Abstract
Background
Bronchiectasis is a neutrophil-driven inflammatory chronic respiratory disease. While neutrophil elastase has been deeply investigated, other neutrophil serine proteases are less studied. The association between airway cathepsin G and clinical outcomes in bronchiectasis has not been described. Is cathepsin G sputum activity associated with clinical outcomes in bronchiectasis?
Methods
This international, multicentre, prospective cohort study enrolled bronchiectasis patients from three bronchiectasis programmes in Milan (Italy), Dundee (UK) and Barcelona (Spain). Participants were stratified into three groups based on sputum cathepsin G activity measured using immunoassays. Data on clinical features, sputum characteristics, exacerbations, hospitalisations and mortality were collected over a 3-year follow-up period.
Results
The median (interquartile range) activity of cathepsin G in sputum was 45.1 ng·mL−1 (1.3–128.8 ng·mL−1). Patients with higher sputum cathepsin G activity demonstrated significantly worse clinical outcomes in terms of disease severity (measured by the Bronchiectasis Severity Index, E-FACED, the modified Reiff score, % predicted forced expiratory volume in 1 s and the prevalence of chronic bacterial infections, particularly Pseudomonas aeruginosa) and disease activity (daily produced sputum volume and quality of life scores). Additionally, high cathepsin G activity was linked to more frequent exacerbations during the 3-year follow-up (incidence rate ratio 1.62, 95% CI 1.29–2.07) and a greater risk of hospitalisation due to severe exacerbations (incidence rate ratio 2.32, 95% CI 1.03–5.21). A strong correlation was found between sputum cathepsin G and neutrophil elastase activities.
Conclusion
Sputum cathepsin G activity relates to disease severity, disease activity and prognosis in bronchiectasis. High cathepsin G levels are associated with more severe clinical outcomes and could serve as a target for future therapeutic interventions, including dipeptidyl peptidase 1 inhibitors.
Shareable abstract
Sputum cathepsin G activity is associated with disease severity, disease activity and prognosis in bronchiectasis. High cathepsin G levels could serve as a target for future therapeutic interventions, including DPP1 inhibitors. https://bit.ly/416Nbc5
Introduction
Bronchiectasis is a chronic respiratory disease characterised by the radiological evidence of irreversible airway enlargement accompanied by productive cough and pulmonary exacerbations [1]. Although once considered rare, bronchiectasis is now recognised as a significant contributor to respiratory morbidity, with rising incidence and prevalence globally and growing awareness of its burden on patient quality of life, healthcare resources and mortality risk [2]. Nevertheless, the treatment landscape remains limited, and commonly prescribed medications are largely focused on symptom control and chest infection prevention [3–5].
Airway inflammation plays a cornerstone role in bronchiectasis pathogenesis, driving the vicious vortex of infections, mucociliary clearance impairment and lung destruction [6, 7]. Specifically, neutrophilic airway inflammation is the most common endotype in bronchiectasis, with dysfunctional neutrophils representing the most abundant immune cells in the airways of these patients [8, 9]. Dysregulated neutrophils can potentially harm the airways through the release of serine proteases [10]. Modulation of serine proteases by dipeptidyl peptidase 1 (DPP1) inhibitors has recently emerged as a novel therapeutic option in bronchiectasis. Successful phase 2 and phase 3 trials have demonstrated a reduction of exacerbation frequency and a slowing of lung function decline in patients with bronchiectasis treated with DPP1 inhibitors [11–13]. Based on these results, the DPP1 inhibitor brensocatib has recently been approved by both the US Food and Drug Administration and the European Medicines Agency for the treatment of bronchiectasis.
Neutrophil elastase (NE) represents the most well-studied neutrophil serine protease, and its sputum activity has been associated with higher disease severity and poor prognosis in previous studies [14, 15]. Cathepsin G (CatG) is a serine protease released from the azurophilic granules of neutrophils, with potent bactericidal and tissue-degrading properties. In vitro studies have demonstrated its capacity to degrade elastin fibres; destroy epithelial cells, causing ciliated cell dysfunction and ineffective Pseudomonas aeruginosa clearance; and sustain the inflammatory response through the upregulation of interleukin (IL)-36, IL-1 and IL-33 [16–19]. These in vitro studies suggest it is likely that CatG is involved in the pathophysiology of bronchiectasis and perpetuates the vicious vortex of disease. Furthermore, DPP1 inhibitors reduce CatG alongside NE [20]. By contrast, clinical trials of selective NE inhibition in bronchiectasis have achieved only modest or inconsistent clinical benefits, suggesting that the compensatory activity of other neutrophil serine proteases, such as CatG, may limit the efficacy of elastase-specific approaches [21, 22]. However, no clear association between CatG activity in sputum and clinical outcomes in bronchiectasis has been demonstrated to date.
