The HDAC inhibitor SAHA does not rescue CFTR membrane expression in Cystic Fibrosis

International audienc

Role of club cells and CCSP in COPD

La protéine CCSP (« Club Cell Secretory Protein »), produite par les cellules Club au niveau de l’épithélium respiratoire, se retrouve déficiente chez les patients atteints de Bronchopneumopathie Chronique Obstructive (BPCO). Le but de ce travail était de comprendre la régulation et les différents rôles de la protéine CCSP afin d’en évaluer son potentiel intérêt thérapeutique. Nous avons dans un premier temps observé les effets du polymorphisme connu de CCSP au niveau de sa région promotrice, la mutation G38A, sur la transcription même du gène. Nous avons constaté in vivo dans une étude clinique prospective sur 1 an comprenant 66 patients souffrant de BPCO, et confirmé in vitro dans un modèle de cellules BEAS-2B transfectées, que la fumée de cigarette était un répresseur de la transcription de CCSP et que ce phénomène était amplifié par la présence de la mutation G38A. De plus, in vitro, certains facteurs de transcription tels que p53 et Nkx2.1, ainsi que les lipopolysaccharides, affectaient l'efficacité du promoteur de CCSP.Ensuite, nous avons caractérisé les cellules qui sécrètent cette protéine dans un modèle ex vivo de culture en interface air-liquide de cellules primaires épithéliales bronchiques. Nous avons observé par microscopie électronique à balayage des cellules en dôme, forme caractéristique des cellules Club, et par microscopie électronique à transmission des cellules contenant des granules de sécrétion contenant la protéine CCSP. Nous avons constaté par immunofluorescence que les cellules marquées CCSP+ étaient également MUC5AC+ (marqueur de cellules à mucus), P63+ (marqueur de cellules basales) ou encore KI-67+ (marqueur de prolifération). Nous suggérons donc que les cellules Club sont des cellules progénitrices, permettant ainsi la régénération de l’épithélium bronchique. Par ailleurs, nous avons évalué l’implication de la protéine CCSP dans le recrutement des neutrophiles, cellules inflammatoires prépondérantes dans la BPCO. Une étude pharmacologique a d’abord permis d’évaluer les effets de CCSP sur des neutrophiles de sujets témoins. Le déplacement des neutrophiles, stimulé par l’IL8 ou le fMLP (tous deux puissants agents chemoattractants), était inhibé par CCSP. Puis, par une étude in vitro, nous avons déterminé la modulation du sécrétome de l’épithélium bronchique par CCSP. Lorsque les sécrétions d’épithélia reconstitués ex vivo à partir de biopsies de fumeur et de BPCO étaient mis en présence de neutrophiles, un chimiotactisme exagéré des neutrophiles étaient constaté. Lorsque les épithélia étaient traités avec la protéine CCSP, à l’état de base ou stimulés par de la fumée de cigarette, ce chimiotactisme exagéré était alors diminué.Enfin, dans une dernière partie, nous nous sommes intéressés à la régulation de la protéine, dans un modèle de culture cellulaire NCI-H292, lignée de cellules bronchiques cultivées en monocouche. Nous avons supplémenté ces cellules en CCSP exogène afin d’analyser les variations du profil protéomique des secrétions engendrées (méthode LC-MS/MS). De façon générale, il s’avérait que la supplémentation en CCSP permettrait une restauration de la « machinerie » du protéasome avec une augmentation des protéines de la famille des tubulines.Ce travail de thèse a démontré que la protéine CCSP était un acteur potentiel de la physiopathologie de la BPCO. L’étude de sa régulation a montré que la synthèse de CCSP était effectivement diminuée dans la BPCO. Ainsi, une supplémentation en CCSP pourrait être une piste thérapeutique.A defective in Club Cell Secretory Protein (CCSP) produced by nonciliated Club cells was observed in COPD (Chronic Obstructive Pulmonary Disease) airways. Our aim was to understand CCSP biological mechanisms of action and its dysregulation in COPD and whether it might be a therapeutic axis in COPD.First, the influence of the CCSP G38A polymorphism on CCSP transcription levels and its regulatory mechanisms were analyzed. Our in vivo study conducted in a 1 year prospective cohort consisting of 66 COPD patients confirmed that circulating CCSP levels were associated with smoking. Moreover, the CCSP G38A polymorphism and the smoking status significantly repressed CCSP serum levels. Our in vitro study conducted in BEAS-2B transfected cells supported those findings as CSE repressed the CCSP transcription of the A carrying transfected cells more intensely than the wild type cells. Noteworthy, LPS, Nkx2.1 and p53 transcription factors also modulated the CCSP promoter efficiency in vitro. Furthermore, CCSP producing cells were characterized in an air-liquid interface (ALI) culture model of bronchial epithelial cells. Transmission electron microscopy, scanning electron microscopy and confocal microscopy confirmed the pseudostratified organization of the reconstituted epithelium. Evidences of full differentiation were identified and labeled with MUC5AC (goblet cells), tubulinIV (ciliated cells), P63 (basal cells) and CCSP (club cells). Moreover, the ex vivo reconstituted COPD epithelium released higher levels of IL8 and MUC5AC. Ki-67 and collocating antibodies with CCSP argued for an accessory stem cell and a transitory differentiating roles for CCSP+ cells.Then, we aimed to investigate whether exaggerated airway neutrophilia was driven by the CCSP-defective COPD airway epithelium. CCSP action on healthy neutrophil chemotaxis was evaluated in a pharmacological study demonstrating that CCSP directly inhibited neutrophil chemotaxis induced by fMLP and IL8. Then, in an in vitro study, ALI-reconstituted COPD airway epithelium in a clean environment promoted an exaggerated neutrophilic chemotaxis compared to smokers and controls at steady state. Treating the airway epithelium with exogenous CCSP prevented baseline and CSE-induced neutrophil chemotaxis.Finally, CCSP regulation was studied in NCI-H292 cells, a human pulmonary cell line. The cells were supplemented with CCSP. Proteomic profile (LC-MS/MS method) of the bronchial epithelium in response to CCSP treatment demonstrated that the proteasome machinery and the tubulin family members were upregulated.This work supported the potential implication of CCSP in the pathophysiology of COPD. CCSP was confirmatively defective in COPD patients, therefore, restoring physiological concentrations of CCSP by exogenous supplementation may be a therapeutic perspective

