Primary ciliogenesis is a crucial step for multiciliated cell determinism in the respiratory epithelium

Abstract

The alteration of the mucociliary clearance is a major hallmark of respiratory diseases related to structural and functional cilia abnormalities such as chronic obstructive pulmonary diseases (COPD), asthma and cystic fibrosis. Primary cilia and motile cilia are the two principal organelles involved in the control of cell fate in the airways. We tested the effect of primary cilia removal in the establishment of a fully differentiated respiratory epithelium. Epithelial barrier integrity was not altered while multiciliated cells were decreased and mucous‐secreting cells were increased. Primary cilia homeostasis is therefore paramount for airway epithelial cell differentiation. Primary cilia‐associated pathophysiologic implications require further investigations in the context of respiratory diseases.

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

“…Primary ciliogenesis is a necessary step during the cell cycle to orchestrate the fate of the two daughter cells: non-differentiated cells (able to form a PC) and differentiated cells (either MCC or secretory cells). Primary cilia are transient in homeostasis but their dysregulation (loss or maintenance) may induce an alteration of cell fate as we recently evidenced during AEC differentiation [ 27 ]. In COPD, the increase of PCC suggested that non-differentiated cells did not progress through the cell cycle and were blocked in G0.…”

“…Micrographs were acquired on a Zeiss AxioImager microscope (20× Ph and 63×) with ZEN software (version 8.1, 2012, Zeiss, Marly le Roi, France) and processed with ImageJ (National Institutes of Health, version 2.2.0, Bethesda, MD, USA) for analysis. We routinely control the specificity of the cilia labelling with co-staining and we perform negative and positive controls for each set of experiment [ 11 , 27 ]. The density of multiciliated cells (MCC) was evaluated with the average pixel density of the fluorescence associated to cilia staining as previously described [ 10 ].…”

Ciliogenesis is intrinsically altered in COPD small airways

“…A correlation between the decreased number of PCC and that of MCC in COPD small airway epithelium could be expected. Indeed, it was previously reported that PC removal of airway undifferentiated cells led to impairment of multiciliogenesis [10] and that murine tracheal MCC originated from PCC [13]. However, in COPD bronchial epithelium, the number of PCC is markedly increased, especially in areas of epithelium remodelling, suggesting the involvement of PC in altered epithelial repair/regeneration processes in COPD [4].…”

“…(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.…”