Airway biomarkersNeutrophilic inflammation

Studies have consistently identified neutrophilic inflammation as a driver of disease severity in bronchiectasis. High levels of neutrophils and neutrophil-associated proteins are observed in bronchiectasis sputum and BAL (figure 2) [28, 29]. Neutrophils are recruited to the airways by inflammatory cytokines in response to infection and, under normal physiological conditions, contribute to host defence primarily through phagocytosis of pathogens [30, 31]. Other neutrophil defence mechanisms include degranulation and release of neutrophil extracellular traps (NETs) which are web-like structures, composed of decondensed chromatin, DNA, histones and granule contents extruded into the extracellular space to disarm and trap pathogens [32, 33].

FIGURE 2

Known interactions between biomarkers of neutrophilic inflammation, infection, epithelial dysfunction and mucociliary clearance in bronchiectasis. MMP: matrix metalloproteinase; SLPI: secretory leukocyte protease inhibitor; AAT: alpha-1 antitrypsin; NETs: neutrophil extracellular traps; NE: neutrophil elastase; PR: proteinase; MPO: myeloperoxidase; PZP: pregnancy zone protein; AZU: azurocidin; ICAM: intracellular adhesion molecule; LTB4: leukotriene B4; TNF: tumour necrosis factor; IL: interleukin. Figure created with biorender.com.

The most clinically accessible and inexpensive neutrophil biomarker in practice is sputum colour. The green colour of purulent sputum is due to haem pigment present in myeloperoxidase (MPO) released into the airways during degranulation and NETosis [34–36]. Sputum purulence is associated with bacterial infection, radiological severity and quality of life in bronchiectasis [36, 37]. In the AIR-BX studies, patients with more cough and purulent sputum were more likely to experience symptomatic improvement in response to inhaled aztreonam [38]. Therefore, sputum colour alone, assessed using tools such as the Murray chart, is a biomarker of neutrophilic airways inflammation with potential to guide therapy [37]. However, it remains a proxy measure. Markers of specific elements of the inflammatory cascade hold potential to further characterise neutrophilic endotypes and aid drug development.

Neutrophil proteases and NETs

NETs and NET-associated proteins are prospective biomarkers of a bronchiectasis endotype defined by excessive, dysregulated neutrophilic inflammation which may respond to antineutrophil therapies. A multicohort observational study found sputum NET concentration, measured using complexes between DNA/histones and neutrophil elastase, was associated with multiple bronchiectasis severity markers including BSI, exacerbations and mortality (table 1). Proteomic analysis also showed that NET-associated proteins including MPO, neutrophil elastase, resistin and azurocidin-1 identified a group of patients with severe disease. Sputum NET concentration has been linked to microbial dysbiosis, and both intravenous antibiotic therapy and macrolide therapy successfully reduced sputum NETs [29].

TABLE 1

Inflammatory, epithelial dysfunction and mucociliary clearance biomarkers in bronchiectasis and clinical associations

Of the NET-associated proteins, neutrophil elastase is furthest advanced in the process of becoming a validated biomarker. Neutrophil elastase is a neutrophil serine protease (NSP) stored in the primary (azurophilic) granules. It is released through both degranulation and NETosis and degrades extracellular matrix proteins and virulence factors of various Gram-negative bacteria. However, it also cleaves proteins on host cells, leading to epithelial damage and airway remodelling as well as inducing mucus hypersecretion and hyperviscosity [75–77]. Sputum neutrophil elastase activity correlates with exacerbations, lung function decline and bacterial infection in bronchiectasis [39–42]. A point-of-care test has been developed (NEATstik) which scores sputum neutrophil elastase concentration on a 10-point scale and is able to identify patients at higher risk of exacerbation [78].

Neutrophil elastase is a particularly clinically relevant biomarker because novel anti-inflammatory therapies targeting NSPs are currently in development. Preliminary trials of direct neutrophil elastase inhibition have shown mixed results [79, 80]. However, dipeptidyl peptidase (DPP)-1 inhibitors are a promising new therapy in development. DPP-1 cleaves and activates NSPs including neutrophil elastase during neutrophil maturation in the bone marrow [81, 82]. In the WILLOW phase 2 trial (clinicaltrials.gov identifier NCT03218917) of the DPP-1 inhibitor brensocatib (10 mg and 25 mg) versus placebo, brensocatib prolonged time to first exacerbation and reduced neutrophil elastase activity in patients with bronchiectasis, and a large phase 3 trial is now underway (clinicaltrials.gov identifier NCT04594369) [27].

