Damien Adam

Jacqueline Roux-Delrieu

Emilie Luczka

Arnaud Bonnomet

Julien Lesage

Jean-Claude Mérol

Myriam Polette

Michel Abély

Christelle Coraux
email: christelle.coraux@univ-reims.fr

Cystic Fibrosis airway epithelium remodelling: involvement of inflammation

Keywords: Cystic Fibrosis, airway epithelium remodelling, regeneration, airway inflammation

INTRODUCTION

Cystic Fibrosis (CF) is the most common lethal autosomal recessively inherited disorder among Caucasians. Mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene are associated with altered ion and fluid transports leading to viscous secretions in the lungs that promote bacterial infection and chronic inflammation and resulting in a decline in lung function [START_REF] Rowe | Cystic fibrosis[END_REF][START_REF] Pillarisetti | Infection, inflammation, and lung function decline in infants with cystic fibrosis[END_REF]. Proteases, oxidants, virulence factors and inflammatory mediators induce airway epithelial damages [START_REF] Chmiel | The role of inflammation in the pathophysiology of CF lung disease[END_REF][START_REF] Halldorsson | Azithomycin maintains airway epithelial integrity during Pseudomonas aeruginosa infection[END_REF][START_REF] Rajan | Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors[END_REF][START_REF] Berger | Inflammation in the lung in cystic fibrosis[END_REF] such as hyperplasia of surface goblet and basal cells [START_REF] Voynow | Basal-like cells constitute the proliferating cell population in cystic fibrosis airways[END_REF][START_REF] Trinh | Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue[END_REF][START_REF] Hubeau | Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa[END_REF][START_REF] Leigh | Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients[END_REF], squamous metaplasia [START_REF] Trinh | Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue[END_REF][START_REF] Leigh | Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients[END_REF][START_REF] Piorunek | Correlation between the stage of cystic fibrosis and the level of morphological changes in adult patients[END_REF], increase in epithelium height [START_REF] Voynow | Basal-like cells constitute the proliferating cell population in cystic fibrosis airways[END_REF][START_REF] Tiddens | Cartilaginous airway wall dimensions and airway resistance in cystic fibrosis lungs[END_REF] as well as cell shedding (7-10, 12, 13). Whether this remodelling is secondary to deleterious infectious and inflammatory mediators, or to CFTR dysfunction remains debated. Indeed, in response to insults, epithelial cell migration, proliferation and differentiation are required for proper tissue repair and regeneration [START_REF] Coraux | Differential expression of matrix metalloproteinases and interleukin-8 during regeneration of human airway epithelium in vivo[END_REF][START_REF] Dupuit | Differentiated and functional human airway epithelium regeneration in tracheal xenografts[END_REF] and some studies have reported a major role for CFTR in the early event of cell migration [START_REF] Trinh | Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue[END_REF][START_REF] Schiller | Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair[END_REF][START_REF] Kirk | CFTR channels and wound healing. Focus on « Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair[END_REF]. While it is well known that some ion channels regulate cell migration [START_REF] Schwab | Cells move when ions and water flow[END_REF][START_REF] Girault | Evidence of K + channel function in epithelial cell migration, proliferation and repair[END_REF], it has been recently demonstrated that CF airway epithelial cells exhibited slower migration than non-CF cells [START_REF] Trinh | Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue[END_REF][START_REF] Schiller | Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair[END_REF]. As CFTR expression or activity inhibition resulted in a significant delay in wound repair, and F508del-CFTR correction significantly improved wound healing [START_REF] Trinh | Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue[END_REF][START_REF] Schiller | Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair[END_REF], it is now clear that the CFTR default modulates airway epithelial cell migration. Moreover, using a humanized xenograft mouse model, we have previously shown that the CF airway epithelial regeneration is impaired even in absence of infection: the regeneration process is delayed in terms of ciliated cell differentiation and the reconstituted epithelium is remodelled, significantly higher with basal cells hyperplasia [START_REF] Hajj | Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis[END_REF].

Chronic inflammation is a hallmark of CF. Inflammation begins early in the course of the disease, even in patients without clinically apparent lung disease [START_REF] Cantin | Cystic fibrosis lung inflammation: early, sustained, and severe[END_REF]. As several studies have reported the presence of increased inflammatory markers in bronchoalveolar lavages (BAL) of infants in apparent absence of CF-related pathogens [START_REF] Khan | Early pulmonary infection in infants with cystic fibrosis[END_REF][START_REF] Balough | The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis[END_REF][START_REF] Konstan | Bronchoalveolar lavage findings in CF patients with stable, clinically mild lung disease suggest ongoing infection and inflammation[END_REF][START_REF] Armstrong | Lower respiratory infection and inflammation in infants with newly diagnosed cystic fibrosis[END_REF], it has been postulated that inflammation may precede infection by some direct contribution of the defective CFTR [START_REF] Konstan | Current understanding of the inflammatory process in cystic fibrosis: onset and etiology[END_REF][START_REF] Bodas | The NFB signalling in cystic fibrosis lung disease: pathophysiology and the therapeutic potential[END_REF]. However, alternative explanations have been proposed, in particular the persistence of the inflammatory response after clearance of bacterial infection [START_REF] Chmiel | The role of inflammation in the pathophysiology of CF lung disease[END_REF][START_REF] Konstan | Current understanding of the inflammatory process in cystic fibrosis: onset and etiology[END_REF]. Regardless of the initial source of stimulation, inflammatory mediators, that can be produced by airway epithelial cells themselves [START_REF] Bonfield | Altered respiratory epithelial cell cytokine production in cystic fibrosis[END_REF][START_REF] Becker | Cytokine secretion by cystic fibrosis airway epithelial cells[END_REF][START_REF] Okada | Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways[END_REF], are elevated in sputa and BALs from CF compared to control patients and exaggeratedly produced in response to infection [START_REF] Khan | Early pulmonary infection in infants with cystic fibrosis[END_REF][START_REF] Armstrong | Lower respiratory infection and inflammation in infants with newly diagnosed cystic fibrosis[END_REF][START_REF] Koller | Cytokine concentrations in sputum from patients with cystic fibrosis and their relation to eosinophil activity[END_REF][START_REF] Bonfield | Inflammatory cytokines in cystic fibrosis lungs[END_REF][START_REF] Muhlebach | Quantitation of inflammatory response to bacteria in young cystic fibrosis and control patients[END_REF][START_REF] Dakin | Inflammation, infection and pulmonary function in infants and young children with cystic fibrosis[END_REF][START_REF] Mango | Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8[END_REF].

In the present study, using primary cultures of non-infected CF and non-CF human airway epithelial (HAE) cells from nasal origin cultured in an air-liquid interface (ALI) with and without inflammatory stimulation along the regeneration process, we demonstrated that the transient hyperinflammatory phenotype of CF cells could be partly responsible of the CF epithelium remodelling.

MATERIALS AND METHODS

Human airway tissues and cell dissociation

The use of human tissues was authorized by the bioethical law n° 2004-800 of the French Public Health Code, after written consent from the patients and the approval of our local Institutional Review Board. Non-infected human airway tissues were collected after nasal polypectomy from 9 CF non-smoker patients (median age 13; range 4-46; genotypes: F508del/F508del: 4; F508del/G85E: 2; F508del/W1282X: 1; F508del/R1158X: 1; F508del/G542A: 1) and 16 non-CF non-smoker subjects who did not suffer from any other disease (asthma, allergy, chronic obstructive pulmonary disease (COPD)) at the time of experiments were carried out (median age: 54; range 30-82). Non-CF and CF human airway epithelial (HAE) cells were obtained after dissociation by 0.05% type XIV collagenase (Pronase E; Sigma Aldrich, St Louis, MO) as described in the online supporting information.

