The SPLUNC1-ßENaC complex prevents Burkholderia cenocepacia invasion in normal airway epithelia.

Cystic fibrosis (CF) patients are extremely vulnerable to Burkholderia cepacia complex (Bcc) infections. However, the underlying etiology is poorly understood. We tested the hypothesis that short palate lung and nasal epithelial clone 1 (SPLUNC1)-epithelial sodium channel (ENaC) interactions at the plasma membrane are required to reduce Bcc burden in normal airways. To determine if SPLUNC1 was needed to reduce Bcc burden in the airways, SPLUNC1 knockout mice and their wild-type littermates were infected with B. cenocepacia strain J2315. SPLUNC1 knockout mice had increased bacterial burden in the lungs compared to wild-type littermate mice. SPLUNC1-knockdown primary human bronchial epithelia (HBECs) were incubated with J2315, which resulted in increased bacterial burden compared to non-transduced HBECs. We next determined the interaction of the SPLUNC1-ENaC complex during J2315 infection. SPLUNC1 remained at the apical plasma membrane of normal HBECs but less was present at the apical plasma membrane of CF HBECs. Additionally, SPLUNC1-ßENaC complexes reduced intracellular J2315 burden. Our data indicate that (i) secreted SPLUNC1 is required to reduce J2315 burden in the airways and (ii) its interaction with ENaC prevents cellular invasion of J2315.

Automated acquisition and analysis of airway surface liquid height by confocal microscopy.

The airway surface liquid (ASL) is a thin-liquid layer that lines the luminal side of airway epithelia. ASL contains many molecules that are involved in primary innate defense in the lung. Measurement of ASL height on primary airway cultures by confocal microscopy is a powerful tool that has enabled researchers to study ASL physiology and pharmacology. Previously, ASL image acquisition and analysis were performed manually. However, this process is time and labor intensive. To increase the throughput, we have developed an automatic ASL measurement technique that combines a fully automated confocal microscope with novel automatic image analysis software that was written with image processing techniques derived from the computer science field. We were able to acquire XZ ASL images at the rate of ∼ 1 image/s in a reproducible fashion. Our automatic analysis software was able to analyze images at the rate of ∼ 32 ms/image. As proofs of concept, we generated a time course for ASL absorption and a dose response in the presence of SPLUNC1, a known epithelial sodium channel inhibitor, on human bronchial epithelial cultures. Using this approach, we determined the IC50 for SPLUNC1 to be 6.53 µM. Furthermore, our technique successfully detected a difference in ASL height between normal and cystic fibrosis (CF) human bronchial epithelial cultures and detected changes in ATP-stimulated Cl(-)/ASL secretion. We conclude that our automatic ASL measurement technique can be applied for repeated ASL height measurements with high accuracy and consistency and increased throughput.