Brensocatib has also been shown to reduce the NSPs proteinase (PR)-3 and cathepsin-G (Cat-G) which are also activated by DPP-1 and have similar proteolytic activity to neutrophil elastase [83, 84]. Both have been linked to disease severity in COPD [85, 86]. Preliminary data suggest that sputum PR-3 is raised in bronchiectasis exacerbations [43]. Further clinical associations with these proteases in bronchiectasis, and their role in response to anti-inflammatory therapies, are yet to be uncovered.

MPO is another NET-associated enzyme responsible for reactive oxygen species generation. As well as its role in sputum purulence it is independently associated with radiological severity and exacerbation risk in bronchiectasis [19, 44]. In a study of 28 bronchiectasis patients randomised to inhaled gentamicin versus control, sputum MPO fell significantly in response to treatment in association with reduced sputum volume and improved peak expiratory flow and 6-min walk distance [45].

Additionally, Finch et al. [47] demonstrated that pregnancy-zone protein (PZP), a serum protein with known antiprotease and T-cell immunosuppressive effects, was present in the cytoplasm of neutrophils and released in NETs. Sputum PZP was linked with high BSI and exacerbation risk, as well as higher airway bacterial load, and was reduced in response to antibiotics. This study determined that PZP is a biomarker of an endotype characterised by NET formation and high bacterial load and is a possible mechanism through which neutrophils alter the adaptive immune response to bacteria by modifying T-cell function.

Protease/antiprotease imbalance

In the normal immune response, the actions of NSPs are tightly regulated by antiproteases. Three key antiproteases that regulate neutrophilic inflammation in the lung are secretory leukocyte protease inhibitor (SLPI), elafin and alpha-1 antitrypsin (AAT). Protease/antiprotease imbalance leading to unopposed NSP activity is probably a contributor to excessive neutrophil-mediated epithelial damage in bronchiectasis [87].

SLPI is an inhibitor of neutrophil elastase and Cat-G, and elafin inhibits both neutrophil elastase and PR-3 [88]. Both are cleaved and degraded by neutrophil elastase, diminishing their ability to mitigate NSP-mediated epithelial damage [89–91]. Deficiency of both these antiproteases is linked to disease severity in COPD and cystic fibrosis [92, 93]. In bronchiectasis, SLPI deficiency is linked to higher BSI, poorer pulmonary function and P. aeruginosa infection (table 1) [26, 48]. In sputum proteomic analysis, SLPI was upregulated in response to antibiotic therapy [29]. SLPI is therefore a potential future tool to identify bronchiectasis endotypes defined by protease/antiprotease imbalance to target anti-inflammatory therapies [87].

Inflammatory cytokines

In addition to neutrophils themselves, multiple inflammatory cytokines contribute to neutrophilic inflammatory pathways in the lung (figure 2). Neutrophils are recruited to the airways by cytokines including leukotriene B4 (LTB4), interleukin (IL)-8, IL-1β and tumour necrosis factor (TNF)-α released by macrophages, endothelial/epithelial cells and other cell types primarily in response to bacteria, but also in the absence of active infection in bronchiectasis [31, 67, 94].

In a small study by Bedi et al. [49], the powerful chemoattractant LTB4 was significantly increased in patients with moderate to severe compared with mild bronchiectasis and healthy controls and correlated with lower FEV1 % predicted and higher number of antibiotic courses for exacerbations. Other studies have shown that sputum IL-8, IL-1β and TNF-α levels correspond to radiological severity and sputum bacterial load and decrease following intravenous and nebulised antibiotic treatment (table 1) [44, 50, 52].