The aim of our study was to describe the associations between CatG activity in the sputum of patients with bronchiectasis and disease severity and activity, as well as clinical outcomes.
Methods
An international, multicentre, prospective, observational cohort study was conducted within three bronchiectasis programmes: the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan (Italy); Ninewells Hospital, Dundee (UK); and the Hospital de la Santa Creu i Sant Pau, Barcelona (Spain). Consecutive adults (≥18 years old) with productive cough with or without recurrent pulmonary exacerbations and with radiological evidence of bronchiectasis in at least one pulmonary lobe on high-resolution computed tomography were enrolled during clinical stability between March 2017 and May 2019 if they were able to produce sputum. Patients were followed up for 3 years. Clinical stability at baseline was defined by the absence of antibiotic treatment for exacerbation for at least 4 weeks prior to the first visit. Patients with a history of cystic fibrosis or those with traction bronchiectasis due to pulmonary fibrosis were excluded from the study. The study was approved by the ethical committees of the three participating hospitals, and all participants provided written informed consent.
Data collection and study definitions
Enrolled patients underwent clinical, radiological, microbiological and functional evaluations according to local standard operating procedures as recommended by the latest international guidelines [3, 4]. Demographics, comorbidities, disease severity and activity, bronchiectasis aetiology, clinical status and radiological, functional and microbiological sputum evaluations were recorded during stable state. Quality of life and clinical outcomes, including mild/moderate exacerbations, severe exacerbations requiring hospital admissions and all-cause mortality, were recorded during a 3-year follow-up period.
Bronchiectasis severity was evaluated according to both the Bronchiectasis Severity Index (BSI) and the E-FACED multidimensional scores [23, 24]. Radiological severity was assessed using the modified Reiff score, which accounts for the number of involved lobes (with the lingula being considered as a separate lobe) and the degree of dilatation (score range: 1–18) [25]. The burden on quality of life was measured through the Quality of Life-Bronchiectasis Respiratory Symptoms Scale (QoL-B RSS) [26]. Exacerbations were defined as a deterioration in three or more key bronchiectasis symptoms (among cough, change in sputum volume and/or consistency, sputum purulence, breathlessness and/or exercise tolerance, fatigue and/or malaise, haemoptysis) for at least 48 h with a required change in treatment determined by the clinician [27]. Exacerbations were considered severe when patients required hospital admission. Patients were considered “frequent exacerbators” if they experienced three or more exacerbations during the year preceding the enrolment [28].
Bacteriology was performed on spontaneous sputum samples during stable state. Chronic infection was defined by the isolation of the same microorganism on sputum by standard microbiology on two or more occasions during steady state, at least 3 months apart over the 1-year period prior to enrolment [1]. Patients unable to provide sputum samples during the 1-year period before enrolment due to absence of productive cough were classified as not having chronic infection for the purpose of the analysis.
Evaluations of CatG and NE activity in sputum
Spontaneous sputum samples were obtained from all patients at baseline. Sputum was diluted 1:8 in PBS, vortexed and centrifuged at 3000×g for 15 min. Samples were stored at −80°C for all subsequent analyses. Evaluation of CatG was performed on the sputum supernatant for all patients using the ProteaseTag Active Cathepsin G Immunoassay (Proaxis, Belfast, UK). Soluble sputum samples were assayed at further dilutions ranging from 1:5 to 1:50 according to the detected activity of CatG. Assays that remained below the lowest limit of detection at 1:5 dilutions were recorded as zero for the purpose of analysis. Once CatG was evaluated, if enough sample was available, a quantification of NE was also performed on the same sputum sample using the ProteaseTag Active Neutrophil Elastase Immunoassay (Proaxis). Both tests were used as per manufacturer's instructions [29].
Study groups and end-points
Patients were divided into three study groups based on the tertiles of active CatG concentration. This approach was chosen to allow comparison across increasing biomarker levels in the absence of validated clinical cut-off values and to avoid outcome-driven threshold selection in this exploratory analysis. The primary end-point of the study was the association of CatG activity with the exacerbation rate during 3-year follow-up. Secondary end-points of the study included the association of CatG activity with markers of disease severity (including BSI, E-FACED, modified Reiff score, forced expiratory volume in 1 s (FEV1), modified Medical Research Council dyspnoea scale (mMRC) score, body mass index (BMI) and the presence of chronic bacterial infections) and activity (including volume of daily produced sputum, quality of life expressed by the QoL-B RSS, exacerbations and hospital admissions due to severe exacerbations) at baseline [7], as well as hospitalisations and all-cause mortality during follow-up. Although disease severity and disease activity represent partially overlapping constructs, they capture distinct dimensions of bronchiectasis. Disease severity markers were selected to reflect overall disease burden and long-term prognosis, whereas disease activity markers were chosen to reflect current inflammatory and symptomatic burden, in line with previous studies and expert consensus [7]. Finally, an analysis of the correlation between CatG and NE activities was performed.
