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Introduction La Mucoviscidose (CF) est une maladie genetique rare a transmission autosomique recessive due a des mutations du gene cftr codant pour le canal chlore AMPc-dependant de faible conductance CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Ce canal est egalement implique dans la plasticite cellulaire, notamment la differenciation. Le gene suppresseur de tumeur fhit (Fragile Histidine Triad) est egalement implique dans la plasticite epitheliale, en particulier dans le controle de la transition epithelio-mesenchymateuse (TEM) au cours de la progression tumorale. FHIT est detecte au pole apical des cellules ciliees bronchiques, une localisation egalement partagee par le canal CFTR. Nous nous sommes interesses aux relations physiques et fonctionnelles potentielles existant entre ces deux proteines au sein de l’epithelium des voies aeriennes humaines. Materiels et methodes Nous avons detecte CFTR et FHIT par immunohistochimie sur des coupes bronchiques humaines afin d’observer une co-localisation potentielle. Apres avoir caracterise l’expression de ces deux proteines par les lignees cellulaires NuLi-1, MM39 et S9, ainsi que par les cellules epitheliales de polypes nasaux sains, des siRNAs specifiques de CFTR et de FHIT ont ete utilises pour etudier leur eventuelle co-regulation. Enfin, nous avons utilise la technique overlay ainsi que la technique de resonance plasmonique de surface afin d’observer une interaction physique directe potentielle CFTR-FHIT. Resultats L’etude par immunohistochimie des coupes bronchiques humaines a revele que CFTR et FHIT presentaient une co-localisation partielle au niveau de la membrane apicale de l’epithelium respiratoire. La diminution d’expression de FHIT par l’utilisation de siRNAs specifiques entraine une augmentation de l’expression de la proteine CFTR, alors que le niveau de transcrits CFTR n’est pas significativement modifie. La technique overlay nous a permis d’observer une interaction directe entre CFTR et FHIT mais cette derniere n’a pas pu etre confirmee par la technique de resonance plasmonique de surface. Conclusion Nos resultats montrent une co-localisation partielle de CFTR et FHIT au niveau de l’epithelium bronchique humain, sur des lignees cellulaire NuLi-1 et MM39 ainsi que sur des polypes nasaux sains. Nous avons demontre par la technique d’interference a ARN, que les proteines CFTR et FHIT ont un lien fonctionnel bien qu’une interaction directe reste a etre confirmee.

Background: An alteration of primary cilia is observed in remodelled airways in the context of COPD. Since this organelle is required to activate Hedgehog (HH) signalling pathway orchestrating cell fate during lung development, we explored HH activation during human airway epithelial cell differentiation and in adult lungs from COPD and non-COPD patients. Methods: Human airway epithelial cells were cultured from nasal polyps and bronchial brushing in air-liquid interface conditions to reconstitute an epithelium representative of the adult airways. ELISA and immunostaining analysis of the principal ligand Sonic Hedgehog (SHH) were performed at different steps of differentiation on airway epithelial cell cultures, bronchial aspirations and formalin-fixed paraffin embedded lung tissues of non-COPD and COPD patients. Results: Non-differentiated airway epithelial cells produced SHH in vitro during the course of differentiation (from 6pg/ml at ALI-0 to 238pg/ml at ALI-35). Depriving culture medium of SHH with a specific antibody induced epithelial remodelling particularly impacting ciliogenesis (2 fold decrease of ciliated cells). SHH was observed on bronchial tissue sections and detected by ELISA with a 2 fold decrease expression for COPD patients. Conclusion: These findings suggest a crucial involvement of HH pathway in airway basal cells to guide differentiation and indicate an alteration of its activation in COPD patients. Measuring HH elements in biological samples may help categorize COPD patients and propose novel diagnostic and therapeutic strategies.