Cell culture

Nasal HAE cells were used at passages 0 (P0) (from 9 non-CF and 9 CF donors) and at passage 1 (P1) (from 4 non-CF and 4 CF donors) (see online supporting information) in order to work with hyperinflamed (P0) and no more inflamed (P1) CF cells, as it has been previously reported [START_REF] Becker | Cytokine secretion by cystic fibrosis airway epithelial cells[END_REF]. The ALI culture method was adapted from Fulcher et al. [START_REF] Fulcher | Well-differentiated human airway epithelial cell cultures[END_REF]. Briefly, HAE cells were cultured on Transwell supports (Transwell-Clear, Corning, Acton, MA) in submerged conditions, then in an ALI for 0 to 35 days (ALI D0 to ALI D35) using, in the basal compartments, a medium consisting in 1:1 DMEM/F12 (Gibco, Paisley, UK) and BEGM (Lonza, Walkersville, MD) with the Lonza supplements for hEGF, epinephrine, BPE, hydrocortisone, insulin, triiodothyronine and transferrin, and supplemented with 200 UI/ml penicillin, 200 µg/ml streptomycin (Gibco), 0.1 nM retinoic acid (Sigma Aldrich) and 1.5 µg/ml bovin serum albumin (BSA; Sigma Aldrich), with or without pro-inflammatory cytokine (cytomix cocktail composed of TNF (1 ng/ml; Sigma Aldrich), IL-1 (1 ng/ml; Abcys, Courtaboeuf, France) and IFN (1 ng/ml; Miltenyl Biotec, Auburn, CA) stimulation as described in the online supporting information.

Histological analysis

Histology of 4 non-CF and 4 CF cultures (genotypes : F508del/F508del: 3; F508del/W1282X: 1) at P0 and at P1 was analysed at ALI D35 on cryosections after hematoxylin-eosin staining as described in the online supporting information. Epithelium height was measured on 20 images per section and 5 different sections per culture using a Nikon Eclipse Ci microscope (Nikon, Champigny sur Marne, France) equipped with a Nikon DS-Fi2 camera, and the dedicated Nis-elements software. The results are expressed in µm.

Immunohistochemistry

To quantify the different cell types composing the non-CF and CF cultures (genotypes: F508del/F508del: 3; F508del/W1282X: 1) at P0 and P1, basal, goblet and ciliated cells were stained by immunohistochemistry on cryosections or on methanol-fixed cultures using antibodies directed against cytokeratin 13 (CK13, basal cell marker; Sigma Aldrich), MUC-5AC (goblet cell marker; Novocastra, Newcastle upon Tyne, UK), MUC-5B (provided by Dr Copin, Lille France) and -tubulin (ciliated cell marker; Chemicon, Temecula, CA). Cycling cells were detected using antibodies anti-Ki67 (Dako, Glostrup, Denmark). Primary antibodies were revealed with goat anti-mouse Alexa Fluor 488 antibodies (Molecular Probe, Eugene, OR) and cell nuclei stained using DAPI (Sigma Aldrich). Negative controls were performed by omitting the primary antibodies. Images were acquired on an AxioImager ZI Microscope (Carl Zeiss, Göttingen, Germany) equipped with a CCD camera (CoolSnap HQ, Photometrics, Tucson, AZ) and using the dedicated Axiovision release 4.8 software (Carl Zeiss). Technical and quantification methods are described in details in the online supporting information. The results are expressed as percentage of positive cells for CK13, MUC-5AC and Ki67, and in number of positive cells per mm 2 for ciliated cells.

RT-qPCR analysis

IL-8 mRNA expression by nasal HAE cells from 7 non-CF and 5 CF donors at P0 (genotypes: F508del/F508del: 3; F508del/W1282X: 1; F508del/R1158X: 1), and from 4 non-CF and 4 CF donors at P1 (genotypes: F508del/F508del: 3; F508del/W1282X: 1) was analysed by RT-qPCR. IL-8 expression was also studied in 9 non-CF and 9 CF cultures (Passage 0; genotypes: F508del/F508del: 4; F508del/G85E: 2; F508del/W1282X: 1; F508del/R1158X: 1; F508del/G542A: 1) at ALI D0, ALI D7, ALI D15, ALI D35, and in 4 non-CF and 4 CF cultures (Passage 1; genotypes: F508del/F508del: 3; F508del/W1282X: 1) at ALI D35.

Total RNA was extracted using the High Pure RNA isolation kit (Roche Diagnostics, Indianapolis, IN), following the manufacturer's instructions. Total RNA was reversetranscribed to cDNA using Transcriptor First Strand cDNA synthesis kit (Roche Applied Science, Mannheim, Germany) and quantitative PCR was performed using the Fast Start Universal Probe Master kit (Roche) as recommended by the manufacturer, in the LightCycler 480 Instrument (Roche). As described in the online supporting information, results were normalized to the expression levels of GAPDH and expressed as IL-8/GAPDH ratios.

Cytokine measurement

At ALI D0, ALI D7, ALI D15, ALI D25 and ALI D35, 4 non-CF and 4 CF (genotypes: F508del/F508del: 3; F508del/W1282X: 1) cultures at passage 0 were rinsed with sterile PBS, then 100 µl of fresh DMEM medium without phenol red (Gibco) were added to the basal compartment of the cultures, in contact with the porous membranes. The cultures were incubated for 4 hours at 37°C. Basal supernatants were collected and stored at -80°C until use.

IL-8 was measured in these supernatants using the Human Interleukin 8 ELISA kit (Emelca Bioscience, Breda, The Netherlands) according to the manufacturer's instruction.

Statistical analysis

All experiments were performed using nasal HAE cells or ALI cultures obtained from, at least, 4 non-CF and 4 CF donors. Results are expressed as mean ± SEM. Data were analysed by the non-parametric Mann-Whitney test for statistical analysis using StatView software (SAS Institute, Inc., NC). Statistical significance was defined as p  0.05.

RESULTS

In absence of exogenous infection and inflammation, the regeneration of the human CF airway epithelium leads to the reconstitution of a remodelled epithelium

Using an in vivo model of humanized xenograft in the mouse, we have previously reported that the CF human airway epithelial regeneration was abnormal and reconstituted a remodelled epithelium [START_REF] Hajj | Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis[END_REF]. In order to free oneself from any exogenous stimulation, we reproduced the experiments in the perfectly defined model of in vitro ALI culture. Non-CF and CF cultures were analysed at ALI D35, when they were approximately covered at 70% by ciliated cells and consequently considered as fully differentiated. As indicated by the measurement of epithelium height, the CF airway epithelium was significantly 32.87 % higher (38.03 ± 1.01 µm) than the non-CF epithelium (28.62 ± 0.75 µm) (Fig. 1C). Moreover, the CF epithelium exhibited basal cell hyperplasia (47.01 % cell number increase), with 39.37 ± 2.07 % of CK13 positive cells, while the non-CF epithelium reached 26.78 ± 0.35 % at the same time (Fig. 1F). Surprisingly, goblet cells were not more numerous in CF cultures compared to non-CF ones, with 0.32 ± 0.027 % and 0.29 ± 0.014 % of MUC-5AC positive cells, respectively (Fig. 1I), all MUC-5AC positive cells being also positive for MUC-5B (Supplementary Fig. 1). Ciliated cells were quantified during the course of their differentiation, from ALI D15 to ALI D35. At ALI D15, the number of -tubulin positive ciliated cells was significantly 47.95 % lower in CF (235.92 ± 30.40 cells / mm 2 ) than in non-CF cultures (453.04 ± 46.25 cells / mm 2 ) (Fig. 1L), as it was also observed at ALI D25 with 349.19 ± 34.98 cells / mm 2 in CF and 642.09 ± 56.05 cells / mm 2 in non-CF cultures (decrease of 45.62 %) (Fig. 1O). In contrast, at ALI D35, both types of cultures exhibited an equivalent amount of ciliated cells with 783.85 ± 20.81 cells / mm 2 in CF and 821.81 ± 37.89 cells / mm 2 in non-CF cultures (Fig. 1R).

The quantification of cycling epithelial cells showed a decrease in the number of Ki67 positive cells during the course of regeneration in both non-CF and CF cultures. Interestingly, the number of proliferating cells was not significantly different in non-CF and CF cultures, whatever the time point analysed (Supplementary Fig. 2).

