Rationale: Recent evidence from clinical studies suggests that neutrophil elastase (NE) released in neutrophilic airway inflammation is a key risk factor for the onset and progression of lung disease in young children with cystic fibrosis (CF). However, the role of NE in the complex in vivo pathogenesis of CF lung disease remains poorly understood.
Objectives: To elucidate the role of NE in the development of key features of CF lung disease including airway inflammation, mucus hypersecretion, goblet cell metaplasia, bacterial infection, and structural lung damage in vivo.
Methods: We used the Scnn1b-Tg mouse as a model of CF lung disease and determined effects of genetic deletion of NE (NE−/−) on the pulmonary phenotype. Furthermore, we used novel Foerster resonance energy transfer (FRET)–based NE reporter assays to assess NE activity in bronchoalveolar lavage from Scnn1b-Tg mice and sputum from patients with CF.
Measurements and Main Results: Lack of NE significantly reduced airway neutrophilia, elevated mucin expression, goblet cell metaplasia, and distal airspace enlargement, but had no effect on airway mucus plugging, bacterial infection, or pulmonary mortality in Scnn1b-Tg mice. By using FRET reporters, we show that NE activity was elevated on the surface of airway neutrophils from Scnn1b-Tg mice and patients with CF.
Conclusions: Our results suggest that NE plays an important role in the in vivo pathogenesis and may serve as a therapeutic target for inflammation, mucus hypersecretion, and structural lung damage and indicate that additional rehydration strategies may be required for effective treatment of airway mucus obstruction in CF.
Recent evidence from observational studies in young children with cystic fibrosis (CF) suggests that neutrophil elastase (NE) is a key risk factor for the onset and progression of CF lung disease. NE is a major neutrophil product that has been implicated in inflammation, mucus hypersecretion, structural lung damage, and host defense. However, the role of NE in the complex in vivo pathogenesis of CF lung disease including its potentially protective versus damaging effects remains unknown.
This study identifies NE as a key molecule in the in vivo pathogenesis of airway neutrophilia, mucus hypersecretion, and structural damage, but not mucus obstruction and spontaneous bacterial infection in a mouse model of CF lung disease. These results suggest NE as a promising therapeutic target to inhibit deleterious effects of inflammation without compromising bacterial host defense in CF and potentially other neutrophilic airway diseases. However, our results also show that airway surface dehydration is sufficient to produce mucus obstruction in the absence of mucus hypersecretion indicating that additional rehydration therapies may be necessary for effective treatment of mucus plugging in CF.
Recent observational studies in infants and young children with cystic fibrosis (CF) diagnosed early by newborn screening identified neutrophil elastase (NE) as a key risk factor for the onset and persistence of bronchiectasis (1–3) and studies in older children with CF demonstrated that increased NE activity was associated with subsequent decline in lung function (4). These results indicate that NE plays an important role in the initiation and progression of CF lung disease. NE is a major product of activated neutrophils and has been implicated in the pathogenesis of key features of CF lung disease, such as chronic airway inflammation, mucus hypersecretion and goblet cell metaplasia, and structural lung damage (5–11). Furthermore, recent evidence suggests that NE degrades and disables CF transmembrane conductance regulator (CFTR) Cl− channels and cleavage-activates the epithelial Na+ channel (ENaC) and thereby aggravates the basic ion transport defects that cause airway surface dehydration in CF airways (12–14). In addition, NE has also been implicated as an important player in the defense against bacterial infection associated with CF airway disease (11, 15, 16). However, the relative roles of potentially protective versus damaging properties of NE in the in vivo pathogenesis of CF lung disease remain poorly defined.
The aim of this study was, therefore, to determine the role of NE in the complex in vivo pathogenesis of airway inflammation, mucus obstruction, bacterial infection, and structural lung damage in chronic neutrophilic airway disease. To achieve this goal, we used the βENaC (Scnn1b)–overexpressing mouse that exhibits airway surface dehydration, a key pathophysiologic mechanism in CF (17, 18), and develops CF-like lung disease characterized by early onset airway mucus plugging associated with bacterial infection and spontaneous mortality in neonatal mice, and chronic neutrophilic airway inflammation, mucus hypersecretion, goblet cell metaplasia, and emphysema with distal airspace enlargement, increased compliance, and reduced lung tissue density in adult survivors (19–24). We crossed Scnn1b-Tg mice with NE-deficient (NE−/−) mice and determined effects of genetic NE deletion on inflammatory cell influx, airway morphology, mucin gene expression, airway mucus content, mucus solids concentration, epithelial ion transport, bacterial infection, and distal airspace enlargement. Furthermore, we used novel Foerster resonance energy transfer (FRET) reporters (25, 26) to localize and quantify NE activity at the surface of neutrophils and cell-free supernatant of bronchoalveolar lavage (BAL) from Scnn1b-Tg mice and sputum from patients with CF. The results of these studies yielded novel insights into the role of NE in the in vivo pathogenesis and as a therapeutic target in CF and potentially other neutrophilic airway diseases. Some of the results of these studies have been previously reported in the form of an abstract (27).
All animal studies were approved by the animal welfare authority responsible for the University of Heidelberg. Scnn1b-Tg (19, 28) and NE−/− mice (15) on the C57BL/6 background were intercrossed to generate Scnn1b-Tg/NE−/−, Scnn1b-Tg, NE−/−, and wild-type (WT) mice. Microbiology studies were performed in 4- to 6-day-old neonatal mice. Ion transport studies were performed on tracheal tissues from 5- to 25-week-old adult mice. All other studies were performed in 6-week-old adult mice. Additional details on experimental animals are provided in the online supplement.
