contributed equally to this article. ** Drs. Wainwright and McColley contributed equally to this article.+Associate Editor, AJRCCM (participation complies with American Thoracic Society requirements for recusal from review and decisions for authored works).
Secretory leucoprotease inhibitor (SLPI) is a neutrophil serine protease inhibitor constitutively expressed at many mucosal surfaces including that of the lung. Originally identified as a serine protease inhibitor, it is now evident that SLPI also has anti-microbial and anti-inflammatory functions, and therefore plays an important role in host defence. Previous work has shown that some host defence proteins such as SLPI and elafin are susceptible to proteolytic degradation. Consequently, we investigated the status of SLPI in the Cystic Fibrosis (CF) lung. A major factor that contributes to the high mortality rate among CF patients is Pseudomonas aeruginosa infection. In this study, we report that P. aeruginosa-positive CF bronchoalveolar lavage fluid (BALF), which contains lower SLPI levels and higher neutrophil elastase (NE) activity compared to P. aeruginosa-negative samples, was particularly effective at cleaving recombinant human SLPI. Additionally, we found that only NE inhibitors were able to prevent SLPI cleavage, thereby implicating NE in this process. NE in excess was found to cleave recombinant SLPI at two novel sites in the NH2-terminal region and abrogate its ability to bind LPS and NF-κB consensus binding sites but not its ability to inhibit activity of the serine protease cathepsin G. In conclusion, this study provides evidence that SLPI is cleaved and inactivated by NE present in P. aeruginosa-positive CF lung secretions and that P. aeruginosa infection contributes to inactivation of the host defence screen in the CF lung.
There is an abundance of antimicrobial peptides in cystic fibrosis (CF) lungs. Despite this, individuals with CF are susceptible to microbial colonization and infection. In this study, we investigated the antimicrobial response within the CF lung, focusing on the human cathelicidin LL-37. We demonstrate the presence of the LL-37 precursor, human cathelicidin precursor protein designated 18-kDa cationic antimicrobial protein, in the CF lung along with evidence that it is processed to active LL-37 by proteinase-3. We demonstrate that despite supranormal levels of LL-37, the lung fluid from CF patients exhibits no demonstrable antimicrobial activity. Furthermore Pseudomonas killing by physiological concentrations of exogenous LL-37 is inhibited by CF bronchoalveolar lavage (BAL) fluid due to proteolytic degradation of LL-37 by neutrophil elastase and cathepsin D. The endogenous LL-37 in CF BAL fluid is protected from this proteolysis by interactions with glycosaminoglycans, but while this protects LL-37 from proteolysis it results in inactivation of LL-37 antimicrobial activity. By digesting glycosaminoglycans in CF BAL fluid, endogenous LL-37 is liberated and the antimicrobial properties of CF BAL fluid restored. High sodium concentrations also liberate LL-37 in CF BAL fluid in vitro. This is also seen in vivo in CF sputum where LL-37 is complexed to glycosaminoglycans but is liberated following nebulized hypertonic saline resulting in increased antimicrobial effect. These data suggest glycosaminoglycan–LL-37 complexes to be potential therapeutic targets. Factors that disrupt glycosaminoglycan–LL-37 aggregates promote the antimicrobial effects of LL-37 with the caveat that concomitant administration of antiproteases may be needed to protect the now liberated LL-37 from proteolytic cleavage.
Rationale: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune and inflammatory processes.Objectives: To investigate the capacity of anaerobes to contribute to cystic fibrosis (CF) airway pathogenesis via SCFAs.Methods: Samples of 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFA levels in anaerobic supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of two SCFA receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings and 16HBE14o 2 and CFBE41o 2 cells were evaluated using reverse transcription polymerase chain reaction, Western blot analysis, laser scanning cytometry, and confocal microscopy. SCFA-induced IL-8 secretion was monitored by ELISA.Measurements and Main Results: Fifty-seven (52.3%) of 109 PWCF were anaerobe positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF younger (n = 24) and older (n = 85) than 6 years of age. All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic, and butyric acids. SCFA levels were higher in BAL samples of adults than in those of children. GPR41 levels were elevated in CFBE41o 2 versus 16HBE14o 2 cells; CF versus non-CF bronchial brushings; and 16HBE14o 2 cells after treatment with cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dosedependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells, with a higher production of IL-8 in CFBE41o 2 than in 16HBE14o 2 cells.Conclusions: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via up-regulated GPR41.Keywords: anaerobic bacteria; cystic fibrosis; inflammation; short-chain fatty acids Correspondence and requests for reprints should be addressed to Bojana Mirković, Ph
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