These results, obtained in standardized cultures, showed that exogenous infection and inflammation characterizing the CF airways could not be considered as the major source of remodelling factors, and that CF epithelium remodelling was not related to an increased epithelial cell proliferation. They also suggested that alterations of CF HAE cells such as their hyperinflammatory phenotype or CFTR default could play a role in the remodelling phenomenon.

Exogenous inflammation leads to the regeneration of a remodelled epithelium in non-CF cultures

To establish the role played by inflammation in the human airway epithelium remodelling, we investigated the consequences of an inflammatory stimulation of nasal non-CF HAE cells all along the ALI culture with a mixture (cytomix) of mediators found in CF airways, i.e. IL-1, TNF and IFN 37).

Cytomix-exposed non-CF cultures were remodelled at ALI D35 (Fig. 2). Inflamed non-CF cultures were significantly 29.17% higher (36.98 ± 0.40 µm) than non-stimulated cultures (28.62 ± 0.75 µm) (Fig. 2C) and showed a significant increase of 34.20 % in the number of basal cells: we quantified 35.94 ± 1.28 % of CK13 positive cells in cytomix-exposed cultures when control cultures were composed of 26.78 ± 0.35 % of basal cells (Fig. 2F). In the case of chronic stimulation with cytomix, the goblet cell differentiation was significantly 131.03 % enhanced, with 0.67 ± 0.029 % of MUC-5AC positive cells in stimulated cultures and 0.29 ± 0.014 % in control cultures (Fig. 2I). In contrast, the number of ciliated cells was not different in inflamed cultures, in comparison to non-stimulated cultures, either at ALI D15 (451.84 ± 42.22 and 453.04 ± 46.25 cells/mm2, respectively) (Fig. 2L), or at ALI D25 (616.05 ± 60.59 and 642.09 ± 56.05 cells/mm2, respectively) (Fig. 2O) or at ALI D35 (790.53 ± 42.49 and 821.81 ± 37.89 cells/mm2, respectively) (Fig. 2R). These data demonstrated that exogenous inflammation was responsible of a remodelling of the human airway epithelium, in terms of increase in epithelium height and in basal and goblet cell number. However, the inflammatory environment did not induce modification of the kinetic of ciliated cell differentiation.

Human CF HAE cells exhibit a hyperinflammatory phenotype which is lost with time

To explore our hypothesis of the involvement of CF HAE cell transient inflammatory memory in the remodelling of the CF airway epithelium, we first verified the hyperinflammatory status of nasal CF HAE cells already reported in the literature for CF bronchial epithelial cells [START_REF] Bonfield | Altered respiratory epithelial cell cytokine production in cystic fibrosis[END_REF][START_REF] Okada | Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways[END_REF]. Expression of the CF inflammation marker IL-8 was assessed by RT-qPCR or ELISA. Despite huge inter-patients variability, IL-8 mRNA expression was significantly more important in CF HAE cells than in non CF HAE cells at P0 without any phase of culture, as shown in figure 3A. In contrast, after a round of amplification, CF cells at P1 did not express more IL-8 mRNA than non-CF cells at the same passage (Fig. 3B). During airway epithelial regeneration by nasal non-CF and CF HAE cells at P0, IL-8 mRNA expression was significantly higher in CF cultures than in non-CF cultures at ALI D0, decreased until ALI D7 then became similar or lower (Fig. 3C). At protein level, the IL-8 secretion by cells at P0 exhibited the same pattern of expression (Fig. 3D). Finally, at ALI D35, IL-8 mRNA expression was similar or lower in CF cultures, in comparison to non-CF cultures, either at P0 (Fig. 3C) or at P1 (Fig. 3E).

These results confirmed the hyperinflammatory status of CF HAE cells at P0 and the loss of this phenotype with time of culture when they were free of any exogenous inflammatory stimulation.

When the inflammatory memory is lost by CF HAE cells, the reconstituted epithelium is not remodelled

As the hyperinflammatory phenotype of CF cells was lost with time, we evaluated the potential remodelling of non-CF and CF epithelia regenerated from nasal HAE cells at P1. At ALI D35, epithelium height was similar in P1-non-CF (29.55 ± 1.54 µm) and P1-CF (30.61 ± 0.96 µm) cultures (Fig. 4C). Quantification of basal cells showed that P1-CF and P1-non-CF cultures exhibited an equivalent number of CK13 positive cells with 30.52 ± 1.30 % and 29.11 ± 0.70 %, respectively (Fig. 4F). Moreover, no difference in the amount of MUC-5AC expressing goblet cells was detect in P1-CF (0.35 ± 0.043 %) and P1-non-CF cultures (0.34 ± 0.038 %) (Fig. 4I). Finally, at ALI D35, both types of culture displayed a comparable number of ciliated cells, with 654.72 ± 71.37 -tubulin positive cells / mm 2 in P1-non-CF and 574.32 ± 83.01 -tubulin positive cells / mm 2 in P1-CF cultures (Fig. 4L).

These results obtained with P1 cultures demonstrated that when CF HAE cells lost their hyperinflammatry phenotype, they reconstituted an epithelium that was not remodelled. These data strengthen our hypothesis that the hyperinflammatory phenotype could play a role in the remodelling phenomenon.

Exogenous inflammation induces only goblet cell number increase during the CF airway epithelium regeneration.

In order to define if exogenous inflammation and inflammatory memory of CF HAE cells could play a cumulative effect on the epithelium remodelling, nasal CF HAE cells were stimulated with cytomix all along the ALI culture (Fig. 5). In terms of epithelium height at ALI D35, no significant difference was observed in cytomix-exposed (36.64 ± 1.24 µm) and control CF cultures (38.03 ± 1.01 µm) (Fig. 5C). The number of basal cells was not enhanced by inflammatory stimulation, with 35.78 ± 2.18 % and 39.37 ± 2.07 % of CK13 positive cells in stimulated and control cultures, respectively (Fig. 5F). In contrast and as already observed above for non-CF cultures, goblet cell differentiation was significantly 96.87 % enhanced in inflamed CF cultures, with 0.63 ± 0.034 % of MUC-5AC positive cells in stimulated cultures and 0.32 ± 0.027 % in control cultures (Fig. 5I). Finally, the number of ciliated cells was not affected by exogenous inflammation in CF cultures, in comparison to non-stimulated CF cultures, either at ALI D15 (256.90 ± 18.25 and 235.92 ± 30.40 cells/mm2, respectively) (Fig. 5L), or at ALI D25 (340.35 ± 19.59 and 349.19 ± 34.98 cells/mm2, respectively) (Fig. 5O) or at ALI D35 (767.78 ± 62.26 and 783.85 ± 20.81 cells/mm2, respectively) (Fig. 5R).

These results showed that exogenous inflammation induced goblet cell hyperplasia in CF cultures but did not play any cumulative effect on the remodelling in terms of epithelium height, basal cell hyperplasia or ciliated cell differentiation delay.

DISCUSSION

We have previously reported that, in absence of exogenous infection, the CF airway epithelial regeneration was abnormal and reconstituted a remodelled epithelium [START_REF] Hajj | Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis[END_REF]. However, the xenograft model we used did not permit to exclude the influence of the inflammatory environment in aberrant CF regeneration. In the present study, using in vitro ALI cultures of primary nasal HAE cells in perfectly defined environmental conditions, we could determine the involvement of inflammation in the human CF airway epithelium regeneration. Our results demonstrate that, even in absence of exogenous inflammation, the regenerated CF epithelium is remodelled, higher than the non-CF counterpart, exhibits basal cell hyperplasia and shows a delay in ciliated cell differentiation. Surprisingly, we do not detect any goblet cell hyperplasia.

All these results are consistent with our previous observations [START_REF] Hajj | Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis[END_REF] and suggest for the first time that the altered regeneration and remodelling of the CF airway epithelium could be mainly due to dysfunctions of the CF HAE cells.

