Cystic Fibrosis Airway Secretions Research Articles

Airway Mucins Inhibit Oxidative and Non-Oxidative Bacterial Killing by Human Neutrophils.

Neutrophil killing of bacteria is mediated by oxidative and non-oxidative mechanisms. Oxidants are generated through the NADPH oxidase complex, whereas antimicrobial proteins and peptides rank amongst non-oxidative host defenses. Mucus hypersecretion, deficient hydration and poor clearance from the airways are prominent features of cystic fibrosis (CF) lung disease. CF airways are commonly infected by Pseudomonas aeruginosa and Burkholderia cepacia complex bacteria. Whereas the former bacterium is highly sensitive to non-oxidative killing, the latter is only killed if the oxidative burst is intact. Despite an abundance of neutrophils, both pathogens thrive in CF airway secretions. In this study, we report that secreted mucins protect these CF pathogens against host defenses. Mucins were purified from CF sputum and from the saliva of healthy volunteers. Whereas mucins did not alter the phagocytosis of Pseudomonas aeruginosa and Burkholderia cenocepacia by neutrophils, they completely suppressed bacterial killing. Accordingly, mucins markedly inhibited non-oxidative bacterial killing by neutrophil granule extracts, or by lysozyme and the cationic peptide, human β defensin-2 (HBD2). Mucins also suppressed the neutrophil oxidative burst through a charge-dependent mechanism that could be reversed by the cationic aminoglycoside, tobramycin. Our data indicate that airway mucins protect Gram-negative bacteria against neutrophil killing by suppressing the oxidative burst and inhibiting the bactericidal capacity of cationic proteins and peptides. Mucin hypersecretion, dehydration, stasis and anionic charge represent key therapeutic targets for improving host defenses and airway inflammation in CF and other muco-secretory airway diseases.

Refinement of metabolite detection in cystic fibrosis sputum reveals heme correlates with lung function decline.

The bacterial growth environment within cystic fibrosis (CF) sputum is complex, dynamic, and shaped by both host and microbial processes. Characterization of the chemical parameters within sputum that stimulate the in vivo growth of airway pathogens (e.g. Pseudomonas aeruginosa) and their associated virulence factors may lead to improved CF treatment strategies. Motivated by conflicting reports of the prevalence and abundance of P. aeruginosa-derived metabolites known as phenazines within CF airway secretions, we sought to quantify these metabolites in sputum using quadrupole time-of-flight mass spectrometry. In contrast to our previous work, all phenazines tested (pyocyanin (PYO), phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide, and 1-hydroxyphenazine) were below detection limits of the instrument (0.1 μM). Instead, we identified a late-eluting compound that shared retention time and absorbance characteristics with PCA, yet generated mass spectra and a fragmentation pattern consistent with ferriprotoporphyrin IX, otherwise known as heme B. These data suggested that UV-vis chromatographic peaks previously attributed to PCA and PYO in sputum were mis-assigned. Indeed, retrospective analysis of raw data from our prior study found that the heme B peak closely matched the peaks assigned to PCA, indicating that the previous study likely uncovered a positive correlation between pulmonary function (percent predicted forced expiratory volume in 1 second, or ppFEV1) and heme B, not PCA or any other phenazine. To independently test this observation, we performed a new tandem mass-spectrometry analysis of 71 additional samples provided by the Mountain West CF Consortium Sputum Biomarker study and revealed a positive correlation (ρ = −0.47, p<0.001) between sputum heme concentrations and ppFEV1. Given that hemoptysis is strongly associated with airway inflammation, pulmonary exacerbations and impaired lung function, these new data suggest that heme B may be a useful biomarker of CF pathophysiology.

The effect of the decoy molecule PA401 on CXCL8 levels in bronchoalveolar lavage fluid of patients with cystic fibrosis

