Valerie Urbach
email: valerie.urbach@inserm.fr

Maëlle Briottet

Khadeeja Sy

Charlie London

Abdel Aissat

Mickael Shum

Virginie Escabasse

Bruno Louis

Valérie Urbach

Valérie Urbach

Specialized pro-resolving mediator Resolvin E1 corrects the altered cystic fibrosis nasal epithelium cilia beating dynamics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Main Text

Introduction

CF is a recessive monogenic disease caused by the mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, encoding the CFTR chloride channel. More than 2000 mutations have been identified, but at least one copy of the F508del mutation, is expressed in 90% of the patients [START_REF] Riordan | Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA[END_REF]. Although, CF is a systemic disease, the airway disease is the main cause of morbidity and mortality. The loss of CFTR protein function leads to defective secretion of chloride and bicarbonate, and sodium hyperabsorption resulting in a dehydration of the airway surface liquid (ASL) layer that contributes to a viscous and sticky mucus and impaired mucociliary clearance. This altered ASL layer favors chronic infections and inflammation leading to progressive lung damage and respiratory failure [START_REF] Bierlaagh | A new era for people with cystic fibrosis[END_REF]. Inflammation response in CF, is described as sustained, exaggerated, and inefficient against infection [START_REF] Khan | Early Pulmonary Inflammation in Infants with Cystic Fibrosis[END_REF][START_REF] Montgomery | Hypoxia and sterile inflammation in cystic fibrosis airways: mechanisms and potential therapies[END_REF][START_REF] Ranganathan | Early Lung Disease in Infants and Preschool Children with Cystic Fibrosis. What Have We Learned and What Should We Do about It?[END_REF][START_REF] Balázs | Mucus obstruction and inflammation in early cystic fibrosis lung disease: Emerging role of the IL-1 signaling pathway[END_REF][START_REF] Rehman | Inflammation as a Regulator of the Airway Surface Liquid pH in Cystic Fibrosis[END_REF]. Acute inflammation is normally followed by an active resolution phase to enable the return to tissue homeostasis. The resolution of inflammation requires the biosynthesis and functions of specialized pro-resolving mediators (SPMs) [START_REF] Serhan | Resolvins in inflammation: emergence of the pro-resolving superfamily of mediators[END_REF]. SPMs such as lipoxins, resolvins, protectins and maresins exert numerous actions including the inhibition of the NF-kB pathway [START_REF] Chiang | Infection regulates pro-resolving mediators that lower antibiotic requirements[END_REF][START_REF] Liao | Resolvin D1 attenuates inflammation in lipopolysaccharide-induced acute lung injury through a process involving the PPARγ/NF-κB pathway[END_REF][START_REF] Serhan | Design of Lipoxin A4 Stable Analogs That Block Transmigration and Adhesion of Human Neutrophils[END_REF] and the inhibition of leucocytes migration [START_REF] Serhan | Design of Lipoxin A4 Stable Analogs That Block Transmigration and Adhesion of Human Neutrophils[END_REF]. SPMs also stimulate the efferocytosis of apoptotic immune cells [START_REF] Dalli | Specific lipid mediator signatures of human phagocytes: microparticles stimulate macrophage efferocytosis and pro-resolving mediators[END_REF][START_REF] Rogerio | Resolvin D1 and Aspirin-Triggered Resolvin D1 Promote Resolution of Allergic Airways Responses[END_REF]. We and others have previously shown that lipoxin A4 (LXA4) levels were significantly reduced in the airway of patients with CF [START_REF] Karp | Defective lipoxin-mediated anti-inflammatory activity in the cystic fibrosis airway[END_REF][START_REF] Ringholz | Reduced 15-lipoxygenase 2 and lipoxin A4/leukotriene B4 ratio in children with cystic fibrosis[END_REF]. More recently, we demonstrated the contribution of CF nasal epithelial cells in the abnormal biosynthesis of LXA4 and several other SPMs [START_REF] Shum | CF Patients' Airway Epithelium and Sex Contribute to Biosynthesis Defects of Pro-Resolving Lipids[END_REF]. Therefore, lower levels of SPMs could contribute to the altered inflammatory response in the CF airway disease. SPMs treatment can improve various epithelial functions in models of CF airway disease [START_REF] Briottet | The Role of Specialized Pro-Resolving Mediators in Cystic Fibrosis Airways Disease[END_REF]. Previous works demonstrated that LXA4 and resolvin D1 (RvD1) were able to restore the ASL layer height in human bronchial epithelial cells [START_REF] Verrière | Lipoxin A4 Stimulates Calcium-Activated Chloride Currents and Increases Airway Surface Liquid Height in Normal and Cystic Fibrosis Airway Epithelia[END_REF][START_REF] Al-Alawi | Physiological levels of lipoxin A4 inhibit ENaC and restore airway surface liquid height in cystic fibrosis bronchial epithelium[END_REF][START_REF] Higgins | Activation of P2RY11 and ATP Release by LXA4 Restores the Airway Surface Liquid Layer and Epithelial Repair in Cystic Fibrosis[END_REF][START_REF] Ringholz | Resolvin D1 regulates epithelial ion transport and inflammation in cystic fibrosis airways[END_REF], suggesting that SPMs could have an impact on the quality of mucociliary clearance. The roles of the two SPMs, resolvin E1 (RvE1, 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-EPA) and lipoxin B4 (LXB4, 5S,14R,15S-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid) have not been explored before on CF airway epithelial cell function [START_REF] Serhan | Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing[END_REF][START_REF] Serhan | On the stereochemistry and biosynthesis of lipoxin B[END_REF].

The mucociliary clearance results from the transport of mucus out of the airways due to the cilia beating at the epithelial cell surface. However, the dynamics of cilia is not well established, especially in a pathological environment including the altered airway surface hydration and mucus content. Primary cultures of nasal epithelial cells at air-liquid interface (ALI) are useful models to mimic physiological environment, as we can obtain a fully differentiated pseudo-stratified epithelium with basal cells, goblet cells that produce mucus, and ciliated cells [START_REF] Shum | CF Patients' Airway Epithelium and Sex Contribute to Biosynthesis Defects of Pro-Resolving Lipids[END_REF][START_REF] García | Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures[END_REF]. In this model, CF primary cultures have shown a reduced and disrupted ASL layer height compared with primary cultures from non-CF donors [START_REF] Tarran | Normal and Cystic Fibrosis Airway Surface Liquid Homeostasis[END_REF]. However, limited methods are available to assess cilia beating on such models. Most cilia beating analysis only measured cilia beating frequency (CBF). However, CBF alone cannot explain the capacity of cilia to propel the mucus layer. Other parameters such as amplitude, cilia beating pattern or coordination are measured in primary cilia dyskinesia studies. But these methods have mainly been used for imaging cilia beating from the side after removal of the cilia environment generated by epithelial cells [START_REF] Bottier | A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part I, experimental analysis[END_REF][START_REF] Blanchon | Deep phenotyping, including quantitative ciliary beating parameters, and extensive genotyping in primary ciliary dyskinesia[END_REF][START_REF] Reula | High-Speed Video Microscopy for Primary Ciliary Dyskinesia Diagnosis: A Study of Ciliary Motility Variations with Time and Temperature[END_REF]). Cicuta's team recently developed an automated method, the multiscale Differential Dynamic Microscopy (multi-DDM) to obtain the CBF as well as the cilia synchronization of cilia beating in intact ALI cultures [START_REF] Chioccioli | Phenotyping ciliary dynamics and coordination in response to CFTR-modulators in Cystic Fibrosis respiratory epithelial cells[END_REF][START_REF] Cicuta | The use of biophysical approaches to understand ciliary beating[END_REF]. This previous study demonstrated that the spatial and temporal coherence of cilia movement was altered in CF samples. Another study developed on ALI cultures characterized a third parameter of cilia movement influenced by mucus environment: the cilia beating orientation [START_REF] Gsell | Hydrodynamic model of directional ciliary-beat organization in human airways[END_REF].

In our work, we aimed to characterize the dynamics of cilia beating on a model mimicking the pathophysiological context of CF airway disease and by considering as many parameters as possible, including the cilia beat frequency (CBF), synchronization, orientation, density as well as the temperature which is a well-known modulator of cilia beating and mucociliary clearance in vitro and in vivo [START_REF] Reula | High-Speed Video Microscopy for Primary Ciliary Dyskinesia Diagnosis: A Study of Ciliary Motility Variations with Time and Temperature[END_REF][START_REF] Sauvalle | Effect of the temperature of nasal lavages on mucociliary clearance: a randomised controlled trial[END_REF]. In order to identify the level of dependency of each parameter and guide their interpretation, we performed a principal component analysis (PCA) which allowed to further characterize a differential cilia beating phenotype in nasal epithelial primary cultures derived from CF patients compared to control subjects (figure 1). Moreover, we investigated the impact of RvE1 and LXB4 on cilia beating in CF and the cellular mechanisms involved.

