation, as often seen in chronic lung diseases, has traditionally been assumed to be largely detrimental, and neutrophils have been regarded as an attractive therapeutic target. However, it is increasingly apparent that neutrophils are far more sophisticated than previously anticipated, and thus, our perception of these cells in the context of disease pathology needs to be re‐evaluated.

We now recognize that neutrophils are highly responsive to environmental cues, exhibiting plasticity and a capacity to modulate their transcriptional profile to instigate a substantial de novo biosynthetic programme receptive to their specific needs [1]. This ability to integrate environmental signals and translate them into distinct effector programmes is accentuated by recognition that neutrophils can exhibit a far greater life span than originally anticipated [2]. The realization of neutrophil plasticity has subsequently led to the concept of neutrophil heterogeneity, whereby we now appreciate that not all neutrophils display comparable phenotype or function. Such heterogeneity is likely not solely driven by variable transcriptional profiles in response to microenvironmental cues, but also by the stage of neutrophil maturity, as the phenotype and function of circulating neutrophils vary considerably over the course of their life span [3]. Studies in mice and humans have demonstrated that senescent neutrophils are characterized by elevated CXCR4 expression, reduced size, hypersegmented nuclei and proclivity to undergo activation and neutrophil extracellular trap (NET) formation (NETosis) [4]. Accordingly, inflammatory, pro‐survival signals in the context of chronic inflammatory diseases may prolong neutrophil life span, whereby ‘aged’ neutrophils may subsequently contribute to disease pathology. Supportive of this concept, the frequency of CXCR4^hi neutrophils is increased in patients with asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) [5]. Conversely, emergency granulopoiesis in response to sustained inflammation can result in the release of immature neutrophils into the circulation. These are characterized by reduced expression of CD16, CD10 and CXCR2, band‐shaped nuclei and reduced granule content [3]. Immature neutrophil populations have been described both in asthma [6] and in COPD [7], but their functional significance in the context of chronic lung diseases is still unclear. Reflective of a growing recognition of neutrophil heterogeneity, discrete neutrophil subsets with divergent phenotypes and functionality have now been reported under both physiological and pathological conditions (Table 1) [3, 8]. To what extent these subsets represent bona fide distinct populations as opposed to a function of variable plasticity and maturity is yet to be clearly defined, but such studies highlight the necessity to no longer consider neutrophils as a sole, uniform population.

TABLE 1.

