Document Type

Dissertation

Date of Degree

Spring 2017

Degree Name

PhD (Doctor of Philosophy)

Degree In

Molecular Physiology and Biophysics

First Advisor

Michael J. Welsh

Abstract

The airway surface contains a number of important defense mechanisms to protect against infection. Antimicrobials found in the thin layer of fluid lining the airways, the airway surface liquid (ASL), rapidly kill bacteria. Another defense mechanism, mucociliary transport, propels foreign particles and mucus out of the airways. These and potentially other host defense properties show a dependence on the pH of the ASL. An acidic ASL pH reduces bacterial killing by cationic antimicrobial peptides, and increases ASL viscosity, potentially effecting mucociliary transport. Consequently, an acidic ASL pH can impair airway host defense.

An example of a disease where an acidic ASL pH impairs airway host defense, is Cystic Fibrosis (CF). The major cause of morbidity and mortality in CF is airway infections. Humans with CF and the CF pig model develop airway infections. But curiously CF mice are spared. Compared to non-CF, people with CF and CF pigs show an abnormally acidic ASL pH. However, the ASL pH in CF mice is not different to that of non-CF. Thus, we hypothesized that CF mice do not show defects in airway host defense because their ASL pH is not acidic compared to non-CF. As pH is a balance between acid and base secretion, we first determined which HCO3- and H+ secreting proteins contribute to ASL pH and the differences between humans, pigs, and mice.

CF is caused by defects in an anion channel, CFTR. We found that in all three species, CFTR secreted HCO3- into the ASL, which was absent when CFTR was defective. To determine how much CFTR is required to rescue ASL pH and host defense properties, we mixed CF and non-CF airway epithelia from newborn pigs in different ratios. HCO3- secretion, ASL pH, and host defense properties showed a direct relationship to CFTR, suggesting that CFTR was the rate-limiting step. As CFTR was limiting, we found that supernormal CFTR expression in a small number of cells could dramatically increase ASL pH, suggesting viral-mediated gene therapy approaches may have benefit for CF. We found that Ca2+ activated Cl- channels also played some role in ASL pH in CF pigs and CF mice. However, as CF pigs develop airway infection, while CF mice do not, this suggested that other factors might be important for differences in ASL pH and consequently the development of disease.

To further investigate the determinants of ASL pH, we examined H+ secretion. Humans and pigs showed 6 times more H+ secretion compared to mice. This acidification occurred through the non-gastric H+/K+ ATPase (ATP12A). ATP12A was also much more highly expressed in human and pig airways compared to mice. Blocking ATP12A in human and pig airways increased ASL pH and consequently improved host defense properties such as bacterial killing and ASL viscosity. Conversely, expressing ATP12A in CF mouse airways acidified ASL, impaired defenses, and increased airway bacteria.

These findings suggest that ASL pH is a balance between HCO3- and H+ secretion. In humans and pigs lacking CFTR, unchecked H+ secretion by the non-gastric H+/K+ ATPase (ATP12A) acidifies the ASL, which impairs airway host defenses. However, as mouse airways expressed little ATP12A and secrete minimal H+, loss of CFTR does not lead to ASL acidification, protecting CF mice. These findings not only

help explain why CF mice are protected from infection, but also nominate ATP12A as a therapeutic target for improving ASL pH and host defense in humans with CF.

Keywords

Acidification, Airway Surface Liquid (ASL), ATP12A, CFTR, Cystic Fibrosis, pH

Pages

xi, 132 pages

Bibliography

Includes bibliographical references (pages 118-132).

Copyright

Copyright © 2017 Viral Shailesh Shah

Included in

Biophysics Commons

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