Results

Characterization of cilia beat phenotype of airway epithelial primary cultures using a multiparametric analysis. We performed a PCA for five quantitative parameters: the density of cilia in movement (movement), the cilia beat frequency (CBF), the synchronization determined by the surface size ( λ² ) below which cilia beat in a homogeneous way, the orientation (the dispersion of cilia beat orientation over the video) and the room temperature during acquisitions (figure 2A). The correlation circles showed that principal components (PC) 1 to 4 explained 33.1 %, 26.21%, 18.80% and 14.98% of the total variance, respectively. Therefore, the first 2 PC could explain 59.31% of variance of the data. The CBF and temperature were the two main contributors of variance for the first PC. Percentage of movement, synchronization (λ²) and orientation of cilia beating appeared rather independent of the CBF and mainly contributes to the second PC (figure 2A, left). The density of movement negatively correlated with the orientation and synchronization of the cilia beating. These last two parameters positively correlate over the 2nd PC. In contrast, cilia beat orientation and synchronization appeared negatively correlated over the 3rd PC, suggesting that they contributed differently to PC2 and PC3 (figure 2A). The synchronization (λ²), participated in 35.45% of the variance explained by PC2 and 32.86% for PC3, whereas orientation participated in 19% for PC2 and 64.3% in PC3. This suggested that synchronization and orientation of the beating could bring complementary information. Taken together, the PCA has shown that the percentage of movement, the cilia beat frequency, synchronization and orientation gave distinct information that could enable a fine characterization of cilia beating phenotype.

Distinct cilia beating phenotypes between non-CF and CF samples. We compared the cilia beating phenotypes between CF and non-CF untreated samples and observed different profiles which are visualized by the ellipses for the two first components (figure 2B and supplemental figure 1 for PC3 and PC4). At 24h, 48h and 72h after washing the mucus, the ellipses from non-CF and CF datasets were not stackable and were oriented in opposite directions (figure 2B). 24h after removing the mucus, the centroids of cilia beating data from non-CF and CF groups were significantly different (p=0.001) while the dispersion of the was similar (p= 0.531). The differences between the CF and non-CF groups were still observed after 48h and 72h. The cilia beating phenotype for the F508del homozygous samples only was not significantly different from the CF group (4 F508del homozygous and 2 heterozygous) (figure 2B, dashed line). The temperature gradient over the scatter plot illustrates how temperature changed along the first PC and that the non-CF and CF experimental sets differentiated more at higher temperature (red) than at lower temperature (yellow) and mostly along the PC2 axis (figure 2B). Since cilia beat density, synchronization (λ²) and orientation were the major contributors to the PC2 axis, these parameters were mainly responsible for the differences between CF and non-CF samples rather than the CBF. This analysis was confirmed when the four parameters were analyzed separately. The CBF values did not show differences between CF and non-CF samples 48h and 72h after mucus removal (figure 2C). The CBF slightly increased in CF hNEC 24h after mucus removal which is consistent with the slight but significant increase in the temperature at 24h (WT: 23.19°C; CF: 23.94°C; p<0.01) since a high correlation between CBF and temperature was observed. In contrast, the percentage of movement, the synchronization (λ²) and orientation of cilia beating were significantly different between CF and non-CF samples. CF samples showed a lower degree of cilia movement for the three time points after removal of mucus (figure 2D). In CF hNEC, the cilia beating was coordinated over a larger area than in non-CF samples (higher λ²) (figure 2E). Finally, the cilia beat orientation showed a higher dispersion for CF samples compared with non-CF samples (Figure 2F). To consider the impact of the temperature revealed by the PCA, we further compared the beating parameters within two subgroups of lower ([19°C-24°C]) and higher (]24°C-28°C]) room temperature, 24h after mucus removal (supplemental figure 2). This confirmed that at the lower temperatures, the beating parameter were not significantly different between CF and non-CF except for the orientation (higher dispersion in CF). At higher temperatures, all the parameters showed significant differences between CF and non-CF samples with decreased CBF and density of movement and increased synchronization and dispersion of cilia in CF. The PCA analysis also showed that the density was not much correlated to the CBF but appeared inversely correlated to synchronization and cilia orientation. Therefore, to minimalize the influence of the quantity of cilia in movement to the other parameters, we tested the restriction of the analysis to the videos containing over 50% of movement in both groups (supplemental figure 3). This resulted in reduced differences of movement between CF and non-CF samples while keeping enough videos. However, despite the restriction, significant differences in cilia synchronization and orientation between CF and non-CF samples were kept (supplemental figure 3). This suggested that the differences in cilia synchronization and orientation between CF and non-CF did not only result from the reduced number of cilia in movement in CF culture. After mucus removal, cilia beat synchronization and orientation did not evolve much over time within the same sample's group (CF or non-CF) (figure 2 and table 2). In contrast, the CBF decreased after mucus removal, especially for the acquisition performed at the higher temperature (]24°C-28°C]) the CBF dropped by 1.7Hz (p<0.0001) and by 1.2Hz (p<0.0001) between 24 and 72h in non-CF and CF samples respectively. For lower acquisition temperatures the CBF for non-CF samples did not change and dropped by 1Hz (p<0.01) in CF samples. Taken together, the cilia beating characteristics of CF and non-CF hNEC were significantly different at any time after mucus removal which is consistent with an impaired cilia beating in hNEC from CF samples. More specifically, while we did not detect significant CBF differences, we showed a reduced cilia density, a higher synchronization and dispersion of cilia orientation in CF hNEC. These differences could at least partially result from the changes in cilia environment between CF and non-CF samples, CF environment restricting the beating orientation and synchronization.

SPMs regulate cilia beating of CF airway epithelium.

We then explored whether SPMs could regulate cilia beating parameters. The CF hNEC, were treated 24h after mucus removal with SPMs (10nM) for 1h, 24h or 48h. Therefore, the samples exposed to SPMs for 24h, accumulated mucus for 48h, and similarly the ones exposed to SPMs for 48h which accumulated mucus for 72h. Figure 3 displays the effects of LXB4 and RvE1 tested on CF hNEC. For each condition we compared CF samples treated with SPMs (in green) to CF control samples treated with the vehicle (in blue), and to non-CF control samples (treated with the vehicle, in grey). We found that LXB4 (10nM) treatment significantly moved the ellipse from the CF beating phenotype toward a non-CF phenotype after 1h and 48h treatments (25h and 72h after mucus removal). RvE1 treatment of hNEC cultures from 5 different patients also tended to modify the cilia beating of CF samples towards a non-CF profile, which was significant at 24h treatment. A further analysis of the RvE1 effects on hNEC from each CF donor separately revealed that responses to RvE1 were donor specific even between donors carrying the same CFTR mutation. The impact of RvE1 on hNEC cultures from two different patients, both homozygous for the F508del mutation CF1 (n=3) and CF2 (n=2) are presented in the figure 4. For the hNEC derived from the first CF patient (CF1), the PCA analysis showed that the RvE1 treatment significantly restored the cilia beating phenotype towards a non-CF phenotype starting from 1h and increasing to 48h post treatment. After 24h and 48h, the beating pattern of the treated samples overlays the non-CF cilia beating pattern (figure 4A). Figure 4B, showed that RvE1 restored cilia beating frequency (increased), and dispersion of cilia orientation (decrease) and tended to restore synchronization (decreased) in hNEC-CF1. LXB4 treatment on hNEC from this patient (CF1) also resulted in improving the cilia beating (Supplemental figure 4). In contrast, for hNEC cultures derived from the other patient CF2, the PCA analysis as well as the separate analysis of cilia beating parameters showed that RvE1 failed to modify any cilia beating parameters (Figure 4C and4D). The comparison of the initial beating profiles of the hNEC from these 2 different patients (CF1 and CF2) showed that they were not similar. Indeed, before treatment, the hNEC that responded to RvE1 (hNEC-CF1), were synchronized over a higher surface of 368µm² and 291µm², with a dispersion of orientation of 54 and 51 degrees, after 48h and 72h of mucus accumulation, respectively. In contrast, the hNEC that did not respond to RvE1 (hNEC-CF2) were synchronized over a smaller surface of 150µm² and 268µm², with a dispersion of orientation of 45.4 and 48 degrees. These last values were close to the values obtained in non-CF samples with a cilia synchronization of 170µm² and 200µm², and a dispersion of orientation of 46 and 47.5 degrees.

The role of TMEM16A in the effect of RvE1 on airway surface hydration and cilia beating.

We studied whether the effect of RvE1 on cilia beating profile could be related to modifications of the cilia environment. Since the CF airway surface dehydration results in a more viscous mucus, we investigated the effect of RvE1 on the hydration of the surface of hNEC. The ASL layer height was visualized using live cell confocal microscopy upon treatment with RvE1 (10nM) or vehicle control. Figure 5A and 5B, showed that in control conditions, the ASL was thin (4.4 ± 0.09 µm) and disrupted. The treatment with RvE1 for 1h significantly enhanced an ASL layer height increase to 8.2 ± 0.35 µm. The role of TMEM16A, a calcium-dependent chloride channel, was investigated by using its specific inhibitor Ani9. Exposure to Ani9 (10µM) completely inhibited the RvE1 effect in increasing the ASL layer height, suggesting that RvE1 restored the ASL layer by stimulating the TMEM16A chloride channel. We then investigated the effect of inhibiting TMEM16A with Ani9 on the cilia beating. The effect of Ani9 was illustrated by the changes in ellipses orientation of the PCA analysis towards a CF phenotype (without SPM) rather than the non-CF phenotype induced by RvE1 (Figure 5C). Ani9 also inhibited the effect of RvE1 on CBF and on cilia orientation especially at 48h (Figure 5D). These results suggest that the impact of RvE1 on cilia beating parameters involves at least partially the effect of this SPM in restoring airway epithelium surface hydration through the stimulation of calcium-dependent chloride secretion by the TMEM16A channel.