Statistical analysis
An ad hoc electronic form extracted from the EMBARC database was used to collect study variables. Absolute and relative (percentage) frequencies and medians (interquartile ranges (IQRs)) were respectively used to summarise qualitative and quantitative variables. Chi-squared or Fisher exact tests were used on qualitative variables, when appropriate, whereas Kruskal–Wallis H test or ANOVA were used on quantitative ones according to data distribution. Correlation of CatG with continuous variables was performed using the Spearman correlation. A negative binomial regression model was used to test the association between CatG activity and exacerbations during follow-up through incidence rate ratios. For all-cause mortality, exploratory Cox proportional hazards models adjusted for age and sex were performed. A two-tailed p-value <0.05 was considered statistically significant. The primary outcome was prespecified and analysed without adjustment for multiple comparisons. For secondary analyses, the false discovery rate (FDR) was controlled using the Benjamini–Hochberg procedure (FDR q=0.05), applied within families of related comparisons, including baseline demographic characteristics, disease severity markers and disease activity markers. Both nominal and Benjamini–Hochberg-adjusted p-values were considered when interpreting the results. The statistical software SPSS version 29.0.2.0 (IBM Corporation, Armonk, NY, USA) was used for all statistical computations, while GraphPad Prism version 10.3.1 (GraphPad Software, LLC, San Diego, CA, USA) was used for plotting. No preliminary studies were found on CatG in bronchiectasis. For this reason, the sample size was based on sample sizes of similar studies published in the literature [14, 15, 30].
Results
Among the 389 patients enrolled (248 female (63.8%), median age: 68.0 years (IQR 59.5–74.0 years)), 167 (42.9%) were from Milan, 156 (40.1%) from Dundee and 66 (17.0%) from Barcelona. The median BSI score was 6.0 (IQR 4.0–9.0), with 113 patients (29.4%) belonging to the severe BSI risk class. The median E-FACED score was 2.0 (IQR 1.0–4.0), with 20 patients (5.2%) reported to have a severe disease based on E-FACED. 186 patients (47.8%) had a chronic bacterial infection, with 88 (22.6%) having chronic P. aeruginosa infection. 143 patients (37.0%) were frequent exacerbators, while 76 (19.6%) had been hospitalised in the previous year for severe exacerbations. Additional features of the study population are reported in table S1.
Distribution of sputum CatG in the study population
The median concentration of active CatG in the sputum of the entire population was 45.1 ng·mL−1 (IQR 1.3–128.8 ng·mL−1). The study population was divided in three groups according to tertiles distribution: patients with CatG <11.0 ng·mL−1 (n=128) were assigned to the “Low CatG” group, those with CatG between 11.0 and 97.0 ng·mL−1 (n=133) to the “Medium CatG” group, and those with CatG >97.0 ng·mL−1 (n=128) to the “High CatG” group. Patient characteristics of the three study groups are reported in table 1. No differences in terms of sex, age and previous or current smoking were found across the study groups.
TABLE 1.
