Background
Cystic fibrosis (CF) lung disease begins silently in infancy and is characterized by infection, chronic inflammation, bronchiectasis, progressive lung function decline and intermittent pulmonary exacerbations [
1‐
5]. Early detection and treatment of pulmonary decline is key to optimal long-term outcome; however, physiologic measures of lung function and radiologic outcomes fail to capture the inflammation and infection that begins in infancy and is often silent in CF [
6‐
8]. In addition, with the recent development of medications targeting the genetic mutations in CF, there is a need for outcome measures suitable for infants and young children for clinical trials. There are limited, non-invasive, objective tools available to help identify those CF infants at risk for a more rapidly progressive pulmonary course. This unmet need could be fulfilled by identifying biomarkers of lower airway inflammation and/or injury that are suitable both as “point-of-care” diagnostics and for monitoring longitudinal changes.
Neutrophilic inflammation, enzymatic protease activity and cytokine release are hallmarks of CF lung disease with the presence of neutrophil elastase (NE) in BALF known to be a risk factor for the development of bronchiectasis [
9]. However, surveillance bronchoscopy with lavage is an invasive procedure that is not routinely performed in the United States, making the measurement of NE in young patients challenging. Biomarkers that reflect CF lung disease in easily obtained biological specimens are more feasible for application in both the clinical and research settings. Three biomarkers – desmosine, club cell secretory protein (CCSP) and cathepsin B have previously been associated with CF lung disease; however, their applicability to CF infants has not been investigated [
10‐
18]. We have shown that sputum CCSP concentration was both higher in adults with CF in the outpatient setting compared to during a CF pulmonary exacerbation and negatively associated with sputum NE concentration [
17]. CCSP is produced by bronchial epithelial cells and known to have anti-inflammatory properties and may reflect CF lung disease activity [
17]. We have also shown that both sputum and urinary desmosine concentration decrease significantly during a CF pulmonary exacerbation [
11,
12]. Previous work in premature infants and animals has suggested CCSP, cathepsin B and desmosine may all play a role in the development of chronic lung disease [
19‐
22]. Desmosine is a breakdown product of elastin and is widely described as a non-specific biomarker of lung injury. Cathepsin B is an elastolytic cysteine protease that is linked to remodeling of extracellular matrix, bacterial infection and biofilm formation in the CF airways, specifically involving
Pseudomonas aeruginosa, a known pathogen in CF [
10,
13‐
16]
. These three targeted biomarkers may provide an objective measure of the degree of inflammation or tissue destruction occurring in the lung, and if so, would be invaluable both for clinical management and as surrogate endpoints for clinical trials.
Our objective was to measure the concentration of desmosine, CCSP and cathepsin B during the first 2 years of life and correlate biomarker concentrations with markers of infection, inflammation and lung function. We also sought to compare biomarker concentrations measured in CF infants with those measured in a cohort of healthy infants and in an adult cohort of patients with CF with more established lung disease [
17]. Some of the results of these studies have been previously reported in the form of an abstract [
23].
Discussion
There is an unmet need in this population, with no objective means by which to identify and follow the progression of lung disease in CF infants. Through the comparison to healthy controls and older subjects with CF, our findings suggest that CCSP and cathepsin B are potential biomarkers of lung disease activity in infants and should be investigated in future longitudinal and mechanistic studies.
The search for biomarkers of CF lung disease in infants and children remains challenging as they do not typically expectorate sputum, traditional lung function testing is not attainable, BALF is not routinely obtained, cumulative radiation exposure is a concern and phlebotomy can be difficult [
35‐
38]. In addition to our prior work establishing a relationship between CCSP and desmosine concentration to CF pulmonary exacerbation in older CF patients, additional work in bronchial secretions in cell culture led us to investigate these three targeted biomarkers in a CF infant cohort [
11,
12,
17,
18]. Utilizing quantitative proteomics to investigate the in vitro bronchial epithelial secretome, uteroglobin-related protein 2 (i.e. CCSP) was found to be decreased and cathepsin B was increased in CF secretions compared to non-CF secretions [
18]. These findings were in the absence of inflammatory cells and pathogens, suggesting altered immune and injury repair proteins related to CFTR dysfunction [
18]. These three biomarkers have shown clinical and biological relevance in CF, providing a foundation for this investigation in infants.