RvE1 modifies secreted mucins organization. To evaluate the possible role of mucins in the differences of cilia beat profile between non-CF and CF hNEC and upon RvE1 treatment, we explored the secretion and transcription of the two secreted mucins MUC5B and MUC5AC. Immunofluorescence labelling of the secreted mucins by hNEC from the CF1 patient and by non-CF samples 72h after mucus removal are displayed in figure 6A. MUC5B (in red) and MUC5AC (in green) partially colocalized and constructed a mesh and different structures could be observed. In non-CF primary cultures, the mucins adopted a structure of linear strands or thin packed bundles. In contrast, in hNEC CF1 primary cultures, the mucins stained thicker than in non-CF cultures. They were more structured as sheets than linear strands (figure 6A). Furthermore, the thickness measurement of the MUC5AC staining showed a thicker mucus layer in CF1 hNEC sample (4.1 ± 0.11 µm) than in the non-CF samples (3.1 ± 0.05µm) (p<0.0001) (figure 6B). The RvE1 exposure of CF1 hNEC for 48h induced a mucins' structure change with more linear strands than in control (vehicle). In addition, this treatment decreased MUC5AC thickness of hNEC toward non-CF values (3.5 ± 0.08 µm) (figure 6A and6B). Finally, exposure to Ani9 prevented the effect of RvE1 in modifying MUC5AC structure and the mucin thickness was significantly increased (4.8 ± 0.11µm) compared with the condition where the cultures were only exposed to RvE1 (figure 6A and6B). At the transcriptional level, MUC5AC and MUC5B levels were higher in CF samples, although the differences only reached significance for MUC5AC. RvE1 (10nM) treatment during 24h did not significantly affect either MUC5B or MUC5AC mean expression level in CF samples (figure 6C). Thus, this further analysis was consistent with the assumption that CF airway epithelia were covered with a thicker mucins layer than non-CF cells and that RvE1 treatment regulated it.

Discussion

This biophysical study provided a global picture of multiple parameters of cilia beating that are altered in CF nasal epithelial primary culture. Moreover, this is the first report showing that cilia beating orientation was more dispersed in CF than in non-CF nasal epithelial cells primary cultures. Our data also demonstrated the role of the SPMs, RvE1 and LXB4 in restoring at least partially, cilia beating in CF nasal epithelial cells and that RvE1 effect involved the calcium-activated chloride channel, TMEM16A.

Decrease of cilia beating quantity in CF airway epithelial cells.

Several papers reported a difference in the number of ciliated cells between CF and non-CF airway epithelial cells. Cell transcriptomic of proximal airway of CF donors at end-stage lung disease displayed a higher proportion of ciliated cells. However, when the samples were cultured at ALI, a reduced quantity of ciliated cells were observed in the CF samples [START_REF] Carraro | A molecular atlas of proximal airway identifies subsets of known airway cell types revealing details of the unique molecular pathogenesis of Cystic Fibrosis[END_REF]. Another study reported a decrease in ciliated cells staining in CF lung tissue and primary cultures [START_REF] Collin | Loss of ciliated cells and altered airway epithelial integrity in cystic fibrosis[END_REF]. A more recent analysis showed that 6 weeks of hNEC cultures at ALI, resulted in differences in ciliated cells differentiation between CF and non-CF donors [START_REF] Paranjapye | Cell function and identity revealed by comparative scRNA-seq analysis in human nasal, bronchial and epididymis epithelia[END_REF]. In our study, we did not strictly quantify the number of ciliated cells, we used instead an automated method to quantify the percentage of movement on each video recorded and we showed that this parameter significantly decreased by >20% in CF samples compared with non-CF ones. The PCA analysis also revealed that the percentage of movement was one of the parameters that discriminated most CF and non-CF samples. For each culture insert, the acquisitions were performed at 6 to 8 different locations, each time trying to maximize the quantity of cilia moving in the region of interest. Therefore, although it cannot be excluded that the cilia movement might have been more difficult to detect in CF samples, our results are consistent with previous reports showing that CF cultures were less densely ciliated.

No change in cilia beat frequency in CF airway epithelial cells. Previous studies performed on CBF in CF airway epithelial cells gave contradictory results. A normal CBF of CF nasal brushing in vitro was initially reported [START_REF] Rutland | Nasal mucociliary clearance and ciliary beat frequency in cystic fibrosis compared with sinusitis and bronchiectasis[END_REF]. In another study, the CBF in CF nasal biopsies and airway epithelial primary culture at ALI was found higher than healthy controls [START_REF] Alikadic | Ciliary beat frequency in nasal and bronchial epithelial cells in patients with cystic fibrosis[END_REF]. In contrast, others reported that CF nasal brushings or ALI airway epithelial primary cultures showed significantly lower CBF and dyskinesia compared with that of healthy subjects [START_REF] Thomas | Ciliary function of the nasal and bronchial epithelium in children with cystic fibrosis[END_REF][START_REF] Schmid | Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation *[END_REF][START_REF] Watson | Defective adenosine-stimulated cAMP production in cystic fibrosis airway epithelia: a novel role for CFTR in cell signaling[END_REF]. Our data are consistent with a more recent study performed with similar tools as the ones we used (ALI primary cultures and multi-DDM method), the CBF ranged from 2.4 to 5.1 Hz with no perceptible differences between CF and non-CF samples [START_REF] Chioccioli | Phenotyping ciliary dynamics and coordination in response to CFTR-modulators in Cystic Fibrosis respiratory epithelial cells[END_REF]. Furthermore, our PCA also suggested that CBF was not the parameter mainly responsible for the differences between CF and non-CF cilia beat profiles, highlighting the interest of exploring other parameters.

Spatial homogeneity of cilia beat frequency increased in CF airway epithelial cells.

We explored the homogeneity of cilia beat by calculating the surface size in which cilia beat at similar frequencies and determine the level of cilia coordination by the λ² value (in µm 2 ) which is accountable for the spatial and temporal organization of cilia beating. Our data obtained on nasal epithelial cells are consistent with previous measurements of this parameter in CF and non-CF bronchial epithelial cell primary cultures showing that, 24h after mucus removal, the λ² in CF samples was higher than in non-CF samples [START_REF] Chioccioli | Phenotyping ciliary dynamics and coordination in response to CFTR-modulators in Cystic Fibrosis respiratory epithelial cells[END_REF]. Since epithelial cells apical surface are between 10 to 100µm2, these results indicated that in CF, cilia beating was homogeneous over 3 to 30 cells and in non-CF cells, cilia beating was homogeneous only over 2 to 10 cells (depending on cell size). Consistent with this individual analysis, the PCA revealed that the synchronization level was also one of the main parameters that discriminated CF and non-CF cilia beat patterns. We also showed that the percentage of movement appeared inversely correlated to synchronization, while it was not much correlated to the CBF. When we are restricting the analysis to videos containing over 50% of movement, the differences of synchronization between CF and non-CF samples were then less significant. This result was consistent with previous studies showing that cilia density influences their organization [START_REF] Gsell | Hydrodynamic model of directional ciliary-beat organization in human airways[END_REF].

Dispersion of cilia beat orientation increased in CF airway epithelial cells.

We introduced a fourth parameter to consider the diversity of cilia beating orientation which has not been previously studied in CF. Our method permitted the calculation of the main cilia direction over square boxes of 16.7µm. Therefore, we looked at cilia main beating orientation in area ~280µm². We thus studied the dispersion of cilia orientation between areas were CF and non-CF beating were no more correlated (λ² = 280µm2). This suggests that the orientation parameters gave other information than λ²; these two parameters being related to 2 different spatial scales of analyses. Furthermore, the PCA indicated that λ² and orientation contributed differently to PC2 and PC3 which was also consistent with our hypothesis that cilia beating orientation is a complementary characteristic of cilia dynamics. Our data provided the first evidence that orientation of cilia beating was more dispersed in CF than in non-CF nasal epithelial cells primary cultures while cilia beating (mainly CBF) was more homogenous over bigger areas for CF samples. In addition, when restricting the analysis to acquisitions where >50% of movement was detected, the dispersion in cilia orientation in CF samples detached further from non-CF samples suggesting that orientation was more independent of the percentage of movement than the λ² (the area within cilia beat at the same frequency). The higher dispersion of cilia orientation in CF cells compared with non-CF could be consistent with a recent study showing that sheets of mucus over 200µm² covering the cell surfaces and compressing cilia in CF cells resulted in disorganized and flattened cilia [START_REF] Morrison | Treatment of cystic fibrosis airway cells with CFTR modulators reverses aberrant mucus properties via hydration[END_REF].