Neutrophil subsets and their relevance in homeostatic and pathological settings

Neutrophil subset	Frequently co‐expressed markers and/or morphological features	Known functions of the markers defining the subset	Prevalence and role in health and disease
CD177⁺	Proteinase 3 (PR3).	CD177 is a glycoprotein expressed on neutrophil plasma membrane and specific granules. It promotes the interaction of neutrophils with endothelial cells and their extravasation. However, genetic ablation in mouse or human neutrophils did not affect cell migratory capacity. PR3 is a serine protease mainly stored in neutrophil azurophilic granules, but it also presents on the cell surface of a subset of neutrophils in the peripheral blood of healthy individuals.	Humans: 45%–65% of circulating neutrophils during homeostasis. Frequency of circulating CD177⁺ neutrophils is augmented in inflammatory conditions, including asthma, IBD, sepsis, severe bacterial infections, cancer and anti‐neutrophil cytoplasmic autoantibody (ANCA)‐dependent vasculitis. In the latter, PR3 is the main target of the autoantibodies.
OLFM4⁺	_	Olfm4 was initially identified as a gene highly induced in myeloid stem cells by G‐CSF treatment. OLFM4 is located in specific granules. It acts as a negative regulator of neutrophil bactericidal activity, and its genetic ablation in mice boosted response against S. aureus or E. coli and improved survival in a model of sepsis.	Humans: 20%–25% of circulating neutrophils during homeostasis. In vitro, OLFM4⁺ neutrophils display a higher ability to produce NETs compared with OLFM4⁻ neutrophils. OLFM4⁺ neutrophils are increased in sepsis and have been suggested to contribute to autoimmune responses in ANCA‐dependent vasculitis. OLFM4 levels are increased in the serum of asthmatic patients and inversely correlate with asthma control state.
CXCR4^hi CD62L^low	CD11b^hi, CD49d^hi; hypersegmented nucleus and reduced size (in senescent neutrophils).	CXCR4 mediates neutrophil retention in the haematopoietic compartment by binding to CXC12, which is constitutively expressed by bone marrow stromal cells. Its expression is low on neutrophils freshly released from the bone marrow, but increases in senescent neutrophils, which are targeted for clearance. CD62L (L‐selectin) is involved in neutrophil attachment to the endothelium at sites of inflammation. It is downregulated with ageing and rapidly shed after neutrophil activation. The integrins CD11b and CD49d are also involved in neutrophil adhesion to the endothelium and migration to sites of inflammation.	Mouse: 55 ± 14% of circulating neutrophils in ZT5 (i.e. 5 h post‐initiation of the light period). Humans: ~60% of circulating neutrophils at night‐time. CXCR4^hi CD62L^low frequency is increased in several pathological settings, including asthma, COPD and CF. CXCR4^hi neutrophils display higher proclivity to form NETs (both in humans and in mice).
CD63⁺ (otherwise known as A2 or ‘GRIM’ neutrophils in CF airways)	CD11b^hi, CD66^hi, MHC II⁺, CD16^low, CD14^low ↑ Arginase 1 [ARG1] (in CF patients)	CD63 is a tetraspanin involved in the retention of neutrophil elastase (NE) into primary granules; CD63 mobilization to the cell surface correlates with extensive NE release to the extracellular space. CD66 is involved in the modulation of neutrophil adhesion and effector function. MHC II is an antigen‐presenting molecule essential for development and activation of CD4+ T cells. CD16 is a low‐affinity Fc receptor. CD14 acts as a co‐receptor for TLR4. ARG1 is an enzyme that metabolizes arginine and is stored in gelatinase granules.	In CF patients, neutrophils isolated from sputum display robust mobilization of CD63 to the surface compared with circulating neutrophils; this is accompanied by an upregulation of CD11b, CD66 and MHC II. Conversely, CD14 and CD16 are downregulated, and this is believed to contribute to the blunted phagocytic ability observed in airway PMNs isolated from CF patients as opposed to healthy individuals. Neutrophils isolated from the BAL of CF patients also feature increased ARG1 activity, which can block T‐cell proliferation. Frequency of CD63⁺ neutrophils is increased in sputum from asthmatic children (compared with healthy children). CD63 surface expression is also increased in senescent/apoptotic neutrophils.
CD64⁺	PD‐L1 (in COVID‐19 patients)	CD64 is the high‐affinity Fcγ‐receptor I; it binds to opsonized pathogens and immune complexes, thus triggering phagocytosis. PD‐L1 binds to PD‐1 on T cells and inhibits their activation, proliferation, cytokine secretion and survival.	CD64 surface expression is increased upon bacterial infection in both mice and humans. It is used as a marker of sepsis. Upregulated on blood neutrophils upon IAV and RSV infection, as well as during the delayed asthmatic response. A subpopulation of immature CD64⁺ PD‐L1⁺ neutrophils has been associated with more severe disease in COVID‐19 patients.
CD49d⁺	Cysteinyl leukotriene receptor 1 [CysLTR1] (in patients with viral respiratory tract infections, including IAV, IBV, RSV HRV, HMPV and HPIV). Can be co‐expressed with CXCR4 (possible link to intrinsic ageing?).	CD49d is the α₄ subunit of the VLA−4 integrin, which mediates the interaction with VCAM−1 on endothelial cells. CysLTR1 is a receptor for cysteinyl leukotrienes.	Mouse: Upon infection with Sendai virus, CD49d⁺ neutrophils induce FcεRI expression on lung dendritic cells. Humans: CD49d and CysLTR1 co‐expressing neutrophils are elevated in nasal lavage from patients with acute respiratory viral infections. CD49d⁺ neutrophil frequency is significantly increased in the peripheral blood and nasal lavage of atopic patients (compared with non‐atopic) and increases further upon allergen challenge.
CD49d^hiCXCR4^hi VEGFR1^hi	↑MMP‐9	VEGFR1 binds to VEGF‐A and VEGF‐B. MMP‐9 is a metalloproteinase involved in the degradation of extracellular matrix.	CD49d^hiCXCR4^hiVEGFR1^hi neutrophils are believed to support angiogenesis during development, normal tissue repair and pathogenic states characterized by tissue hypoxia. For instance, they are involved in the revascularization of transplanted tissues, as well as in tumour neovascularization, where MMP‐9 secretion facilitates vessel penetration.
ICAM‐1^hiCXCR1^low	↑ROS production	ICAM‐1 is an adhesion molecule involved in leucocyte trafficking. CXCR1 is a receptor for ELR⁺ chemokines.	ICAM‐1^hiCXCR1^low neutrophils are able to reverse transmigrate from the extravascular space back into the circulation. Their frequency is increased in rheumatoid arthritis and ischaemia/reperfusion damage. They have been suggested to contribute to systemic propagation of inflammation. They have been observed also in patients with COPD.
Low‐density neutrophils (LDNs), multiple subsets	CD15^hi, CD14^low, CD10^variable, CD16^variable; CD11b^hi, CD66b^hi (in SLE, HIV, asthma and cancer); CD33^hi (in cancer).	CD15, also known as Lewis x, is expressed in mature human neutrophils. It is considered to be involved in cell–cell interactions, phagocytosis and stimulation of degranulation. CD16 is the low‐affinity FCγRIII; its expression is acquired at the band cell stage during neutrophil differentiation, but it is downregulated during apoptosis and activation. CD10, also known as neutral endopeptidase, starts being expressed at the segmented stage. CD10 is a marker of neutrophil maturation. CD66b, also known as CEACAM8, is GPI‐anchored, highly glycosylated protein belonging to the carcinoembryonic Ag supergene family. It is located in lipid rafts, where it physically associates with CD11b, and it is considered an activation marker. CD33 (Siglec‐3) is a transmembrane receptor whose binding by sialic acids leads to activation of ITIM on the cytosolic portion of the protein and inhibition of apoptosis.	Opposite to standard neutrophils (normal density neutrophils), LDNs appear in the low‐density Ficoll gradient fraction together with mononuclear cells. They present as a mixed population of variable maturity. LDNs are present in a variety of pathological settings characterized by chronic inflammation and/or immunosuppression, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, cancer, psoriasis, asthma, malaria, tuberculosis, HIV infection, obesity and pregnancy. LDNs are also observed in septic patients, as well as in those with fungal (e.g. Aspergillus fumigatus and C. albicans) and viral infections (particularly from HBV and HCV). LDN properties appear to be context‐dependent. In SLE and psoriasis, LDNs display heightened inflammatory activity, with enhanced cytokine secretion and NETosis, but reduced phagocytic ability. In cancer and HIV, as well as in pregnant women, they show an immunosuppressive phenotype, characterized by low arginase activity and impaired capacity to drive CD8 proliferation and IFN‐γ production.
PMN‐I and PNM‐II (in the context of infection by methicillin‐resistant Staphylococcus aureus [MRSA])	PMN‐I: CD49d⁺, CD11b⁻, TRL5⁺, TRL8⁺, secrete IL‐12 and CCL3, drive classical activation of macrophages. PMN‐II: CD49d⁻, CD11b⁺, TRL7⁺, TRL9⁺, secrete IL‐10 and CCL2, drive alternative activation of macrophages.	_	Mouse: PMN‐I were isolated from MRSA‐resistant hosts, whereas PMN‐II were isolated from MRSA‐sensitive hosts.

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For a more extensive discussion of different neutrophil subsets, refer to references [1, 3, 4, 8].