BAL was performed as previously described (20). Total cell counts were determined and keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, and DNA concentrations were measured in the cell-free supernatant by ELISA and the DNA stain EvaGreen, respectively. After BAL, lungs were used for measurements of lung volume, mean linear intercepts, destructive index, airway mucus obstruction, and goblet cell counts. Additional information is provided in the online supplement.
Histology and morphometric analyses of airway mucus content and goblet cell metaplasia were performed as previously described (20) and detailed in the online supplement.
Real-time polymerase chain reaction was performed using inventoried TaqMan gene expression assays for Gob5 (Mm01320697_m1), Muc5ac (Mm01276718_m1), Muc5b (Mm00466391_m1), and Gapdh (Mm99999915_g1) following the manufacturer’s instructions (Applied Biosystems, Darmstadt, Germany) and analyzed as previously described (20) and detailed in the online supplement.
Airway mucus was sampled from mouse trachea and wet and dry weights and percent solids were determined as previously described (19) and detailed in the online supplement.
Transepithelial ion transport studies were performed in freshly excised mouse tracheal tissues using perfused micro-Ussing chambers as previously described (29) and detailed in the online supplement.
BAL was performed in 4- to 6-day-old neonatal mice (23), cultured on universal and selective plates, and colonies were counted and classified by morphology and MALDI TOF mass-spectroscopy as described in the online supplement.
Sputum studies in patients with CF and healthy volunteers were approved by the ethics committee of the University of Heidelberg and informed written consent was obtained from all subjects, their parents, or legal guardians. Sputum was obtained from 12 patients with CF and 9 healthy nonsmoking volunteers (non-CF). Sputum and BAL cells were processed as previously described (31). Membrane-associated NE activity on cells isolated from BAL or sputum was determined using the lipidated FRET reporter NEmo-2, and soluble NE activity was determined in cell-free BAL or sputum supernatant using the soluble FRET reporter NEmo-1 (25, 26, 32). The antiprotease capacity of BAL was determined by adding increasing volumes of BAL supernatant to purified human NE in assay buffer. Additional information is provided in the online supplement.
Data were analyzed with GraphPad Prism Version 4.0 (GraphPad Software Inc., La Jolla, CA) and are reported as mean ± SEM. Log-transformed data are shown as scatter plots with median ± interquartile range. Statistical analyses were performed using unpaired Student t test, one-way analysis of variance, Mann-Whitney test, Fischer t test, and Kaplan-Meier analysis as appropriate, and P less than 0.05 was accepted to indicate statistical significance.
To determine the role of NE in chronic airway inflammation, we compared BAL inflammatory cell counts, levels of free DNA, and the neutrophil-chemoattractants KC and MIP-2 in 6-week-old adult Scnn1b-Tg/NE−/− mice with Scnn1b-Tg, WT, and NE−/− littermates (Figure 1). As expected from previous studies, neutrophils were rarely detected in WT and NE−/− mice, but were markedly elevated in Scnn1b-Tg mice (Figure 1B) (19, 20). Airway neutrophilia in Scnn1b-Tg mice was associated with increased levels of free DNA (Figure 1E) (33). In Scnn1b-Tg/NE−/− mice BAL neutrophils and free DNA were significantly reduced compared with Scnn1b-Tg mice (Figures 1B and 1E). Macrophage, eosinophil, and lymphocyte counts were not different between experimental groups (Figures 1A, 1C, and 1D). Neutrophilic airway inflammation in Scnn1b-Tg mice was associated with increased levels of KC and MIP-2 in BAL (Figures 1F and 1G) as previously described (19). In Scnn1b-Tg/NE−/− mice KC and MIP-2 concentrations were reduced when compared with Scnn1b-Tg mice, but remained substantially elevated compared with WT and NE−/− mice (Figures 1F and 1G). Taken together, these results demonstrate that lack of NE reduced chronic neutrophilic airway inflammation in Scnn1b-Tg mice even in the presence of elevated KC and MIP-2 suggesting that NE is important for neutrophil migration from the blood into the airway lumen in this mouse model of CF lung disease.
Besides chronic inflammation, CF airway disease is characterized by mucus hypersecretion and airway mucus obstruction (9, 10). Because NE has been implicated in goblet cell metaplasia and mucin hypersecretion (34, 35), we compared goblet cell counts, mucin transcript expression, and airway mucus content in 6-week-old WT, NE−/−, Scnn1b-Tg, and Scnn1b-Tg/NE−/− mice. Goblet cell numbers and transcript levels of the goblet cell marker Gob5 and the secreted mucins Muc5ac and Muc5b were significantly increased in Scnn1b-Tg mice compared with WT and NE−/− mice (Figures 2A–2E). In Scnn1b-Tg/NE−/− mice goblet cell metaplasia was abrogated and expression of Gob5, Muc5ac, and Muc5b was reduced to near normal levels (Figures 2A–2E). However, mucus plugging remained unchanged and neonatal mortality did not differ in Scnn1b-Tg/NE−/− compared with Scnn1b-Tg mice (Figures 2A, 2F, and 2G).