Background The regulation of mucociliary clearance is a key part of the defense mechanisms developed by the airway epithelium. If a high aggregate quality of evidence shows the clinical effectiveness of nasal irrigation, there is a lack of studies showing the intrinsic role of the different irrigation solutions allowing such results. This study investigated the impact of solutions with different pH and ionic compositions, eg, normal saline, non-diluted seawater and diluted seawater, on nasal mucosa functional parameters. Methods For this randomized, controlled, blinded, in vitro study, we used airway epithelial cells obtained from 13 nasal polyps explants to measure ciliary beat frequency (CBF) and epithelial wound repair speed (WRS) in response to 3 isotonic nasal irrigation solutions: (1) normal saline 0.9%; (2) non-diluted seawater (Physiomer®); and (3) 30% diluted seawater (Sterimar). The results were compared to control (cell culture medium). Results Non-diluted seawater enhanced the CBF and the WRS when compared to diluted seawater and to normal saline. When compared to the control, it significantly enhanced CBF and slightly, though nonsignificantly, improved the WRS. Interestingly, normal saline markedly reduced the number of epithelial cells and ciliated cells when compared to the control condition. Conclusion Our results suggest that the physicochemical features of the nasal wash solution is important because it determines the optimal conditions to enhance CBF and epithelial WRS thus preserving the respiratory mucosa in pathological conditions. Non-diluted seawater obtains the best results on CBF and WRS vs normal saline showing a deleterious effect on epithelial cell function.

International audience; Interleukin (IL)-22 plays a critical role in regulating the maintenance of the mucosal barrier. As airway epithelial regeneration is abnormal in cystic fibrosis (CF), we investigated IL-22 integrity in CF. We first demonstrated, using Il-22-/- mice, that IL-22 is important to prevent lung damage induced by the CF pathogen Pseudomonas aeruginosa. Next, IL-22 receptor was found normally expressed at the airway epithelial surfaces of CF patients. In wound-healing assays, IL-22-treated CF cultures had higher wound-closure rate than controls, suggesting that IL-22 signaling per se could be functional in a CF context. However, persistence of neutrophil-derived serine-proteases is a major feature of CF airways. Remarkably, IL-22 was found altered in this protease-rich inflammatory microenvironment; the serine protease-3 being the most prone to fully degrade IL-22. Consequently, we suspect an acquired deficiency of the IL-22 pathway in the lungs of CF patients due to IL-22 cleavage by the surrounding neutrophil serine-proteases.

Background: Airway remodelling is a hallmark of COPD but its physiopathology remains unclear. Since Hedgehog (HH) pathway has been shown to regulate repair and regeneration in the mouse lung, we investigated its role in human airway epithelial cell differentiation in the context of COPD. Methods: Sections from formalin-fixed paraffin embedded lung tissues of non-COPD and COPD patients were stained to characterize the localization and activation of HH pathway elements. Human airway primary cells obtained from nasal polyps were differentiated in vitro at the air-liquid interface. RT-qPCRs, western-blots and immunofluorescent studies were performed during the kinetic of epithelial cell differentiation with or without HH inhibitors. Results: A comparison of immunostaining analysis on tissue sections pointed towards an alteration of HH pathway in COPD patients including a decrease of Gli2 expression. Hedgehog pathway was activated in the course of airway epithelial cells differentiation in vitro as shown by the increase of the transcription factors Gli1 and Gli2 (protein and RNA expression). Hedgehog inhibitors induced epithelial remodelling characterized by a 2 fold decrease of ciliated cells and a 2 fold increase of secretory cells. Conclusion: These findings indicate that HH pathway contributes to airway epithelial cell plasticity and suggest its involvement in airway remodelling in COPD. Restoring HH signalling may help correct the remodelling observed in COPD patients.

Accumulating data now suggest that ZO-1, once delocalized from tight junctions, could be implicated in the regulation of tumor-promoting genes. Because of their major implication in different steps of tumor progression, we investigated here the influence of ZO-1 on chemokines expression in breast cancer cells. Using GeneArray analysis to compare chemokine mRNA expression in breast tumor cells transfected with a siRNA against ZO-1, we identified CXCL-8IL-8 as a major potential target of ZO-1 signaling, being strongly downregulated following ZO-1 siRNA transfection. Examining further the relationship between ZO-1 and interleukin-8 (CXCL8/IL-8), we first showed that CXCL8/IL-8 expression correlates with a relocalization of ZO-1 in several breast cancer cell lines. Moreover, CXCL8/IL-8 is downregulated in invasive BT549 cells transfected with three different ZO-1 siRNA and overexpressed in noninvasive BT20 and SKBR3 cells transfected with vectors expressing ZO-1. We also provide evidence for an activation of the CXCL8/IL-8 promoter by ZO-1. Finally, we show that the regulation of CXCL8/IL-8 by ZO-1 is independent of the β-catenin pathway. Our results thus clearly show an implication of ZO-1 in CXCL8/IL-8 regulation. Because of the major implications of CXCL8/IL-8 in tumor invasion, such a regulation could play an important role in breast cancer progression. Mol Cancer Res; 10(1); 121–32. ©2011 AACR.