Temperature's increase stimulated cilia beat in CF airway epithelial cells. Several studies have shown that increasing temperature had a positive effect on mucociliary clearance. The mucociliary clearance rates of nasal fluid measured in vivo was higher at 37°C than 20°C [START_REF] Sauvalle | Effect of the temperature of nasal lavages on mucociliary clearance: a randomised controlled trial[END_REF]. The mucociliary clearance measured in vitro on tracheal ovine model was also reduced at lower temperature [START_REF] Kilgour | Mucociliary function deteriorates in the clinical range of inspired air temperature and humidity[END_REF]. In primary cilia dyskinesia, cilia beat pattern is more easily observed as dyskinetic at 22-24°C than at 4°C when measured on brushed cells [START_REF] Reula | High-Speed Video Microscopy for Primary Ciliary Dyskinesia Diagnosis: A Study of Ciliary Motility Variations with Time and Temperature[END_REF]. In these studies, whether the effect of temperature on mucociliary clearance was only due to improvement of CBF or also due to modifications of cilia coordination was not clear. However, a linear positive dependence of CBF with temperature has been reported, notably between 19°C and 32°C, when measured on nasal epithelial brushings [START_REF] Green | The effect of temperature on nasal ciliary beat frequency[END_REF]. Consistent with this previous data, our study showed a strong positive correlation between temperature and CBF and that increasing temperature (from 20°C to 28°C) stimulated CBF significantly (p<0.0001) in both CF and non-CF hNEC. Temperature also had an impact on the other parameters of cilia beating. Higher temperatures increased the density of movement of non-CF hNEC but decreased it in CF hNEC. Increasing temperatures significantly decreased synchronization and orientation in non-CF hNEC but had less effect on CF cells. Our study also revealed that at the higher working temperature (24°C-28°C), CF samples showed a lower CBF and density of movement and a higher synchronization and orientation than non-CF ones. In contrast, at the lower temperatures (19°C-24°C) cilia beating phenotype for non-CF and CF cultures were more alike. It has been previously reported that decreasing temperature from 37°C to 29°C for a couple of hours, stimulated F508del CFTR trafficking to the plasma membrane resulting in the rescue of chloride transport [START_REF] Rennolds | Low temperature induces the delivery of mature and immature CFTR to the plasma membrane[END_REF][START_REF] Sondo | Rescue of the mutant CFTR chloride channel by pharmacological correctors and low temperature analyzed by gene expression profiling[END_REF]. Although in our experiments the epithelial cells moved from 37°C to room temperature for 10-15 min for the acquisitions, it cannot be excluded that a CFTR rescuing process at lower temperature could explain the lack of cilia beating differences between CF and non-CF samples. Taken together, our data suggested that acquisitions performed at body temperature could reveal larger cilia beating differences between CF and non-CF epithelial cells.

Mucus accumulation altered the cilia beat in CF epithelial cells.

Early studies reported a normal CBF of cilia from CF nasal brushing in vitro together with an abnormal nasal mucociliary clearance measured in vivo in the same patients [START_REF] Rutland | Nasal mucociliary clearance and ciliary beat frequency in cystic fibrosis compared with sinusitis and bronchiectasis[END_REF]. This suggested the role of mucus abnormalities in the altered mucociliary clearance. Indeed, in this previous study, CBF was analyzed on cells resuspended in an aqueous solution that diluted the mucus and facilitated cilia beating [START_REF] Rutland | Nasal mucociliary clearance and ciliary beat frequency in cystic fibrosis compared with sinusitis and bronchiectasis[END_REF]. Organization of cilia beating over long distances has been explained by hydrodynamic coupling [START_REF] Gsell | Hydrodynamic model of directional ciliary-beat organization in human airways[END_REF][START_REF] Bruot | Realizing the Physics of Motile Cilia Synchronization with Driven Colloids[END_REF][START_REF] Hamilton | Motile cilia hydrodynamics: entrainment versus synchronization when coupling through flow[END_REF][START_REF] Pellicciotta | Cilia density and flow velocity affect alignment of motile cilia from brain cells[END_REF][START_REF] Pellicciotta | Entrainment of mammalian motile cilia in the brain with hydrodynamic forces[END_REF]. In numeric modelling viscosity of cilia environment has been found to modulate their organization [START_REF] Gsell | Hydrodynamic model of directional ciliary-beat organization in human airways[END_REF]. In another model, it was shown that cilia are only synchronized locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium [START_REF] Ringers | Local synchronization of cilia and tissue-scale cilia alignment are sufficient for global metachronal waves[END_REF]. Therefore, the differences of CBF between CF and non-CF measured in our model, where mucus and ASL layer hydration spontaneously evolved are consistent with the role of mucus concentration on CBF, as previously reported. In addition, we observed that CBF values decreased overtime with mucus accumulation and that the CBF differences between CF and non-CF samples were no longer detectable at later stages, suggesting that mucus accumulation was a strong limiting factor of the CBF. In contrast, the size of the area in which CBF appeared homogenous (λ²) did not evolve much over time during mucus accumulation. This suggested that 24h was enough to secrete the amount of mucus necessary to constrain the spatial organization of cilia beating. Further mucus accumulation did not modify the spatial organization of cilia movements while this accumulation contributed to slow down the beating (decreased CBF). Cilia orientation did not evolve much neither during mucus accumulation. Recent reports indicated that orientation of cilia beat is not predefined and that that cilia can reorient from 35° to 40° in 24h to align to the mucus flow while when mucus is removed, their direction is more dispersed [START_REF] Schneiter | Multi-scale alignment of respiratory cilia and its relation to mucociliary function[END_REF][START_REF] Gsell | Hydrodynamic model of directional ciliary-beat organization in human airways[END_REF]. In addition, a few studies reported that in healthy conditions cilia are not perfectly aligned and that small heterogeneity in cilia orientation optimized mucus transport [START_REF] Schneiter | Multi-scale alignment of respiratory cilia and its relation to mucociliary function[END_REF][START_REF] Ramirez-San Juan | Multi-scale spatial heterogeneity enhances particle clearance in airway ciliary arrays[END_REF]. In our study, we observed a thicker mucus layer above CF cells and increased MUC5AC transcription level which were consistent with previous reports showing a hyper-concentration of mucins, especially MUC5B and MUC5AC in CF airways resulting in a compressed periciliary layer [START_REF] Collin | Loss of ciliated cells and altered airway epithelial integrity in cystic fibrosis[END_REF][START_REF] Derichs | Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching[END_REF][START_REF] Button | A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia[END_REF][START_REF] Henderson | Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure[END_REF][START_REF] Peters-Hall | Quantitative Proteomics Reveals an Altered Cystic Fibrosis In Vitro Bronchial Epithelial Secretome[END_REF]. Moreover, in CF tracheobronchial epithelial cells, mucus structure is also altered, and defective maturation of mucins after their secretion leads to a compact structure instead of a linear one [START_REF] Abdullah | Defective postsecretory maturation of MUC5B mucin in cystic fibrosis airways[END_REF]. In BALs from young CF patients, bigger and less soluble flakes, made of densely packed mucins MUC5B and MUC5AC were described [START_REF] Esther | Mucus accumulation in the lungs precedes structural changes and infection in children with cystic fibrosis[END_REF]. These changes in mucus concentration and structure have been mainly related to the altered airway hydration because of ion transport defect in CF.