Mucociliary dysfunction and airway mucus obstruction in CF have been linked to mucus hyperconcentration caused by airway surface dehydration (17–19) and/or increased mucus viscosity caused by elevated free DNA released from airway neutrophils (36). Because DNA concentration in BAL was increased in Scnn1b-Tg, but not in Scnn1b-Tg/NE−/− mice (Figure 1E), we assessed mucus hyperconcentration as an alternative cause of mucus plugging in the absence of mucus hypersecretion in Scnn1b-Tg/NE−/− mice. The mucus solids concentration was approximately 7% in WT, remained normal in NE−/− mice, and was significantly increased to approximately 14% in Scnn1b-Tg mice (19). In contrast to DNA, mucus concentration was elevated to similar levels in Scnn1b-Tg/NE−/− and Scnn1b-Tg mice (Figure 2H). Measurements of epithelial ion transport across freshly excised tracheal tissues demonstrated that the amiloride-sensitive Isc reflecting ENaC-mediated Na+ absorption was increased to similar levels in Scnn1b-Tg/NE−/− and Scnn1b-Tg mice compared with WT and NE−/− control animals (Figure 2I). Furthermore, cAMP-induced Cl− secretory responses and inhibition by the CFTR inhibitor CFTRinh-172 and bumetanide were not different in Scnn1b-Tg/NE−/− compared with Scnn1b-Tg mice (see Figure E1 in the online supplement). These results show that lack of NE had no effect on the hyperabsorptive ion transport phenotype of Scnn1b-Tg mice. Collectively, these results demonstrate that NE plays an important role in the in vivo pathogenesis of mucin hypersecretion and goblet cell metaplasia, and suggest that dehydration of constitutively secreted mucus is sufficient to produce mucus stasis and plugging even in the absence of mucin hypersecretion and elevated DNA.
Mucociliary dysfunction is associated with spontaneous bacterial infection in neonatal Scnn1b-Tg mice (23). Because NE plays an important role in bacterial killing (15, 16), we determined if lack of NE compromised host defense and aggravated spontaneous airway infection in neonatal Scnn1b-Tg mice. Culturing of BAL from 4- to 6-day-old mice revealed low numbers of bacteria from the oropharyngeal flora in WT and NE−/− mice (0–100 cfu per lung). Bacterial counts were significantly increased to similar levels in Scnn1b-Tg and Scnn1b-Tg/NE−/− mice, with up to 5 × 105 cfu per lung (Figures 3A and 3B). Actinobacillus muris and Streptococcus acidominimus were the dominant species (Figure 3C). Although the gram-negative A. muris was most abundant in BAL from WT and NE−/− mice, spontaneous bacterial infection in Scnn1b-Tg and Scnn1b-Tg/NE−/− mice was associated with a significantly higher relative abundance (>50%) of the gram-positive species S. acidominimus (Figure 3C). These results indicate that lack of NE did not aggravate spontaneous bacterial infection caused by airway mucus dehydration and plugging.
Previous studies in Scnn1b-Tg mice, and studies using cigarette smoke exposure, showed that chronic neutrophilic airway inflammation is associated with emphysema in mice (20, 21, 37). To determine if NE contributes to spontaneous emphysema in Scnn1b-Tg mice, we compared lung volume, distal airspace morphology, mean linear intercepts, and the destructive index as a measure of alveolar destruction in 6-week-old Scnn1b-Tg/NE−/− mice with Scnn1b-Tg mice, and WT and NE−/− control animals. Alveolar architecture, lung volume, mean linear intercepts, and destructive index did not differ in WT versus NE−/− mice, but alveolar architecture appeared abnormal and quantitative emphysema parameters were significantly increased in Scnn1b-Tg mice (Figure 4). In Scnn1b-Tg/NE−/− mice, elevated lung volume, mean linear intercepts, and destructive index were significantly reduced compared with Scnn1b-Tg mice (Figures 4B–4D). Taken together, these results indicate that NE released in chronic neutrophilic airway inflammation plays an important role in structural lung damage in CF-like lung disease in mice.
To localize and quantify NE activity in neutrophilic airway inflammation in mice and patients with CF, we used a set of highly sensitive NE-specific ratiometric FRET reporters of the NEmo series developed to measure cell surface-bound (NEmo-2) and soluble (NEmo-1) NE activity (25, 26). Using NEmo-2, we detected NE activity on the surface of BAL neutrophils from 6-week-old Scnn1b-Tg mice, but not on neutrophils from WT mice or NE−/− and Scnn1b-Tg/NE−/− control animals (Figures 5A and 5B). Conversely, no NE activity was detected in BAL supernatant from Scnn1b-Tg mice using either NEmo-1 or zymography (Figure 5C; see Figure E2A). Instead, BAL supernatant from mice of all experimental groups (i.e., WT, NE−/−, Scnn1b-Tg, and Scnn1b-Tg/NE−/−) inhibited the activity of purified NE in a dose-dependent fashion (Figure 5D). These results indicate that mice express high levels of endogenous NE inhibitors that neutralize the activity of secreted NE in the airways (Figure 5D). Because elastin zymography detected lysis bands in the range of the molecular size of several matrix metalloproteinases (see Figure E2A), we also determined matrix metalloproteinases activity in BAL supernatant using a substrate cleaved by a broad spectrum of matrix metalloproteinases (see online supplement). Matrix metalloproteinases activity did not differ in BAL supernatants from WT, NE−/−, Scnn1b-Tg, and Scnn1b-Tg/NE−/− mice indicating that NE had no impact on matrix metalloproteinase activity in Scnn1b-Tg mice (see Figure E2B).
