Quantitative Proteomics Reveals an Altered Cystic FibrosisIn VitroBronchial Epithelial Secretome

Abstract

Alterations in epithelial secretions and mucociliary clearance contribute to chronic bacterial infection in cystic fibrosis (CF) lung disease, but whether CF lungs are unchanged in the absence of infection remains controversial. A proteomic comparison of airway secretions from subjects with CF and control subjects shows alterations in key biological processes, including immune response and proteolytic activity, but it is unclear if these are due to mutant CF transmembrane conductance regulator (CFTR) and/or chronic infection. We hypothesized that the CF lung apical secretome is altered under constitutive conditions in the absence of inflammatory cells and pathogens. To test this, we performed quantitative proteomics of in vitro apical secretions from air-liquid interface cultures of three lifeextended CF (DF508/DF508) and three non-CF human bronchial epithelial cells after labeling of CF cells by stable isotope labeling with amino acids in cell culture. Mass spectrometry analysis identified and quantitated 666 proteins across samples, of which 70 exhibited differential enrichment or depletion in CF secretions (61.5-fold change; P , 0.05). The key molecular functions were innate immunity (24%), cytoskeleton/extracellular matrix organization (24%), and protease/antiprotease activity (17%). Oxidative proteins and classical complement pathway proteins that are altered in CF secretions in vivo were not altered in vitro. Specific differentially increased proteins-MUC5AC and MUC5B mucins, fibronectin, and matrix metalloproteinase-9-were validated by antibody-based assays. Overall, the in vitro CF secretome data are indicative of a constitutive airway epithelium with altered innate immunity, suggesting that downstream consequences of mutant CFTR set the stage for chronic inflammation and infection in CF airways.

Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance

“…Approximately half of these proteins (53%) were significantly different in abundance between genotypes, with 80% being increased in CF. This finding is similar to those of a recent study evaluating the ASL proteome of CF and non-CF HBE cultures, which demonstrated that the majority of differentially expressed proteins was elevated in CF (26). An unexpected finding was the inclusion of many intracellular proteins and membrane-associated proteins ( Figure 1D), with only 25.6% (z668 protein) of the total 2,608 proteins being classified as known secreted proteins (27) (Figure 1E, Table E2B).…”

Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis

“…CF ASN is characterized by an abnormal molecular composition (including altered lactate, small metabolite, and protein contents), itself caused by altered epithelial function, and the sustained presence of bacteria and PMNs in the CF airway lumen . It is interesting to note that at concentrations similar to those used to promote PMN transmigration and conditioning in our model, CF ASN induces T cell apoptosis .…”

Inflammation and its genesis in cystic fibrosis

“…CF mouse models, which often lack airway obstruction and spontaneous infection, consistently demonstrate heightened and prolonged inflammatory responses in the airway after a disease relevant challenge (e.g., bacteria or bacterial products), supporting the concept of primary alterations in host response . Likewise, a reductionist approach to this question, through use of isolated cells studied in vitro, has repeatedly shown altered inflammatory signaling both with and without infectious challenge . Human CF fetal tracheal grafts implanted into an animal model produced eight times more IL‐8, a potent neutrophil chemoattractant, than did non‐CF fetal tracheal grafts .…”