SPMs restored airway surface hydration, mucus organization and cilia beat. Before our study, the effect of SPMs on cilia beating had not been explored. We found that RvE1 and LXB4 were able to restore CF cilia beating parameters, in CF samples where cilia beating was impaired. Furthermore, the effect of RvE1 in enhancing the ASL height and decreasing the thickness of mucus was consistent with an improvement of cilia beating. Indeed, proper hydration determines the quality of the mucociliary clearance. The hydration maintains a distinct mucus and periciliary layer which are necessary for efficient mucociliary clearance [START_REF] Button | A Periciliary Brush Promotes the Lung Health by Separating the Mucus Layer from Airway Epithelia[END_REF][START_REF] Tildy | Therapeutic Options for Hydrating Airway Mucus in Cystic Fibrosis[END_REF]. The inhibitory effect of Ani9 on the RvE1 effect on hydration, on mucus thickness and on cilia beating, suggested that these effects were due to the stimulation of the TMEM16A channel. TMEM16A is a calcium-dependent chloride channel which has been considered as a therapeutic target to bypass the loss of chloride secretion by CFTR in CF. It was reported that TMEM16A expression is diminished in CF and that increase of TMEM16A expression, and stimulation improved chloride secretion and mucus transport rate [START_REF] Danahay | TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis[END_REF][START_REF] Sonneville | MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology[END_REF]. However, whether TMEM16A should be activated has been controverted. Indeed, the TMEM16A contribution to chloride secretion was found to be marginal and an increase in TMEM16A in goblet cells and mucus production have been observed during inflammation responses [START_REF] Kunzelmann | TMEM16A in Cystic Fibrosis: Activating or Inhibiting?[END_REF]. On another hand, TMEM16A expression in goblet cells could also be beneficial to hydrate newly secreted mucins [START_REF] Galietta | TMEM16A (ANO1) as a therapeutic target in cystic fibrosis[END_REF]. The simultaneous effect of RvE1 in hydration, in decreasing mucus thickness and restoring cilia beat is consistent with a beneficial role of TMEM16A. Changes in mucins structure in CF have been linked principally to airway hydration and to a lesser extent extracellular pH and bicarbonate concentration [START_REF] Abdullah | Defective postsecretory maturation of MUC5B mucin in cystic fibrosis airways[END_REF][START_REF] Garcia | Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion[END_REF]. Bicarbonate has been shown to play a role in mucin expansion [START_REF] Garcia | Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion[END_REF][START_REF] Chen | A new role for bicarbonate in mucus formation[END_REF][START_REF] Ermund | Hyper-osmolarity and calcium chelation: Effects on cystic fibrosis mucus[END_REF][START_REF] Gorrieri | Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release[END_REF]. TMEM16A is permeable to bicarbonate [START_REF] Han | HCO3-Transport through Anoctamin/Transmembrane Protein ANO1/TMEM16A in Pancreatic Acinar Cells Regulates Luminal pH[END_REF][START_REF] Jun | Pore dilatation increases the bicarbonate permeability of CFTR, ANO1 and glycine receptor anion channels[END_REF] and was found to regulate goblets cells exocytosis [START_REF] Benedetto | TMEM16A is indispensable for basal mucus secretion in airways and intestine[END_REF][START_REF] Centeio | Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease[END_REF]. This novel effect of RvE1 at nanomolar concentration in restoring cilia beating parameters by increasing ASL height and reducing the thickness of the mucus layer is consistent with its role in airway innate immunity reported in other models [START_REF] Aoki | Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma[END_REF][START_REF] Haworth | Resolvin E1 regulates interleukin-23, interferon-γ and lipoxin A4 to promote resolution of allergic airway inflammation[END_REF][START_REF] Kebir | Resolvin E1 promotes phagocytosis-induced neutrophil apoptosis and accelerates resolution of pulmonary inflammation[END_REF]. Indeed, RvE1 improves lung inflammation, airway mucus and airway hyper-reactivity in a murine model of asthma [START_REF] Haworth | Resolvin E1 regulates interleukin-23, interferon-γ and lipoxin A4 to promote resolution of allergic airway inflammation[END_REF]. RvE1 promotes oral mucosal surface clearance of neutrophils via its epithelial receptor ChemR23 [START_REF] Campbell | Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution[END_REF]. In our study, we did not explore the role of ChemR23 in the effect of RvE1 on cilia beating phenotype. However, ChemR23 was found highly expressed in the lung and its role in damping inflammation has been reported in this tissue [START_REF] Owman | Molecular cloning and tissue distribution of cDNA encoding a novel chemoattractant-like receptor[END_REF][START_REF] Provoost | Pro-and Anti-Inflammatory Role of ChemR23 Signaling in Pollutant-Induced Inflammatory Lung Responses[END_REF]. At higher concentrations, RvE1 effects can be mediated by BLT1, which is also expressed in human airway epithelial cells [START_REF] Arita | Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1 and ChemR23 to regulate inflammation[END_REF][START_REF] Xiong | BLT1 signaling in epithelial cells mediates allergic sensitization via promotion of IL-33 production[END_REF]. Taken together our finding could open novel therapeutic strategies for CF patients that are not eligible or do not respond to CFTR modulators.

Benefit of the method of multiparametric analysis developed.

Our method developed to observe cilia beating in their pathophysiological environment, including periciliary layer and mucus permitted to study five parameters related to cilia beating using an automatic analysis of video microscopy records. The PCA allowed to determine the dependency level of each parameter. The PCA and the separate analysis for each parameter were consistent with each other. However, the PCA analysis appeared to be more sensitive and allowed to reveal significant differences of the cilia beat profiles between non-CF and CF samples as well as the impact of SPMs. Such analyses might be useful to study other diseases of the airway track or related to other organs with motile cilia and to screen the patient dependent responses to drugs.

Conclusion

This multi-parametric study further described the alteration of cilia beating in CF. Furthermore, we provided evidence for the role of RvE1 and LXB4 in restoring cilia beating, with RvE1 changing mucins spatial organization and enhancing ASL layer hydration through TMEM16A channel stimulation. The method also allowed to identify responsive and non-responsive donors' nasal epithelial cultures despite their same CFTR mutation.

Materials and Methods

Study population

Nasal polyp or mucosa samples from nine non-CF and six CF donors of the Centre Hospitalier Intercommunal de Créteil (CHIC, France) were excised after informed consent (Table 1). All experiments were performed in accordance with the Declaration of Helsinki and the Huriet-Serusclat and Jardet law on human research ethics. The study of these human samples was approved by the Comité de Protection des Personnes Ile de France (ID-RCB: 2011A00118-33).

Primary culture

The human nasal epithelial cells (hNEC) from patients were cultured under ALI in Pneumacult medium (StemCell) as previously described [START_REF] Shum | CF Patients' Airway Epithelium and Sex Contribute to Biosynthesis Defects of Pro-Resolving Lipids[END_REF]. The apical mucus secretions were washed twice a week by gently adding 200µL of warm medium from the basolateral compartment for one minute. Furthermore, to study cilia beating over mucus accumulation, the mucus was washed rigorously 24h before the first video acquisitions (83) (Supplemental for details).

High speed video microscopy acquisitions

Videos of cilia were acquired from above using an inverted Axiovert 200M microscope (Carl Zeiss) at magnification 40x (NEOFLUAR 40x/0.75) and a high-speed video camera PL-A741 (PixeLink). The videos of 400 x 400 pixels were recorded at 158fps, for 10s [START_REF] Blanchon | Deep phenotyping, including quantitative ciliary beating parameters, and extensive genotyping in primary ciliary dyskinesia[END_REF]. The cilia beating was acquired in 6 to 8 different locations of the insert. The inserts were kept at 37°C between acquisitions. The SPM (10nM) or vehicle solution were applied on the basolateral side. For every culture insert, videos were taken before SPM's treatment and after 1, 4, 24 and 48 hours. Ani9 (10µM) was applied 30min before SPM's treatment (Supplemental for details).

Calculation of cilia beating parameters

The density of cilia beating was evaluated using the standard deviation projection of the video and considering all pixels above a threshold of 30, for 8-bit encoded images. The frequency and the coordination were calculated using multiscale Differential Dynamic Microscopy (multi-DDM) [START_REF] Chioccioli | Phenotyping ciliary dynamics and coordination in response to CFTR-modulators in Cystic Fibrosis respiratory epithelial cells[END_REF]. The coordination described as the surface (λ² in µm²) below which cilia beat in a homogeneous way was calculated by dividing the video in square boxes ranging from 2.7 µm to 133.6µm. The orientation was calculated as the standard deviation of principal beating direction over square boxes of 50 pixels (17 µm) (Supplemental for details).

Multiparametric analysis using Principal Component Analysis

The PCA was calculated over 1659 videos recorded for untreated non-CF samples to be compared with untreated or SPMs treated CF samples, using the Stats package in R. Videos were taken at different time points up to 72h after thorough mucus washing. We analyzed five quantitative parameters: the density of movement (Movement), the temperature of acquisition (Temperature), the cilia beating frequency (CBF), the synchronization of cilia beating (λ²), the cilia orientation (Orientation).

ASL layer height analysis

The ASL layer height was measured as previously described using Texas red®-dextran (10,000MW, Invitrogen, Auckland, NZ) [START_REF] Verrière | Lipoxin A4 Stimulates Calcium-Activated Chloride Currents and Increases Airway Surface Liquid Height in Normal and Cystic Fibrosis Airway Epithelia[END_REF]. Epithelial cells were stained using Calcein-AM (Invitrogen, Auckland, New Zealand). Epithelia were treated in the basolateral compartment with RvE1 (10nM) for 1 hour. Z-scans of the epithelia were acquired with a Zeiss LSM 510 Meta (40X). For each culture insert, the ASL height was measured at 27 different locations.

Secreted mucins visualization

Culture inserts used for video-microscopy were fixed with Carnoy solution and permeabilized. Mouse monoclonal anti-MUC5AC (Abcam ab212636) 1:1000, rabbit polyclonal anti-MUC5B (Thermofisher PA5-82342) 1: 500, rat monoclonal anti-α-tubulin (YL1/2) (Thermofisher MA1-80017) 1:500 were used as primary antibodies. Images were acquired using an inverted confocal microscope (Zeiss Axio Observer 7 with LSM 900 Airyscan 2, 20x objective) and analyzed using FIJI. For each acquisition, 50 measures of the MUC5AC layer thickness were taken.

Mucin transcripts quantification

Mucin transcripts were evaluated with RT-qPCR on CF and non-CF samples. The cDNA sequence amplification was achieved using Taqman Master Mix (Thermofisher 4444557) and the primers for mucins MUC5B (Hs00861595_m1), MUC5AC (Hs01365616_m1) and the housekeeping gene beta-2-microglobulin (Hs00984230_m1).

Reagents

LXB4 was purchased from Cayman (90420) and RvE1 from VINRESOL Co (Budapest, Hungary, 20010207), kept at -80°C and used at 10nM. Ani9 (Sigma, SML1813) was used at 10µM.

Statistical analysis

Number of samples used are indicated in Table 1. The centroids of the PCA "clusters" and data dispersion were compared using the PERMANOVA test [START_REF] Mcardle | Fitting multivariate models to community data: A comment on distance-based redundancy analysis[END_REF] and PERMDISP test [START_REF] Anderson | Multivariate dispersion as a measure of beta diversity[END_REF] respectively from the vegan package on R. On bar graphs, data are represented as mean values ± SEM. Statistical significances were tested using an unpaired t-test or an equivalent non-parametric test, Mann-Whitney, using GraphPad InStat software. Statistical significance was defined as p < 0.05.