In this in vitro study, we first show an increase in the CF epithelium height. Although detected in airway tissues, this kind of epithelial remodelling was neither described nor explained previously in in vitro well-differentiated cultures of primary CF HAE cells from nasal, tracheal or bronchial origin. Indeed, the increase in epithelium height has been previously reported in CF lungs, compared to lungs from subjects without chronic airway disease [START_REF] Voynow | Basal-like cells constitute the proliferating cell population in cystic fibrosis airways[END_REF][START_REF] Hays | Characterizing mucous cell remodeling in cystic fibrosis. Relationship to neutrophils[END_REF] or from patients suffering from COPD [START_REF] Tiddens | Cartilaginous airway wall dimensions and airway resistance in cystic fibrosis lungs[END_REF]. Airway epithelium height is also greater in these latter patients than in non-COPD subjects, at nasal and bronchial levels [START_REF] Vachier | Inflammatory features of nasal mucosa in smokers with and without COPD[END_REF]. In the same way, we highlight basal cell hyperplasia in CF ALI cultures, as it has been reported in CF [START_REF] Voynow | Basal-like cells constitute the proliferating cell population in cystic fibrosis airways[END_REF][START_REF] Hubeau | Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa[END_REF] and COPD lungs [START_REF] Jeffery | Comparison of the structural and inflammatory features of COPD and asthma. Giles F. Filley lecture[END_REF]. Interestingly, although these two pathologies have different aetiologies, genetic in CF and mainly environmental in COPD, they are both characterized by chronic airway inflammation. This observation suggests that inflammation could be involved in the observed remodelling. Interestingly, stimulation of non-CF HAE cells with a cocktail of proinflammatory cytokines all along the regeneration process leads to an increase in epithelium height and in the number of basal cells at values comparable to those measured in CF cultures, while in CF cultures this stimulation does not induce more remodelling than that seen in control counterparts (Table 1). Surprisingly, CF HAE cells that lost their hyperinflammatory phenotype (P1 cells) no longer reconstitute a remodelled epithelium. Taken together, these results strongly support that the CF epithelium remodelling could be due to the hyperinflammatory phenotype of CF HAE cells that appears to be sufficient to induce such histological alterations. Our experiments with nasal HAE cells confirm this expected characteristic of hyperinflammatory phenotype of bronchial CF HAE cells and its loss with time [START_REF] Okada | Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways[END_REF][START_REF] Ribeiro | Chronic airway infection/inflammation induces a Ca 2+ i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia[END_REF]. In our study, the exaggerated inflammation of CF HAE cells is still detectable until 7 days of ALI culture and disappears thereafter, as attested by the transient expression of IL-8. This observation suggests that a one week-hyperinflammation of CF HAE cells is sufficient to induce their capacity to reconstitute a remodelled epithelium. The hypothesis of pro-remodelling effect of the inflammatory memory of CF cells is consolidated by the description of airway epithelium remodelling by intrinsically-produced inflammatory mediators such as IL-1. Indeed, in a transgenic mouse model allowing inducible expression of human IL-1 in lung epithelium, IL-1 increases thickness of the conducting airways [START_REF] Lappalainen | Interleukin-1 causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung[END_REF]. The higher airway epithelium height could be related to the increased number of basal cells. Hyperplasia has been reported in airways of CF or COPD patients as well as of smokers [START_REF] Herfs | Proinflammatory cytokines induce bronchial hyperplasia and squamous metaplasia in smokers[END_REF]. Whereas this hyperplasia was not characterized at first [START_REF] Leigh | Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients[END_REF], it was later demonstrated that cell proliferation was restricted to basal cells [START_REF] Voynow | Basal-like cells constitute the proliferating cell population in cystic fibrosis airways[END_REF][START_REF] Herfs | Proinflammatory cytokines induce bronchial hyperplasia and squamous metaplasia in smokers[END_REF][START_REF] Demoly | Cell proliferation in the bronchial mucosa of asthmatics and chronic bronchitics[END_REF] shown to retain transit-amplifying cell properties and ability to restore a fully differentiated mucociliary epithelium [START_REF] Hajj | Basal cells of the human adult airway surface epithelium retain transit-amplifying cell properties[END_REF]. This basal cell proliferation is induced by proinflammatory mediators such as TNF-, IL-1 and IL-6, and reduced by anti-TNF-, anti-IL-1and anti-IL-6R therapies in a model of mouse lung inflammation triggered by cigarette smoke [START_REF] Herfs | Proinflammatory cytokines induce bronchial hyperplasia and squamous metaplasia in smokers[END_REF]. In our experiences, we postulate that a transient hyperexpression of inflammatory mediators by CF cells could be involved in basal cell hyperplasia and epithelium height increase. Although such kinds of remodelling are described in the literature, their physiopathological impact remains to be elucidated.

In another way, we confirm the interesting result obtained previously in vivo, i.e. absence of goblet cell hyperplasia in the CF regenerated epithelium [START_REF] Hajj | Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis[END_REF]. Analysis of the expression of MUC-AC that stains all mucus-secreting goblet cells of the airway surface epithelium [START_REF] Hajj | Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis[END_REF] shows an increase in the number of goblet cells, only in inflamed non-CF and CF cultures. It is known that goblet cell hyperplasia is regulated by some inflammatory mediators such as Th2 cytokines [START_REF] Elias | Airway remodeling in asthma[END_REF] or neutrophil elastase (47) and by infectious endotoxin such as lipopolysaccharide from Pseudomonas aeruginosa [START_REF] Yanagihara | Lipopolysaccharide induces mucus cell metaplasia in mouse lung[END_REF]. Our results together with the fact that MUC-5AC overexpression is induced by some inflammatory or infectious mediators [START_REF] Voynow | Regulation of mucin genes in chronic inflammatory airway diseases[END_REF], lead to conclude that goblet cell hyperplasia in CF is only due to exogenous factors, and that hyperinflammatory phenotype of CF HAE cells is not sufficient to trigger mucus cell hyperplasia.

In this study, we also confirm our previous observation of a delay in ciliated cell differentiation in CF, and show that this phenomenon is independent of the inflammatory environment of the cultures (Table 1). While a lot of ultrastructural alterations of cilia in CF epithelium are consensually reported [START_REF] Piorunek | Correlation between the stage of cystic fibrosis and the level of morphological changes in adult patients[END_REF][START_REF] Dovey | Ultrastructural morphology of the lung in cystic fibrosis[END_REF], few data are available concerning a default in the number of airway ciliated cells. CF children exhibit less bronchial ciliated cells than children suffering from non-suppurative lung disease [START_REF] Thomas | Persistent disruption of ciliated epithelium following paediatric lung transplantation[END_REF]. Moreover, Danel et al. found less ciliated cells in CF samples recovered by bronchial brushing than in control samples, and hypothesized it was due to the high susceptibility of ciliated cells to be damage [START_REF] Danel | Quantitative assessment of the epithelial and inflammatory cell populations in large airways of normal and individuals with cystic fibrosis[END_REF]. As we have shown that ciliated cell differentiation delay in nasal CF cultures is not related to infection or inflammation, and together with recent data showing a down regulation of cilia genes in native nasal epithelium of CF patients (52), we propose that the decreased number of ciliated cells in CF epithelium could be due to a permanent regeneration process rather than a fragility of these cells and to a failure of differentiation related to the basic CFTR default.

In conclusion, we show that exogenous infection and inflammation characterizing the CF lung pathology are not the exclusive factors leading to airway epithelium remodelling that is partly regulated by the alterations of CF HAE cells. This could explain the CF deleterious loop: infection and inflammation are hallmarks of CF so that airway epithelium is continuously damaged and epithelial cells constantly stimulated by resident inflammation maintaining in this way their hyperinflammatory phenotype. Moreover, being in permanent regeneration, the reconstituted epithelium is remodelled, with goblet cell hyperplasia and a diminished number of ciliated cells conducting to worsen the mucociliary clearance impairment that leads to infection and inflammation.