Next, we used NEmo-1 and NEmo-2 to analyze NE activity on neutrophils and in supernatant of sputum from patients with CF and healthy nonsmoking volunteers (non-CF). As expected from previous studies (38), absolute and relative neutrophil counts were substantially higher in human sputum compared with murine BAL (Figure 1B), and were significantly increased in CF (77.8 ± 20.6 × 105 neutrophils per gram of sputum; 88.2 ± 3.4%) versus non-CF sputum (17.8 ± 4.6 × 105 neutrophils per gram of sputum; 60.5 ± 6.2%; P < 0.05 for absolute neutrophil counts per gram of sputum and P < 0.001 for relative neutrophil counts) (see Table E1 and Figure E3). NEmo-2 detected a significant increase of NE activity on the surface of CF neutrophils compared with non-CF control subjects (Figures 5E and 5F). Furthermore, measurements with NEmo-1 demonstrated that soluble NE activity was also increased in the supernatant of CF sputum compared with non-CF control subjects (Figure 5G). Collectively, these FRET reporter studies demonstrate that NE activity is increased at the surface of airway neutrophils from Scnn1b-Tg mice and patients with CF, and suggest that extracellular NE activity is shielded by antiproteases in moderate airway neutrophilia observed in Scnn1b-Tg mice and that antiprotease activity may be exceeded in more severe neutrophilia in patients with CF with chronic lung disease.
Neutrophilic airway inflammation is a hallmark of CF lung disease, NE released from activated neutrophils has been implicated in several key pathologies present in CF airways, and recent clinical studies identified increased NE activity as main risk factor for the onset and progression of lung disease in children with CF (1, 2, 4, 5, 12). However, it remains unknown which of the diverse functions of NE contribute to the association between NE activity and severity of CF lung disease in vivo. In the present study, we therefore determined the effects of genetic deletion of NE on airway inflammation, mucus obstruction, bacterial infection, and structural lung damage using Scnn1b-Tg mice as a model of CF lung disease (19, 22). Our results provide several novel insights into the role of NE in the complex in vivo pathogenesis that may have important implications for the understanding of early CF lung disease and the development of effective therapies.
First, we confirm that NE plays an important role in neutrophilic airway inflammation. Similar to patients with CF, airway surface dehydration and mucociliary dysfunction in Scnn1b-Tg mice is associated with early onset airway neutrophilia and increased levels of free DNA and the CXC chemokines KC and MIP-2 in BAL (Figure 1) (19, 22). We show that genetic deletion of NE reduced neutrophilia and abrogated elevated DNA in BAL from Scnn1b-Tg/NE−/− mice, even in the presence of elevated KC and MIP-2 (Figure 1). Consistent with previous reports (6, 39), our studies using NEmo FRET reporters detected high levels of NE activity at the surface of activated BAL neutrophils from Scnn1b-Tg mice (Figures 5A–5C). These data support an important role of membrane-associated NE activity for transmigration of neutrophils from the blood into the airway lumen in response to the neutrophil chemoattractants KC and MIP-2 secreted by epithelial cells and macrophages in chronic mucostatic airway disease. Of note, previous studies demonstrated that NE was not required for neutrophil recruitment into the lung when mice were challenged with high doses of Pseudomonas aeruginosa (>106 cfu per mouse) or LPS (40). These data suggest that signals released in acute gram-negative infection may induce other factors involved in neutrophil transmigration, such as adhesion molecules, integrins, and cadherins, which may enable neutrophil recruitment even in the absence of NE. Collectively, these results suggest that pharmacologic inhibition of NE activity may be a promising therapeutic strategy to limit neutrophil recruitment and detrimental effects of neutrophil products in the airways of patients with CF who are not infected with P. aeruginosa and potentially in patients in whom infection is controlled.