Although we did not find that CCSP or desmosine concentration changed significantly over the first 2 years of life, we identified differences in cathepsin B concentration between our CF infants, healthy controls and older subjects with CF. We are the first to report the presence of cathepsin B in the urine and BALF of infants with CF. Although previous work has identified the presence of NE in CF infant BALF, ours is the first to document the presence of a cysteine protease in the CF infant airway [
9]. Cathepsin B concentration was found to be increased in BALF from baboons who subsequently developed BPD, suggesting a role in the development of chronic lung disease [
21]. CF infants had a significantly higher urinary cathepsin B concentration than normal, healthy infants without CF. Lung disease in CF is known to have its onset early in life and the higher concentration of cathepsin B is a potential indication of early airway damage. In addition, the CF infants had significantly lower concentrations of cathepsin B in their urine and BALF compared to an older cohort of subjects with CF who had more established lung disease. Only 5 of the 33 infants with CF in our study were infected with
P. aeruginosa, and these subjects did not have a higher concentration of urinary or BALF cathepsin B, confirming a previous finding that cathepsins are not reliable markers of
P. aeruginosa infection in CF [
39]. Urinary cathepsin B concentration was positively associated with urinary desmosine concentration, confirming the presence of these two deleterious proteins in CF infants. An elevated urinary desmosine concentration in premature infants has been suggested as a biochemical marker of damage to the developing lung, and also has been shown to decrease during inpatient treatment for a CF pulmonary exacerbation in older patients [
12,
22,
40]. Cathepsin B in the airway of subjects with CF is thought to be a marker of inflammation; however, our study was not designed to investigate mechanism of action.
CCSP concentration was significantly increased in infant BALF compared to our older subjects with CF. In addition, BALF CCSP concentration was negatively associated with IL-8 and positively correlated with blood CCSP concentration. Our previous longitudinal studies of sputum CCSP in CF have shown increased concentrations during times of stability compared to hospitalization for a CF pulmonary exacerbation [
17]. CCSP has anti-inflammatory properties and is primarily produced by non-ciliated Club Cells in the conducting airways. Club cells secrete CCSP in very high concentrations in the epithelial lining fluid where it modulates the production and activity of phospholipase A
2, interferon-γ and tumor necrosis factor-α, serving to protect the lung against oxidative stress [
41‐
43]. Given that infants still have relatively healthy airways compared to adults with CF, they may have a higher density of intact Club Cells in the airways, leading to a higher measureable concentration of CCSP. Interestingly, CCSP has been studied in premature infants in the context of the development of bronchopulmonary dysplasia (BPD). CCSP concentration in tracheal aspirates from premature infants increases with age and in response to infection [
19,
44] . Airway CCSP concentration was lower and not inducible in those premature infants who developed BPD or died, compared to those infants who did not develop BPD [
20]. Although we do not have airway CCSP concentration in healthy control infants for comparison, infants with CF may have a surge of CCSP during the first year of life as an anti-inflammatory, anti-infectious response to the development of CF lung disease. The positive correlation between BALF and plasma CCSP concentration suggests that plasma levels may be a surrogate for airway CCSP concentration. Plasma CCSP concentration was also positively associated with bacteria not thought to be pathogenic in CF. Although the mechanistic role of CCSP in the development of CF lung disease is unclear, our findings suggest CF infants have significantly more of this biomarker detected in their airways and this was associated with less airway inflammation.
Our study is not without limitation. Our cohort of CF infants was small, and the subset who received a bronchoscopy with lavage had even more limited numbers. We compared biomarker concentration in two airway specimens (BALF and expectorated sputum) and in two CF populations (adults and children). Comparing the concentration of cellular and non-cellular components in BALF with sputum introduces a dilution factor; however, “normalization” of BALF for epithelial lining fluid (ELF) is not recommended due to a lack of a reliable method for its quantification [
45]. Previous work in CF has determined that sputum may be a sample more highly concentrated in inflammatory mediators than BALF [
46]. In our study, the CCSP concentration in BALF was significantly higher than sputum, and the difference in cathepsin B concentration between BALF and sputum was strengthened by a similar difference in urinary cathepsin B concentration. Although comparing an older CF cohort with CF infants may seem unorthodox, other validated biomarkers of CF lung disease reflect disease activity, regardless of age. Both sputum NE and IL-8 concentrations are negatively associated with lung function and are correlates of disease severity [
36,
47]. We anticipate that CCSP and cathepsin B concentrations may behave in a similar fashion. This study was not designed to elucidate the mechanisms of action of the three targeted biomarkers; instead, we identified differences in cohorts of CF and healthy subjects that can serve to generate hypotheses for further longitudinal and mechanistic studies. Finally, we measured cathepsin B using an ELISA and did not measure protease activity.
Acknowledgements
The authors wish to thank the adults with CF and the families of infants with and without cystic fibrosis who allowed their children to participate in this study. The authors did not receive any financial assistance to conduct this study or write this manuscript.