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HMPV, human metapneumovirus; HPIV, human parainfluenza virus; HRV, human rhinovirus; IAV, influenza A virus; IBD, irritable bowel disease; IBV, infectious bronchitis virus; ICAM‐1, intercellular adhesion molecule‐1; OLFM4, olfactomedin 4; PMN, polymorphonuclear cells; RSV, respiratory syncytial virus; VCAM‐1, vascular cell adhesion molecule‐1; VEGF, vascular endothelial growth factor; VEGFR1, vascular endothelial growth factor receptor 1; VLA‐4, very late antigen‐4.

Another concept that has been debunked is that neutrophils are insular cells, which act swiftly and then disappear without impacting on the immune response beyond the acute phase of inflammation. It is now clear that neutrophils exhibit pleiotropic roles in shaping the phenotype and scale of an elicited immune response and ensuing repair programmes through engaging in complex crosstalk with both immune and structural cells via cell–cell contact and release of soluble mediators [9] (Figure 1). The depth of such interactions is beyond the scope of this review, but we highlight specific examples to demonstrate the marked capacity of neutrophils to modulate diverse and disparate components of the host's response. As frequent first responders, neutrophils are a rich source of the chemokines CCL2, CCL3 and CCL20, which operate to potentiate ensuing monocyte influx [10]. Neutrophil‐derived granule proteins have subsequently been shown to augment macrophage phagocytosis [11] and cytokine secretion [12], whilst their cathelicidins have been shown to be potent inducers of NLRP3 inflammasome activation and IL‐1β cytokine production by macrophages [13]. Moreover, neutrophil‐derived IL‐18 has been demonstrated to promote NK cell activation and IFN‐γ production [14]. Neutrophils can also profoundly potentiate facets of the host's adaptive response. Neutrophil‐derived products have been demonstrated to facilitate recruitment of dendritic cells, whilst additionally promoting dendritic cell maturation and antigen presentation [9]. Neutrophils themselves can even upregulate MHC II and costimulatory molecules and operate as antigen‐presenting cells [15, 16], whilst also acting as a source of signals that drive the recruitment, activation and differentiation of Th1 and Th17 cells [17, 18, 19]. Neutrophils have additionally been shown to exhibit profound effects on stromal cells, with their products capable of modifying epithelial/endothelial barrier function and cytokine production [20, 21, 22], fibroblast proliferation, differentiation and extracellular matrix (ECM) generation [23, 24]. Thus, it is clear that overexuberant neutrophilia, as seen in chronic lung diseases, can potentiate pathological inflammation and tissue remodelling.

Neutrophil interactions with immune and structural cells. Neutrophils can engage in complex crosstalk with structural and immune cells, either by directly interacting with them via cell surface molecules or by secreting a variety of mediators. This figure provides examples whereby neutrophils modulate the phenotype and function of different cell types. Depending on variable microenvironmental cues, age and maturity, neutrophils can exhibit divergent roles in regulating both the initiation and the resolution of inflammation, as well as driving tissue repair or pathological tissue remodelling. DC‐SIGN, dendritic cell‐specific intercellular adhesion molecule‐3 grabbing non‐integrin; FGF2, fibroblast growth factor 2; HB‐EGF, heparin binding‐epidermal growth factor; mCRAMP, murine cathelicidin‐related antimicrobial peptide; MIP, macrophage inflammatory protein; NO, nitric oxide; VEGF, vascular endothelial growth factor

However, it is increasingly recognized that neutrophils are not solely pro‐inflammatory but also exhibit extensive anti‐inflammatory, pro‐resolving and repair functions. Neutrophils have been described as a source of anti‐inflammatory cytokines TGF‐β and IL‐10, whilst also operating to scavenge pro‐inflammatory cytokines/chemokines through the production of decoy receptors or receptor antagonists [25]. Whilst neutrophils have the capacity to promote adaptive immunity, they also have the potential to suppress lymphocyte responses, such as through their ability to deplete extracellular L‐arginine and reduce T‐cell proliferation [26]. Persistent neutrophilia is not the norm, and endogenous feedback loops function to suppress their own activation and limit the toxicity of their most potent effector mechanisms, as well as dictating the production of pro‐resolving lipid mediators that retard further neutrophil recruitment [27]. Moreover, neutrophils are inherently programmed to undergo apoptosis within a defined period and, upon their phagocytosis by tissue‐resident macrophages, can impart negative feedback regulatory mechanisms to suppress granulocyte colony‐stimulating factor (G‐CSF)‐mediated neutrophil granulopoiesis [28], whilst also reprogramming macrophages to a reparative, M2‐like phenotype characterized by production of IL‐10 [29]. A role for neutrophils in promoting tissue repair has become increasingly apparent, and they can operate as a potent source of growth factors and pro‐angiogenic mediators [25, 30]. Given our new appreciation of the multifaceted roles of neutrophils, it is important to revisit our view of these cells in the pathophysiology of chronic lung diseases. This will aid in the design of improved therapeutic strategies, which consider the nuances of their behaviour rather than assuming their contribution is solely destructive.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE

COPD is a progressive inflammatory disease of the lungs and a leading cause of morbidity and mortality worldwide [31]. Cigarette smoking remains the most definitive risk factor for COPD onset in developed countries [31, 32], with prolonged exposure to noxious agents, primarily associated with biomass smoke, largely responsible for disease inception in non‐smokers from low‐ and middle‐income countries [33]. Once established, COPD is characterized by irreversible airflow limitation owing to small airway fibrosis (obstructive bronchiolitis) and alveolar destruction (emphysema) [34], which, in conjunction with acute bacterial‐ and viral‐induced exacerbations [35], culminate in patient dyspnoea, persistent cough, excessive sputum production, significantly impaired quality of life and death [36].