Demographics, disease severity and disease activity characteristics of the study population according to sputum CatG activity
| Overall | Sputum CatG | p-value | Adjusted p-value# | |||
|---|---|---|---|---|---|---|
| Low | Medium | High | ||||
| Total participants, n | 389 | 128 | 133 | 128 | ||
| Demographics | ||||||
| Females | 248 (63.8) | 72 (56.3) | 93 (69.9) | 83 (64.8) | 0.07 | 0.18 |
| Age, years | 68.0 (59.5–74.0) | 68.0 (59.0–75.0) | 67.0 (57.5–73.0) | 68.0 (60.3–75.0) | 0.45 | 0.52 |
| Age >65 years | 226 (58.1) | 78 (60.9) | 72 (54.1) | 76 (59.4) | 0.52 | 0.52 |
| BMI, kg·m−2 | 24.1 (20.8–27.5) | 25.0 (22.7–29.9) | 24.2 (21.1–26.8) | 22.0 (19.0–26.0) | <0.001 | <0.001 |
| Former or current smoker | 160 (41.5) | 61 (48.0) | 52 (39.7) | 47 (36.7) | 0.17 | 0.28 |
| Disease severity | ||||||
| BSI score | 6.0 (4.0–9.0) | 5.0 (4.0–8.0) | 6.0 (4.0–9.0) | 8.0 (5.0–12.0) | <0.001 | <0.001 |
| BSI risk class | <0.001 | <0.001 | ||||
| Mild | 124 (32.2) | 53 (42.4) | 44 (33.1) | 27 (21.3) | ||
| Moderate | 148 (38.4) | 50 (40.0) | 53 (39.8) | 45 (35.4) | ||
| Severe | 113 (29.4) | 22 (17.6) | 36 (27.1) | 55 (43.3) | ||
| E-FACED score | 2.0 (1.0–4.0) | 2.0 (1.0–3.0) | 2.0 (1.0–3.0) | 3.0 (1.0–5.0) | <0.001 | <0.001 |
| E-FACED risk class | <0.001 | <0.001 | ||||
| Mild | 277 (72.3) | 102 (82.3) | 99 (75.6) | 76 (59.4) | ||
| Moderate | 86 (22.5) | 16 (12.9) | 31 (23.7) | 39 (30.5) | ||
| Severe | 20 (5.2) | 6 (4.8) | 1 (0.8) | 13 (10.2) | ||
| Reiff score | 4.0 (2.0–6.0) | 3.0 (2.0–4.0) | 4.0 (2.0–6.0) | 4.0 (3.0–8.3) | <0.001 | 0.002 |
| FEV1 % predicted (mean±sd) | 75.2 (25.0) | 76.0 (24.8) | 78.6 (22.8) | 71.0 (27.0) | 0.04 | 0.06 |
| FEV1 <50% predicted | 66 (17.2) | 19 (15.1) | 15 (11.5) | 32 (25.0) | 0.01 | 0.02 |
| FEV1 <35% predicted | 25 (6.5) | 10 (7.9) | 3 (2.3) | 12 (9.4) | 0.05 | 0.06 |
| mMRC score | 1.0 (0.0–2.0) | 1.0 (0.0–2.0) | 1.0 (0.0–2.0) | 1.0 (0.0–2.0) | 0.14 | 0.16 |
| mMRC 3–4 | 66 (17.0) | 26 (20.5) | 14 (10.5) | 26 (20.3) | 0.05 | 0.06 |
| Chronic bacterial infection | 186 (47.8) | 35 (27.3) | 61 (45.9) | 90 (70.3) | <0.001 | <0.001 |
| Pseudomonas aeruginosa | 88 (22.6) | 8 (6.3) | 26 (19.5) | 54 (42.2) | <0.001 | <0.001 |
| Staphylococcus aureus | 24 (6.2) | 5 (3.9) | 12 (9.0) | 7 (5.5) | 0.24 | 0.26 |
| Haemophilus influenzae | 62 (15.9) | 17 (13.3) | 24 (18.0) | 21 (16.4) | 0.59 | 0.59 |
| Disease activity | ||||||
| Daily sputum volume, mL | 14.5 (5.0–30.0) | 10.0 (5.0–20.0) | 12.0 (5.0–45.0) | 17.5 (5.0–42.5) | 0.005 | 0.03 |
| QoL-B RSS score | 66.7 (51.0–77.8) | 70.4 (59.3–77.8) | 70.4 (51.9–77.8) | 66.7 (40.7–74.1) | 0.04 | 0.09 |
| Exacerbations in the previous year, n | 2.0 (1.0–3.0) | 1.0 (1.0–3.0) | 2.0 (1.0–3.0) | 2.0 (1.0–3.0) | 0.74 | 0.74 |
| ≥3 exacerbations in the previous year | 143 (37.0) | 44 (34.6) | 49 (37.1) | 50 (39.4) | 0.19 | 0.24 |
| ≥1 hospitalisation previous year | 76 (19.6) | 23 (18.1) | 20 (15.2) | 33 (25.8) | 0.08 | 0.13 |
Data are presented as median (interquartile range) or n (%), unless otherwise indicated. BMI: body mass index; BSI: Bronchiectasis Severity Index; CatG: cathepsin G; FEV1: forced expiratory volume in 1 s; mMRC: modified Medical Research Council dyspnoea scale; QoL-B RSS: QoL-B Respiratory Symptoms Scale. #: adjusted for multiple comparisons using the Benjamini–Hochberg procedure (false discovery rate 5%).
