Gel-forming mucins, the primary macromolecular components of airway mucus, facilitate airway clearance by mucociliary transport. In cystic fibrosis (CF) altered mucus properties impair mucociliary transport. Airways primarily secrete two closely related gel-forming mucins, MUC5B and MUC5AC. However, their morphologic structures and associations in airways that contain abundant submucosal glands and goblet cells are uncertain. Moreover, there is limited knowledge about mucins in airways not affected by inflammation, infection, or remodeling or in CF airways. Therefore, we examined airways freshly excised from newborn non-CF pigs and CF pigs before secondary manifestations develop. We found that porcine submucosal glands produce MUC5B, whereas goblet cells produce predominantly MUC5AC plus some MUC5B. We found that MUC5B emerged from submucosal gland ducts in the form of strands composed of multiple MUC5B filaments. In contrast, MUC5AC emerged from goblet cells as wispy threads and sometimes formed mucin sheets. In addition, MUC5AC often partially coated the MUC5B strands. Compared with non-CF, MUC5B more often filled CF submucosal gland ducts. MUC5AC sheets also accumulated in CF airways overlying MUC5B strands. These results reveal distinct morphology and interactions for MUC5B and MUC5AC and suggest that the two mucins make distinct contributions to mucociliary transport. Thus, they provide a framework for understanding abnormalities in disease.mucus | cystic fibrosis | lung | asthma | COPD

The pulmonary airways are continuously exposed to bacteria. As a first line of defense against infection, the airway surface liquid (ASL) contains a complex mixture of antimicrobial factors that kill inhaled and aspirated bacteria. The composition of ASL is critical for antimicrobial effectiveness. For example, in cystic fibrosis an abnormally acidic ASL inhibits antimicrobial activity. Here, we tested the effect of pH on the activity of an ASL defensin, human β-defensin-3 (hBD-3), and the cathelicidin-related peptide, LL-37. We found that reducing pH from 8.0 to 6.8 reduced the ability of both peptides to kill Staphylococcus aureus. An acidic pH also attenuated LL-37 killing of Pseudomonas aeruginosa. In addition, we discovered synergism between hBD-3 and LL-37 in killing S. aureus. LL-37 and lysozyme were also synergistic. Importantly, an acidic pH reduced the synergistic effects of combinations of ASL antibacterials. These results indicate that an acidic pH reduces the activity of individual ASL antimicrobials, impairs synergism between them, and thus may disrupt an important airway host defense mechanism.

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR -/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

People with cystic fibrosis (CF) exhibit growth defects. That observation has been attributed, in part, to decreased insulin-like growth factor 1 (IGF1) levels, and the reduction has been blamed on malnutrition and pulmonary inflammation. However, patients with CF already have a reduced weight at birth, a manifestation not likely secondary to poor nutrition or inflammation. We found that, like humans, CF pigs were smaller than non-CF littermates and had lower IGF1 levels. To better understand the basis of IGF1 reduction, we studied newborn pigs and found low IGF1 levels within 12 h of birth. Moreover, humerus length and bone mineral content were decreased, consistent with less IGF1 activity in utero. These findings led us to test newborn humans with CF, and we found that they also had reduced IGF1 levels. Discovering lower IGF1 levels in newborn pigs and humans indicates that the decrease is not solely a consequence of malnutrition or pulmonary inflammation and that loss of cystic fibrosis transmembrane conductance regulator function has a more direct effect. Consistent with this hypothesis, we discovered reduced growth hormone release in organotypic pituitary slice cultures of newborn CF pigs. These findings may explain the long-standing observation that CF newborns are smaller than non-CF babies and why some patients with good clinical status fail to reach their growth potential. The results also suggest that measuring IGF1 levels might be of value as a biomarker to predict disease severity or the response to therapeutics. Finally, they raise the possibility that IGF1 supplementation beginning in infancy might be beneficial in CF.

Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAREx7 and CAREx8). We found low-level expression of the CAREx8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAREx7, CAREx8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAREx7, CAREx8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAREx8 providing a potential mechanism for the apical localization of CAREx8 in airway epithelial. In summary, apical localization of CAREx8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.