A novel method for live imaging of human airway cilia using wheat germ agglutinin

Multiciliated epithelial cells in the airway are essential for mucociliary clearance. Their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. Epithelial surfaces were observed using an epi-fluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.

Scientific RepoRtS | (2020) 10:14417 | https://doi.org/10.1038/s41598-020-71049-z www.nature.com/scientificreports/ et al. successfully showed perturbation of the ciliary beating direction in an Odf2 gene-mutated mouse 5 . However, the human trachea is sufficiently thick to obscure light, making it difficult to observe ciliary movement. Labeling cilia with fluorescent materials may be necessary to observe the movement of human airway cilia in situ. The surface of the airway cilia is covered with a mesh-like network of membrane-tethered mucin and cell surface proteoglycans, which are considered to be important feature for maintaining appropriate physiological properties for mucociliary clearance. This implies the possibility that agglutinins could be used for labeling cilia 12,13 . MUC1 and 4 and heparan and keratan sulfate proteoglycans have been detected around airway cilia 12,14 . Sugar chains in these molecules contain sialic acid and N-acetyl glucosamine, which can be a target of wheat germ agglutinin (WGA) [15][16][17][18][19] . Okada et al. have shown that WGA can be attached to the cilia of cultured airway epithelial cells. This strongly suggests the potential usefulness of WGA for airway cilia visualization 14 .
In this study, we found that WGA) attaches to the surface of airway epithelial cilia in murine and human tracheae. WGA conjugated with a fluorescent dye enabled live imaging of airway epithelial cilia. Herein, we report a method for live imaging of cilia, and show aberrant ciliary movement in 2 tracheal samples with abnormal cilia, obtained from patients who underwent radiation therapy and tracheotomy.

Results
Binding capacity of WGA to airway cilia. We initially used mouse tracheae to determine the capability of WGA to stain the cilia of living airway epithelial cells. Since mouse tracheae are sufficiently thin so as to observe ciliary movement through transmitted light, live images of WGA-stained cilia can be compared with those obtained using transmitted light. An epifluorescence microscope equipped with a × 60 water-immersion lens and a high-speed camera were used for observation (Fig. 1).
Tracheal epithelial cilia were successfully visualized with fluorescein isothiocyanate-WGA (FITC-WGA) in 5 out of 5 mice ( Fig. 2A, Movie 1). A large proportion of the cilia observed by transmitted light were positive for FITC-WGA, and the movements of cilia stained with FITC-WGA were similar to those observed via transmitted light. The movement of FITC-WGA-stained cilia was observed as a directional beat of hair-like shapes. The timing of ciliary beat initiation differed in ciliated cells in close proximity to each other. In addition, epithelia stained with FITC-WGA still exhibited the capacity to transport fluorescent micro-beads (data not shown). These results suggested that metachronal ciliary beating and transport functions are maintained even after FITC-WGA treatment. On the other hand, ciliary beat frequency (CBF) in the FTC-WGA-treated epithelium was approximately 15% lower than that in untreated control epithelium (Fig. 2B). It may be possible that the aggregation of carbohydrates and WGA hampers the ciliary beat, at least to some extent.
Visualization of human airway cilia movement using fluorescence-labeled WGA . FITC-WGA staining also enabled the observation of ciliary movement in all 13 human tracheal samples. A representative movie of the ciliary movement is shown in Movie 2, and the captured frame of Movie 2 and scanning electron microscopy (SEM) image of the same sample are shown in Fig. 3. The mean CBF of ciliated cells in the tracheal sample was approximately 12 Hz. The directionality of the ciliary beating was discernible. Therefore, these data tracheal epithelial ciliary movement in patients who underwent radiation therapy and tracheotomy. A tracheal sample, obtained from a patient who had been irradiated for cancer treatment, had damage to the epithelium. Some regions consisted of ciliated cells with normal and abnormally short cilia (Fig. 4).   To evaluate the disorganization of directionality, the standard deviation of the ciliary beat direction was determined. The standard deviation in the irradiated sample was higher than that in the normal epithelium (1.29 vs. 0.07). A tracheal sample was obtained from a patient who underwent tracheotomy approximately 1 month prior to sampling. In some regions, the epithelium was mainly covered by short cilia (Fig. 5). Due to the shortness of the cilia, it was unclear where the power stroke of cilia was directed; however, the axis of the cilium stroke appeared to be disorganized.
These results suggested that using fluorescence-labeled WGA may reveal dysfunctions in ciliary directionality after airway epithelial damage.