Neutrophils are recognized as the central cellular mediator in COPD pathogenesis. Irrespective of underlying clinical phenotype [37], the airways of patients with COPD are dominated by neutrophilic inflammation, directly correlating with severity of airway obstruction and accelerated decline in lung function based on forced expiratory volume in 1 second (FEV₁) [38]. Inhalation of noxious agents and acute exacerbations trigger the NF‐κB and p38 MAPK‐dependent release of neutrophil chemotactic mediators by activated airway epithelial cells and macrophages [39], collectively promoting neutrophil mobilization, recruitment and activation within the airways. Ensuing degranulation results in the accumulation of serine proteases, myeloperoxidase (MPO) and matrix metalloproteinase (MMP)‐8 and MMP‐9 [40, 41, 42], which consequently drive pathological features of COPD by triggering proteolytic degradation of the airway ECM and driving emphysematous disease. Enzymatic breakdown of the ECM additionally liberates the neutrophil chemo‐attractant proline–glycine–proline (PGP) [43] within the airways of patients with COPD, acting in synergy with cigarette smoke to perpetuate a vicious cycle of CXCR2‐mediated neutrophilic chemotaxis and ensuing clinical disease (Figure 2) [44, 45]. It is also noteworthy that early‐life NF‐κB‐induced activation of neutrophils and NE accumulation within the lungs of pre‐clinical models contribute towards lifelong impairments in lung architecture and predisposition to COPD development during adulthood [46].

Putative mechanisms by which neutrophils and their products may directly drive pathological features of chronic lung disease. Neutrophils and their products are considered major drivers of pathology in multiple chronic lung diseases. Direct evidence demonstrating a role for neutrophils and their products in driving tissue destruction and hallmark features of pathological remodelling in patients is often lacking. Accordingly, putative pathways defined within this figure are based, in part, upon circumstantial data or extrapolated from in vitro studies or a specific disease state – but could conceivably have relevance to multiple chronic lung diseases. AcPGP, acetylated proline–glycine–proline; ASM, airway smooth muscle; BLT1, high‐affinity LTB₄ receptor; MUC5AC, mucin 5AC; MUC5B, mucin 5B

In parallel with conventional pathways, the role of NETosis is an evolving mechanism in COPD pathogenesis. Elevated concentrations of NETs are observed in sputum from stable and acutely exacerbating COPD (AECOPD) patients, positively correlating with severity of airflow limitation and exacerbation frequency [47]. Moreover, NETosis induced by Haemophilus influenzae (a key pathogen involved in stable colonization and AECOPD [35]) amplifies pro‐inflammatory IL‐6 trans‐signalling within the airways, driving persistent sputum neutrophilia within a subset of patients with COPD [48]. Interestingly, this highlights a potential causal relationship between NET formation and Haemophilus dominance within the COPD lung microbiome [47].

Despite their enhanced activity, neutrophils from patients with COPD demonstrate multiple features of impaired functionality. Sapey and colleagues [49] first identified this compromised state whereby peripheral neutrophils from patients with COPD exhibit aberrant chemotactic responses towards an IL‐8 or GROα gradient, likely associated with enhanced phosphoinositide 3‐kinase (PI3K; an enzyme vital in regulating chemotactic directionality) signalling [50]. These defective migratory dynamics may explain the protease‐mediated collateral tissue damage caused upon neutrophil recruitment to the lungs of patients with COPD following upregulation of adhesion molecules [51]. Compounding this is the impaired phagocytic activity of neutrophils from patients with COPD [47], although conflicting results are reported based on bacterial species [52]. Nevertheless, direct inhibition of phagocytosis appears to be a consequence of elevated NET complexes within COPD sputum [47] and is exaggerated by the ability of cigarette smoke to suppress neutrophil phagocytosis via the disruption of caspase‐3‐mediated homeostatic regulation [53], likely contributing towards the persistent bacterial colonization and increased risk for exacerbation‐induced morbidity and mortality [54].

The centrality of neutrophils in COPD pathogenesis has prompted a concerted effort to therapeutically target these effector cells, albeit largely disappointingly. Inhaled corticosteroids (ICS) are widely prescribed during both stable disease and episodic exacerbations, yet ICS prolong neutrophil survival [55] and have limited capacity to reduce exacerbation frequency in patients with COPD [56]. NETosis is additionally resistant to corticosteroid treatment, with suggestions the common ICS fluticasone propionate may inadvertently increase sputum NETosis and further diminish phagocytic capacity [47]. Such caveats are compounded by the heightened risk of secondary bacterial infection and airway mucus hypersecretion in patients with COPD prescribed ICS during respiratory viral‐induced exacerbations [57], collectively highlighting ICS as a modestly effective treatment for all disease states.

Small molecule compounds targeting neutrophil signalling pathways are being investigated as a more precise means of therapeutic intervention (Table 2). CXCR2 antagonists routinely limit neutrophil infiltration in the lungs and periphery of patients with moderate‐to‐severe COPD [58, 59] although improvements in FEV₁ are infrequent and protection is not always observed in a clinical setting following initial pre‐clinical promise [60]. Moreover, increased exacerbation risk, neutropenia and pneumonia are reported in an unacceptable proportion of patients following intervention [58, 59, 60], thus mitigating clinical benefit. Likewise, inhibitory compounds targeting neutrophil elastase (NE) [61], TNF‐α [62] and leukotriene B₄ (LTB₄) synthesis [63] repeatedly show limited capacity for clinically meaningful protection in patients with COPD, raising several questions regarding dose, intervention time‐points and the appropriate use of pre‐clinical models in determining novel therapeutic efficacy. Recent pre‐clinical studies targeting NETosis employ the use of mucolytics (deoxyribonuclease I) for degrading cigarette smoke‐induced NETs within the airways [64]. Murine COPD models of cigarette smoke‐ and non‐typeable H. influenza‐induced respiratory inflammation have additionally identified anti‐PD‐1 treatment as a strategy to limit airway neutrophilic influx and alveolar destruction [65]. Whilst these novel approaches support effective disease mitigation, the aforementioned studies suggest a high degree of scrutiny concerning immunosuppression should be applied if progression to clinical trials ensues. Restoring neutrophil chemotactic accuracy via the selective inhibition of hyperactive PI3K signalling is being explored as a low toxicity alternative to the mitigation of pro‐inflammatory activity, and shows initial promise without altering migratory speed [50]. PI3Kδ inhibition further limits MMP‐9 and reactive oxygen species (ROS) secretion from stable and AECOPD neutrophils [66], although the beneficial impact appears most prominent in stable patients [67, 68].