Legends

Figure 1. Graphical abstract of the study. We developed a new method of multiparametric signal analysis of cilia beating on air-liquid interface primary cultures of nasal epithelial cells from patients with cystic fibrosis (CF). The combined study of the frequency, synchronisation, orientation and quantity of cilia beating as well as of the ASL height and secreted mucins allowed to describe a cilia dyskinesia in CF that can be corrected by the specialized pro-resolving lipid mediators, resolvin E1. Similarly, videos were acquired at different times after RvE1exposure: at 1h (58 with vehicle and 46 with RvE1), at 24h (59 with vehicle and 49 with RvE1) and at 48h (39 with vehicle and 32 with RvE1). Scatter plot on the first two PC is displayed, each point corresponding to one video acquisition. PERMANOVA statistical tests were performed to compare cilia beat phenotype (centroid) recorded on CF hNEC exposed to the vehicle (blue) and CF hNEC exposed to SPMs (green); with p-values >0.5 considered as not significant (NS). PCA analysis from non-CF samples exposed to the vehicle is displayed in grey.

Figure 2 .

 2 Figure 2. Distinct cilia beating phenotypes of CF and non-CF hNEC. The principal component analysis (PCA) was performed over 1659 videos with 5 quantitative parameters: the percentage of movement (Movement), the temperature of acquisition (T°C), the cilia beat frequency (CBF), synchronization (λ²) and orientation (Orientation). A. The correlation circles are displayed for the 4 first PC. B. Cilia beating from CF and non-CF cultures (treated with vehicle), were recorded at different time after washing out the mucus: at 24h (116 video of non-CF and 272 of CF hNEC), at 48h (120 non-CF and 75 CF) and 72h (91 non-CF and 42 CF). Scatter plot on the first two PC is displayed. Ellipses were drawn using stat_ellipse function (package ggplot2) and calculated using multivariate Student distribution and 95% confidence interval. PERMANOVA statistical tests were performed to compare cilia beat phenotype (centroid) recorded on CF (blue) and non-CF hNEC (grey). Ellipses for the F508del homozygous samples only are also displayed (dotted line). The points are colored according to the temperature of acquisition from 21°C to 27°C. The analysis of cilia beating individual parameters was performed on the same records. C. Cilia beat frequency (CBF). D. Percentage of the video area covered by cilia beating (movement). E. Synchronization defined as the surface size below which cilia beat in a homogeneous way (λ²). F. Dispersion of cilia beat orientation. One point per video acquisition. Mean ± SEM. Mann-Whitney tests, * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3 .

 3 Figure 3. Effects of LXB4 and RvE1 on cilia beat phenotype. LXB4 (10nM) and RvE1 (10nM) or vehicle (ethanol) was added, 24h after mucus accumulation, into the basolateral compartment of culture inserts from 4 (for LXB4 experiment) and 5 (for RvE1 experiment) CF patients. Video were acquired at different times after LXB4 exposure: at 1h (35 videos with vehicle and 16 with LXB4), at 24h (39 with vehicle and 26 with LXB4) and at 48h (20 with vehicle and 15 with LXB4).Similarly, videos were acquired at different times after RvE1exposure: at 1h (58 with vehicle and 46 with RvE1), at 24h (59 with vehicle and 49 with RvE1) and at 48h (39 with vehicle and 32 with RvE1). Scatter plot on the first two PC is displayed, each point corresponding to one video acquisition. PERMANOVA statistical tests were performed to compare cilia beat phenotype (centroid) recorded on CF hNEC exposed to the vehicle (blue) and CF hNEC exposed to SPMs (green); with p-values >0.5 considered as not significant (NS). PCA analysis from non-CF samples exposed to the vehicle is displayed in grey.

Figure 4 .

 4 Figure 4. Distinct effects of RvE1 on hNEC from two F508del homozygous donors. PCA analysis (PC1 and PC2) corresponding to hNEC from CF1 (A) and from CF2 patients (C) treated with vehicle and RvE1 (10nM), as well as the non-CF hNEC beating phenotypes. Times correspond to the RvE1 exposure treatment duration. Corresponding values of cilia beating frequency (CBF), synchronization (λ²) and orientation for the 2 groups (untreated and RvE1 treated samples) over time for hNEC from CF1 patient (B) and from CF2 patient (D). Mean ± SEM; * p<0.05, **p<0.01,***p<0.001,****p<0.0001. PERMANOVA statistical tests were performed to compare cilia beat phenotype (centroids) recorded on CF hNEC exposed to the vehicle (blue) and CF hNEC exposed to RvE1 (green). Mean ± SEM, * p<0.05, ****p<0.0001.

Figure 5 .

 5 Figure 5. Role of the TMEM16A in the effect of RvE1 on the ASL height and the cilia beating phenotype. A. Representative orthogonal views of confocal microscopy z-stacks showing the ASL for a CF sample in control condition (vehicle) and treated with RvE1 (10nM). ASL is labelled using

Figure 6 .

 6 Figure 6. RvE1 modified mucins secretion and structure. A. Representative confocal projection images of MUC5AC (green) and MUC5B (red) secreted apically by non-CF and CF hNECs cultures treated with vehicle solution or RvE1(10nM) ± Ani9 (10µM). Culture inserts were fixed after 48h of treatment. Orthogonal views (Z) of MUC5AC. White scale bar = 10μm. B. Average thickness of MUC5AC structures measured on hNEC from 2 CF donors and 4 non-CF donors (2 culture inserts by condition and 3 to 5 acquisitions). C. Mean transcription levels of MUC5B and MUC5AC in untreated hNEC from 8 non-CF donors and 6 CF donors and in hNEC derived from 4 CF donors and treated for 24h with RvE1 (10nM). Transcription levels expressed as delta Ct (Ct of the mucin minus Ct of the housekeeping gene). Mean ± SEM. T-test, *p<0.05, ****p<0.0001.

Table 1 : Samples donor distribution for cilia beating acquisitions.

 1 Median age in years, 1st and 3rd quartiles in brackets.

Table 2 .

 2 Cilia beating parameters changes over time after mucus removal in CF and non-CF hNEC primary cultures. Values are expressed as Mean ± SEM.

Acknowledgments

The authors thank the imaging platform of the Institut Mondor de Recherche Médicale (Créteil, France) and the support of X. Decrouy. This study was funded by the French National Institute of Health (INSERM), the CF patient association Vaincre la Mucoviscidose and the doctoral school of Life Science and Health in Créteil (France).


Maxime Masson

Christian Sallaberry
email: christian.sallaberry@univ-pau.frmarie-noelle.bessagnet

Rodrigo Agerri
email: rodrigo.agerri@ehu.eus

Marie-Noelle Bessagnet

Philippe Roose

Annig Le

Parc Lacayrelle
email: annig.lacayrelle@univ-pau.fr

A domain-independent method for thematic dataset building from social media: the case of tourism on Twitter

Keywords: Social Media, Dataset Building, Social Web Analysis, Computational Social Science, Natural Language Processing

In this article, we propose a generic method to build thematic datasets from social media. Many research works gather their data from social media, but the extraction processes used are mostly ad hoc and do not follow a formal or standardized method. We aim at extending the processes currently used by designing an iterative, generic and domain-independent approach to build thematic datasets from social media with three modulable dimensions at its core: spatial, temporal and thematic. We experiment our method using data extracted from Twitter to build a thematic dataset about tourism in a highly touristic region. This dataset is then evaluated using both quantitative and qualitative metrics to highlight the value of this method. The application to this use case shows the effectiveness of our domain-independent method to generate thematic datasets from Twitter data.

Introduction

Recently, we have seen a significant growth in available data sources covering many topics and the rise of user-generated content. Tourism in particular represents one of the biggest economic sectors in the world in which accurate thematic datasets are critical to better analyze and decipher trends [START_REF] Aguiar | Big data and tourism: opportunities and aplications in tourism destination management[END_REF].

In the domain of tourism, large datasets can have many practical use cases. On the one hand, they can be used for the purpose of supporting the decisionmaking process of tourism stakeholders for the improvement, development and planning of touristic cities and areas. This is done by analyzing the data to better understand the practice and needs of tourists. Such analysis is particularly useful for companies specialized in tourism marketing (such as Destination Marketing Organization, DMO) where understanding the desires and expectations of tourists is key. On the other hand, tourism data can be analyzed for tourists themselves by building recommender systems. Those systems analyze the types of practices of a large number of tourists to be able to recommend better suited places, activities or tourist itineraries. Furthermore, different types of sources can be used to extract touristic data. Historically, these are mainly databases with 2 categories that stand out: [START_REF] Aguiar | Big data and tourism: opportunities and aplications in tourism destination management[END_REF] commercial databases, such as those from Online Travel Agencies (OTAs) and, (2) public databases, for example government issued or crowd sourced ones. The latter is a part of what is called User-Generated Content (UGC). In recent years, this category of data has grown significantly, ranging from social media (Twitter, FourSquare) to review sites (TripAdvisor ).