Second, our results demonstrate that NE activity is a potent stimulus for mucus hypersecretion in CF-like lung disease in vivo. As expected from previous studies (34), neutrophilia was associated with goblet cell metaplasia and elevated transcript levels of the goblet cell marker Gob5 and secreted mucins Muc5ac and Muc5b in airways from Scnn1b-Tg mice (20). Of note, goblet cell metaplasia and increased mucin expression were abrogated in Scnn1b-Tg/NE−/− mice (Figures 2A–2E). However, even in the absence of goblet cell metaplasia and elevated mucin expression, Scnn1b-Tg/NE−/− mice showed severe mucus plugging and mortality similar to Scnn1b-Tg littermates (Figures 2F and 2G). Measurements of the concentration of solids demonstrated that airway mucus was dehydrated to similar levels in Scnn1b-Tg and Scnn1b-Tg/NE−/− mice (Figure 2H). Furthermore, bioelectric studies demonstrated that ENaC-mediated Na+ absorption was increased to similar levels and that cAMP-induced Cl− secretion remained normal in native airway tissues from Scnn1b-Tg and Scnn1b-Tg/NE−/− mice, indicating that lack of NE had no effect on net airway hyperabsorption of ions and fluid in Scnn1b-Tg mice (Figure 2I; see Figure E1). Of note, previous studies demonstrated that free NE activity increased Na+ absorption by proteolytic activation of ENaC and inhibited Cl− secretion by degradation of CFTR (12, 13, 41). In our study, we did not detect free NE activity in BAL from Scnn1b-Tg mice (Figures 5C and 5D), suggesting that the absence of differences in ENaC- and CFTR-mediated currents between Scnn1b-Tg and Scnn1b-Tg/NE−/− airways may be explained by the lack of free NE activity in Scnn1b-Tg mice. Additionally, previous studies demonstrated that airway-specific overexpression of the βENaC subunit in Scnn1b-Tg mice resulted in the expression of a large subpopulation of αβ ENaC channels (42). In contrast to αβγ channels that constitute the dominant population in WT airways, αβ ENaC channels are constitutively active and do not require proteolytic activation by NE or other extracellular proteases (41–43), thus providing an alternative explanation why airway Na+ transport was increased to similar levels in Scnn1b-Tg/NE−/− compared with Scnn1b-Tg mice (Figure 2I). Taken together, these results indicate that ENaC-mediated Na+ hyperabsorption, probably leading to airway surface dehydration, is sufficient to impair clearance of constitutively secreted mucus and produce airway mucus plugging even in the absence of mucus hypersecretion (Figure 2). The importance of proper hydration for effective mucociliary clearance is supported by recent studies of effects of mucus concentration on the biophysical properties of the mucus layer covering airway epithelia (44), and by the lung phenotype of mice deficient of the SLC26A9 Cl− channel that develop mucus plugging caused by a reduced capacity to increase Cl− and fluid secretion in parallel to mucin hypersecretion in allergic airway inflammation (29, 45). These results predict that mucus rehydration by osmotically active agents (17, 46), or restoration of epithelial ion and fluid transport by pharmacologic modulation of CFTR, ENaC, or alternative Cl− channels (47, 48), may be more effective in the treatment of mucus plugging than inhibition of mucus hypersecretion triggered by neutrophilic airway inflammation.
Third, our results provide important information on the in vivo role of NE in bacterial host defense of mucostatic airways. Besides mucociliary clearance, neutrophils constitute an important part of the airways’ innate defense system and NE has been shown to contribute to antibacterial defense against P. aeruginosa (15, 16). Conversely, overwhelming NE activity can cleave complement and chemokine receptors on immune cells, thereby compromising their capacity to kill bacteria (49). We, therefore, tested the impact of genetic deletion of NE on spontaneous bacterial infection associated with mucociliary dysfunction in neonatal Scnn1b-Tg mice (23). Absolute bacterial counts in BAL were low in WT and NE−/− mice and were significantly increased in Scnn1b-Tg mice (Figure 3). Interestingly, similar to patients with CF (50), the relative abundance of species was also changed with S. acidominimus being the most prevalent species in Scnn1b-Tg mice, whereas A. muris was most abundant in WT mice (Figure 3). Of note, neither the absolute nor the relative abundance of bacteria was different in lungs from Scnn1b-Tg/NE−/− compared with Scnn1b-Tg mice (Figure 3). These results demonstrate that lack of NE did not aggravate spontaneous bacterial airway infection associated with mucociliary dysfunction in Scnn1b-Tg mice and indicate that other innate defense mechanisms, such as antimicrobial peptides or macrophage-mediated killing (51), are sufficient to limit bacterial infection in mucostatic airways. These results also suggest that therapeutic targeting of NE as an antiinflammatory strategy may not aggravate bacterial infection in CF lung disease. Of note, in a previous study using an acute pneumonia model, NE was shown to contribute to killing of P. aeruginosa via degradation of the major outer membrane protein F (16), a mechanism that may become relevant in patients with CF infected with P. aeruginosa. Therefore, future preclinical and clinical trials with emerging small molecule NE inhibitors (52–54) are required to exclude potentially adverse effects on host defense in patients with CF.
Fourth, this study demonstrates that NE is implicated in emphysema-like lesions associated with dehydration-induced airway disease in Scnn1b-Tg mice. Increased NE activity has been linked to emphysema pathogenesis based on observations in patients with α1-antitrypsin deficiency and animal studies using intrapulmonary elastase instillation and cigarette smoke exposure in NE−/− mice (37, 55, 56). The present study demonstrates that genetic deletion of NE resulted in a significant reduction of emphysema-like changes in Scnn1b-Tg mice (Figure 4). Of note, in contrast to patients with CF (2, 57), probably because of anatomic differences including a much lower number of airway branching in the mouse compared with the human lung, mice with chronic neutrophilic airway disease do not develop bronchiectasis (20, 21, 37). However, our results (Figure 4), together with results obtained from NE instillation and cigarette smoke exposure (37), indicate that emphysema is a robust read-out for NE-mediated structural lung damage in mice. In this context, our results support the concept that NE plays an important role in the in vivo pathogenesis and is a promising therapeutic target to reduce structural damage associated with neutrophilic airway inflammation, and suggest the Scnn1b-Tg mouse as a useful model for preclinical testing of novel NE inhibitors (54).
