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.

Keywords: CBF; CiliOPD; airway epithelial cells; chronic obstructive pulmonary disease; cilia.

Figure 1

Cilia beat frequency of multiciliated cells is decreased in COPD patients. Dot plot (mean +/− SD) showing the CBF in non-COPD (black, n = 10) and COPD (red, n = 10) AEC. *, p < 0.05 non-COPD vs. COPD.

Figure 2

Multiciliated cell generation is decreased in COPD patients. (a) Examples of micrographs taken from AEC cultures from non-COPD and COPD patients at ALI-21 and ALI-35 showing motile cilia (Arl13b, red). Nuclei are stained in blue (DAPI). (b) Dot plot (mean +/− SD) represents the mean grey values of cilia-associated fluorescence at ALI-21 and ALI-35 in non-COPD AEC-derived ALI cultures (black, n = 10) and COPD AEC-derived ALI cultures (red, n = 10). ** p < 0.01, *** p < 0.001 non-COPD vs. COPD.

Figure 3

Primary ciliogenesis is altered in COPD-derived AEC ALI cultures. (a) Examples of micrographs taken from AEC cultures from non-COPD and COPD patients at ALI-14 showing motile cilia and primary cilia (Arl13b, red). Nuclei are stained in blue (DAPI). Magnification corresponding to the selected area is shown. White arrowheads indicate primary cilia. Dot plot (mean +/− SD) represents the percentage of MCC (b), the percentage of primary ciliated cells (c), and the length of PC (d) at ALI-14 in non-COPD AEC-derived ALI cultures (black, n = 6) and COPD AEC-derived ALI cultures (red, n = 8). * p < 0.05, ** p < 0.01 non-COPD vs. COPD.