International audience; Chronic inflammation is a hallmark of cystic fibrosis (CF) lung disease and airway epithelium damage and remodelling are important components of lung pathology progression in CF. Whether this remodelling is secondary to deleterious infectious and inflammatory mediators, or to alterations of CF human airway epithelial (HAE) cells, such as their hyper inflammatory phenotype or their basic cystic fibrosis transmembrane conductance regulator (CFTR) default, remains debated. In this study, we evaluated the involvement of alterations of CF HAE cells in airway epithelium remodelling. HAE cells from non‐CF and CF patients were cultured in an air–liquid interface, with and without inflammatory stimulation, along the regeneration process, and the remodelling of the reconstituted epithelium was analysed. We confirmed that CF HAE cells showed a hyperinflammatory phenotype which was lost with time. In comparison to non‐CF epithelium, CF epithelium regeneration in the absence of exogenous inflammation was higher and exhibited basal cell hyperplasia. This remodelling was mimicked by inflammatory stimulation of non‐CF cells and was absent when CF HAE cells were no longer hyperinflamed. Moreover, the number of goblet cells was similar in non‐CF and CF cultures and increased equally under inflammatory stimulation. Finally, whatever the inflammatory environment, CF cultures showed a delay in ciliated cell differentiation. In conclusion, alterations of CF HAE cells partly regulate airway epithelium remodelling following injury and regeneration. This remodelling, together with goblet cell hyperplasia induced by exogenous inflammation and alteration of ciliated cell differentiation, may worsen mucociliary clearance impairment, leading to injury

Introduction La mucoviscidose (CF) est la maladie genetique la plus frequente. Les perspectives de therapie pro-regeneratrice, cellulaire et/ou genique sont nombreuses dans la CF. La production in vitro d’epithelium respiratoire non-CF mais surtout CF, en grande quantite, pourrait fournir un modele preclinique humain pour le criblage de molecules therapeutiques. Le but de la presente etude est d’obtenir, a partir de cellules souches embryonnaires humaines (hES), des progeniteurs epitheliaux en preambule a leur differenciation en cellules epitheliales respiratoires. Methodes Les cellules hES (lignee Sahlgrenska 1 [SA-O1]), sont des cellules provenant d’un embryon surnumeraire issu de fecondation in vitro et ne faisant plus l’objet d’un projet parental. Ces cellules sont cultivees sur des cellules nourricieres dont la proliferation est stoppee par un traitement a la mitomycine C (STO) ou sur des substrats tels que le matrigel et la fibronectine plasmatique, dans un milieu commercial optimise pour les cellules epitheliales respiratoires (BEGM) en presence de 0,5 nM de BMP4 et de 0,3 mM d’acide ascorbique (AA) ou retinoique (AR) en continu, ou pendant 3 jours ou 6 jours. La presence de cellules basales progenitrices epitheliales (CK13 positives) a ete examinee par detection immunocytochimique. Resultats Nos resultats montrent par immunocytochimie la presence de cellules CK13 positives des 20 jours de traitement continu AA/BMP4 sur cellules STO. Sur matrigel, nous observons une forte presence de cellules exprimant la CK13 des 30 jours de culture avec 6 jours de traitement AA/BMP4. Ce pourcentage diminue avec 3 jours de traitement AA/BMP4 et s’approche de zero en condition de traitement AR/BMP4. Apres 6 jours de traitement AA/BMP4, la fibronectine plasmatique semble etre un substrat moins efficace que les cellules STO ou le matrigel pour l’induction de la differenciation des cellules CK13 positives. Conclusion Il apparait qu’une stimulation de 6 jours avec la combinaison AA/BMP4 sur matrigel soit suffisante pour induire la differenciation efficace des cellules hES en cellules basales epitheliales. Il apparait egalement que l’AR soit un inducteur moins puissant que l’AA, et que les cellules hES se developpent mieux sur le matrigel que sur la fibronectine plasmatique. Il sera interessant par la suite de discriminer un ou plusieurs marqueurs membranaires permettant d’isoler les cellules d’interet CK13 positives par cytometrie en flux afin de les amplifier specifiquement. Le devenir de ces cellules pourra alors etre evalue in vitro, in vivo ou ex vivo afin de determiner leur capacite a reconstituer un epithelium complet, mature et fonctionnel.