Unanswered questions for markers of neutrophilic inflammation

The described studies have established a clear link between neutrophilic inflammation and bronchiectasis severity and identified multiple candidate biomarkers for development of targeted antineutrophil therapies. Ongoing research must clarify the relationships between these biomarkers and whether all are equally representative of neutrophilic inflammation or whether multiple neutrophilic endotypes exist defined by different neutrophil proteins or processes.

There is evidence that neutrophils in bronchiectasis have altered function. Notably, Bedi et al. [46] reported impaired phagocytosis, delayed apoptosis and prolonged lifespan in neutrophils from bronchiectasis patients compared with controls [46, 95, 96].

A possible mechanism for altered neutrophil function is neutrophil heterogeneity. Discrete neutrophil subsets, identifiable by cell surface markers, are now recognised. Well-characterised neutrophil subsets include the CD63+, CD177+, CXCR4hiCD62Llow subset, and CD64+ subsets. CD63 is a tetraspanin involved in retaining neutrophil elastase in neutrophil granules and surface CD63 expression correlates with extracellular neutrophil elastase release [97, 98]. The CD177+ neutrophil subset expresses PR-3 on the cell surface, which is proteolytically active and more resistant to inhibition than soluble PR-3 [99, 100]. The CXCR4hiCD62Llow subset is indicative of “aged” neutrophils marked for clearance to the bone marrow [100] and Zhang et al. [101] demonstrated that aged neutrophils display increased NETosis. CD64 is a complement receptor responsible for phagocytosis of opsonised pathogens. Additionally, the CD63+ and CXCR4hiCD62Llow subsets both have increased expression of Cd11b, an integrin molecule involved in neutrophil migration and attachment [100].

Changes in neutrophil subsets have been reported in several inflammatory diseases including asthma, COPD and coronavirus disease 2019, but their role in neutrophilic inflammation and clinical outcomes in bronchiectasis has not yet been studied [79, 102–105]. Bedi et al. [46] found increased neutrophil cell-surface CD11b and decreased CD62L expression in severe bronchiectasis. However, other studies have found no difference in neutrophil phagocytosis or CD11b expression in bronchiectasis suggesting further study is needed [95, 96, 106].

Eosinophilic inflammation

Neutrophilic inflammation has been considered the predominant inflammatory endotype in bronchiectasis. However, it has been recognised recently that eosinophilic inflammation also plays a role, at least in a subset of bronchiectasis patients.

Eosinophils are markers of type 2 T-helper (Th2) inflammation which plays a well-established role in atopic and allergic lung diseases [107]. Sputum eosinophilia is the gold standard for identifying asthma and COPD endotypes with improved response to inhaled corticosteroids [108]. In bronchiectasis patients without asthma, sputum eosinophilia has been linked to greater bronchodilator reversibility and to other sputum markers of Th2 response including IL-13 [109, 110].

In a European multicohort study of 1007 patients with bronchiectasis, Shoemark et al. [53] found that 22.6% of patients with bronchiectasis had raised sputum eosinophils >3%. Importantly, they showed that a blood eosinophil count >300 cells·µL−1 correlated with sputum eosinophilia, making it a credible biomarker of airway Th2 inflammation. Low blood eosinophil counts were linked to disease severity and mortality and, following antibiotic treatment for P. aeruginosa, eosinophilic patients had shorter time to first exacerbation compared to non-eosinophilic patients.

Exhaled nitric oxide fraction (FENO) is an additional marker of airway Th2 inflammation already routinely used as a point-of-care test in asthma [111]. In an observational study of 249 bronchiectasis patients, Oriano et al. [55] identified that a Th2-high endotype defined by increased FENO ≥25 dpp and blood eosinophils >300 cells·μL−1 was present in 31% of bronchiectasis patients without asthma. In this study, Th2 inflammation was associated with higher BSI score, lower FEV1 % predicted and increased dyspnoea.

Existence of an eosinophilic phenotype identifiable by easily obtainable blood and FENO tests has important implications for treatment. Treatments used for Th2-mediated lung disease, including corticosteroids and biological agents, are not currently recommended in bronchiectasis [1, 2]. Post hoc analysis of a randomised control trial of inhaled fluticasone in bronchiectasis found that patients with blood eosinophils >3% had a greater improvement in quality of life compared with noneosinophilic patients [54]. A case series of 12 patients showed improvement in lung function, chronic symptoms and exacerbations following treatment with the monoclonal antibodies mepolizumab and benralizumab in bronchiectasis patients with blood eosinophils >300 cells·μL−1 [112]. Large-scale prospective studies are required to confirm these reports. Further investigation of the interaction between Th1 and Th2 inflammatory endotypes and their respective biomarkers is also needed to guide therapies targeting Th2 inflammation.