Rôle des cellules Club et de CCSP dans la Bronchopneumopathie Chronique Obstructive (BPCO)

A defective in Club Cell Secretory Protein (CCSP) produced by nonciliated Club cells was observed in COPD (Chronic Obstructive Pulmonary Disease) airways. Our aim was to understand CCSP biological mechanisms of action and its dysregulation in COPD and whether it might be a therapeutic axis in COPD.First, the influence of the CCSP G38A polymorphism on CCSP transcription levels and its regulatory mechanisms were analyzed. Our in vivo study conducted in a 1 year prospective cohort consisting of 66 COPD patients confirmed that circulating CCSP levels were associated with smoking. Moreover, the CCSP G38A polymorphism and the smoking status significantly repressed CCSP serum levels. Our in vitro study conducted in BEAS-2B transfected cells supported those findings as CSE repressed the CCSP transcription of the A carrying transfected cells more intensely than the wild type cells. Noteworthy, LPS, Nkx2.1 and p53 transcription factors also modulated the CCSP promoter efficiency in vitro. Furthermore, CCSP producing cells were characterized in an air-liquid interface (ALI) culture model of bronchial epithelial cells. Transmission electron microscopy, scanning electron microscopy and confocal microscopy confirmed the pseudostratified organization of the reconstituted epithelium. Evidences of full differentiation were identified and labeled with MUC5AC (goblet cells), tubulinIV (ciliated cells), P63 (basal cells) and CCSP (club cells). Moreover, the ex vivo reconstituted COPD epithelium released higher levels of IL8 and MUC5AC. Ki-67 and collocating antibodies with CCSP argued for an accessory stem cell and a transitory differentiating roles for CCSP+ cells.Then, we aimed to investigate whether exaggerated airway neutrophilia was driven by the CCSP-defective COPD airway epithelium. CCSP action on healthy neutrophil chemotaxis was evaluated in a pharmacological study demonstrating that CCSP directly inhibited neutrophil chemotaxis induced by fMLP and IL8. Then, in an in vitro study, ALI-reconstituted COPD airway epithelium in a clean environment promoted an exaggerated neutrophilic chemotaxis compared to smokers and controls at steady state. Treating the airway epithelium with exogenous CCSP prevented baseline and CSE-induced neutrophil chemotaxis.Finally, CCSP regulation was studied in NCI-H292 cells, a human pulmonary cell line. The cells were supplemented with CCSP. Proteomic profile (LC-MS/MS method) of the bronchial epithelium in response to CCSP treatment demonstrated that the proteasome machinery and the tubulin family members were upregulated.This work supported the potential implication of CCSP in the pathophysiology of COPD. CCSP was confirmatively defective in COPD patients, therefore, restoring physiological concentrations of CCSP by exogenous supplementation may be a therapeutic perspective.La protéine CCSP (« Club Cell Secretory Protein »), produite par les cellules Club au niveau de l’épithélium respiratoire, se retrouve déficiente chez les patients atteints de Bronchopneumopathie Chronique Obstructive (BPCO). Le but de ce travail était de comprendre la régulation et les différents rôles de la protéine CCSP afin d’en évaluer son potentiel intérêt thérapeutique. Nous avons dans un premier temps observé les effets du polymorphisme connu de CCSP au niveau de sa région promotrice, la mutation G38A, sur la transcription même du gène. Nous avons constaté in vivo dans une étude clinique prospective sur 1 an comprenant 66 patients souffrant de BPCO, et confirmé in vitro dans un modèle de cellules BEAS-2B transfectées, que la fumée de cigarette était un répresseur de la transcription de CCSP et que ce phénomène était amplifié par la présence de la mutation G38A. De plus, in vitro, certains facteurs de transcription tels que p53 et Nkx2.1, ainsi que les lipopolysaccharides, affectaient l'efficacité du promoteur de CCSP.Ensuite, nous avons caractérisé les cellules qui sécrètent cette protéine dans un modèle ex vivo de culture en interface air-liquide de cellules primaires épithéliales bronchiques. Nous avons observé par microscopie électronique à balayage des cellules en dôme, forme caractéristique des cellules Club, et par microscopie électronique à transmission des cellules contenant des granules de sécrétion contenant la protéine CCSP. Nous avons constaté par immunofluorescence que les cellules marquées CCSP+ étaient également MUC5AC+ (marqueur de cellules à mucus), P63+ (marqueur de cellules basales) ou encore KI-67+ (marqueur de prolifération). Nous suggérons donc que les cellules Club sont des cellules progénitrices, permettant ainsi la régénération de l’épithélium bronchique. Par ailleurs, nous avons évalué l’implication de la protéine CCSP dans le recrutement des neutrophiles, cellules inflammatoires prépondérantes dans la BPCO. Une étude pharmacologique a d’abord permis d’évaluer les effets de CCSP sur des neutrophiles de sujets témoins. Le déplacement des neutrophiles, stimulé par l’IL8 ou le fMLP (tous deux puissants agents chemoattractants), était inhibé par CCSP. Puis, par une étude in vitro, nous avons déterminé la modulation du sécrétome de l’épithélium bronchique par CCSP. Lorsque les sécrétions d’épithélia reconstitués ex vivo à partir de biopsies de fumeur et de BPCO étaient mis en présence de neutrophiles, un chimiotactisme exagéré des neutrophiles étaient constaté. Lorsque les épithélia étaient traités avec la protéine CCSP, à l’état de base ou stimulés par de la fumée de cigarette, ce chimiotactisme exagéré était alors diminué.Enfin, dans une dernière partie, nous nous sommes intéressés à la régulation de la protéine, dans un modèle de culture cellulaire NCI-H292, lignée de cellules bronchiques cultivées en monocouche. Nous avons supplémenté ces cellules en CCSP exogène afin d’analyser les variations du profil protéomique des secrétions engendrées (méthode LC-MS/MS). De façon générale, il s’avérait que la supplémentation en CCSP permettrait une restauration de la « machinerie » du protéasome avec une augmentation des protéines de la famille des tubulines.Ce travail de thèse a démontré que la protéine CCSP était un acteur potentiel de la physiopathologie de la BPCO. L’étude de sa régulation a montré que la synthèse de CCSP était effectivement diminuée dans la BPCO. Ainsi, une supplémentation en CCSP pourrait être une piste thérapeutique