Figure 1 :

 1 Figure 1: In absence of exogenous infection and inflammation, the regeneration of CF

Figure 2 :

 2 Figure 2: Exogenous inflammation leads to the regeneration of a remodelled epithelium

Figure 3 :

 3 Figure 3: Nasal CF HAE cells exhibit a hyperinflammatory phenotype which is lost with

Figure 4 :

 4 Figure 4: When the inflammatory memory is lost by CF HAE cells, the reconstituted

Figure 5 :

 5 Figure 5: Exogenous inflammation induces only goblet cell number increase during the

Table 1 .

 1 52) Clarke LA, Sousa L, Barreto C, et al. Changes in transcriptome of native nasal epithelium expressing F508del-CFTR and intersecting data from comparable studies. Respir Res 2013; 14: Features of non-CF and CF epithelia regenerated in vitro from passage 0 nasal HAE cells in absence or presence of inflammatory stimulation

	38.

ACKNOWLEDGEMENTS

We thank all the ENT who provided us with CF and non-CF tissues: Dr. C. Ruaux, Clinique Mutualiste de la Sagesse (Rennes, France); Dr. R. Nicollas, Hôpital de la Timone (Marseille, France); Dr. T. Nasser, Polyclinique Courlancy (Reims, France); Pr. C. Debry, Hôpital Hautepierre (Strasbourg) and Dr. C. Ferron, Hôpital Hôtel Dieu (Nantes). We also thank the Cell and Tissue Imaging Platform (PICT) of the University of Reims Champagne-Ardenne. This work was supported by the "Association Vaincre la Mucoviscidose", the "Contrat de Plan Etat-Région", The American Committee and the Friends of the American Memorial Hospital of Reims, the University Hospital Centre and the University of Reims Champagne-Ardenne (PHU Rinnopari) and the INSERM.

LIST OF SUPPORTING INFORMATION

-Supplementary materials and methods -Supplementary Figure 1 -Supplementary Figure 2 SUPPORTING INFORMATION Cystic Fibrosis airway epithelium remodelling: involvement of inflammation

MATERIALS AND METHODS

Human airway tissues and cell dissociation

Human tissues were transferred to our laboratory in RPMI 1640 medium supplemented with 20 mM HEPES (Gibco) and antibiotics (200 UI/ml penicillin, 200 µg/ml streptomycin; Gibco).

Collected tissues were then dissociated by type XIV collagenase incubation in RPMI-HEPES overnight at 4°C. The dissociated nasal human airway epithelial (HAE) cells, called cells at passage 0 (P0), were counted and used for air-liquid interface (ALI) culture. They were also analysed to assess their inflammatory status by RT-qPCR after a pre-plating step in order to remove all contaminant blood cells.

Cell culture

Nasal HAE cells (P0) were suspended in CnT17 medium (CELLnTEc, Bern, Switzerland) and seeded (5x10 4 cells per membrane) on type IV collagen (Sigma Aldrich)-coated porous 12 mm diameter Transwell polyester membranes. Cells were grown in liquid-liquid conditions in CnT17 medium until confluent (approximately 1 week). Then the culture medium was definitively removed from the upper compartment (ALI D0) and the epithelium was allowed to differentiate at the ALI with supplemented BEGM/DMEM/F12 medium in the basal compartment of the culture chamber. All the cultures were incubated at 37°C in a 100% humidified incubator in the presence of 5% CO2. In some experiments, the ALI medium was supplemented with a cocktail of pro-inflammatory cytokines called cytomix from ALI D0 to ALI D35. These cultures were examined at ALI D0, ALI D7, ALI D15, ALI D25 and ALI D35.

In a set of experiments, nasal non-CF and CF HAE cells were plated on type IV collagen-coated plastic flasks for cell proliferation in CnT17 medium. When confluent, the cells were detached by tryspine/EDTA (Gibco) treatment and called cells at passage P1 (P1). They were used for RT-qPCR experiments and seeded on type IV collagen-coated porous 12 mm Transwell membranes and grown as described above. These P1 cultures were examined at ALI D35.

Histological analysis

ALI cultures were embedded in optimum cutting temperature (O.C.T) compound (Tissue-Tek, Zoeterwoude, The Netherlands), frozen in liquid nitrogen and stored at -80°C until use for histology and immunohistochemistry.

Seven-micrometer frozen sections were cut with a microtome, collected on superfrost slides (Thermo Scientific, Braunschweig, Germany), and either stained with hematoxylin-eosin using Rapid-Chrome Frozen Sections Staining Kit (Thermo Shandon Inc., Pittsburgh, PA) and mounted in Eukitt (Electron Microscopy Sciences, Hartfield, PA) for histology or frozen at -20°C for immunohistochemistry.

Immunohistochemistry

Slides were air-dried, and sections were blocked with phosphate buffered saline (PBS; Gibco)-BSA 10% for 30 minutes at room temperature (RT) to prevent unspecific bindings. ALI D35 sections were incubated with mouse anti-human CK13 (dilution 1/1000), mouse anti-MUC-5AC (dilution 1/100) or mouse anti-MUC5B (dilution 1/100) antibodies, and ALI D0, D7, D15 and D35 culture sections with mouse anti-human Ki67 antibodies (dilution 1/10), diluted in PBS-BSA 3 % overnight at 4°C. After washes with PBS at RT, sections were blocked again with PBS-BSA 10 % for 30 minutes at RT, then incubated with the goat anti-mouse Alexa Fluor washed with PBS, incubated with 4', 6'-diamino-2-phenylindole (DAPI, 200 ng/ml in PBS; Sigma Aldrich) for 15 minutes at RT, washed with PBS and finally mounted with coverslides in Aquapolymount solution (Polysciences, Warrington, PA). For quantification of basal cells, goblet cells and cycling cells, 6 different sections per culture were analysed. The number of positive cells was determined as a percentage of the total number of cells visualized with DAPI.

For goblet cells double immunostaining, sections were stained with anti-MUC-5B antibodies, exposed to Alexa Fluor 488-goat anti-mouse antibodies, then incubated with goat anti-mouse Fab (H+L) fragments (30 minutes at RT; Jackson Immunoresearch, PA) to block free sites of mouse anti-MUC-5B antibodies, and finally stained with anti-MUC-5AC antibodies followed by incubation with Alexa Fluor 594-goat anti-mouse antibodies (dilution 1/200 in PBS/BSA 3%; Molecular Probe, Eugene, OR). For the quantification of ciliated cells, cultures at ALI D15, ALI D25 and ALI D35 were rinsed with PBS containing CaCl2 and MgCl2 (Gibco), fixed with cold methanol for 10 minutes at -20°C, washed with PBS at RT, blocked with PBS-BSA 10% for 30 minutes at RT, incubated with mouse anti-human -tubulin (dilution 1/1000 in PBS/BSA 3%) overnight at 4°C. After washes with PBS, membranes were blocked again with PBS-BSA 10% for 30 minutes at RT, then incubated with goat anti mouse Alexa Fluor 488 secondary antibodies (dilution 1/200 in PBS/BSA 3%), washed with PBS, incubated with DAPI (200 ng/ml in PBS) for 15 minutes at RT, washed with PBS and finally mounted between slides and coverslides in Aquapolymount solution. Ten images (objective x10) per culture were analysed in order to cover approximately 80% of the total surface of cultures. Using ImageJ software (http://rsb.info.nih.gov/ij/) functionalities, the average number of ciliated cells per mm 2 was calculated after fluorescent signal treatment.

RT-qPCR analysis

Specific primers used (Eurogentec, Seraing, Belgium) were as follow for IL-8: forward, 5'-AGACAGCAGAGCACACAAGC-3'; reverse, 5'-ATGGTTCCTTCCGGTGGT-3'. Results were normalized to the expression levels of GAPDH. Primers for GAPDH were: forward, 5'-ACCAGGTGGTCTCCTCTGAC-3'; reverse, 5'-TGCTGTAGCCAAATTCGTTG-3'.Each sample was quantified using a standard curve made of serial dilutions of a batch of human placental cDNA made with the Transcriptor First Strand cDNA synthesis kit (Roche). Results were expressed as IL-8/GAPDH ratios.