Systemic inflammatory markers

A number of studies have examined “routine” blood tests and their relationship to disease characteristics (table 1). Blood white cell and neutrophil counts were shown to rise in acute bronchiectasis exacerbations, fall in response to therapy, and correlate with radiological severity and poorer pulmonary function [17, 63, 68, 69]. Additionally, a study of 802 patients from the Spanish Registry of Bronchiectasis (RIBRON) found that chronically raised C-reactive protein (CRP) was associated with increased risk of severe exacerbations [113]. Raised CRP has also been found to predict bacterial infection, decrease in response to antibiotic therapy and correlate with radiological disease severity [64, 65, 114]. However, these markers are induced by multiple inflammatory processes and lack specificity to identify distinct bronchiectasis endotypes that will respond to targeted anti-inflammatory therapies.

Fibrinogen, a key component of the coagulation cascade as well as acute phase reactant, has been suggested as a biomarker of a pro-inflammatory phenotype in COPD [115, 116]. In bronchiectasis, Lee et al. [72] found that fibrinogen was associated with increased BSI and FACED scores, as well as increased exacerbation risk. Principal component analysis of 31 proteins from 90 bronchiectasis patients conducted by Saleh et al. [73] also distinguished fibrinogen as a marker of disease severity, driven primarily by poorer lung function and P. aeruginosa colonisation. Similarly, erythrocyte sedimentation rate, which is dependent primarily on the concentration of circulating fibrinogen, has been shown to mark exacerbations and treatment response in bronchiectasis [67, 68].

Aα-Val360, produced specifically when fibrinogen is cleaved by neutrophil elastase, has been measured in the plasma of COPD patients and found to correlate with multiple severity markers and rise during exacerbations, although it has not yet been studied in bronchiectasis [117, 118]. Another degradation product, desmosine, which is released when elastin (a key component of the extracellular matrix in blood vessels and the lung) is degraded by neutrophil elastase, was found to correlate with both sputum neutrophil elastase and severe exacerbations in bronchiectasis [39, 119]. In cardiovascular disease, plasma desmosine is associated with greater atherosclerotic burden and poorer outcomes after myocardial infarction [120, 121]. In bronchiectasis, Huang and co-workers [74, 120, 121] demonstrated a link between serum desmosine and increased cardiovascular mortality, including when other risk factors such as age and comorbidities were adjusted for.

Systemic inflammation is also associated with platelet aggregation at sites of infection [122]. In a study by Aliberti et al. [70] of 1771 bronchiectasis patients, those with thrombocytosis experienced more severe disease, poorer quality of life, higher exacerbation risk severity and higher 3- and 5-year mortality. Méndez et al. [71] found that soluble p-selectin, a marker of platelet activation also involved in leukocyte recruitment, was increased in severe bronchiectasis, further supporting the association between platelet aggregation and activation and bronchiectasis severity.

Levels of intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 are also upregulated through inflammatory pathways in bronchiectasis [67, 123]. In particular, circulating ICAM-1, which is released by endothelial cells and immune cells and regulates neutrophil transmigration into the airways, is associated with radiological severity and sputum bacterial load and decreases following treatment with intravenous and nebulised antibiotics [44, 50, 52, 124]. Both ICAM-1 and VCAM-1 are linked to vascular inflammation and cardiovascular disorders [125].

Bronchiectasis is associated with increased cardiovascular risk. ∼30% of patients with bronchiectasis have a cardiovascular comorbidity and this risk increases with disease severity [12, 126, 127]. These markers provide insight into possible mechanisms behind the relationship between neutrophilic lung inflammation, systemic inflammation, exacerbations and cardiovascular disease and may in future aid risk stratification and targeted interventions to address cardiovascular risk in bronchiectasis [128].