BackgroundThe chemokine interleukin-8 (CXCL8) is a key mediator of inflammation in airways of patients with cystic fibrosis (CF). Glycosaminoglycans (GAGs) possess the ability to influence the chemokine profile of the CF lung by binding CXCL8 and protecting it from proteolytic degradation. CXCL8 is maintained in an active state by this glycan interaction thus increasing infiltration of immune cells such as neutrophils into the lungs. As the CXCL8-based decoy PA401 displays no chemotactic activity, yet demonstrates glycan binding affinity, the aim of this study was to investigate the anti-inflammatory effect of PA401 on CXCL8 levels, and activity, in CF airway samples in vitro. MethodsBronchoalveolar lavage fluid (BALF) was collected from patients with CF homozygous for the ΔF508 mutation (n=13). CXCL8 in CF BALF pre and post exposure to PA401 was quantified by ELISA. Western blot analysis was used to determine PA401 degradation in CF BALF. The ex vivo chemotactic activity of purified neutrophils in response to CF airway secretions was evaluated post exposure to PA401 by use of a Boyden chamber-based motility assay. ResultsExposure of CF BALF to increasing concentrations of PA401 (50–1000pg/ml) over a time course of 2–12h in vitro, significantly reduced the level of detectable CXCL8 (P<0.05). Interestingly, PA401 engendered release of CXCL8 from GAGs exposing the chemokine susceptible to proteolysis. Subsequently, a loss of PA401 was observed (P<0.05) due to proteolytic degradation by elastase like proteases. A 25% decrease in neutrophil chemotactic efficiency towards CF BALF samples incubated with PA401 was also observed (P<0.05). ConclusionPA401 can disrupt CXCL8:GAG complexes, rendering the chemokine susceptible to proteolytic degradation. Clinical application of a CXCL8 decoy, such as PA401, may serve to decrease the inflammatory burden in the CF lung in vivo.

Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure.

The pathogenesis of mucoinfective lung disease in cystic fibrosis (CF) patients likely involves poor mucus clearance. A recent model of mucus clearance predicts that mucus flow depends on the relative mucin concentration of the mucus layer compared with that of the periciliary layer; however, mucin concentrations have been difficult to measure in CF secretions. Here, we have shown that the concentration of mucin in CF sputum is low when measured by immunologically based techniques, and mass spectrometric analyses of CF mucins revealed mucin cleavage at antibody recognition sites. Using physical size exclusion chromatography/differential refractometry (SEC/dRI) techniques, we determined that mucin concentrations in CF secretions were higher than those in normal secretions. Measurements of partial osmotic pressures revealed that the partial osmotic pressure of CF sputum and the retained mucus in excised CF lungs were substantially greater than the partial osmotic pressure of normal secretions. Our data reveal that mucin concentration cannot be accurately measured immunologically in proteolytically active CF secretions; mucins are hyperconcentrated in CF secretions; and CF secretion osmotic pressures predict mucus layer-dependent osmotic compression of the periciliary liquid layer in CF lungs. Consequently, mucin hypersecretion likely produces mucus stasis, which contributes to key infectious and inflammatory components of CF lung disease.

Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure

Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure

Pseudomonas aeruginosa biological functions are inhibited by normal airway epithelial secretions: implications for cystic fibrosis (P3074)

Abstract P. aeruginosa does not colonize healthy individuals, but it is the leading cause of death in patients with cystic fibrosis (CF). The ability of P. aeruginosa to readily colonize CF-lungs is at least partially related to alterations in airway secretions caused by the disease. In addition to being dehydrated, these secretions have reduced antimicrobial activity compared to that found in normal airways. Thus, we proposed that normal airway secretions contain one or more molecules that reduce P. aeruginosa virulence whereas CF airway secretions do not. To test this hypothesis in vitro, we collected apical secretions from polarized CFTR+ and CFTR- Calu-3 cell monolayers. We applied these secretions at various concentrations to the P. aeruginosa strain PA14 and examined the effect of the secretions on two key biological activities associated with P. aeruginosa virulence: motility and biofilm formation. Secretions from CFTR+ (“normal”) cells dose-dependently inhibited both PA14 flagellar motility and biofilm formation. However, secretions from CFTR- (“CF-like”) cells failed to inhibit either biological function. Our preliminary identification experiments suggest that the inhibitory agent(s) in the CFTR+ secretions is a heat-labile, large (&amp;gt;50 kDa) molecule, probably a protein. Thus, it is likely that normal epithelial secretions play a direct role in inhibiting P. aerugionsa virulence and that these secretions are dysfunctional in patients with CF.

145 A novel tool to study neutrophil elastase activity on neutrophils from cystic fibrosis airway secretions

145 A novel tool to study neutrophil elastase activity on neutrophils from cystic fibrosis airway secretions

Concentration of the antibacterial precursor thiocyanate in cystic fibrosis airway secretions