LEGEND OF SUPPLEMENTARY FIGURES


Joël Belmin
email: j.belmin@aphp.fr

Cristiano Donadio

Witold Jarzebowski

) Kamran

Lucie Valembois

Carmelo Lafuente-Lafuente

The value of BNP plasma levels in very old persons with chronic peripheral edema

Keywords: chronic heart failure, elderly, BNP, brain natriuretic peptide, chronic peripheral edema

Background: Chronic peripheral edema (CPE) is frequent in old patients and very often results from multiple causes. Aims: To investigate if BNP plasma dosage helped diagnose etiologies of CPE. Methods: Cross-sectional observational study conducted in geriatric hospital wards (intermediate and long-term care) on consecutive in-hospital patients > 75 years having CPE and no dyspnea. From medical history, physical examination, routine biological tests and chest radiography, two investigators determined the etiologies of edema with special attention to recognize chronic heart failure (CHF). This reference diagnosis was compared to the clinical diagnosis mentioned in the medical chart. BNP plasma levels were measured soon after the investigators visit.

Results: Among the 141 patients (113 women and 28 men) aged 86 +/-6 years, a single cause was identified in 53 (38%), and multiple causes in 84 (60%). Main causes were venous insufficiency (69%), CHF (43%), hypoprotidemia (38%) and drug induced edema (26%). CHF was frequently misdiagnosed by attending clinicians, missed in 18 cases and wrongly diagnosed in 14. BNP was significantly higher in patients with CHF than in those without (median and interquartile range: 490 [324-954] and 137 pg/mL respectively, p<0.0001). ROC curve showed that the level of 274 pg/mL was appropriate to diagnose CHF with specificity of 0.89 and sensitivity of 0.82. BNP level above this cut-off was significantly and independently associated to the diagnosis of CHF. Conclusion: CHF is frequently misdiagnosed in old patients with CPE and BNP dosage helped to improve diagnosis of this condition and identify CHF. * Chest radiography was missing in 6 patients with CHF and 15 patients without CHF ** Values are median and interquartile range † Echocardiography was not performed in 22 patients (5 with CHF, 17 without) Ejection fraction < 50% † 33 (28) †

Chronic peripheral edema (CPE) is frequent in old patients and very often results from multiple mechanisms, including elevated peripheral or central venous pressure, decreased oncotic plasma pressure, increased capillary permeability related to drugs or to vascular inflammation or damage [START_REF] Gorman | Swollen lower limb-1: general assessment and deep vein thrombosis[END_REF][START_REF] Topham | Chronic lower limb oedema[END_REF]. The diseases responsible for CPE are numerous, and the appropriate diagnosis is often a challenging task for clinicians [START_REF] Blankfield | Etiology and diagnosis of bilateral leg edema in primary care[END_REF][START_REF] Cho | Peripheral edema[END_REF][START_REF] Ely | Approach to leg edema of unclear etiology[END_REF][START_REF] Mortimer | Swollen lower limb-2: lymphoedema[END_REF][START_REF] Yale | Approach to diagnosing lower extremity edema[END_REF][START_REF] Young | The swollen leg. Clinical significance and differential diagnosis[END_REF]. In very old patients, the diagnosis is more difficult due to several reasons [START_REF] Thaler | Bilateral leg edema in the elderly. Clinical consideration and treatment options[END_REF]. First, multiple etiologies are often encountered in the same patient due to the high prevalence of chronic diseases which make the diagnosis more complex [START_REF] Thaler | Bilateral leg edema in the elderly. Clinical consideration and treatment options[END_REF]; the diagnosis of heart failure is often more difficult due to reduced physical activity and/or atypical symptoms; chronic venous disease is very frequent and often lead to miss another concomitant etiology of CPE.

Appropriate management of CPE mainly depends on the etiology of edema. The identification of heart failure in patients with CPE is of crucial importance [START_REF] Cho | Peripheral edema[END_REF][START_REF] Ely | Approach to leg edema of unclear etiology[END_REF][START_REF] Shah | Peripheral edema due to heart disease: diagnosis and outcome[END_REF].

Among patients with CPE, mortality was three fold higher in those with heart failure than in those without [START_REF] Shah | Peripheral edema due to heart disease: diagnosis and outcome[END_REF]. Heart failure requires an appropriate treatment which is able improve symptoms and prognosis.

Brain natriuretic peptide (BNP) determination was found to help the diagnosis of heart failure in patients with acute dyspnea [START_REF] Davis | Plasma brain natriuretic peptide in assessment of acute dyspnea[END_REF][START_REF] Maisel | Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure[END_REF][START_REF] Mueller | Use of B-type natriuretic peptide in the evaluation and management of acute dyspnea[END_REF] including elder persons [START_REF] Mason | Utility of biomarkers in the differential diagnosis of heart failure in older people: findings from the heart failure in care homes (HFinCH) diagnostic accuracy study[END_REF] and its dosage is becoming widely available. We hypothesized that in very old patients, BNP dosage could help the clinician for determining etiologies of CPE not associated with dyspnea.

Methods

Design and patients

This prospective observational cross-sectional study was conducted in 4 geriatric wards of a French hospital (Hôpital Charles Foix, Ivry-sur-Seine), in intermediate care and long term care settings. Consecutive patients were included if they were 75 years or older, had peripheral edema for more than 15 days, had no dyspnea or documented acute heart failure and were not in end-of-life situation.

For each patient, we recorded from the medical chart the etiologies of edema determined by the physician responsible for the patient care. We also discussed with him/her to record additional information about etiologies of CPE not mentioned in the medical chart. In the following this referred to the "clinical diagnosis".

Reference diagnosis of CPE etiologies

Two investigators (a geriatrician and a cardiologist) developed a systematic approach to determine etiologies of CPE derived from literature [START_REF] Cho | Peripheral edema[END_REF][START_REF] Ely | Approach to leg edema of unclear etiology[END_REF]. They searched history of impaired exercise tolerance, dyspnea, heart, pulmonary or liver disease, venous thromboembolic disease. They examined patients to search for pulmonary crackles, hepatojugular reflux or jugular distension, varicose veins (in upright position), lower limb hemosiderin skin deposits or venous ulcers, skin thickening or changes. They also recorded drugs, realized ECG and analyzed it to search for repolarization abnormalities, atrial fibrillation and signs of prior myocardial infarction. The most recent chest radiograph of the medical chart was analyzed to search for pleural effusion, alveolar or interstitial pulmonary edema and to calculate cardiothoracic index. Recent blood biochemical analysis for creatinine, TSH, proteins, albumin available in the medical records were also provided, with the exception of brain natriuretic peptide (BNP) if it had been measured before.

Echocardiography and venous ultrasound were performed at the discretion of the attending clinician and, when available, were also considered by the investigators. Using all these elements, each investigator separately determined the etiologies of edema for each patient. The diagnosis of heart failure was based on the 2008 guidelines of the European Society of Cardiology for heart failure [START_REF] Dickstein | Committee for Practice Guidelines (CPG) ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008[END_REF]. Symptoms and signs compatible with heart failure were defined as: (i) breathlessness, graded as New York Heart Association (NYHA) class II, III, or IV; (ii) moderate edema (up to the knees bilaterally); or (iii) mild edema (confined to the ankles) + one other clinical sign (see below); (iv) mild edema and taking at least furosemide 40 mg (or equivalent) per day; or (v) two or more other signs [raised jugular venous pressure (JVP), lung crackles, respiratory rate >20/min, third heart sound].

In cases where the diagnosis remains uncertain, the response to treatment directed towards heart failure was taken into account. The diagnosis of chronic venous disease was retained if patient had a history of lower limb venous thrombosis or venous ulcer, extensive varicose veins and skin hemosiderin deposit and/or ultrasound examination showing venous insufficiency. Hypoproteinemia was considered to be involved in CPE if plasma proteins were < 55 g/L and albumin < 25 g/L. Drug-induced CPE was considered if the patient received calcium channel blockers, non-steroidal anti-inflammatory drugs, corticosteroid or other drug known to induce leg edema. Lymphedema was considered when skin thickening was present. Finally, results of both investigators were confronted and discrepancies were solved by discussion. For each patient, the list of etiologies retained after this process was considered as the "reference diagnosis".

