DOI

10.17077/etd.ao84lcg9

Document Type

Dissertation

Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Molecular Physiology and Biophysics

First Advisor

Michael J. Welsh

First Committee Member

Michael Anderson

Second Committee Member

Christopher Benson

Third Committee Member

Charles Harata

Fourth Committee Member

Amy Lee

Fifth Committee Member

Andrew Russo

Abstract

Cystic fibrosis (CF) is a common lethal hereditary disease resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR dysfunction affects multiple organ systems and most morbidity and mortality in CF results from lung disease. The CF lung appears healthy at birth, but spontaneously develops airway disease characterized by infection, inflammation, mucus plugging and airway remodeling. A CF pig model was recently generated to determine the events that initiate lung disease. CF pigs recapitulate many findings seen in humans with CF, including the spontaneous development of lung disease. I used newborn CF pigs to investigate two leading hypotheses regarding CF disease initiation: abnormal airway surface liquid (ASL) composition and defective mucociliary transport (MCT). I developed an assay to study ASL composition and found that CF ASL contained similar sodium concentrations, elevated potassium concentrations, and a decreased fraction of volatile material. I developed an assay to measure MCT in vivo. By tracking individual particles in 3-dimensions I found that newborn pigs exhibit a ventrally directed cilia orientation in the trachea. I also found that MCT is highly heterogeneous and particles traveled at different speeds within airways and between airways, challenging the classic view that airway mucus exists as continuous blanket. Comparing particle transport revealed that non-CF and CF newborn pigs exhibit similar basal particle clearance and speeds. Cholinergic stimulation induces mucus and fluid secretion. Particles became stuck in newborn CF pigs after cholinergic stimulation and stasis persisted with tissue submersion. This challenged the leading hypothesis that attributes CF airway disease pathogenesis to ASL depletion. I hypothesized that adherent mucus impairs mucociliary transport in CF airways and I developed an assay to visualize mucus stasis in submerged tracheal segments ex vivo. CF trachea stimulated in vivo exhibited highly adhesive mucus entities that emerged exclusively from submucosal gland ducts. These adherent entities impaired MCT even with extremely high ASL depths. Non-CF trachea with combinatorial disruption of HCO3- and Cl- transport reproduced the defect in CF signifying that anion transport disruption was responsible for adherent mucus. These data suggest that CFTR disruption directly produces multiple host defense defects, including defective bacterial killing and abnormally adherent mucus. Therapeutic targeting of the described defects may provide new opportunities to intervene early and improve the lives of those with CF.

Public Abstract

Cystic fibrosis (CF) is a common lethal inherited disease that affects multiple organ systems, but most morbidity and mortality results from lung disease. The CF lung appears healthy at birth and spontaneously develops an airway disease characterized by infection, inflammation, and airway mucus plugging. However, the origins of CF airway disease are unknown. A CF pig model was generated to determine the events that initiate lung disease. Using newborn CF pigs, I investigated two leading hypotheses regarding CF disease initiation: abnormal composition of the thin layer of fluid lining the airways known as airway surface liquid (ASL) and defective transport of particles out of the lung by mucus and cilia through a process described as mucociliary transport (MCT). Studies of ASL composition found no differences in sodium concentration between non-CF and CF pigs. To study MCT, I developed a method to measure transport of individual particles in vivo in newborn pigs. Non-CF and CF newborn pigs exhibited similar basal particle clearance and speeds, but particles became stuck in CF pigs after stimulating secretion of mucus and fluid. I hypothesized that adherent mucus in CF airways caused particles to become stuck. To test this hypothesis, I developed a method to visualize mucus secretion in living tissue and found that mucus fails to detach from mucus production sites in CF airways thereby disrupting MCT. Mucus detachment failure may represent a CF disease initiator and an early therapeutic target.

Keywords

Cilia, Cystic Fibrosis, Mucociliary Transport, Mucus, Pigs

Pages

xvi, 127 pages

Bibliography

Includes bibliographical references (pages 116-127).

Copyright

Copyright © 2015 Mark Jeffrey Hoegger

Included in

Biophysics Commons

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