A recently discovered enzyme system produces antibacterial hypothiocyanite (OSCN−) in the airway lumen by oxidizing the secreted precursor thiocyanate (SCN−). Airway epithelial cultures have been shown to secrete SCN− in a CFTR-dependent manner. Thus, reduced SCN− availability in the airway might contribute to the pathogenesis of cystic fibrosis (CF), a disease caused by mutations in the CFTR gene and characterized by an airway host defense defect. We tested this hypothesis by analyzing the SCN− concentration in the nasal airway surface liquid (ASL) of CF patients and non-CF subjects and in the tracheobronchial ASL of CFTR-ΔF508 homozygous pigs and control littermates. In the nasal ASL, the SCN− concentration was ~30-fold higher than in serum independent of the CFTR mutation status of the human subject. In the tracheobronchial ASL of CF pigs, the SCN− concentration was somewhat reduced. Among human subjects, SCN− concentrations in the ASL varied from person to person independent of CFTR expression, and CF patients with high SCN− levels had better lung function than those with low SCN− levels. Thus, although CFTR can contribute to SCN− transport, it is not indispensable for the high SCN− concentration in ASL. The correlation between lung function and SCN− concentration in CF patients may reflect a beneficial role for SCN−.

Nebulized Hypertonic Saline Decreases IL-8 in Sputum of Patients with Cystic Fibrosis

Inflammation within the cystic fibrosis (CF) lung is mediated by inflammatory chemokines, such as IL-8. IL-8 is protected from proteolytic degradation in the airways by binding to glycosaminoglycans, while remaining active. Evidence that increased hypertonicity of airway secretions induced by hypertonic saline treatment alters levels of IL-8 is lacking. To investigate the antiinflammatory effect of hypertonic saline (HTS) treatment within the CF lung by focusing on IL-8. Degradation of IL-8 in CF lung secretions after treatment with glycosaminoglycan lyases and HTS was analyzed by Western blot analysis and ELISA. The ex vivo chemotactic activity of purified neutrophils in response to CF airway secretions was evaluated post nebulization of HTS (7% saline). In vivo CF bronchoalveolar lavage fluid (BALF) IL-8 levels were significantly higher than the control group (P < 0.05). Digesting glycosaminoglycans in CF BALF displaced IL-8 from glycosaminoglycan matrices, rendering the chemokine susceptible to proteolytic cleavage. High sodium concentrations also liberate IL-8 in CF BALF in vitro, and in vivo in CF sputum from patients receiving aerosolized HTS, resulting in degradation of IL-8 and decreased neutrophil chemotactic efficiency. Glycosaminoglycans possess the ability to influence the chemokine profile of the CF lung by binding and stabilizing IL-8, which promotes neutrophil chemotaxis and activation. Nebulized hypertonic saline treatment disrupts the interaction between glycosaminoglycans and IL-8, rendering IL-8 susceptible to proteolytic degradation with subsequent decrease in neutrophil chemotaxis, thereby facilitating resolution of inflammation.

Nutrient Availability as a Mechanism for Selection of Antibiotic Tolerant Pseudomonas aeruginosa within the CF Airway

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues.

The Inhibition of Superoxide Production by the Soluble Phase of Cystic Fibrosis Sputum

BackgroundCystic fibrosis (CF) lung disease is characterized by an infiltration of activated neutrophils. Oxidative radical formation and neutrophil proteases may interact with the components of CF airway secretions. This study examined the soluble factors in CF airway secretions (sol) and determined their effect on the production of superoxide radicals.MethodsNeutrophils were isolated from three normal subjects. Each preparation of neutrophils can be used in several experiments on the same day of preparation. Using a standardized cytochrome c reduction method, the effect of pooled CF sol isolated from nine CF patients, on neutrophil superoxide production was studied.ResultsCF sol phase significantly attenuates the production of superoxide by phorbol stimulated control neutrophils in a time and concentration dependent manner. When CF sol is boiled, the inhibitory effect on superoxide was reversed. CF soluble heat labile factors clearly attenuated superoxide production, independent of elastase concentration. We conclude that the presence of heat labile soluble factors in CF sputum, serve to attenuate superoxide production by human neutrophils.

Analysis of fungal-bacterial community interactions in cystic fibrosis airway secretions

Analysis of fungal-bacterial community interactions in cystic fibrosis airway secretions

MUC5AC and MUC5B Mucins Increase in Cystic Fibrosis Airway Secretions during Pulmonary Exacerbation

