The hypoxic environment of cystic fibrosis airways allows the persistence of facultative anaerobic bacteria, which can produce short-chain fatty acids (SCFAs) through fermentation. However, the relevance of SCFAs in cystic fibrosis lung disease is unknown. We show that SCFAs are present in sputum samples from cystic fibrosis patients in millimolar concentrations (mean±sem1.99±0.36 mM).SCFAs positively correlated with sputum neutrophil count and higher SCFAs were predictive for impaired nitric oxide production. We studied the effects of the SCFAs acetate, propionate and butyrate on airway inflammatory responses using epithelial cell lines and primary cell cultures. SCFAs in concentrations present in cystic fibrosis airways (0.5–2.5 mM) affected the release of granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin (IL)-6. SCFAs also resulted in higher IL-8 release from stimulated cystic fibrosis transmembrane conductance regulator (CFTR) F508del-mutant compared to wild-type CFTR-corrected bronchial epithelial cells. At 25 mM propionate reduced IL-8 release in control but not primary cystic fibrosis epithelial cells. Low (0.5–2.5 mM) SCFA concentrations increased, while high (25–50 mM) concentrations decreased inducible nitric oxide synthase expression. In addition, SCFAs affected the growth ofPseudomonas aeruginosain a concentration- and pH-dependent manner.Thus, our data suggest that SCFAs contribute to cystic fibrosis-specific alterations of responses to airway infection and inflammation.

Neutrophils release DNA-based extracellular traps to capture and kill bacteria. The mechanism(s) and proteins that promote neutrophil extracellular trap (NET)-mediated bacterial trapping are not clearly established. Surfactant protein D (SP-D) is an innate immune collectin present in many mucosal surfaces. We hypothesized that SP-D can bind both the pathogens and NETs to augment NET-mediated bacterial trapping. To test this hypothesis, we used LPS and Pseudomonas aeruginosa pneumonia mouse models and performed in vivo and ex vivo assays. In this study, we show that NETs are produced by the neutrophils recruited to the airways in response to the bacterial ligand. Notably, NETs are detected as short fragments of DNA–protein complexes in the airways as opposed to the long stringlike structures seen in ex vivo cultures. SP-D recognizes both the short NET fragments and the long NET DNA structures. SP-D–NET copurification studies further show that SP-D can simultaneously recognize NETs and carbohydrate ligands in vivo. Similar to the LPS model, soluble DNA–protein complexes and increased amounts of SP-D are detected in the murine model of P. aeruginosa pneumonia. We then tested the effect of SP-D on NET-mediated trapping of P. aeruginosa by means of Western blots, fluorescence microscopy, and scanning electron microscopy. Results of these experiments show that SP-D microagglutinates P. aeruginosa and allows an efficient bacterial trapping by NETs. Collectively, these findings provide a unique biological relevance for SP-D–DNA interactions and places SP-D as an important innate immune protein that promotes bacterial trapping by NETs during neutrophil-mediated host defense.

Rationale: Airway nitric oxide concentrations are reduced in cystic fibrosis (CF). Arginases compete for L-arginine, the substrate of nitric oxide synthesis. Objectives: We hypothesized that increased arginase activity may be one factor contributing to nitric oxide deficiency in CF. Measurements: We therefore studied sputum arginase activity, exhaled nitric oxide, and pulmonary function in patients with cystic fibrosis. Results: Mean (± SEM) sputum arginase activity was significantly higher in patients admitted for pulmonary exacerbation compared with patients with stable disease (1.032 ± 0.148 vs. 0.370 ± 0.091 U/mg protein, p = 0.004). Fourteen days of intravenous antibiotic treatment resulted in significantly decreased sputum arginase activity in all patients (p = 0.0002). However, arginase activity was still significantly (p = 0.0001) higher in CF sputum after treatment for exacerbation compared with induced sputum from healthy control subjects (0.026 ± 0.006 U/mg protein). Negative correlations were found for sputum arginase activity at admission with FEV₁ (r = −0.41, p = 0.01), as well as changes in arginase activity with percent change in FEV₁ during antibiotic therapy (r = −0.4, p < 0.01) in CF. Exhaled nitric oxide in CF was positively correlated to FEV₁ (r = 0.34, p = 0.007), and in patients admitted for pulmonary exacerbation negatively correlated to sputum arginase activity (r = −0.45, p = 0.03). Conclusions: These data suggest that increased sputum arginase activity contributes to nitric oxide deficiency in CF lung disease and may be relevant in the pathogenesis of CF airway disease.