BNP determination

BNP plasma levels were determined by immunoassay (Biosite, Triage) within days after the investigators' visit, when blood was collected for routine medical care. Results were not available for the investigators at the time of their diagnosis.

Statistics

Comparison of qualitative variables used test khi-2 test. For quantitative variables, comparisons used the Student t test if they were normally distributed or the Mann-Whitney U test otherwise. Differences were considered significant if p< 0.05. For each type of CPE etiologies, agreement between clinical diagnosis and reference diagnosis was studied by Kappa coefficient (κ). Agreement was considered as good if κ > 0.80, moderate if κ was comprised between 0.60 and 0.80, or poor if κ < 0.60. We used a Receiver Operating Characteristic (ROC) curve to compare the different sensitivities and specificities according to the values of BNP for the reference diagnosis of chronic heart failure. We used the Youden index to select the BNP threshold. Additionally, we calculated the Net Reclassification Improvement (NRI) of the best BNP threshold value for the diagnosis of CHF [START_REF] Leening | Net reclassification improvement: computation, interpretation, and controversies: a literature review and clinician's guide[END_REF]. The respective values of BNP and other variables recorded for the diagnosis of chronic heart failure were studied as follows. First, by univariate analysis, we compared the variables of patients having chronic heart failure according to reference diagnosis to those without CHF. We run univariate and multivariable logistic regression analysis using chronic heart failure (yes/no) as the dependent variable, and BNP (below/above threshold) and all the variables with p<0.10 in the univariate analysis as independent variables, and excluding variables related to chest radiography because of missing data. Concerning sample size, we did not estimate a required sample size, simply intended to include as many patients as possible. A post-hoc calculation shows that our study had 93% power to detect BNP sensitivity and specificity greater than 0.80, given the observed prevalence of heart failure. All calculations were made using STATA software (v 13, StataCorp LP, USA)

Results

Patients and chronic peripheral edema etiologies

Hundred and forty-one patients (28 males and 113 females, aged 86 +/-6 years) were included in the study and their characteristics are showed in Table 1. Echocardiography was available in 119 patients (84.4%). The distribution of CPE etiologies and their type are listed in the Table 2. Multiple CPE etiologies were found in 84 patients (60%) and single etiology in 53 (38%). The number of CPE etiologies identified by investigators were significantly greater that those identified by clinicians (260 versus 185, p<0.0001).

Discrepancies between investigators diagnosis occurred in 9 patients, mostly related to missing extraction of data from the medical records, leading to an easy resolvation. As compared to the reference diagnosis, clinical diagnosis recorded a greater percentage of patients with undetermined etiology or with a single CPE etiology (p<0.0001). Conversely, the percentage of patients with multiple CPE etiologies was significantly less. Agreement between clinical diagnosis and reference diagnosis was poor for most etiologies (κ < 0.60).

The role of hypoproteinemia and that of drugs were frequently and widely unrecognized.

They were mainly related to malnutrition and calcium channel blockers respectively.

Agreement between clinical and reference diagnosis for chronic heart failure is detailed in Table 3. In 32 patients (23%), the clinical diagnosis for CHF was different from the reference. Chronic heart failure was missed in 18 patients with chronic heart failure (13%) and was diagnosed in 14 (10%) without chronic heart failure.

BNP and other factors related to chronic heart failure

Characteristics of patients with chronic heart failure were compared to those of patients without chronic heart failure (Table 4). Patients with chronic heart failure had more frequently hypertension, history of acute heart failure, bilateral crackles, jugular distension or hepatojugular reflux, ECG and chest radiography abnormalities. In addition, systolic blood pressure and plasma BNP values in this group were significantly greater than in patients without CHF, whereas plasma creatinine values were significantly less (Table 4).

We examined how were related BNP values to the reference diagnosis of chronic heart failure by a receiver operating characteristic (ROC) curve plotting 1-specificity against sensitivity for each value of BNP. The area under the ROC curve is 0.883. A BNP value of 274 pg/mL was selected from the maximal value of the Youden index. 0.89 specificity and 0.82 sensitivity This threshold BNP value had, for the diagnosis of heart failure in these patients, an Event NRI of 11.5% (i.e. proportion of patients without an initial clinical diagnosis of CHF in whom BNP correctly diagnosed CHF), a Non-event NRI of 6.3% (i.e. patients with an initial clinical diagnosis of CHF in whom BNP accurately eliminated CHF), and an overall NRI of 0.18 (p = 0.037) (Supplementary data, Table S1).

Then we created a new discrete variable named high BNP: above/below this threshold (274 pg/mL). To examine respective associations of all the variables related to the reference diagnosis of CHF, we ran a first logistic regression model using chronic heart failure (yes/ no) as dependent variable, and the following variables as independent variables: high BNP, age, hypertension, history of acute heart failure, systolic blood pressure, crackles, jugular distension, hepatojugular reflux, ECG repolarization abnormalities, atrial fibrillation, ECG signs of prior myocardial infarction, plasma creatinine. The variables independently and significantly related to chronic heart failure were High BNP, systolic blood pressure, pulmonary cracles and hepatojugular reflux (Table 5). High BNP was a major and significant contributor to explain variance in the model.

Comments

This study showed that BNP dosage is relevant to improve the etiological diagnosis of CPE in very old patients, in particular to identify CPE related to chronic heart failure. In addition, it showed that most of these patients had 2 or more etiologies of CPE and that their correct identification is far to be perfect in the clinical field. Recognition of chronic heart failure is a major step to implement appropriate treatments and to improve the prognosis of patients with CPE of cardiac origin.

The clinical utility of BNP dosage has been extensively studied in patients with acute dyspnea, and especially in the context of emergencies [START_REF] Davis | Plasma brain natriuretic peptide in assessment of acute dyspnea[END_REF][START_REF] Maisel | Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure[END_REF][START_REF] Mueller | Use of B-type natriuretic peptide in the evaluation and management of acute dyspnea[END_REF]. A large consensus established that values below 100 pg/mL rule out the diagnosis of heart failure, and that values above 400 pg/mL is a strong argument for this diagnosis [START_REF] Maisel | State of the art: using natriuretic peptide levels in clinical practice[END_REF]. However, studies investigating the relevance of BNP in the context of primary care for non-acute patients are less numerous and generally have found a much lower specificity for the diagnosis of heart failure. In these studies, patients were much younger than those in our study and most of the patients had dyspnea [START_REF] Cowie | Value of natriuretic peptides in assessment of patients with possible new heart failure in primary care[END_REF][START_REF] Fuat | The diagnostic accuracy and utility of a B-type natriuretic peptide test in a community population of patients with suspected heart failure[END_REF][START_REF] Gustafsson | Diagnostic and prognostic performance of N-terminal proBNP in primary care patients with suspected heart failure[END_REF][START_REF] Kelder | Quantifying the added value of BNP in suspected heart failure in general practice: an individual patient data meta-analysis[END_REF][START_REF] Kelder | Clinical utility of three Btype natriuretic peptide assays for the initial diagnostic assessment of new slow-onset heart failure[END_REF][START_REF] Landray | Measuring brain natriuretic peptide in suspected left ventricular systolic dysfunction in general practice: cross-sectional study[END_REF][START_REF] Wright | Plasma amino-terminal pro-brain natriuretic peptide and accuracy of heart-failure diagnosis in primary care: a randomized, controlled trial[END_REF]. To our knowledge our study is the first conducted among very old patients with CPE and without dyspnea. This clinical picture is known to be a quite common presentation of chronic heart failure in very old persons, probably because impaired exercise tolerance is often masked due to lack of physical activity or low reporting due to cognitive troubles [START_REF] Mason | Utility of biomarkers in the differential diagnosis of heart failure in older people: findings from the heart failure in care homes (HFinCH) diagnostic accuracy study[END_REF][START_REF] Hancock | High prevalence of undetected heart failure in long-term care residents: findings from the Heart Failure in Care Homes (HFinCH) study[END_REF]. Heart failure is known to be often misdiagnosed in such population. A recent study in the UK shown cardiologists systematically assessed elder institutionalized persons and detected heart failure in 22% of them [START_REF] Hancock | High prevalence of undetected heart failure in long-term care residents: findings from the Heart Failure in Care Homes (HFinCH) study[END_REF]. The diagnosis was ignored in 9 of 10 patients and conversely, in 3 of 4 patients with previously diagnosed heart failure, the diagnosis was not confirmed. In our study, we also found that heart failure was widely misdiagnosed. In addition, numerous causes of CPE can be associated in the same old patient, which make their identification more complex, including that of chronic heart failure.