Sputum CatG and disease severity
An analysis of the association between CatG activity in sputum and disease severity is provided in table 1 and figure 1. CatG activity was associated with an increase in both the BSI (Low CatG 5.0 (IQR 4.0–8.0) versus Medium CatG 6.0 (IQR 4.0–9.0) versus High CatG 8.0 (IQR 5.0–12.0); p<0.001) and the E-FACED (Low CatG 2.0 (IQR 1.0–3.0) versus Medium CatG 2.0 (IQR 1.0–3.0) versus High CatG 3.0 (IQR 1.0–5.0); p<0.001) scores. Similarly, the proportion of patients with severe scores in both the both BSI (Low CatG 17.6% versus Medium CatG 27.1% versus High CatG 43.3%; p<0.001) and E-FACED (Low CatG 4.8% versus Medium CatG 0.8% versus High CatG 10.2%; p<0.001) increased according to CatG activity.
FIGURE 1.
Distribution of disease severity and activity markers according to cathepsin G (CatG) activity distribution. a) Bronchiectasis Severity Index (BSI). b) E-FACED. c) Modified Reiff score. d) Forced expiratory volume in 1 s (FEV1) % predicted. e) Body mass index (BMI). f) Chronic Pseudomonas aeruginosa infection. g) Daily produced sputum volume. h) Quality of Life-Bronchiectasis Respiratory Symptoms Scale (QoL-B RSS). Unadjusted p-values are reported.
In terms of radiological severity, CatG activity was associated with an increase in the modified Reiff score (Low CatG 3.0 (IQR 2.0–4.0) versus Medium CatG 4.0 (IQR 2.0–6.0) versus High CatG 4.0 (IQR 3.0–8.3); p<0.001). In terms of functional impairment, higher CatG activity was associated with lower FEV1 % predicted at nominal analysis (Low CatG 76.0±24.8% versus Medium CatG 78.6±22.8% versus High CatG 71.0±27.0%; p=0.04); however, this association did not remain statistically significant after correction for multiple comparisons. No difference in the mMRC score was detected between the groups (Low CatG 1.0 (IQR 0.0–2.0) versus Medium CatG 1.0 (IQR 0.0–2.0) versus High CatG 1.0 (IQR 0.0–2.0); p=0.14). The proportion of patients with chronic bacterial infection (Low CatG 27.3% versus Medium CatG 45.9% versus High CatG 70.3%; p<0.001), and especially P. aeruginosa (Low CatG 6.3% versus Medium CatG 19.5% versus High CatG 42.2%; p<0.001), increased according to CatG activity. Finally, patients in the Low CatG group had a higher BMI (25.0 kg·m–2, IQR 22.7–29.9 kg·m–2) than patients belonging to the Medium CatG (24.2 kg·m–2, IQR 21.1–26.8 kg·m–2) and High CatG (22.0 kg·m–2, 19.0–26.0 kg·m–2) groups (p<0.001). Similarly, the proportion of underweight patients was higher in the High CatG group (18.8%) compared to the Medium CatG (8.4%) and the Low CatG (5.6%) groups (p=0.002).
Sputum CatG and disease activity
An analysis of the association between CatG activity in sputum and markers of disease activity is provided in table 1 and figure 1. Increased CatG activity was associated with an increase in daily sputum volume (Low CatG 10.0 mL (IQR 5.0–20.0 mL) versus Medium CatG 12.0 mL (IQR 5.0–45.0 mL) versus High CatG 17.5 mL (IQR 5.0–42.5 mL); p=0.005). Also, higher CatG activity was associated with worse QoL-B RSS scores at nominal analysis (Low CatG 70.4 (IQR 59.3–77.8) versus Medium CatG 70.4 (IQR 51.9–77.8) versus High CatG 66.7 (IQR 40.7–74.1); p=0.036); however, this association did not remain statistically significant after correction for multiple comparisons. No clear association between CatG activity and either exacerbations or severe exacerbations in the previous year was found (table 1).
Sputum CatG and NE
NE was evaluated in 279 patients in our cohort. No differences in terms of BSI score were detected between patients with (6.0, IQR 4.0–9.0) and without (6.0, IQR 4.0–10.0) NE data available (p=0.83). The median activity of NE was 8.9 μg·mL−1 (IQR 1.6–22.7 μg·mL−1). A strong correlation between CatG and NE was found for patients in whom both biomarkers were evaluated (Spearman rho: 0.81, p<0.001; figure 2). Analyses of the association between NE activity in the sputum and population features, disease severity and disease activity markers were performed and are provided in the supplement.
FIGURE 2.
Spearman correlation between neutrophil elastase (NE) and cathepsin G (CatG). R=0.81, p<0.0001. The red box highlights the area containing samples with low NE and high CatG, defined based on the medians of the study population.