TABLE 2.

Clinical trials targeting neutrophilic inflammation in chronic lung disease

Treatment	Target	Stratified on neutrophils	Patients	Duration of treatment	Outcomes	Ref.
COPD
MK‐7123	CXCR2	No	GOLD stage II‐III FEV₁ 30%−70% predicted FEV₁/FVC ≤70% Stable	≥26 weeks (primary analysis). ≥52 weeks (optional safety extension).	Reduced sputum neutrophils at 3 months. Reduced sputum MMP‐9. Reduced blood neutrophils. Reduced plasma MMP‐9, MPO and CXCL5. Neutropenia in 2·6%–18·4% of treatment groups. Improved FEV₁	[58]
AZD5069	CXCR2	No	GOLD stage II‐III FEV₁ ≥30% and <80% predicted FEV₁/FVC <70% Stable	4 weeks	Reduced peripheral blood neutrophils. Neutropenia in 4%−10% of treatment groups. No improvement in clinical end‐points.	[59]
GKS1325756 (Danirixin)	CXCR2	No	FEV₁ >40% predicted FEV₁/FVC <70%	24 weeks	No change in blood neutrophils. No improvement in clinical end‐points.	[60]
AZD9668	NE	No	GOLD stage II‐III	12 weeks	No change in urine desmosine (biomarker for lung matrix degradation) concentration. No improvement in clinical end‐points.	[61]
Infliximab	TNF‐α	No	GOLD stage I‐IV Stable	24 weeks	No treatment benefits. Higher rate of pneumonia in the treatment group. No improvement in clinical end‐points.	[62]
BAYx1005	LTB₄	No	FEV₁ <65% predicted Stable	2 weeks	Reduced sputum LTB₄ Clinical outcomes not assessed.	[63]
GSK045	PI3Kδ	No	GOLD stage II–III FEV₁/FVC <70% Stable and AECOPD	In vitro stimulation	Reduced ROS release from sputum neutrophils in stable patients. Reduced MMP‐9 and ROS release from blood neutrophils in stable and AECOPD patients. Clinical outcomes not assessed.	[66]
BIRB796	P38 MAPK	No	GOLD stage II–III FEV₁/FVC <70% Stable and AECOPD	In vitro stimulation	Reduced MMP‐9 release from blood neutrophils in stable patients. Clinical outcomes not assessed.	[66]
GSK2269557 (Nemiralisib)	PI3Kδ	No	GOLD stage II‐III FEV₁ 40%−80% predicted FEV₁/FVC <70% Stable	2 weeks	No change in sputum neutrophils. Reduced sputum IL‐6 and IL−8. No improvement in clinical end‐points.	[67]
GSK2269557 (Nemiralisib)	PI3Kδ	No	GOLD stage II‐III FEV₁ ≤80% predicted FEV₁/FVC <70% Stable and AECOPD	2 weeks	Upregulation of genes associated with sputum neutrophil migration in stable patients only. Inhibition of NE and ROS from stimulated blood neutrophils from AECOPD patients. Improved FEV₁	[68]
Asthma
SCH527123	CXCR2	Yes	Severe asthma >40% sputum neutrophils	4 weeks	Reduced sputum and blood neutrophils. Fewer mild exacerbations. No improvement in ACQ or FEV₁. No change in sputum MPO, IL‐8 or elastase.	[111]
AZD5069	CXCR2	Yes	Uncontrolled, persistent asthma Blood neutrophil counts greater than 2·7 × 10⁹/L	6 months	Reduced peripheral blood neutrophils. No improvement in clinical end‐points.	[112]
LY293111	LTB₄	No	Atopic asthma	7 days (allergen challenge model)	Reduced BAL neutrophils. No improvement in clinical end‐points.	[113]
MK‐0633	LTB₄/Cysteinyl LTs	No	Chronic asthma FEV₁ >45 and <85% predicted	6 weeks	Modest improvement in baseline FEV₁. Changes in neutrophil numbers not reported. Elevated aspartate aminotransferase and alanine aminotransferase. Benefit–risk ratio did not support clinical use.	[114]
GSK2190915	LTB₄/Cysteinyl LTs	Yes	Asthma Raised sputum neutrophils at 2 visits (>50% and >45%)	14 days	Reduced sputum LTB₄. No effect on sputum neutrophils. No improvement in clinical end‐points.	[115]
JNJ‐40929837	LTB₄	No	Mild, atopic asthmatics	7 days (bronchial allergen challenge model)	Reduced sputum LTB₄. Changes in neutrophil numbers not reported. No improvement in clinical end‐points.	[116]
AMG 827 (Brodalumab)	IL‐17RA	No	Inadequately controlled moderate‐to‐severe asthma	12 weeks	Changes in neutrophil numbers not reported. No improvement in clinical end‐points.	[117]
Etanercept	TNF‐α	No	Moderate‐to‐severe persistent asthma	12 weeks	Changes in neutrophil numbers not reported. No improvement in clinical end‐points.	[118]
Golimumab	TNF‐α	No	Uncontrolled, severe persistent asthma	24 weeks	Changes in neutrophil numbers not reported. No improvement in clinical end‐points. Unfavourable risk–benefit profile led to early discontinuation.	[119]
Bronchiectasis
Brensocatib	DPP1	No	Bronchiectasis Frequent exacerbations	24 weeks	Increased time to first exacerbation. Reduced sputum NE.	[128]
AZD9668	NE	No	Bronchiectasis	4 weeks	No change in sputum neutrophils. Improved FEV₁ by 100 mL.	[129]
BAY 85‐8501	NE	No	Bronchiectasis	4 weeks	Reduced NE activity. No improvement in clinical end‐points.	[130]
Cystic fibrosis
AZD9668	NE	No	Cystic fibrosis	4 weeks	No effect on sputum neutrophils or NE. Reduced sputum cytokines. No improvement in clinical end‐points.	[139]
Alpha1‐antitrypsin	Protease/antiprotease balance	No	Cystic Fibrosis	4 weeks	Decreased levels of elastase activity. Decreased neutrophils and pro‐inflammatory cytokines.	[140]
BIIL 284 BS	LTB₄	No	Cystic Fibrosis	24 weeks	Increased pulmonary exacerbations. No clinical benefit.	[141]

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Abbreviation: FVC, forced vital capacity.

