Impaired Ciliary Beat Frequency and Ciliogenesis Alteration during Airway Epithelial Cell Differentiation in COPD

Abstract

Chronic obstructive pulmonary disease (COPD) is a frequent respiratory disease. However, its pathophysiology remains partially elucidated. Epithelial remodeling including alteration of the cilium is a major hallmark of COPD, but specific assessments of the cilium have been rarely investigated as a diagnostic tool in COPD. Here we explore the dysregulation of the ciliary function (ciliary beat frequency (CBF)) and differentiation (multiciliated cells formation in air-liquid interface cultures) of bronchial epithelial cells from COPD (n = 17) and non-COPD patients (n = 15). CBF was decreased by 30% in COPD (11.15 +/− 3.37 Hz vs. 7.89 +/− 3.39 Hz, p = 0.037). Ciliary differentiation was altered during airway epithelial cell differentiation from COPD patients. While the number of multiciliated cells decreased (p < 0.005), the number of primary ciliated cells increased (p < 0.05) and primary cilia were shorter (p < 0.05). Altogether, we demonstrate that COPD can be considered as a ciliopathy through both primary non-motile cilia modifications (related to airway epithelial cell repair and remodeling) and motile cilia function impairment (associated with decrease sputum clearance and clinical respiratory symptoms). These observations encourage considering cilia-associated features in the complex COPD physiopathology and highlight the potential of cilia-derived biomarkers for diagnosis.

Ciliogenesis is intrinsically altered in COPD small airways

“…(forward-5'-CTTTACCTGGGACGTGAATGA-3', reverse-5'-GGAAGTCCTTTCACCCCTTT-3'), RFX3 (forward-5'-ACCTCAACCGTGTCGACTTT-3', reverse-5'-GCTGCTGAAGCATCTTGAAGT-3'), SCGB1A1, FOXJ1, MUC-5AC, HEATR2, MCIDAS and RFX2, all previously described [3,10]. Results were normalized to the housekeeping gene GAPDH expression [3,10]. Relative expression (ΔΔCt) was expressed as fold change from ALI D0 values of each gene in each group.…”

“…GMNC, E2F4, FOXJ1, RFX2, RFX3 and HEATR2, showed expression that plateaued after ALI D15 in COPD cultures, unlike their expression in non-COPD cultures. MCC originate from primary ciliated cells (PCC) [10,13] and we previously reported a dysregulation of PC in COPD bronchial epithelium [3,4]. We therefore analysed primary ciliogenesis in bronchiolar epithelium.…”

“…To the Editor, Chronic obstructive pulmonary disease (COPD) is characterised by a progressive and irreversible airflow limitation due to airway obstruction and emphysema [1]. We and others showed that bronchial epithelial remodelling in COPD is characterised by alteration of ciliogenesis and cilia function [2,3], as well as a dysregulation of non-motile primary cilia (PC) [4]. In COPD, the main site of obstruction is located in small airways [5].…”

“…Cell proliferation was assessed using TOX8 kit (Sigma, St Louis, MO). Expression of ciliogenesis effectors in cultures (ALI D0, D7, D15 and D25) was assessed by RT-qPCR as previously described [3]. Specific primers (Eurogentec, Seraing, Belgium) were P63 (forward-5'-CGCCATGCCTGTCTACAA-3', reverse-5'-TGACTAGGAGGGGCAATCTG-3'), E2F4 (forward-5'-ATCAAGGCAGACCCCACA-3', reverse-5'-GGGCAAACACTTCTGAGGAC-3'), GMNC (forward-5'-ACGGAGACTTGGGTCTCTTTC-3', reverse-5'-TCCGGAAGAGGAAAATTTGA-3'),…”

Whole-Exome Sequencing of Bronchial Epithelial Cells Reveals a Genetic Print of Airway Remodelling in COPD

“…The molecular alteration of airway basal cells, which are the progenitors of secretory and ciliated cells, is central to the pathogenesis of COPD [ 6 , 7 , 8 ]. We previously demonstrated the dysregulation of primary cilia associated with the presence of epithelial remodelling in lung tissues or airway epithelial cells obtained from COPD patients [ 9 , 10 ].…”