Cystic fibrosis (CF) is believed to be associated with mucus hypersecretion; thus, the principal airway gel-forming mucins, MUC5AC and MUC5B, are also expected to be increased relative to non-CF secretions. However, we have shown that these mucins are decreased during stable CF disease. In this study, we determine if these mucins increase during a pulmonary exacerbation of CF. Expectorated sputum was collected from 11 adults with CF during stable disease and then during a pulmonary exacerbation and from 12 healthy control subjects. MUC5AC and MUC5B proteins were measured by Western blot. DNA content was measured using microfluorimetry. MUC5AC protein increased by 908% and MUC5B by 59% (p < 0.05 for both) during an exacerbation compared with periods of stable disease. During stable disease, the vol/vol quantity of MUC5AC protein was 89% less than normal mucus, and the mucin-associated sugars, measured using a lectin binding assay, were 46% less compared with normal mucus. The concentration of DNA in CF sputum did not increase during an exacerbation. During a CF exacerbation, concentration of secreted mucin increased to the amount found in mucus from normal subjects, suggesting that the capacity to secrete mucin in response to an infection or inflammatory stimulus is preserved in CF airways. This might help to protect the airway from injury.

CFTR is a modulator of airway inflammation

although it has been 17 years since the identification of the cystic fibrosis transmembrane ion receptor (CFTR) gene and protein, it remains enigmatic how abnormalities in CFTR can cause chronic and persistent pulmonary infection and inflammation that lead to bronchiectasis and end-stage lung

Serum albumin enhances polyethylenimine‐mediated gene delivery to human respiratory epithelial cells

The interaction of polyethylenimine (PEI) polyplexes with proteins in cystic fibrosis (CF) airway secretions poses a significant hurdle to this nonviral delivery system. The aim of this study was to evaluate whether albumin may increase the efficiency of PEI complexes in mediating gene transfer into respiratory epithelial cells in the presence of CF mucus. PEI (25 kDa) was complexed to DNA in the presence of human serum albumin (HSA) and used to transfect confluent A549 and 9HTEo- cells. Alternatively, albumin was added to preformed PEI-DNA complexes. The cytotoxicity of complexes was analysed by the LDH (lactate dehydrogenase) assay. CF CFT1-C2 cells were allowed to polarise and were transfected either with luciferase- or CFTR-expressing plasmids. To evaluate the effect of CF respiratory secretions on transfection efficiency, confluent cells were transfected in the presence of sputum obtained from two CF patients. The ternary PEI-HSA complexes increased luciferase expression in confluent cultures in a dose-dependent fashion up to 100 times as compared to PEI-DNA. The number of GFP-expressing cells, as evaluated by epifluorescence, was augmented several-fold. When HSA was added to preformed PEI-DNA complexes, a further 5-10-fold increase in gene expression was observed. No significant cytotoxicity was observed with either PEI or PEI-HSA polyplexes. The ternary complexes determined detectable CFTR gene transfer and expression at the apical membrane in polarised CFT1-C2 cells, as evaluated by confocal microscopy. CF sputum inhibited PEI-mediated gene transfer by 7-186-fold. Although luciferase expression mediated by PEI-HSA was still inhibited by CF sputum, these levels were 18-83.8-fold higher than with PEI. Our results demonstrate that albumin increases PEI gene transfer efficiency in confluent and polarised respiratory epithelial cells and can allow CFTR gene expression in the appropriate cellular compartment. PEI-HSA complexes display a higher efficiency than PEI also in the presence of CF sputum, indicating that albumin-containing polyplexes may help overcome barriers imposed by CF airway secretions.

Antioxidant properties of cystic fibrosis sputum

Oxidative stress is a likely contributor to the pathogenesis of cystic fibrosis (CF) lung disease. However, hydrogen peroxide (H(2)O(2)), a physiological oxidant, is not elevated in CF exhalates. H(2)O(2) may be neutralized by antioxidants in CF airway secretions. The H(2)O(2)-detoxifying capacity of CF airway secretions, obtained via sputum induction, was studied in an in vitro H(2)O(2) cytotoxicity model. 16HBE14o- cells were exposed to H(2)O(2) in culture medium containing either 0 or 10% fetal bovine serum (FBS) or 10% CF sputum supernatant (extracted without use of dithiothreitol). The efficiency of H(2)O(2) neutralization was estimated by measuring intracellular oxidant levels (dihydrorhodamine 123) after 2 h and cell viability (propidium iodide) after 24 h of H(2)O(2) exposure. Furthermore, the presence of reduced thiols (DTNB assay) and reduced glutathione (recycling assay) in CF sputum samples was evaluated. CF sputum extracts completely prevented intracellular oxidant accumulation seen in cells incubated with H(2)O(2) in both control media (i.e., 0 or 10% FBS). Furthermore, CF sputum abolished cell death in 16HBE14o- cells exposed to up to 1 mM H(2)O(2). In contrast, there was 100% cytotoxicity in cells exposed to 600 microM H(2)O(2) in both control media. The H(2)O(2)-detoxifying potential of CF sputum was sustained after catalase and heme peroxidases were inactivated by sodium azide, which does not affect glutathione peroxidase. In addition, reduced protein thiols were found in abundance in CF sputum. In conclusion, CF sputum is capable to neutralize H(2)O(2) and abundant reduced thiols and/or glutathione peroxidase are fully sufficient to detoxify H(2)O(2).