We now understand that neutrophils are a heterogeneous population with a repertoire of variable immune modulatory and tissue remodelling functions, and this therefore needs to be considered when evaluating neutrophils and their role in COPD pathogenesis. The development of new biologics targeting specific neutrophil subsets and their respective functions/products, and subsequently stratifying patient groups on neutrophil composition, may thereby enable a personalized medicine approach to disease mitigation, whilst circumventing many of the off‐target effects of current strategies.

ASTHMA

Asthma is a chronic respiratory disease characterized by airway inflammation and pathological airway remodelling (subepithelial fibrosis, increased smooth muscle mass, goblet cell and mucous gland hyperplasia, neovascularization and epithelial alterations), resulting in variable symptoms of wheeze, shortness of breath, chest tightness and cough. Subacute flare‐ups of symptoms termed exacerbations represent a major source of disease morbidity and a significant economic burden. Asthma is an extremely common condition affecting 358 million people worldwide and resulting in 495 000 annual deaths [69]. It has classically been considered an allergic, Th2‐driven pathology characterized by airway eosinophilia [70]. However, it is now recognized that asthma is a heterogeneous disease with as many as 50% of asthmatic patients presenting with non‐Th2 inflammation [71]. Accordingly, variable symptomatic burden, airway inflammation and remodelling have led to the realization of distinct asthma endotypes whereby specific pathological mechanisms are clustered.

Increasingly, the concept of a ‘neutrophilic endotype’ has gained traction, whereby elevated numbers of neutrophils in patient sputum, peripheral blood, bronchoalveolar lavage and lung biopsies frequently associate with severe, corticosteroid‐refractory disease, exacerbations, patient hospitalizations, comorbidities and fatalities [72, 73, 74, 75, 76]. Whilst patients presenting with severe neutrophilic disease are often considered non‐atopic, neutrophils are still largely present in the airways of allergic ‘eosinophilic’ patients, where they are readily increased in number during symptomatic manifestations, such as allergen challenge, viral‐induced exacerbation or nocturnal crisis [77]. Neutrophils are capable of displaying marked plasticity, and those present in asthmatic patients have been reported to exhibit an altered phenotype, and increased activation and functionality [78]. Given our growing awareness of neutrophil heterogeneity and plasticity, more precise phenotyping of neutrophils in asthma patients will be important in delineating potentially variable contributions of neutrophil subsets to disease pathologies and in identifying novel therapeutic strategies. A recent study assessing in‐depth single‐cell analysis of granulocytes in the airway of asthmatic patients and healthy controls highlighted the likely presence of distinct neutrophil subsets [79].

Elevated neutrophil numbers in asthmatic patients are associated with an increase in classical pro‐neutrophilic factors such as CXCL8 and LTB₄ [80, 81]. Transcripts of IL17A, a classical inducer of CXCL8, were found to be elevated in sputum of asthmatic patients, correlating with CXCL8 transcripts, neutrophilic inflammation and disease severity [82]. Accordingly, studies in patients and mice have highlighted a role for both Th1/IFN‐γ and Th17/IL‐17A responses in severe asthma and associated airway neutrophilia [82, 83, 84, 85]. Elevated TNF‐α levels have also been reported in the airways of asthmatic patients associating with neutrophils [86], with hierarchical clustering analysis of sputum transcriptomics showing a neutrophil cluster to be associated with expression of IFN and TNF family members [87]. Accumulating evidence highlights a strong association between airway infection and the presence of neutrophils in asthmatic patients. It has been postulated that neutrophilic asthma may manifest as a consequence of a dysbiosis of the airway microbiome whereby there are increased levels of bacteria but reduced bacterial diversity [88]. Similarly, respiratory viral infections, such as rhinovirus (RV), are a frequent cause of asthma exacerbations and concomitant increases in airway neutrophilia, whilst fungal pathogens also associate with neutrophilic, severe asthma [88]. Other factors have also been associated with the appearance of a neutrophilic endotype of asthma, including environmental factors such as cigarette smoke and pollution [89, 90, 91], and comorbid factors such as obesity and gastroesophageal reflux disease [92, 93]. Given the capacity of corticosteroids to inhibit neutrophil apoptosis and promote their survival [55], debate has arisen as to whether neutrophilic asthma reflects a true endotype of disease or is merely a consequence of the treatment itself [94]. However, others argue that treatment strategies do not ubiquitously underlie this endotype and neutrophils can still be observed in the absence of corticosteroid therapy [95, 96].