Finally, we used a set of NE FRET reporters to assess free (NEmo-1) and neutrophil-bound (NEmo-2) NE activity in the context of CF lung disease (Figure 5). In BAL from Scnn1b-Tg mice showing moderate airway neutrophilia similar to levels observed in early lung disease and infants and young children with CF (i.e., 5–30% neutrophils) (1, 19, 22), NE activity was significantly elevated at the neutrophil surface compared with WT mice, but no free NE activity was detected in cell-free BAL supernatant (Figures 5A–5C). Dose-dependent inhibition of the activity of purified NE by BAL supernatant indicated that NE released from activated neutrophils of Scnn1b-Tg mice was neutralized by antiproteases (Figure 5D). We also show that NEmo-2 enabled detection of increased NE activity on the surface of sputum neutrophils from patients with CF (Figures 5E and 5F). As expected from previous reports (4, 38), the absolute and relative neutrophil counts in sputum from older children and adults with CF included in our study (see Table E1) were substantially higher than in BAL from infants and young children with CF or Scnn1b-Tg mice (1, 19, 22), and higher neutrophil counts were associated with elevated free NE activity in supernatant from CF sputum compared with healthy control subjects (Figure 5G). Collectively, these results are consistent with previous studies that localized high levels of NE activity at the surface of activated blood neutrophils and suggest that free NE activity is inhibited in the extracellular compartment, as long as the antiproteolytic shield of NE inhibitors, such as α1-antitrypsin, is not overwhelmed (6, 39, 58). Furthermore, our results suggest that surface-bound NE activity may play a critical role in tissue destruction in CF lung disease and that NEmo-2 may be a sensitive novel tool to detect increased membrane-bound NE activity in clinical specimens (BAL and sputum) and identify individual patients with CF who may be at greatest risk to develop structural lung damage. However, future studies in a larger number of patients including correlation with severity of lung disease are required to determine the value of membrane-bound NE activity as an inflammation biomarker in CF and potentially other neutrophilic airway diseases.
In summary, our study demonstrates for the first time that NE plays key roles in the in vivo pathogenesis of CF-like lung disease in mice including egression of neutrophils into the airway lumen, goblet cell metaplasia, mucus hypersecretion, and structural abnormalities. Conversely, genetic deletion of NE had no effect on spontaneous bacterial infection, and abrogation of NE-mediated mucus hypersecretion did not prevent dehydration-induced airway mucus obstruction in Scnn1b-Tg mice. These results suggest that NE is a promising therapeutic target to reduce inflammation, mucus hypersecretion, and structural lung damage in CF and potentially other neutrophilic airway diseases without comprising antibacterial host defense. However, our results also indicate that additional rehydration therapies may be necessary for effective treatment of airway mucus plugging.
The authors thank R. Pepperkok (Advanced Light Microscopy Facility) at European Molecular Biology Laboratory, Heidelberg for expert assistance in microscopy; S. Zimmermann for helpful advice for microbiology studies; L. Malleret for performing zymography; and M. Wiebel, C. Prat-Knoll, C. Joachim, and A. Hövel for assistance in sputum collection.
|1.||Sly PD, Brennan S, Gangell C, de Klerk N, Murray C, Mott L, Stick SM, Robinson PJ, Robertson CF, Ranganathan SC; Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST-CF). Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening. Am J Respir Crit Care Med 2009;180:146–152.|
|2.||Sly PD, Gangell CL, Chen L, Ware RS, Ranganathan S, Mott LS, Murray CP, Stick SM; AREST CF Investigators. Risk factors for bronchiectasis in children with cystic fibrosis. N Engl J Med 2013;368:1963–1970.|
|3.||Grasemann H, Ratjen F. Early lung disease in cystic fibrosis. Lancet Respir Med 2013;1:148–157.|
|4.||Sagel SD, Wagner BD, Anthony MM, Emmett P, Zemanick ET. Sputum biomarkers of inflammation and lung function decline in children with cystic fibrosis. Am J Respir Crit Care Med 2012;186:857–865.|
|5.||Voynow JA, Fischer BM, Zheng S. Proteases and cystic fibrosis. Int J Biochem Cell Biol 2008;40:1238–1245.|
|6.||Owen CA. Roles for proteinases in the pathogenesis of chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 2008;3:253–268.|
|7.||Pham CT. Neutrophil serine proteases: specific regulators of inflammation. Nat Rev Immunol 2006;6:541–550.|
|8.||Lee WL, Downey GP. Leukocyte elastase: physiological functions and role in acute lung injury. Am J Respir Crit Care Med 2001;164:896–904.|
|9.||Fahy JV, Dickey BF. Airway mucus function and dysfunction. N Engl J Med 2010;363:2233–2247.|
|10.||Gibson RL, Burns JL, Ramsey BW. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med 2003;168:918–951.|