As part of a local project, we needed to extract, analyze and present data focused on a specific theme defined by a domain expert (in our case, tourism). While many research works concentrate on proposing fully generic and adaptable processing pipelines to extract knowledge from flows of social media data [START_REF] Sathick | A generic framework for extraction of knowledge from social web sources (social networking websites) for an online recommendation system[END_REF] (such as NLP modules calibrated for short, informal messages), they do not propose a generic methodology to build such thematic datasets. In contrast to those previous approaches, the work presented here focuses on the upstream step of the data collection process. It aims at consolidating existing solutions when it comes to social media based Information Extraction (IE). Indeed, this contribution consists of proposing a domain-independent method to obtain high-quality data from social media to build thematic datasets. It is generic and based on an iterative and multi-dimensional (spatial, temporal, thematic) filtering process. It was hypothesized that representing the theme of the dataset to be built up in the form of a vocabulary can contribute to (1) an efficient thematic filtering of posts and to (2) the development of a domain independent process.

The article is organized as follows. Firstly, we introduce our project's motivations and the requirements our extraction method must meet. Secondly, we review the state of the art of the approaches used to gather data out of social media with a focus on Twitter data. Thirdly, our contribution is presented: a domain-independent, generic method for thematic dataset building from social media. We finally experiment and evaluate our method on a local touristic case.

Motivation

This work, carried out in the framework of a multilingual (English, French, Spanish and Basque) project, aims to collect, process, analyze and then value social media data related to the practice of tourism, visitor flows and the use of cultural heritage in the Basque Country, a cross-border highly touristic area.

We decided to adopt a trajectory-based analysis and therefore build multidimensional trajectories from local visitors. Our trajectory model has 3 dimensions at its core. The spatial dimension (where) (1) refers to the set of places (municipalities, natural areas, etc.) visited by tourists. The temporal dimension (when) (2) can be seen at different levels: the period of the trip (season, year, month, time interval etc.) as well as the date and duration of each activity performed. Finally, the thematic dimension (what) (3) is purely semantic, it describes the "what" of the tourist practice, such as the activities performed, the conditions of the trip, the tourist's feelings, whether they were accompanied or not, etc. We aim at moving beyond the well-known concept of spatial tracks and instead focus on trajectories where the thematic dimension has a greater weight than the spatial one, where places could be represented by themes and region by cluster of themes. Fig. 1 shows an example of a trajectory in a tourist thematic space. With thousands of trajectories, we could run pattern analysis on it to detect affinities between types of tourist activities, categories of cultural heritage or even recurring sequences of activities. To build these thematic trajectories, social media is chosen as our primary source of data for several reasons: (1) the ease of access, no need for time-consuming collection campaigns, (2) the diversity, most tourism aspects are covered and (3) the massive amount of data available.

Although our project is mostly focused on the tourism domain, we have already identified future needs for the generation of datasets related with other specific domains such as wine culture or education. It therefore appeared necessary to move towards a method that is as independent of the domain as possible.

Related Work

Due to the extensive amount of research work on the subject, we focus on Twitter data that we have chosen as a main data source for our project. For the spatial dimension [START_REF] Aguiar | Big data and tourism: opportunities and aplications in tourism destination management[END_REF], two different approaches are generally used and sometimes combined to obtain better results. First, filtering can be performed on the spatial metadata attached to the post, either by providing the social media API with a bounding box encompassing the area of study, or by basing it on a precise location (referenced with a latitude/longitude) with a radius around it (location nearness) [START_REF] Scholz | Evaluating geo-tagged twitter data to analyze tourist flows in styria, austria[END_REF]. The advantage of this metadata-based approach is that it is extremely accurate, as the location is generally determined using the GPS of the device. The biggest problem that arises is the low amount of posts that have metadata attached. For Twitter, it was estimated that around 1% of tweets have spatial metadata attached to them [START_REF] Sloan | Who tweets with their location? understanding the relationship between demographic characteristics and the use of geoservices and geotagging on twitter[END_REF]. Thus, relying solely on metadata misses many potentially relevant posts. The second method is applied to the content of the post and is done via toponym filtering [START_REF] Shimada | Analyzing tourism information on twitter for a local city[END_REF][START_REF] Scholz | Evaluating geo-tagged twitter data to analyze tourist flows in styria, austria[END_REF]. A list of place names and their associated abbreviations is compiled and all posts containing them are extracted. The big drawback of this method is the potentially large amount of noise. Disambiguation approaches are used to mitigate this problem, for example by combining this list of toponyms with exclusion lists [START_REF] Zenasni | Spatial Information Extraction from Short Messages[END_REF].

For the temporal dimension ( 2), the extraction is usually done according to the timestamp. It is an easy method to implement and usually quite accurate. Exchanges on social media are often done in real time, so it is not necessarily mandatory to set up a complex temporal entity extraction system.

When it comes to the thematic dimension (3), multiple approaches are used. Content-based approaches use thematic keywords directly related to the theme of study (such as event names [START_REF] Shimada | Analyzing tourism information on twitter for a local city[END_REF]), the use of too specific keywords can restrict too much the number of returned posts, so some research works associate several words together to establish filtering rules. Some social media have the concept of hashtag allowing to identify topics of discussion, which have often been used as a filtering tool [START_REF] Cignarella | Overview of the task on stance detection in italian tweets[END_REF]. Other thematic filtering methods are applied to the metadata and include: the language of the post, its source, etc. To reduce noise, some research work use only the posts from a pre-selected list of accounts known to validate certain desired criteria (e.g., known for speaking regularly about a specific topic [START_REF] Chiruzzo | Haha 2019 dataset: A corpus for humor analysis in spanish[END_REF]). Associated replies and comments can also be extracted [START_REF] Cignarella | Overview of the task on stance detection in italian tweets[END_REF].

Although, in recent years, various efforts have been made to design more generic processing techniques for social media data [START_REF] Sathick | A generic framework for extraction of knowledge from social web sources (social networking websites) for an online recommendation system[END_REF], when it comes to dataset building, each research project usually comes with its own extraction and filtering flow. While some common techniques are shared, there is not yet a fully generic and domain-independent procedure for dataset building from social media.

A new method for thematic dataset building from social media

We propose a method for creating thematic datasets from social media. It aims at formalizing a generic approach to extract social media-sourced data related to a given domain and possibly a given time period or spatial area. This method is designed around the following properties:

-Multi-dimensional: the method is based on 3 dimensions: spatial, temporal, thematic. One can combine two, three of them or only use one. -Generic: it can be implemented with any social media that has a post system. This genericity also extends to languages. -Domain-independent: it supports any target theme for the dataset.

-Iterative, incremental: the method is designed around an iterative and incremental process, namely, each iteration aims at refining the following iterations to have a dataset as qualitative as possible with a minimal noise.

Before proceeding with the collection process (the steps presented in Fig. 2), it is necessary to define the future dataset along the spatial (territorial footprint of the data), temporal (temporal scope) and thematic (the theme, the semantics as of a vocabulary, thesaurus or ontology) dimensions. According to our review of the state of the art, these dimensions cover the majority of use cases in dataset building. The presence of all these dimensions is not mandatory. We could therefore have only tempo-thematic or spatio-thematic datasets.

Our method being iterative and incremental, users can refine the dimensions later in the process, until they are satisfied with the obtained dataset. This process is semi-automatic. After this preliminary definition step, it is recommended to define a calibration dataset, It is a subset of the main one with a more restrictive definition used to calibrate the main, wider collection process.

Filtering the Flow of Posts

Once the dataset have been defined, we can now move on to the collection process. It is applied sequentially to 2 sets of posts with different features.

1. Geotagged posts: posts whose authors have activated GPS location (about 2% [START_REF] Sloan | Who tweets with their location? understanding the relationship between demographic characteristics and the use of geoservices and geotagging on twitter[END_REF] for Twitter). This reduced set of posts is handled first (Fig. 2, 1 ○). 2. All posts: posts containing a text (no image-only posts) (Fig. 2,2 ○).

The flow of associated media (Fig. 2, 3 ○) is a future research axis and will therefore not be discussed in this article.

Each set is extracted following the procedure described in Fig. 2. The order of the steps is only indicative. In this sense, it is necessary to think about which steps can be delegated to the internal filtering system of the social media and which ones must necessarily be carried out locally (the latter have to be processed last). There is no universal answer to this question, it depends on the extent of the search functionality of the social media and the dimensions' complexity.

Pre-processing aims to exclude accounts, keywords or hashtags that we know should not appear in our final dataset while being prone to fit within our dimensions, for example: excluding professional, institutional or promotional accounts, excluding problematic keywords, or excluding certain languages. This step is especially useful to exclude places with the same name but actually unrelated to each other (toponymic homonyms), which could distort the spatial filtering process. Usually, these criteria are not known at the beginning of the process so this step is empty, although it will be filled in future iterations.

The following steps are optional and depend on the dataset definition. For a more efficient collection process, it is advisable to order the dimensions from the one believed to be the most restrictive, to the less restrictive (in order to process as few unnecessary posts as possible) and to delegate as much filtering as possible to the social media native API system. Temporal filtering is done using the timestamp of the posts. This method has been extensively used and covers most of the use cases. Indeed, social media are instantaneous informal exchange zones, i.e. users generally talk about the present moment. In specific cases, a temporal entity detection system could be applied on the content of the posts. This would allow to be more precise in cases where users talk about past or future events (yesterday, next week ).