Given the destructive capacity of neutrophils, it is perhaps not surprising that they are considered to be protagonists of pathology in severe asthma. Manipulation of neutrophils in animal models of asthma has gone some way to support a role in disease progression [97, 98, 99, 100, 101]—though these limited studies have sometimes been confounded by a lack of physiological relevance to the disease they are seeking to recapitulate and/or the specificity of neutrophil targeting strategies. Moving forward, it is critical that the pre‐clinical animal models utilized to understand the pathogenesis of ‘neutrophilic asthma’ and test novel therapeutic strategies recapitulate the key aforementioned aetiological, clinical, inflammatory and remodelling aspects of disease observed in these patients. As such, many of the models utilized to date do not capture the steroid‐resistant, severe neutrophilic disease with low Th2 inflammation seen in a frequently non‐atopic patient group. Assertions as to how neutrophil products may participate in pathological processes in asthma are frequently based upon circumstantial data, or extrapolated from in vitro studies or other disease states, and their direct relevance to asthma pathogenesis should therefore be viewed with caution. MMPs, such as MMP‐8 and MMP‐9, are elevated in asthmatic patients, associating with neutrophilic inflammation and poor prognosis [102, 103, 104, 105]. Accordingly, mice deficient in MMP‐9 show ameliorated inflammation, and bronchial hyper‐responsiveness following allergen exposure [106]. NE is also elevated in the airways of asthmatic patients, associating with adverse outcomes. Largely indirect evidence highlights the capacity of NE to evoke pathological features of asthma [78], whilst chronic exposure of mice to NE results in pulmonary eosinophilia and mucus cell metaplasia [107]. Peripheral blood neutrophils derived from asthmatics produce enhanced levels of superoxide upon stimulation, with levels correlating with airway hyper‐responsiveness and inversely with FEV₁, whilst ROS have been shown to be capable of inducing airway smooth muscle contraction, goblet cell metaplasia and mucus hypersecretion [78]. Neutrophils have also been proposed as a source of pro‐fibrotic TGF‐β [108], and epithelial barrier modulating cytokine oncostatin M (Figure 2) [109], whilst NETs have been purported to be essential to the pathology of RV‐induced exacerbations in a murine model [110].

Much of the data supporting a pathological role for neutrophils in asthma remains primarily circumstantial with a lack of convincing evidence directly demonstrating the mechanisms by which they participate in pathological processes, leading to counterarguments that their presence is inconsequential or even beneficial, and that strategies that seek to broadly block their infiltration carry inherent risks. Strategies that have sought to manipulate neutrophils in a clinical setting in asthmatic patients have been largely unsuccessful in ameliorating disease [111, 112, 113, 114, 115, 116, 117, 118, 119] (Table 2), and thus have done little to support the notion that neutrophils are truly pathological and a viable therapeutic target. Whilst, in some instances, suboptimal patient enrolment could have contributed to a lack of efficacy in these trials, it could also suggest that neutrophils are inconsequential in the context of asthma pathology. Conversely, given our evolving view of neutrophils, is it feasible that these cells, or at least a subset of these cells, could actually be beneficial in the context of asthma and that their presence is reflective of an effort to control inflammation or promote tissue repair? The presence of neutrophil chemokines in a subset of patients with severe asthma is associated with better clinical outcome [98], whilst increased intraepithelial neutrophils in the lungs of children with severe therapy‐resistant asthma correlated with better lung function [120]. Depletion of neutrophils in a mouse model of allergic airway disease inadvertently augmented type 2 inflammation [121]. In this context, neutrophil ablation resulted in a perturbation of neutrophil‐mediated regulatory pathways in the periphery culminating in G‐CSF accumulation, which in turn boosted type 2 innate lymphoid cell (ILC2) function [121]. Similarly, adoptive transfer of neutrophils has also been demonstrated to ameliorate Th2 inflammation in murine models. Intriguingly, biologics shown to ameliorate neutrophilic inflammation in asthmatic patients can cause increased G‐CSF [122, 123] and percentage sputum eosinophils [111]. Similarly, whilst some studies have suggested key pathological roles for neutrophil‐derived products in asthma, others have demonstrated a putative beneficial role for MMP‐9 in limiting and resolving allergic airway inflammation [124] and for NE in destroying mucus plugs [98] that cause airway obstruction.

BRONCHIECTASIS

Bronchiectasis is a heterogeneous chronic lung condition with multiple aetiological causes. Disease pathogenesis is believed to be driven by microbial dysbiosis and chronic infection associated with impaired mucociliary clearance. This leads to persistent airway neutrophilic inflammation, which contributes to airway tissue damage through release of proteases. Numerous defects in neutrophil function have been reported in the bronchiectasis literature including impaired bacterial phagocytosis and killing [125], greater presence of necrotic neutrophils in the airways (suggesting perturbed resolution of inflammation) [126], spontaneous neutrophil apoptosis [125] and increased NETosis [127]. It remains unclear as to whether these defects are inherent to bronchiectasis or whether neutrophil dysfunction occurs secondary to airway inflammatory milieu. The importance of neutrophil serine proteases as a driver of pathology in bronchiectasis was recently confirmed by results from the WILLOW phase 2 clinical trial, which evaluated brensocatib, a dipeptidyl peptidase‐1 (DPP1) inhibitor [128] (Table 2). This drug acts to prevent combined activation of NE, proteinase 3 and cathepsin G and demonstrated the beneficial clinical effect of significantly prolonging time to next exacerbation compared with placebo. Conversely, other smaller studies that have evaluated more selective NE inhibitors have reported lack of effects on a range of clinical outcomes [129, 130]. Larger independent trials are now needed to further investigate the effects of therapies targeting neutrophil dysfunction in bronchiectasis.

CYSTIC FIBROSIS

CF is an important genetic cause of bronchiectasis due to defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Chronic infection and inflammation induce lung damage in patients with CF with changes beginning early in life. The CF airway is dominated by a neutrophil‐mediated inflammatory process. A range of neutrophil functional abnormalities are recognized in CF. As for bronchiectasis, neutrophils from patients with CF have reduced phagocytic capacity, potentially due to diminished surface expression of key phagocytosis receptors CD14 and CD16 [131, 132], and also demonstrate greater oxidative burst and MPO release [133, 134]. These functional attributes contribute in part to the classification of two distinct neutrophil populations observed in the CF airways, namely the CD63^loCD16^hi A1 and CD63^hiCD16^lo A2 (otherwise known as the ‘GRIM’ phenotype due to their granule releasing, immunoregulatory and metabolic attributes [135]) subsets, based primarily on low and high levels of NE‐rich primary granule exocytosis, respectively [131, 136]. MPO concentrations correlate with lung function decline and CF disease severity [137]. Neutrophil proteases including NE and MMPs are also markedly upregulated in the CF airway with MMP‐9 previously shown to correlate with lung function and airway inflammation [138]. Clinical trials of neutrophil‐focussed therapies (Table 2) have largely been unsuccessful in CF, including inhaled NE inhibitors and recombinant human alpha‐1 antitrypsin therapy, which both showed no effect on lung function or clinical outcomes [139, 140]. An LTB₄ receptor antagonist BIIL 284 BS has actually been shown to have detrimental effects including increased exacerbation frequency and worsened lung function [141]. Conversely, nebulized DNase is a widely used efficacious therapy that cleaves DNA released from neutrophils, reduces mucous viscosity and improves lung function. Several trials have proven its efficacy in patients with mild and more advanced disease [142]. More recently, CFTR modulator therapy has been added to the therapeutic repertoire and emerging evidence indicates that these drugs can modulate neutrophil function including beneficial changes in markers of activation [143] and increased bacterial killing [144].