|11.||Taggart CC, Greene CM, Carroll TP, O’Neill SJ, McElvaney NG. Elastolytic proteases: inflammation resolution and dysregulation in chronic infective lung disease. Am J Respir Crit Care Med 2005;171:1070–1076.|
|12.||Le Gars M, Descamps D, Roussel D, Saussereau E, Guillot L, Ruffin M, Tabary O, Hong SS, Boulanger P, Paulais M, et al. Neutrophil elastase degrades cystic fibrosis transmembrane conductance regulator via calpains and disables channel function in vitro and in vivo. Am J Respir Crit Care Med 2013;187:170–179.|
|13.||Caldwell RA, Boucher RC, Stutts MJ. Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport. Am J Physiol Lung Cell Mol Physiol 2005;288:L813–L819.|
|14.||Mall MA. Role of the amiloride-sensitive epithelial Na+ channel in the pathogenesis and as a therapeutic target for cystic fibrosis lung disease. Exp Physiol 2009;94:171–174.|
|15.||Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, Shapiro SD. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med 1998;4:615–618.|
|16.||Hirche TO, Benabid R, Deslee G, Gangloff S, Achilefu S, Guenounou M, Lebargy F, Hancock RE, Belaaouaj A. Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa. J Immunol 2008;181:4945–4954.|
|17.||Boucher RC. Airway surface dehydration in cystic fibrosis: pathogenesis and therapy. Annu Rev Med 2007;58:157–170.|
|18.||Mall MA. Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction: lessons from mouse models. J Aerosol Med Pulm Drug Deliv 2008;21:13–24.|
|19.||Mall M, Grubb BR, Harkema JR, O’Neal WK, Boucher RC. Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 2004;10:487–493.|
|20.||Mall MA, Harkema JR, Trojanek JB, Treis D, Livraghi A, Schubert S, Zhou Z, Kreda SM, Tilley SL, Hudson EJ, et al. Development of chronic bronchitis and emphysema in β-epithelial Na+ channel-overexpressing mice. Am J Respir Crit Care Med 2008;177:730–742.|
|21.||Wielpütz MO, Eichinger M, Zhou Z, Leotta K, Hirtz S, Bartling SH, Semmler W, Kauczor HU, Puderbach M, Mall MA. In vivo monitoring of cystic fibrosis-like lung disease in mice by volumetric computed tomography. Eur Respir J 2011;38:1060–1070.|
|22.||Zhou Z, Duerr J, Johannesson B, Schubert SC, Treis D, Harm M, Graeber SY, Dalpke A, Schultz C, Mall MA. The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease. J Cyst Fibros 2011;10:S172–S182.|
|23.||Livraghi-Butrico A, Kelly EJ, Klem ER, Dang H, Wolfgang MC, Boucher RC, Randell SH, O’Neal WK. Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation. Mucosal Immunol 2012;5:397–408.|
|24.||Mall MA, Graeber SY, Stahl M, Zhou-Suckow Z. Early cystic fibrosis lung disease: Role of airway surface dehydration and lessons from preventive rehydration therapies in mice. Int J Biochem Cell Biol (In press)|
|25.||Gehrig S, Mall MA, Schultz C. Spatially resolved monitoring of neutrophil elastase activity with ratiometric fluorescent reporters. Angew Chem Int Ed Engl 2012;51:6258–6261.|
|26.||Hu HY, Gehrig S, Reither G, Subramanian D, Mall MA, Plettenburg O, Schultz C. FRET-based and other fluorescent proteinase probes. Biotechnol J 2014;9:266–281.|
|27.||Gehrig S, Dalpke A, Schultz C, Mall MA. Neutrophil elastase modulates neutrophilic inflammation, goblet cell metaplasia and emphysema in chronic obstructive lung disease of βENaC-overexpressing mice [abstract]. Am J Respir Crit Care Med 2013;187:A3491.|
|28.||Johannesson B, Hirtz S, Schatterny J, Schultz C, Mall MA. CFTR regulates early pathogenesis of chronic obstructive lung disease in βENaC-overexpressing mice. PLoS ONE 2012;7:e44059.|
|29.||Anagnostopoulou P, Dai L, Schatterny J, Hirtz S, Duerr J, Mall MA. Allergic airway inflammation induces a pro-secretory epithelial ion transport phenotype in mice. Eur Respir J 2010;36:1436–1447.|
|30.||Saetta M, Shiner RJ, Angus GE, Kim WD, Wang NS, King M, Ghezzo H, Cosio MG. Destructive index: a measurement of lung parenchymal destruction in smokers. Am Rev Respir Dis 1985;131:764–769.|
|31.||Hector A, Jonas F, Kappler M, Feilcke M, Hartl D, Griese M. Novel method to process cystic fibrosis sputum for determination of oxidative state. Respiration 2010;80:393–400.|
|32.||Cobos-Correa A, Trojanek JB, Diemer S, Mall MA, Schultz C. Membrane-bound FRET probe visualizes MMP12 activity in pulmonary inflammation. Nat Chem Biol 2009;5:628–630.|
|33.||Kirchner KK, Wagener JS, Khan TZ, Copenhaver SC, Accurso FJ. Increased DNA levels in bronchoalveolar lavage fluid obtained from infants with cystic fibrosis. Am J Respir Crit Care Med 1996;154:1426–1429.|
|34.||Voynow JA, Fischer BM, Malarkey DE, Burch LH, Wong T, Longphre M, Ho SB, Foster WM. Neutrophil elastase induces mucus cell metaplasia in mouse lung. Am J Physiol Lung Cell Mol Physiol 2004;287:L1293–L1302.|
|35.||Voynow JA, Young LR, Wang Y, Horger T, Rose MC, Fischer BM. Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. Am J Physiol 1999;276:L835–L843.|
|36.||Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci USA 1990;87:9188–9192.|