For spatial filtering the approach to be used differs depending on the two sets of posts described above. In the case of geotagged posts, we propose to check whether the position of the post is contained within a bounding box. This method is highly accurate because it is based on the devices' GPS. Moreover, it can usually be offsetted to the social media API. For the posts without spatial metadata attached, the approach is a bit different. We rely on the post content to do the selection. A list of toponyms contained within the area of study is provided as input and is matched within post content to determine which ones are about this area. The risk of noise is higher with this approach, but our iterative method allows to refine this step further in the process.

The objective of thematic filtering is to keep only the posts which are related to the theme, with the latter being defined using a given vocabulary. It is done by aligning this vocabulary with the post content (entity linking [START_REF] Cossin | Iam at clef ehealth 2018: Concept annotation and coding in french death certificates[END_REF]).

Dataset Preview and Iteration

Each time an iteration of this process is performed, a dataset is obtained. We can then preview the resulting dataset and evaluate it, this step is usually performed by a domain specialist (Fig. 2,4 ○). Their role is to determine by reviewing a set of randomly selected posts, whether there is too much noise or not enough posts and try to identify certain types of recurring posts to exclude or that are missing. The criteria to add, remove, extend or narrow are then decided and a new iteration of the process can start using those refined criteria, and so on until the final dataset is deemed satisfactory (feedback loop).

Experimentation and Evaluation

To experiment our methodology, we chose the social media Twitter3 . Concerning the preliminary definition step, we rely on the Thesaurus on Tourism & Leisure of the WTO4 . This resource covers roughly 1,300 touristic concepts. For the purpose of this experiment, we reduce the spatial extent of the data. We wish to gather content from only one specific sub-area which will serve as our spatial dimension: the French Basque Coast, which is broadly considered to be among the most touristic places in the region. Also, in an effort to show the full range of our dimensions, we will focus solely on the summer of 2019.

We compute quantitative statistics on the collected data such as the number of users, tweets, detected concepts or locations. These quantitative metrics will allow us to determine if our method can be implemented on Twitter data and if a consistent number of tweets is collected without having too few posts or excessive noise. Then, we want to evaluate the effectiveness of our thematic filtering process. We take as reference the context annotations generated by Twitter. These are labels that Twitter automatically attaches to tweets based on their content. The approach used to create those is not known but among these annotations, one is about Travel. After reviewing them, we realized that these annotations were usually quite accurate but that many relevant tweets did not have them (e.g., Twitter annotates well but doesn't annotate enough tweets) making it difficult to build large datasets relying solely on them. It therefore seemed relevant to calculate what proportion of tweets suggested as related to travel by Twitter our system select. Lastly, we needed to evaluate whether all other tweets not tagged as "Travel " we import are relevant or just noise. We therefore perform a qualitative analysis on the resulting dataset. We randomly sampled 20, 50, and 100 posts of the resulting datasets and calculated the thematic accuracy @20, @50, and @100 to check the reliability of our process at each iteration. Two tourism experts will manually analyze their content and annotate whether or not they relate to tourism or not. This qualitative metric is meant to demonstrate the role of our method's multiple iterations in improving the accuracy and evaluate the overall quality of the results.

Method Implementation

The whole process (Table 1) was developed with the Tweepy5 library used for the interaction with the Twitter API. We performed 3 refinement iterations and we only collected tweets in French, English and Spanish posted in summer 2019.

Table 1. Application of the method using our dataset requirements.

For geotagged tweets, we simply filter on the bounding box of the area of study and get about 7,000 tweets. Those are then thematically filtered using the whole thesaurus vocabulary in conjunction with the IAM Entity Linker [START_REF] Cossin | Iam at clef ehealth 2018: Concept annotation and coding in french death certificates[END_REF], a dictionary-based approach for semantic annotation. We get 3,447 tweets as output. For non-geotagged tweets, we use 625 multilingual toponyms extracted from OSM6 contained within the area of study. In the first iteration, we use all of them indiscriminately to filter and therefore retrieve too many tweets (more than 2.5 million). We decide to stop the process and refine it at the next iteration.

The feedback from the 1st iteration leads us to blacklist professional or institutional accounts (not interesting for our analysis). We also refine the toponym list and remove 46 common place names (such as Roman Theater ). Lastly, the tourism thesaurus is reduced to exclude some branches deemed irrelevant by our project domain experts. We get about 60,000 tweets at the end of the 2nd iteration and notice a large number of tweets about the G7 an important event taking place in 2019 in the region. A 3rd iteration is carried out with additional pre-processing filters to blacklist G7-related keywords and hashtags. The final dataset is made of about 2,098 geotagged tweets, 25,281 tweets in total belonging to 15,000 users and 458 unique concepts have been found in these tweets. Out of those: 1,668 have the Twitter "Travel " annotation. In other words, only about 6% of the tweets collected were identified as relating to tourism by Twitter, that is not much and it is why we aim at collecting more than those.

Results Analysis

Fig. 3 shows the different sets of tweets of the 3rd (last) iteration (union of both geotagged non-geotagged ones) and among these sets of tweets, the proportion of those annotated with the "Travel " context annotation by Twitter. We observe that among the tweets from the area of study (66,005), most of those tagged as "Travel " by Twitter are selected by our system (1,668 selected, 217 excluded). We get an accuracy of 0.884. We also notice a high number of selected tweets which are not tagged by Twitter (25,711) but that we select. It means that our process detects many more potentially relevant items. This can be rather a positive aspect as Twitter context annotations seem to be missing from a lot of tourism-related tweets. However, we now need to determine whether all those are just noise or other tourism-related tweets that Twitter has not annotated.

For this purpose, a qualitative analysis is set up. 100, 50 and 20 tweets are extracted randomly from the datasets at different stages of the method and manually evaluated by experts to determine whether they have been correctly or wrongfully selected. Table 2 shows the result of the evaluation done by two experts on 20, 50 and 100 tweets, randomly picked just after the thematic filtering for both sets of tweets. We compute the mean thematic accuracy between those two experts and, for the 3rd iteration @100, the associated Cohen's Kappa (κ) which measures the degree of agreement between them to mitigate subjectivity. The question asked was "Is this tweet related to Tourism? ". We obtain a mean thematic accuracy ranging from 0.83 (@20) to 0.74 (@100) for geotagged tweets and 0.72 (@20) to 0.65 (@100) for non-geotagged ones. That means, by extrapolation, potentially 65% to 83% of the tweets not selected by Twitter might actually be relevant to the topic of tourism and our assumption that Twitter is not annotating enough content is correct.

We also observe a thematic accuracy @100 starting at 0.52 at the 1st iteration for geotagged tweets which increases to 0.59 and then 0.74 in iterations 2 and 3 which clearly highlight the effect of filter refinement and feedback loop between each iterations. Overall, experts seem to agree on the outcome as shown by the relatively high κ score. Thematic accuracy on all non-geotagged tweets is slightly lower but follow a similar increasing trend. The final dataset thematic accuracy is acceptable but could have been increased even more by doing more iterations, we limited ourself to 3 for this experiment. To go further, we observed the same sample of incorrect tweet from the first iteration throughout the method to see what proportion of it would get removed in future ones. We use the set of tweets used for the @100 thematic accuracy measure of the 1st iteration. The accuracy is average (0.52) which means: out of 100 tweets evaluated, 48 were incorrectly selected. The 2nd iteration removes 27 of them, so 21 are remaining. Then finally, the last iteration leaves 15 remaining. This is consistent with the accuracy we calculated on random samples previously (≈ 70% of correct tweets).

Conclusion

We proposed a domain-independent and generic method for building thematic datasets from social media based on 3 dimensions. The objective is to move away from ad hoc collection processes and to propose a robust method to build focused datasets. We are thinking of going further by extending our dimensions by taking inspiration from the 5W1H [START_REF] Han | Counseling dialog system with 5w1h extraction[END_REF] dimensions. The 5W1H is a framework widely used in problem solving and question answering based on the 6 interrogative words: Who, What, When, Where, Why, How. We already have the When (temporal), Where (spatial) and What (thematic) but we could imagine other dimensions for the Who (the users, the persons they are referring to), the why (reasoning behind an action) or the how (in what way is it carried out).

Fig. 1 .

 1 Fig. 1. Trajectory of a visitor in what could be the tourism thematic space.

Fig. 2 .

 2 Fig. 2. Overview of the extraction process.

Fig. 3 .

 3 Fig. 3. Number of tweets annotated "Travel " by Twitter among those we select.

Table 2 .

 2 Qualitative analysis of the results.

		Iteration 1	Iteration 2	Iteration 3
		Geotagged All Geotagged All Geotagged	All
	(@ 20) (@ 50) Accuracy (@ 100) Thematic	0.75 0.64 0.52	0.60 0.60 0.59	0.30 0.30 0.35 0.74 (κ 0.74) 0.65 (κ 0.48) 0.83 0.72 0.77 0.74

https://developer.twitter.com/en/products/twitter-api/academic-research

https://www.e-unwto.org/doi/book/10.18111/9789284404551

https://www.tweepy.org

https://www.openstreetmap.org/