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.

Keywords: airway epithelium; cell differentiation; cilia.

FIGURE 1

Loss of PC induces an alteration of motile ciliogenesis during AEC differentiation. A, Examples of micrographs taken from AEC cultures at ALI‐7 (upper panel) and ALI‐14 (bottom panel) showing PC (Arl13b, red; γ‐tubulin, green). Nuclei are stained in blue (DAPI). B, Dot plot (mean ±SEM) showing PC number per mm² at ALI‐7 (n = 8) and ALI‐14 (n = 6) in control (CTL) condition (black) and CH (red)‐treated cells. **P < 0.01 CH vs CTL. C, Histograms representing the assessment of fold‐change (log2) in the normalized expression to GAPDH during ALI cultures by RT‐qPCR (n = 11) for ciliogenesis markers FOXJ1, MCIDAS and HEATR2 at ALI‐2; ALI‐7 and ALI‐14. Results show mean ±SEM, **P < 0.005, *P < 0.05 CH vs CTL. D, Examples of micrographs taken from AEC cultures at ALI‐14 (left panel) and ALI‐35 (right panel) showing MC (Arl13b, red). Nuclei are stained in blue (DAPI). E, Dot plot (mean ±SEM) represents the mean grey values of MC‐associated fluorescence in CTL condition (black; n = 4) and CH‐treated cells (red; n = 5) at ALI‐14 and ALI‐35. **P < 0.01 CH vs CTL and *P < 0.05 CH vs CTL. F, Histograms representing the assessment of fold‐change CH/CTL (log2) in the normalized expression to GAPDH during ALI cultures by RT‐qPCR (n = 10) for ciliogenesis markers FOXJ1, MCIDAS and HEATR2 at ALI‐35. Results show mean ±SEM, **P < 0.005, *P < 0.05 CH vs CTL

FIGURE 2

Loss of PC induces global epithelial remodelling during AEC differentiation. A, Histograms representing the TEER (log2, normalized to CTL, n = 6) of CH‐treated ALI cultures (n = 6). Results show mean ±SEM, ***P < 0.001, **P < 0.01, *P < 0.05 CH vs CTL. B, Examples of micrographs taken from AEC cultures at ALI‐35 showing cleaved caspase‐3 (red) and Zonula occludens‐1 (ZO1, green). Nuclei are stained in blue (DAPI). C, Histogram representing mean grey values of the cleaved caspase‐3‐associated fluorescence of CH‐treated AEC (n = 3) in log 2 ratio CTL/CH at ALI‐35. *P < 0.05 CH vs CTL. D, Histograms representing the assessment of fold‐change (log2) in the normalized expression to GAPDH during ALI cultures by RT‐qPCR (n = 11) for non‐differentiated cell markers (CK5, SOX2, SOX9), and secretory cell markers (SPDEF, SCGB1A1, MUC5AC, MUC5B) at ALI‐35. Results show mean ±SEM, *P < 0.05 CH vs CTL. E, Examples of micrographs taken from AEC cultures at ALI‐35 showing mucins (Muc5b, red; Muc5ac, green). Nuclei are stained in blue (DAPI). F, Histogram representing the relative mean grey values of the mucins‐associated fluorescence normalized to CTL at ALI‐35 of CH‐treated cells for Muc5b and Muc5ac (n = 9). *P < 0.05 CH vs CTL. G, Illustration summarizing in vitro AEC remodelling upon PC inhibition