|37.||Shapiro SD, Goldstein NM, Houghton AM, Kobayashi DK, Kelley D, Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. Am J Pathol 2003;163:2329–2335.|
|38.||Mayer-Hamblett N, Aitken ML, Accurso FJ, Kronmal RA, Konstan MW, Burns JL, Sagel SD, Ramsey BW. Association between pulmonary function and sputum biomarkers in cystic fibrosis. Am J Respir Crit Care Med 2007;175:822–828.|
|39.||Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell EJ. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J Cell Biol 1995;131:775–789.|
|40.||Hirche TO, Atkinson JJ, Bahr S, Belaaouaj A. Deficiency in neutrophil elastase does not impair neutrophil recruitment to inflamed sites. Am J Respir Cell Mol Biol 2004;30:576–584.|
|41.||Diakov A, Bera K, Mokrushina M, Krueger B, Korbmacher C. Cleavage in the γ-subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near-silent channels. J Physiol 2008;586:4587–4608.|
|42.||Mall MA, Button B, Johannesson B, Zhou Z, Livraghi A, Caldwell RA, Schubert SC, Schultz C, O’Neal WK, Pradervand S, et al. Airway surface liquid volume regulation determines different airway phenotypes in liddle compared with betaENaC-overexpressing mice. J Biol Chem 2010;285:26945–26955.|
|43.||Fyfe GK, Canessa CM. Subunit composition determines the single channel kinetics of the epithelial sodium channel. J Gen Physiol 1998;112:423–432.|
|44.||Button B, Cai LH, Ehre C, Kesimer M, Hill DB, Sheehan JK, Boucher RC, Rubinstein M. A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 2012;337:937–941.|
|45.||Anagnostopoulou P, Riederer B, Duerr J, Michel S, Binia A, Agrawal R, Liu X, Kalitzki K, Xiao F, Chen M, et al. SLC26A9-mediated chloride secretion prevents mucus obstruction in airway inflammation. J Clin Invest 2012;122:3629–3634.|
|46.||Graeber SY, Zhou-Suckow Z, Schatterny J, Hirtz S, Boucher RC, Mall MA. Hypertonic saline is effective in the prevention and treatment of mucus obstruction, but not airway inflammation, in mice with chronic obstructive lung disease. Am J Respir Cell Mol Biol 2013;49:410–417.|
|47.||Becq F, Mall MA, Sheppard DN, Conese M, Zegarra-Moran O. Pharmacological therapy for cystic fibrosis: from bench to bedside. J Cyst Fibros 2011;10:S129–S145.|
|48.||Zhou Z, Treis D, Schubert SC, Harm M, Schatterny J, Hirtz S, Duerr J, Boucher RC, Mall MA. Preventive but not late amiloride therapy reduces morbidity and mortality of lung disease in betaENaC-overexpressing mice. Am J Respir Crit Care Med 2008;178:1245–1256.|
|49.||Hartl D, Latzin P, Hordijk P, Marcos V, Rudolph C, Woischnik M, Krauss-Etschmann S, Koller B, Reinhardt D, Roscher AA, et al. Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease. Nat Med 2007;13:1423–1430.|
|50.||Tunney MM, Field TR, Moriarty TF, Patrick S, Doering G, Muhlebach MS, Wolfgang MC, Boucher R, Gilpin DF, McDowell A, et al. Detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis. Am J Respir Crit Care Med 2008;177:995–1001.|
|51.||Bals R, Weiner DJ, Wilson JM. The innate immune system in cystic fibrosis lung disease. J Clin Invest 1999;103:303–307.|
|52.||Griese M, Kappler M, Gaggar A, Hartl D. Inhibition of airway proteases in cystic fibrosis lung disease. Eur Respir J 2008;32:783–795.|
|53.||Elborn JS, Perrett J, Forsman-Semb K, Marks-Konczalik J, Gunawardena K, Entwistle N. Efficacy, safety and effect on biomarkers of AZD9668 in cystic fibrosis. Eur Respir J 2012;40:969–976.|
|54.||Groutas WC, Dou D, Alliston KR. Neutrophil elastase inhibitors. Expert Opin Ther Pat 2011;21:339–354.|
|55.||Churg A, Wright JL. Proteases and emphysema. Curr Opin Pulm Med 2005;11:153–159.|
|56.||Shapiro SD. Animal models for COPD. Chest 2000; 117(5, Suppl 1)223S–227S.|
|57.||Wielpütz MO, Puderbach M, Kopp-Schneider A, Stahl M, Fritzsching E, Sommerburg O, Ley S, Sumkauskaite M, Biederer J, Kauczor HU, et al. Magnetic resonance imaging detects changes in structure and perfusion, and response to therapy in early cystic fibrosis lung disease. Am J Respir Crit Care Med 2014;189:956–965.|
|58.||Korkmaz B, Poutrain P, Hazouard E, de Monte M, Attucci S, Gauthier FL. Competition between elastase and related proteases from human neutrophil for binding to alpha1-protease inhibitor. Am J Respir Cell Mol Biol 2005;32:553–559.|
*M.A.M. and C.S. contributed equally as senior authors.
Supported in part by grants from the Deutsche Forschungsgemeinschaft (MA2081/4-1 to M.A.M. and SFB938/TP E to A.H.D.) and the European Commission (Seventh Framework Program Project No. 603038 CFMatters to M.A.M., and LSHG-CT-2003-503259 to C.S.).
Author Contributions: Conception and design of the study, S.G., A.H.D., C.S., and M.A.M. Acquisition, analysis, and interpretation of data, all authors. Drafting the article or revising it critically for important intellectual content, S.G., J.D., C.J.W., M.W., A.B., A.H.D., C.S., and M.A.M.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201311-1932OC on March 28, 2014