During tumor invasion, tumor epithelial cells acquire migratory and invasive properties involving important phenotypic alterations. Among these changes, one can observe reorganization or a loss of cell–cell adhesion complexes such as tight junctions (TJs). TJs are composed of transmembrane proteins (occludin, claudins) linked to the actin cytoskeleton through cytoplasmic adaptor molecules including those of the zonula occludens family (ZO-1, -2, -3). We here evaluated the potential role of ZO-2 in the acquisition of invasive properties by tumor cells. In vivo, we showed a decrease of ZO-2 expression in bronchopulmonary cancers, with a preferential localization in the cytoplasm. In addition, in vitro, the localization of ZO-2 varied according to invasive properties of tumor cells, with a cytoplasmic localization correlating with invasion. In addition, we demonstrated that ZO-2 inhibition increases invasive and migrative capacities of invasive tumor cells. This was associated with an increase of MT1-MMP. These results suggest that ZO-2, besides its structural role in tight junction assembly, can act also as a repressor of tumor progression through its ability to reduce the expression of tumor-promoting genes in invasive tumor cells.

Introduction : Lors de la progression metastatique, il a ete montre que les jonctions serrees disparaissent et que les molecules les constituant sont soit absentes soit relocalisees dans la cellule tumorale. En effet, dans certaines conditions, ZO-2 (zonula occludens- 2), proteine constitutive des jonctions intercellulaires, peut etre delocalise et meme gagner le noyau. Une etude a egalement suggere un role direct de ZO-2 sur la regulation de la transcription. L’objectif de notre etude est d’evaluer le role potentiel de ZO-2 lors de la migration et de l’invasion de cellules tumorales, et notamment au niveau de la regulation de l’expression de MMP exprimees par les cellules tumorales. Methodes : L’expression de ZO-2 a ete etudiee en immunofluorescence et en Western Blot dans des lignees de cellules tumorales invasives et non-invasives. Au vu des resultats obtenus, nous avons etudie la regulation potentielle de certaines MMP par ZO-2. Pour ce faire, nous avons module l’expression de ZO-2 par transfection de l’ADNc de ce gene et par la technologie des siRNA (small interfering RNA) dans des lignees de cellules tumorales invasives : BT549 (cellules mammaires) et BZR (cellules bronchiques). Pour evaluer la regulation potentielle de certaines MMP ainsi que la variation des capacites invasives des cellules tumorales, des etudes par RT-PCR, Western Blot et des tests d’invasion en chambre de Boyden sont realisees. Resultats : Nous avons observe que ZO-2 est exprime par toutes les lignees etudiees et est retrouve au niveau de la membrane plasmique des cellules non-invasives, et dans le cytoplasme et le noyau des cellules invasives. Les resultats ont egalement montre que ZO- 2, par transfection d’ADNc et de siRNA, module l’expression de certaines MMP (principalement de la MMP-3 et de la MT1- MMP). De plus, des resultats preliminaires ont montre que la modulation de l’expression de ZO-2 dans des cellules tumorales entraine une variation de leur capacite invasive. Conclusion : Ces resultats suggerent que ZO-2 joue un role dans le processus d’invasion tumorale en modulant l’expression de MMP. Travail soutenu par l’Inserm et la Region Champagne-Ardenne