MUC5AC and MUC5B Mucins Are Decreased in Cystic Fibrosis Airway Secretions.

Cystic fibrosis (CF) is characterized by progressive airway obstruction. Although it has been postulated that this is due in part to mucus hypersecretion, there are no published data showing an increase in the gel-forming mucins MUC5AC or MUC5B in CF secretions. We used confocal microscopy to assess the amount of mucin-like glycoprotein and DNA in CF sputum and found more mucin in bronchitis sputum and a much greater amount of DNA in CF sputum. We then used antibodies to MUC5AC and MUC5B with Western gels and dot-blot to quantify mucin in sputum from 12 patients with CF and 11 subjects without lung disease. There was a 70% decrease in MUC5B and a 93% decrease in MUC5AC in CF sputum (P < 0.005 for both). We conclude that the vol/vol concentration of MUC5AC and MUC5B are decreased in the CF airways relative to normal mucus. This may be due to a relative increase in other components of sputum in the CF airway or to a primary defect in mucin secretion in CF.

CCL20 is an inducible product of human airway epithelia with innate immune properties.

Chemokine ligand 20 (CCL20) and human beta-defensins (HBDs) share structural and functional properties, including antiparallel beta-pleated sheet core structures, charge distribution, and signaling to adaptive immune cells via the highly selective CCR6 receptor. Because of their similarities, we hypothesized that in addition to its known adaptive immune signaling functions, CCL20 has antimicrobial properties and participates in pulmonary innate immunity. We found that primary cultures of human airway epithelial and cultured fetal lung explants expressed CCL20 mRNA. Expression of CCL20 transcripts were significantly induced by interleukin (IL)-1beta and tumor necrosis factor-alpha, and inhibited by dexamethasone. Primary cultures of airway epithelia secreted CCL20 both apically and basolaterally, and CCL20 abundance was increased over 30-fold with IL-1beta stimulation, achieving an estimated concentration of 167 ng/ml in airway surface liquid. CCL20 abundance in bronchoalveolar lavage fluid from patients with cystic fibrosis was nearly 90-fold higher compared with bronchoalveolar lavage fluid from healthy volunteers. Interestingly, CCL20 exhibited salt-sensitive antimicrobial activity, mainly against Gram-negative bacteria in low mug/ml concentrations. Additionally, apical washings from IL-1beta-stimulated primary cultures of human airway epithelia had significantly more antimicrobial activity than unstimulated controls. CCL20 rapidly permeabilized bacterial membranes with a time course intermediate to HBD-2 and HBD-3. Thus, CCL20 is a bi-functional peptide with both innate and adaptive immune properties that is regulated by inflammatory mediators, expressed by airway epithelia, and increased in cystic fibrosis airway secretions.

Improved activity of an actin-resistant DNase I variant on the cystic fibrosis airway secretions.

In cystic fibrosis (CF), actin and DNA originating from inflammatory cells contribute to the thickness of airway secretions. Actin can bind to DNA-rich fibers and potently inhibit the enzymatic activity of rhDNase. The in vitro effects of the actin-resistant rhDNase variant (A114R) were analyzed and compared with those of the wild-type rhDNase. Frozen and thawed CF airway secretions were incubated for 30 min with different concentrations (0.1, 0.5, 1, 5, or 10 microg/ml) of either actin-resistant rhDNase or wild-type rhDNase. We observed that both the wild-type and the actin-resistant rhDNase significantly decreased (p < 0.05 and p < 0.001, respectively) the airway secretion viscosity. The decrease in airway secretion viscosity was significant even at low concentrations (0.1 microg/ml) of the actin-resistant variant. Incubation with the actin-resistant variant resulted in a significant decrease (p < 0.02) of the airway secretion contact angle and cough transport. A significantly higher (p < 0.01) increase in contact angle and cough transport of airway secretions was observed at 10 microg/ml with the actin-resistant variant as compared with the wild-type rhDNase. The present study had demonstrated that the actin-resistant rhDNase variant (A114R) has an enhanced capacity to improve the physical properties and cough transport of airway secretions from patients with cystic fibrosis.

Identification of MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosis airway secretions

Identification of MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosis airway secretions