CatG and long-term outcomes
Long-term outcome data were available for 352 of 389 patients (90.5%) for exacerbations, 265 patients (68.1%) for hospitalisations and 374 patients (96.1%) for all-cause mortality. Enrolled patients experienced 2.0 (IQR 1.0–5.0) exacerbations during the 3-year follow-up period. Patients with high CatG activity showed a higher rate of exacerbations during follow-up than patients with low CatG (incidence rate ratio (IRR) 1.62, 95% confidence interval (CI) 1.29–2.07; table 2). A significant statistical interaction was found for the centre in which patients were enrolled (p<0.001). Moreover, those belonging to the High CatG group had higher rates of severe exacerbations requiring hospital admissions than those in the Low CatG group (IRR 2.32, 95% CI 1.03–5.21; table 2). 31 patients (8.8%) died during the study period, with reported mortalities of 10.2% in the Low CatG group, 4.1% in the Medium CatG group and 10.5% in the High CatG group. In multivariable negative binomial regression models adjusted for BSI risk classes and sex, patients in the highest CatG tertile had a significantly higher exacerbation rate than those in the lowest tertile (IRR 1.28, 95% CI 1.00–1.64, p=0.047; table 3).
TABLE 2.
Long-term outcomes of patients enrolled in the study according to CatG activity
| IRR (95% CI) | p-value | |
|---|---|---|
| Exacerbations | ||
| Low CatG | 1.0 (reference) | – |
| Medium CatG | 1.19 (0.93–1.52) | 0.18 |
| High CatG | 1.62 (1.29–2.07) | <0.001 |
| Severe exacerbations (hospitalisations) | ||
| Low CatG | 1.0 (reference) | – |
| Medium CatG | 1.10 (0.48–2.52) | 0.82 |
| High CatG | 2.32 (1.03–5.21) | 0.04 |
CatG: cathepsin G; CI: confidence interval; IRR: incidence rate ratio.
TABLE 3.
Multivariable negative binomial regression models linking CatG activity and future exacerbation risk adjusted for BSI risk classes and sex
| Exacerbation IRR (95% CI) |
p-value | |
|---|---|---|
| CatG | ||
| Low CatG | 1.0 (reference) | – |
| Medium CatG | 1.06 (0.84–1.35) | 0.62 |
| High CatG | 1.28 (1.00–1.64) | 0.047 |
| BSI | ||
| Low BSI | 1.0 (reference) | – |
| Medium BSI | 1.12 (0.88–1.43) | 0.34 |
| High BSI | 2.03 (1.58–2.61) | <0.001 |
| Sex | ||
| Female | 1.0 (reference) | – |
| Male | 0.91 (0.74–1.11) | 0.36 |
BSI: Bronchiectasis Severity Index; CatG: cathepsin G; CI: confidence interval; IRR: incidence rate ratio.
Kaplan–Meier curves showed no association between CatG activity and all-cause mortality (figure 3). In exploratory Cox regression analyses adjusted for age and sex, sputum CatG activity was not independently associated with all-cause mortality (medium versus low CatG: hazard ratio 0.52, 95% CI 0.18–1.49; high versus low CatG: hazard ratio 1.34, 95% CI 0.61–2.96; table S2). These findings were consistent with analyses conducted on NE (tables S3 and S4).
FIGURE 3.
Kaplan–Meier curves depicting mortality during the 3-year follow-up period according to the cathepsin G (CatG) activity groups.
Discussion
Our study highlights that CatG measured in sputum is associated with disease severity, disease activity and risk of future exacerbations in bronchiectasis. We have described for the first time CatG sputum activity in a large cohort of bronchiectasis patients from both Northern and Southern European countries. We found a clear association between elevated CatG activity at baseline and increased risk of both exacerbations and hospitalisation during a 3-year follow-up. Furthermore, we found CatG to be related to several markers of both disease severity (BSI, E-FACED, mReiff, FEV1 and chronic bacterial infections, including P. aeruginosa) and disease activity (daily sputum volume and quality of life) at baseline. Finally, we demonstrated a strong correlation between CatG and NE, emphasising the interlaced role of neutrophil serine proteases in bronchiectasis pathogenesis and strengthening the biological plausibility of our results.
We speculate that the observed association between high CatG activity and increased exacerbation risk might be attributed to the proteolytic activity of CatG contributing to tissue destruction, mucus overproduction and impaired bacterial clearance [16–18]. The lack of an association between NE and all-cause mortality, which was demonstrated by Chalmers et al. [14], might be explained by the different thresholds used for defining groups and by the advent of the COVID-19 pandemic during our follow-up. Also, it should be acknowledged that all-cause mortality is a hard end-point, possibly influenced by many external factors, and that having only 31 events during our follow-up may have impacted our analysis. In this context, while NE activity has previously shown discriminatory ability for 3-year all-cause mortality, CatG may primarily reflects airway inflammatory activity and exacerbation susceptibility rather than the complex, multifactorial determinants of all-cause mortality in bronchiectasis. The lack of association between CatG activity and all-cause mortality persisted after adjustment for age and sex, suggesting that age remains the dominant driver of mortality risk in this cohort.
