LPS decreases CFTR open probability and mucociliary transport through generation of reactive oxygen species
Do Yeon Cho,
Shaoyan Zhang,
Ahmed Lazrak,
Daniel Skinner,
Harrison M. Thompson,
Jessica Grayson,
Purushotham Guroji,
Saurabh Aggarwal,
Zsuzsanna Bebok,
Steven M. Rowe,
Sadis Matalon,
Eric J. Sorscher,
Bradford A. Woodworth
Affiliations
Do Yeon Cho
Department of Otolaryngology Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA; Division of Otolaryngology, Department of Surgery, Veterans Affairs, Birmingham, AL, USA
Shaoyan Zhang
Department of Otolaryngology Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
Ahmed Lazrak
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL, USA
Daniel Skinner
Department of Otolaryngology Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
Harrison M. Thompson
Department of Otolaryngology Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL, USA
Jessica Grayson
Department of Otolaryngology Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL, USA
Purushotham Guroji
Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA
Saurabh Aggarwal
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL, USA
Zsuzsanna Bebok
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA
Steven M. Rowe
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Sadis Matalon
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL, USA
Eric J. Sorscher
Department of Pediatrics, Emory University, Atlanta, GA, USA
Bradford A. Woodworth
Department of Otolaryngology Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA; Corresponding author. Department of Otolaryngology – Head and Neck Surgery, University of Alabama at Birmingham, 1155 Faculty Office Tower, 510 20th Street South, Birmingham, AL, 35233, USA.
Lipopolysaccharide (LPS) serves as the interface between gram-negative bacteria (GNB) and the innate immune response in respiratory epithelial cells (REC). Herein, we describe a novel biological role of LPS that permits GNB to persist in the respiratory tract through inducing CFTR and mucociliary dysfunction. LPS reduced cystic fibrosis transmembrane conductance regulater (CFTR)-mediated short-circuit current in mammalian REC in Ussing chambers and nearly abrogated CFTR single channel activity (defined as forskolin-activated Cl- currents) in patch clamp studies, effects of which were blocked with toll-like receptor (TLR)-4 inhibitor. Unitary conductance and single-channel amplitude of CFTR were unaffected, but open probability and number of active channels were markedly decreased. LPS increased cytoplasmic and mitochondrial reactive oxygen species resulting in CFTR carbonylation. All effects of exposure were eliminated when reduced glutathione was added in the medium along with LPS. Functional microanatomy parameters, including mucociliary transport, in human sinonasal epithelial cells in vitro were also decreased, but restored with co-incubation with glutathione or TLR-4 inhibitor. In vivo measurements, following application of LPS in the nasal cavities showed significant decreases in transepithelial Cl- secretion as measured by nasal potential difference (NPD) – an effect that was nullified with glutathione and TLR-4 inhibitor. These data provide definitive evidence that LPS-generated reactive intermediates downregulate CFTR function in vitro and in vivo which results in cystic fibrosis-type disease. Findings have implications for therapeutic approaches intent on stimulating Cl- secretion and/or reducing oxidative stress to decrease the sequelae of GNB airway colonization and infection.
