Intraflagellar Transport Gene Expression Associated with Short Cilia in Smoking and COPD

Justina Hessel,Michelle R Staudt,Ronald G Crystal,Christopher S Stevenson,Gerhard Wolff,Sreekumar Pillai,Robert J Kaner,Holly Hilton,Charleen Hollmann,Jonna Heldrich,Hans Bitter,Ann E Tilley,Jennifer Fuller,Ben-Gary Harvey,Sudha Visvanathan,Jacqueline Salit,Jenny Yee-Levin,Sharon Radisch,Srinivas Sridhar ,Jay S Fine

doi:10.1371/journal.pone.0085453

Abstract

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.

Highlights

The lung is constantly exposed to inhaled particles, pathogens, irritants and toxins
In exploring how smoking might impact cilia length, based on published observations of the central role of IFT in cilia length, we hypothesized that smoking causes decreased expression of intraflagellar transport genes in the human airway epithelium and that this reduced expression is associated with the shorter cilia observed in smokers. We addressed these hypotheses by assessing cilia length in the large and small airway epithelium of nonsmokers, healthy smokers, and smokers with chronic obstructive pulmonary disease (COPD), and by quantifying expression of 40 IFT genes in the airway epithelium of these groups
Study Population Cilia length was assessed in 228 airway epithelium samples, including 120 LAE samples (n = 25 healthy nonsmokers, n = 25 healthy smokers, and n = 70 COPD smokers [n = 34 GOLD I, n = 29 GOLD II, n = 7 GOLD III]) and 108 SAE samples (n = 20 healthy nonsmokers, n = 32 healthy smokers, and n = 56 COPD smokers [n = 18 GOLD I, n = 29 GOLD II, n = 8 GOLD III, n = 1 GOLD IV]) (Table 1)

Summary

Introduction

The lung is constantly exposed to inhaled particles, pathogens, irritants and toxins. In chronic obstructive pulmonary disease (COPD), mucociliary clearance is dysfunctional for a variety of reasons, which may include alterations in mucus composition and generation, and adverse effects of cigarette smoke on cilia structure and function [5,6]. A number of theoretical mechanistic explanations have been proposed These include the molecular ruler model, in which a protein with a physical length matching the cilia length controls cilia length [8,9]; the limited precursor model, in which cilia length is limited by limited quantities of necessary precursor molecules [9,10]; the cumulated strain model, in which cilia length is controlled by binding energy changes induced by changes in conformation with increasing length [9,11]; a feedback control model, involving length sensing and signal transduction [9,12]; and the balance point model, which is based on the knowledge that ciliary disassembly at the tip is ongoing at steady-state and appears to be length-independent, and which states that there is specific length at which this disassembly process is balanced with the rate of assembly, dependent on the rate of intraflagellar transport of components to the cilia tip [9,13]

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Results

Conclusion