Why should we care about CatG as a biomarker in bronchiectasis if we already have strong data on NE?
The relevance of CatG lies in its potential to complement and refine our understanding of neutrophilic inflammation, which has been traditionally centred on the measurement of NE. Our findings suggest that CatG may offer nonredundant, clinically relevant information for a variety of reasons. Firstly, our data reveal that some patients exhibit elevated levels of CatG despite having low or undetectable NE. This observation implies that CatG could capture traits of neutrophilic inflammation that are not depicted by NE measurement alone. Such a scenario is particularly relevant considering that a quarter of patients enrolled in the WILLOW trial exhibited clinical improvement when treated with the DPP1 inhibitor brensocatib, despite having undetectable NE [11]. Our data sustain the hypothesis that CatG may represent the pivot of an alternative neutrophilic inflammatory pathway. Secondly, our data suggest a potential relationship between CatG activity and symptomatic burden on quality of life, which was evident at nominal analysis but did not remain statistically significant after adjustment for multiple comparisons. This observation should therefore be interpreted as hypothesis-generating. Nonetheless, this association was not proven for NE either in our cohort (see supplementary material) or in other cohorts described in the literature [14, 15]. These findings suggest that CatG might be more disease-specific than NE and are consistent with the results of Cipolla et al. [20], who demonstrated that CatG is the neutrophilic serine protease whose activity is most affected by treatment with brensocatib. Thirdly, the interplay between NE and CatG may reflect a synergistic inflammatory response, as suggested by previous studies conducted in a mouse model of emphysema, in which the combined activity of CatG and NE causes more elastin degradation than either of them independently [31]. Fourthly, the discrepancies between the inconsistent results obtained by clinical trials conducted on specific NE inhibitors in the last decade [21, 22] and the positive results obtained by trials conducted on DPP1 inhibitors (that modulate both NE and CatG) [11, 13] might be interpreted considering the relationship shown in this study between CatG and bronchiectasis disease. For all these reasons, although we acknowledge that the likelihood of CatG being implemented in routine clinical practice in the near future is low, we advocate for the relevance of these data in a translational research context to enrich our understanding of bronchiectasis pathophysiology. Ultimately, the inclusion of CatG within the framework of neutrophilic inflammation contributes to a more comprehensive and nuanced understanding of this complex inflammatory landscape. The intended clinical role of CatG measurement is not as a standalone bedside test, but as a prognostic biomarker reflecti.ng neutrophil-driven disease activity and susceptibility to future exacerbations. Our findings suggest that CatG may be particularly useful for risk stratification and prognostic enrichment of patients with highly active disease.
Why are our data on CatG important if treatments with DPP1 inhibitors show efficacy in frequent exacerbators regardless of serine protease evaluation?
Despite recent DPP1 inhibitor trials not considering serine protease activity as inclusion/exclusion criteria, our data on CatG are relevant for several reasons. Firstly, DPP1 inhibitors are unlikely to exhibit uniform effectiveness in a real-world setting, and identifying both non-responders and super-responders through the measurement of serine proteases, including CatG, could refine our understanding of who benefits most from these treatments. Secondly, our findings align well with the beneficial outcomes observed with DPP1 inhibitor treatments, providing biological plausibility for the efficacy of these interventions and suggesting that FEV1, daily sputum volume and the QoL-B score might be useful tools to predict response to treatment, because they were all related to CatG activity. Thirdly, while DPP1 inhibitors represent a significant advancement, it is conceivable that future immunomodulatory therapies with more precise mechanisms of action will emerge. The characterisation of CatG and its relationship with clinical outcomes could therefore provide the scientific community with valuable insights for developing novel therapeutic strategies targeting different components of airway neutrophilic inflammation. Finally, emerging evidence has highlighted the presence of alternative inflammatory profiles, such as epithelial-driven, mixed or T-helper 2-dominant inflammation, in patients with bronchiectasis [8]. Such patients are likely to exhibit suboptimal responses to DPP1 inhibitors, which predominantly target neutrophil-driven inflammation. Identifying these individuals through biomarkers such as CatG could facilitate a more tailored therapeutic approach, ensuring that the right treatment is administered to the right patient.
