Culture with apically applied healthy or disease sputum alters the airway surface liquid proteome and ion transport across human bronchial epithelial cells.

Maximillian Woodall,Robert Tarran,Mehmet Kesimer,Boris Reidel,Deborah L. Baines

doi:10.1152/ajpcell.00234.2021

Abstract

Airway secretions contain many signaling molecules and peptides/proteins that are not found in airway surface liquid (ASL) generated by normal human bronchial epithelial cells (NHBEs) in vitro. These play a key role in innate defense and mediate communication between the epithelium, the immune cells, and the external environment. We investigated how culture of NHBE with apically applied secretions from healthy or diseased (cystic fibrosis, CF) lungs affected epithelial function with a view to providing better in vitro models of the in vivo environment. NHBEs from 6 to 8 different donors were cultured at air-liquid interface (ALI), with apically applied sputum from normal healthy donors (normal lung sputum; NLS) or CF donors (CFS) for 2–4 h, 48 h, or with sputum reapplied over 48 h. Proteomics analysis was carried out on the sputa and on the NHBE ASL before and after culture with sputa. Transepithelial electrical resistance (TEER), short circuit current (Isc), and changes to ASL height were measured. There were 71 proteins common to both sputa but not ASL. The protease:protease inhibitor balance was increased in CFS compared with NLS and ASL. Culture of NHBE with sputa for 48 h identified additional factors not present in NLS, CFS, or ASL alone. Culture with either NLS or CFS for 48 h increased cystic fibrosis transmembrane regulator (CFTR) activity, calcium-activated chloride channel (CaCC) activity, and changed ASL height. These data indicate that culture with healthy or disease sputum changes the proteomic profile of ASL and ion transport properties of NHBE and this may increase physiological relevance when using in vitro airway models.

Highlights

Airway secretions contain signaling molecules and peptides/proteins that play a key role in innate defense and mediate communication between the external environment, inflammatory cells, and the epithelium
One hundred and thirty-six proteins were common to all samples, whereas 22 proteins were exclusively found in NLS, 19 in cystic fibrosis (CF) donors (CFS), and 805 in Normal human bronchial epithelial cells (NHBEs) airway surface liquid (ASL) (Fig. 1B)
The proteins exclusively found in CFS were tightly correlated with immunity pathways including regulated exocytosis, neutrophil degranulation, immune response, cell activation, and transport

Summary

Introduction

Airway secretions contain signaling molecules and peptides/proteins that play a key role in innate defense and mediate communication between the external environment, inflammatory cells, and the epithelium. Reciprocal activity of the cystic fibrosis transmembrane regulator (CFTR) anion channel and the epithelial Na þ channel (ENaC) is important for maintaining the correct hydration of the airway surface liquid (ASL), mucociliary clearance, and innate immune function of the airways. In respiratory diseases such as cystic fibrosis (CF), aberrant CFTR transport results in an altered luminal environment. ASL volume, pH, protease activity, pathogenic bacteria and their toxins, inflammatory cells, and inflammatory mediators all contribute to the development of a thick viscous sputum [1,2,3,4,5] These changes can in turn, further modify CFTR and ENaC activity. These findings indicate that factors present in the disease sputum can have short-term and long-term effects on ion channel activity in vitro

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Results

Conclusion