Discussion
The current study showed FITC-WGA enabled observation of the human tracheal ciliary beat, and to discern the directionality or axis of such beating in normal and damaged epithelia. Recent studies have shown that the directionality of airway ciliated cells is regulated by planar cell polarity (PCP) signaling. PCP is a property that coordinates the patterning of cells and the orientation of their structures along tissue planes 20 . In each ciliated airway cell, the distal-to-proximal ciliary movement is organized by the proximal localization of Vangl, and the distal localization of Frizzled 21,22 . However, the mechanisms regulating tissue-level planar polarity in the airway epithelium are largely unknown. Moreover, re-organization and maintenance of PCP during regeneration and cellular turn-over processes of the airway epithelium have rarely been investigated. Our method may be useful for providing further insights into PCP regulation in the airway epithelium.
It may be beneficial to recognize epithelial condition by FITC-WGA staining. In the airway, epithelial stem/ progenitor cells exhibit great proliferation capacity after damage 23,24 . The morphological properties of regenerated airway epithelia are similar to those of undamaged epithelia, except in certain chronic respiratory diseases. However, it is largely unexplored whether ciliary beating is restored after the regeneration process 24,25 . Our method can locate damaged regions, and analyze CBF and directionality in situ.
Mouse tracheae stained with FITC-WGA exhibited lower CBF than unstained ones. Hence, CBF measured in FITC-WGA-stained samples was not comparable with unstained samples. It should also be noted that the sensory function of airway cilia may be hampered by WGA interactions. It has been reported that airway cilia are chemosensory, and WGA reduces the responses of olfactory cilia to odorants 26,27 .
In summary, fluorescence-labeled WGA bound to the surface of human airway epithelial cilia. The movement of fluorescence-labeled airway cilia was captured using a high-speed camera at 125 fps. Additionally, fluorescence-labeling rendered the directionality of ciliary beating discernible. Therefore, using fluorescencelabeled WGA may be useful for analyzing the movement of human airway epithelial cilia.

Methods
Mouse trachea. Five mice (C57BL/6 strain, 12 weeks old, female) were euthanized with approval from the Animal Care and Use Committee of Kyoto University by cervical dislocation, and tracheae were obtained. All animal experiments were performed in accordance with the institutional Guidelines for Animal Experiments, the Japanese Government Law Concerning the Protection and Control of Animals, and the Japanese Government Notification on Feeding and Safekeeping of Animals.
Human trachea. Human tracheae were obtained from 13 patients who underwent laryngectomy for treatment of dysphagia, malignant tumors in the pharynx, larynx, esophagus and tongue, and subglottic stenosis. Before laryngectomy, 4 and 4 of patients received cervical radiation, and tracheotomy within 2 years, respectively. Written informed consent were obtained from all the patients before the surgery, and this study was approved by the Kyoto University Hospital Ethics Community (R1204-2) and conducted in accordance with the Kyoto University Hospital Institutional Guidelines. observation of cilia movement. An upright microscope (BX-51; Olympus, Tokyo, Japan), equipped with a × 60 water-immersion objective lens (Olympus), and a high-speed camera (FASTCAM mini UX50; Photron, Tokyo, Japan), was used to observe ciliary movement (Fig. 1). Mouse and human tracheae stained with FITC-WGA were placed in phenol red-free DMEM/F-12 (Nacalai Tesque, Kyoto, Japan), and the luminal surfaces of the tracheae were observed through transmitted light and/or epi-fluorescent illumination. Images of ciliary movement were captured using a high-speed camera at frame rates of 50 or 125 fps.
Analysis of ciliary beat frequency. Ciliary movement in human trachea was recorded at a frequency of 125 fps. The mean CBF in the recorded region was analyzed using the ImageJ plugin ciliaFA (National Institutes of Health, Bethesda, MD, USA) 28 . Statistical differences between CBF in unstained and FITC-WGA-stained mouse tracheae were analyzed by Student's t-test. P < 0.05 was considered significantly different.
Analysis of the ciliary beat direction and axis. Directions and axes of ciliary movement in each ciliated cell were measured using ImageJ. Measurements were performed for at least 15 ciliated cells in one field of view (approximately 210 × 170 µm). Rose diagrams were created using Octave (https ://www.gnu.org/softw are/ octav e/) to show the distributions of directions and axes. The standard deviation of the ciliary beat direction was determined using the following equations 29 : where ciliary beat directions were regarded as unit vectors, v is the unit vector in the complex plane, θ is the ciliary beat direction, i is the imaginary unit, R is the length of the mean of unit vectors, N is the number of measured cells, and S is the standard deviation.

Data availability
All of the data in this study can be obtained from corresponding author upon reasonable request.