Genetic defects in cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene cause CF. Infants with CFTR mutations show a peribronchial neutrophil infiltration prior to the establishment of infection in their lung. The inflammatory response progressively increases in children that include both upper and lower airways. Infectious and inflammatory response leads to an increase in mucus viscosity and mucus plugging of small and medium-size bronchioles. Eventually, neutrophils chronically infiltrate the airways with biofilm or chronic bacterial infection. Perpetual infection and airway inflammation destroy the lungs, which leads to increased morbidity and eventual mortality in most of the patients with CF. Studies have now established that neutrophil cytotoxins, extracellular DNA, and neutrophil extracellular traps (NETs) are associated with increased mucus clogging and lung injury in CF. In addition to opportunistic pathogens, various aspects of the CF airway milieux (e.g., airway pH, salt concentration, and neutrophil phenotypes) influence the NETotic capacity of neutrophils. CF airway milieu may promote the survival of neutrophils and eventual pro-inflammatory aberrant NETosis, rather than the anti-inflammatory apoptotic death in these cells. Degrading NETs helps to manage CF airway disease; since DNAse treatment release cytotoxins from the NETs, further improvements are needed to degrade NETs with maximal positive effects. Neutrophil-T cell interactions may be important in regulating viral infection-mediated pulmonary exacerbations in patients with bacterial infections. Therefore, clarifying the role of neutrophils and NETs in CF lung disease and identifying therapies that preserve the positive effects of neutrophils, while reducing the detrimental effects of NETs and cytotoxic components, are essential in achieving innovative therapeutic advances.

BACKGROUND: The airways of obese asthmatics have been shown to be NO deficient, and this contributes to airway dysfunction and reduced response to inhaled corticosteroids. In cultured airway epithelial cells, L-citrulline, a precursor of L-arginine recycling and NO formation, has been shown to prevent asymmetric dimethyl arginine–mediated (ADMA-mediated) NO synthase (NOS2) uncoupling, restoring NO and reducing oxidative stress. METHODS: In a proof-of-concept, open-label pilot study in which participants were analyzed before and after treatment, we hypothesized that 15 g/d L-citrulline for 2 weeks would (a) increase the fractional excretion of NO (FeNO), (b) improve asthma control, and (c) improve lung function. To this end, we recruited obese (BMI >30) asthmatics on controller therapy, with a baseline FeNO of ≤30 ppb from the University of Colorado Medical Center and Duke University Health System. RESULTS: A total of 41 subjects with an average FeNO of 17 ppb (95% CI, 15–19) and poorly controlled asthma (average asthma control questionnaire [ACQ] 1.5 [95% CI, 1.2–1.8]) completed the study. Compared with baseline, L-citrulline increased whereas ADMA and arginase concentration did not (values represent the mean Δ and 95% CI): plasma L-citrulline (190 μM, 84–297), plasma L-arginine (67 μM, 38–95), and plasma L-arginine/ADMA (ratio 117, 67–167). FeNO increased by 4.2 ppb (1.7–6.7 ppb); ACQ decreased by –0.46 (–0.67 to 0.27 points); the forced vital capacity and forced exhalation volume in 1 second, respectively, changed by 86 ml (10–161 ml) and 52 ml (–11 to 132 ml). In a secondary analysis, the greatest FEV ₁ increments occurred in those subjects with late-onset asthma (>12 years) (63 ml [95% CI, 1–137]), in females (80 ml [95% CI, 5–154]), with a greater change seen in late-onset females (100 ml, [95% CI, 2–177]). The changes in lung function or asthma control were not significantly associated with the changes before and after treatment in L-arginine/ADMA or FeNO. CONCLUSION: Short-term L-citrulline treatment improved asthma control and FeNO levels in obese asthmatics with low or normal FeNO. Larger FEV ₁ increments were observed in those with late-onset asthma and in females. TRIAL REGISTRATION: ClinicalTrials.gov NCT01715844. FUNDING: NIH NHLBI R01 HL146542-01.