IDIOPATHIC PULMONARY FIBROSIS

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic lung disease of unknown cause. It is irreversible and responsible for ~1 in every 100 deaths each year in the UK. In early studies, neutrophil numbers [145], the neutrophil chemokine CXCL8/IL‐8 [146] and the neutrophil regulatory cytokine G‐CSF [147] have all been reported to be increased in bronchoalveolar lavage (BAL) fluid from subjects with IPF. BAL neutrophil numbers also independently predict mortality associated with the disease [145] providing suggestive evidence for a role in disease pathogenesis. This may occur through augmented release of proteases such as NE, which has been shown to degrade a range of ECM proteins. In bleomycin models, mice deficient in NE [148] or treated with a NE inhibitor [149] are protected from lung fibrosis. There is also evidence from animal studies that aged neutrophils can cause fibrotic interstitial lung disease if not restrained by B cells, which ordinarily act to limit their capacity to drive lung pathology [150]. Neutrophil MMP‐2, MMP‐8 and MMP‐9 can degrade all components of the ECM and other non‐matrix proteins. A range of MMPs are reported to be upregulated in IPF [151, 152, 153], and gene‐targeted deletion of Mmp3 [154], Mmp7 [155] and Mmp8 [156] reduces development of pulmonary fibrosis in bleomycin mouse models. Further work is needed to define methods of targeting MMPs as existing MMP inhibitors have had poor efficacy in lung cancer trials and are associated with dose‐limiting side‐effects such as musculoskeletal pain [157].

CONCLUSIONS

It is clear that neutrophils are a prominent feature of the airway inflammation observed in multiple chronic lung diseases, frequently associating with a worse prognosis. However, neutrophil targeting biologics entering clinical trials have yielded largely disappointing results to date. Accordingly, it is clear that in many instances there remains much still to learn about what underlies the influx of neutrophils, how precisely neutrophils are contributing to disease pathogenesis, and how, or even if, we should be targeting these cells therapeutically. Moving forward, it is important that we incorporate better pre‐clinical animal models that truly reflect the pathology we are seeking to simulate, and that we are rigorous in our patient selection for clinical trial enrolment. Increasingly, we are recognizing the heterogeneity within neutrophil populations, and the important immune modulatory, regulatory and repair roles of these cells. It is also clear that neutrophils must be considered in the context of comorbidities and intrinsic and extrinsic confounders. It is feasible, and perhaps probable, that the neutrophil exhibits pleiotropic and potentially conflicting roles in defining pathophysiology of chronic lung disease—some almost certainly detrimental and some potentially even beneficial—that may be variable in a context‐ and patient‐specific setting. More precise phenotyping of neutrophils in patients and a greater awareness of their heterogeneity, complexity and plasticity will be instrumental in defining more targeted therapeutic approaches for manipulating neutrophils and their effector functions in patients. Accordingly, therapeutic manipulation of neutrophils with a broad sword approach may not be the answer. Rather, we should seek to understand their complex and multifaceted roles in the disease state and target them within clearly stratified patient populations with the same subtleties and specificity that they themselves exhibit.

CONFLICT OF INTERESTS

The authors confirm they have no competing interests.

AUTHOR CONTRIBUTIONS

KTM, NB, AS and RJS contributed equally to the planning and writing of the manuscript. KTM and NB prepared figures and tables within the manuscript.

PERMISSION TO REPRODUCE

The authors provide full permission to reproduce content of the article where relevant.

Mincham KT, Bruno N, Singanayagam A, Snelgrove RJ. Our evolving view of neutrophils in defining the pathology of chronic lung disease. Immunology. 2021;164:701–721. 10.1111/imm.13419

Funding information

RJS is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences (209458/Z/17/Z).

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PERMALINK

Our evolving view of neutrophils in defining the pathology of chronic lung disease

Kyle T Mincham

Nicoletta Bruno

Aran Singanayagam

Robert J Snelgrove

Abstract

OUR EVOLVING VIEW OF NEUTROPHILS

TABLE 1.

FIGURE 1.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE

FIGURE 2.

TABLE 2.

ASTHMA

BRONCHIECTASIS

CYSTIC FIBROSIS

IDIOPATHIC PULMONARY FIBROSIS

CONCLUSIONS

CONFLICT OF INTERESTS

AUTHOR CONTRIBUTIONS

PERMISSION TO REPRODUCE

REFERENCES

ACTIONS

PERMALINK

RESOURCES

Cite

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PERMALINK

Our evolving view of neutrophils in defining the pathology of chronic lung disease

Kyle T Mincham

Nicoletta Bruno

Aran Singanayagam

Robert J Snelgrove

Abstract

OUR EVOLVING VIEW OF NEUTROPHILS

TABLE 1.

FIGURE 1.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE

FIGURE 2.

TABLE 2.

ASTHMA

BRONCHIECTASIS

CYSTIC FIBROSIS

IDIOPATHIC PULMONARY FIBROSIS

CONCLUSIONS

CONFLICT OF INTERESTS

AUTHOR CONTRIBUTIONS

PERMISSION TO REPRODUCE

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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