This study has several strengths. Firstly, it is an international, multicentre, prospective cohort study involving a large number of consecutive patients evaluated in third-level bronchiectasis programmes from both Northern and Southern European countries with a 3-year follow-up period. Secondly, the use of highly specific assays for both CatG and NE activities provided reliable measures of protease activity in sputum samples. Finally, CatG and NE were evaluated on the same sputum sample for every patient. However, there are also some limitations that we would like to acknowledge. The inclusion of only patients able to provide a sputum sample might have had an impact on the results, possibly representing a selection bias. Furthermore, CatG was only evaluated on a single occasion, and fluctuations of its active concentration on a day-to-day basis, similarly to what has been described for NE [32], could be expected. Finally, routine systemic inflammatory markers were not systematically collected, precluding assessment of the relationship between sputum CatG activity and standard laboratory markers of systemic inflammation. We acknowledge that this study was not designed to develop or validate a comprehensive clinical prediction model, and we have clarified that formal evaluation of model performance should be addressed in future studies.
The results of our study suggest that CatG activity is associated with greater disease severity, higher disease activity and poorer prognosis in people with bronchiectasis. Despite being strongly associated with NE, an evaluation of CatG activity might help provide a more detailed snapshot of the neutrophilic inflammatory landscape in certain patients. Whether CatG has unique pathophysiological effects that could be targeted independently from NE and other neutrophilic serine proteases remains to be seen and should be addressed in future studies.
Footnotes
Provenance: Submitted article, peer reviewed.
Conflict of interest: A. Gramegna reports consultancy fees from Vertex Italia and Vertex International; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Vertex Italia, Vertex International and Chiesi Italia; support for attending meetings and/or travel from Neupharma; participation on a data safety monitoring or advisory board for Vertex Italia; leadership role with the Italian Association for People with Bronchiectasis and the ECFS Standards of Care committee; and is a European Respiratory Society (ERS) Officer. A. Shoemark reports that this work was supported by the ERS through the EMBARC3 consortium (EMBARC3 is supported by project partners Armata, AstraZeneca, Boehringer Ingelheim, Chiesi, CSL Behring, Grifols, Insmed, Janssen, Lifearc, Novartis and Zambon); grants or contracts from AstraZeneca and Lifearc; consultancy fees from Spirovant, Translate Bio and ReCode Therapeutics; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Translate Bio, Ethris and Insmed; and leadership roles with ERS Clinical Research Collaborations (EMBARC, BEATPCD and AMR Lung). F. Blasi reports grants from AstraZeneca, Insmed and GSK; consultancy fees from Menarini, OM Pharma and Boehringer; and payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca, Chiesi, GSK, Grifols, Insmed, Menarini, OM Pharma, Pfizer, Sanofi, Vertex, Viatris and Zambon. J.D. Chalmers reports support for the present manuscript from the Innovative Medicines Initiative and The European Federation of Pharmaceutical Industries and Associations companies under the European Commission-funded Horizon 2020 Framework Program, and by Inhaled Antibiotic for Bronchiectasis and Cystic Fibrosis (grant 115721); grants from AstraZeneca, Boehringer Ingelheim, Genentech, Gilead Sciences, GlaxoSmithKline, Grifols, Biologicals, Inc., Insmed, Inc. and Novartis; consultancy fees from Zambon and Insmed, Inc.; leadership roles with the ERS, British Thoracic Society and the Scottish Government Chief Scientist Office Translational Clinical Studies Committee; and is an associate editor of this journal. S. Aliberti reports grants from Insmed Incorporated, Chiesi, Fisher & Paykel and GSK; royalties or licences from McGraw Hill; consultancy fees from Insmed Incorporated, Insmed Italy, Insmed Ireland Ltd, Insmed Netherlands BV, Zambon S.p.A., AstraZeneca UK Limited, AstraZeneca, Pharmaceutical LP, CSL Behring GmbH, Grifols, Fondazione Internazionale, Menarini, Moderna Italy, Moderna TX, Boehringer Ingelheim, Chiesi Farmaceutica S.p.A., MSD Italia S.r.l., Vertex Pharmaceuticals, Brahms GmbH, Physioassist SAS, AN2 Therapeutics and GlaxoSmithKline S.p.A.; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from GlaxoSmithKline S.p.A., Thermofisher Scientific, Insmed Italy, Insmed Ireland Ltd, Boehringer Ingelheim, Zambon, Vertex Pharmaceuticals and Fondazione Internazionale Menarini; and participation on a data safety monitoring or advisory board for Insmed Incorporated, Insmed Italy, AstraZeneca UK Ltd., MSD Italia S.r.l and Verona Pharma plc. The remaining authors have nothing to disclose.
Support statement: The publication fee for this work was covered by the Italian Ministry of Health's “Ricerca Corrente” funding to the IRCCS Humanitas Research Hospital.
Supplementary material
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