In our study, diagnostic performance of BNP was excellent using 274 pg/mL value as the cut-off point. This is value is quite close to the classic cut-off points of 100 and 400 pg/mL.

It is also consistent with BNP cut-off values found by others for non-acute heart failure diagnosis [START_REF] Leening | Net reclassification improvement: computation, interpretation, and controversies: a literature review and clinician's guide[END_REF][START_REF] Abadsa | Proposal for a discriminant level of BNP in very elderly persons with heart failure[END_REF][START_REF] Lebourgeois | Plasma brain natriuretic peptide measured in stable conditions is related to mortality in frail and very old patients[END_REF]. In fact, aging process is associated with an age-associated increase in secretion of heart natriuretic peptides [START_REF] Charloux | Dysregulation of pulsatility in aging IV. Pulsatile signaling and cardiovascular aging: functions and regulation of natriuretic peptide signaling[END_REF][START_REF] Tan | The effect of volume expansion on atrial natriuretic peptide and cyclic guanosine monophosphate levels in young and aged subjects[END_REF], and also with a decrease in their renal elimination, but the effect is more pronounced on NT-proBNP than on BNP [START_REF] Leening | Net reclassification improvement: computation, interpretation, and controversies: a literature review and clinician's guide[END_REF].

In our study, we observed that not only heart failure, but also other conditions responsible for CPE were improperly identified in the clinical field. In particular, the role of drugs and hypoproteinemia/malnutrition were widely underestimated. Also these conditions are modifiable by changes in drug regimen, nutritional support and physical activity.

As limitations, our study was single-center and included a limited number of patients, so it is not is not sure that our sample were representative of the general population of elderly patients presenting with CPE. Also, 16% of the included patients had not a recent echocardiography, so the reference diagnosis of heart failure in these patients was based in clinical criteria and that could have lead to some incorrect reference diagnosis of heart failure. However, the combined sensitivity and specificity of the Framingham clinical criteria, which we employed in those patients, is good (sensitivity 92%, specificity 79%) and were applied only to a minority of patients. Finally, external validation in an independent study would be needed to confirm these results.

Overall, our study suggests that very old persons with CPE should be systematically assessed by a careful clinical approach, ECG, chest radiography and a single dosage of plasma BNP. By these simple means, it is possible to identify many conditions, including chronic heart failure, which might be improved by appropriate treatments.

Legend of Figure

ROC curve of plasma BNP for the diagnosis of chronic heart failure among very old patients

with chronic peripheral edema. Arrow shows the threshold of 274 pg/mL having 0.89 specificity (Spe), 0.82 sensitivity (Se), 0.85 positive predictive value (PPV) and 0.86 negative predictive value (NPV). The corresponding values for classic BNP cut-off values (100 and 400 pg/mL) have been calculated.

Table 1 :

 1 Characteristics of the patients. Results are expressed as mean +/-SD or numbers and percentage in brackets, except creatinine, creatinine clairance and BNP which are expressed as median and interquartile range in brackets.

	Variable	Total
		(n=141)
	Demography and history
	Age (years)	86.5 +/-6.0
	Males	28 (20)
	Hypertension	96 (68)
	Diabetes	31 (22)
	Acute heart failure	33 (23)
	Venous ulcer	24 (17)
	Venous thromboembolism	23 (16)
	Physical examination
	Systolic BP (mmHg)	129 +/-19
	Diastolic BP (mmHg)	72 +/-12
	Bilateral pulmonary crackles	33 (23)
	Jugular distension	22 (16)
	Hepatojugular reflux	48 (34)
	Bilateral edema	128 (91)
	Local leg inflammation	17 (12)
	Varicose veins	53 (38)
	Skin hemosiderin	49 (35)
	Electrocardiogram
	Repolarization abnormalities	54 (38)
	Atrial fibrillation	45 (32)
	Prior myocardial infarction	17 (12)
	Chest radiography*
	Cardiothoracic index > 0.50*	92 (77)**
	Pleural effusion*	18 (16)**
	Alveolar or interstitial edema*	29 (26)**
	Echocardiography (n = 119) †
	Mean left ventricular ejection fraction (±	56 (±12) †
	SD) †

Table 2 :

 2 Distribution and types of chronic peripheral edema etiologies diagnosed by clinicians and investigators (reference). Results are expressed as numbers and percentage in brackets. Agreement between clinical and reference diagnosis for each type of etiology was assessed by kappa coefficient.

Table 3 :

 3 Agreement between the diagnoses of chronic heart failure (CHF) done by clinicians and that done by investigators (reference). Kappa coefficient was 0.53, indicating a poor agreement.

		Clinical diagnosis
		Pts with CHF	Pts without
	Reference diagnosis	(n=57)	CHF (n=84)
	Pts with CHF (n=61)	43	18
	Pts without CHF	14	66
	(n=80)

Table 4 :

 4 Characteristics of patients having chronic heart failure compared with those of patients without chronic heart failure (CHF). Results are expressed as mean +/-SD or numbers and percentage in brackets, except creatinine, creatinine clearance and BNP which are expressed as median and interquartile range in brackets. Chest radiography was missing in 6 patients with CHF and 15 patients without CHF ** Values are median and interquartile range

	Variable	With CHF	Without CHF	P
		(n=61)	(n=80)
	Demography and history
	Age (years)	87.4 +/-6.2	85.7 +/-5.7	0.09
	Males	14 (23)	14 (18)	0.55
	Hypertension	48 (79)	48 (60)	0.03
	Diabetes	17 (28)	14 (17)	0.20
	Acute heart failure	23 (38)	10 (12)	0.0008
	Venous ulcer	12 (20)	12 (15)	0.61
	Venous thromboembolism	9 (15)	14 (17)	0.83
	Physical examination
	Systolic BP (mmHg)	133 +/-21	127 +/-18	0.04
	Diastolic BP (mmHg)	73 +/-14	71 +/-10	0.26
	Bilateral pulmonary crackles	25 (41)	8 (10)	0.0001
	Jugular distension	19 (31)	3 (4)	<0.0001
	Hepatojugular reflux	37 (61)	11 (14)	<0.0001
	Bilateral edema	58 (95)	70 (87)	0.21
	Local leg inflammation	6 (10)	11 (14)	0.65
	Varicose veins	18 (30)	35 (44)	0.12
	Skin hemosiderin	23 (37)	26 (352)	0.64
	Electrocardiogram
	Repolarization abnormalities	31 (51)	23 (29)	0.012
	Atrial fibrillation	34 (56)	11 (14)	<0.0001
	Prior myocardial infarction	13 (21)	4 (5)	0.003
	Chest radiography*
	Cardiothoracic index > 0.50*	51 (93)*	41 (63)*	0.0003
	Pleural effusion*	15 (28)*	3 (5)*	0.002
	Alveolar or interstitial edema*	23 (43)*	6 (10)*	0.0001

*

Table 5 :

 5 Bivariate and multivariable logistic regression analysis for associations with the reference diagnosis of chronic heart failure in the 141 participants.

	OR : odds-ratio ; 95%CI : 95% confidence interval

Acknowledgments: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Statement of Conflict of Interest:

The authors have no conflict of interest.

Supplementary Data

The value of BNP plasma levels in very old persons with chronic peripheral edema Belmin J et al.