Club cells and CC16: another “smoking gun”? (With potential bullets against COPD)

International audienc

LIFE BEYOND LIFE - An Easy Way to Derive Lung Fibroblasts from Cadavers

International audienceSeveral protocols have illustrated the possibility of deriving cells, such as fibroblasts, from different organs. These techniques generally concern organs sampled from living persons, but have already been described for cadavers, especially concerning the skin and tendons. We present, for the first time, an easy way to derive pulmonary fibroblasts from a lung tissue sampled from a cadaver and directly culture plated. The fibroblast output was checked daily. We obtained lung fibroblasts from 3 (60%) cadavers and 2 (100%) living persons. The fibroblast output took about 3 days for cells from living persons and took up to 39 days for those from cadavers. We did not clearly identify any parameters that could explain these differences. Nevertheless, these derived cells had the same features as the source cells, especially in terms of morphology and proliferation, and could potentially be used in different research domains such as forensic or regeneration medicine

Supplementing Defect in Club Cell Secretory Protein Attenuates Airway Inflammation in COPD

International audienceBACKGROUND Club cell secretory protein (CCSP) is a protective biomarker associated with annual decline in lung function. COPD progression results from an imbalance between injury and repair initially triggered by cigarette smoking. OBJECTIVE We investigated the effect of CCSP as a therapeutic strategy to restore the balance between injury and repair in COPD simultaneously, validating an ex vivo air-liquid interface (ALI) culture of human bronchial epithelial cells. METHODS Endobronchial biopsy specimens (EBBs) were obtained from 13 patients with COPD, eight smokers, and eight control subjects. Morphometric analysis of the initial EBBs was performed. ALI cultures derived from the same EBBs were exposed to cigarette smoke extract (CSE) with or without exogenous recombinant human CCSP (rhCCSP) supplementation. CCSP and IL-8 concentrations were assessed at steady state and after CSE exposure. RESULTS Morphometric analysis of the initial EBBs showed increased cell density but decreased immunostaining of CCSP+ cells in EBBs of patients with COPD (P = .03 vs control subjects). At steady state, lower CCSP (P = .04) and higher IL-8 levels (P < .0001) were found in COPD ALI epithelium. Exogenous rhCCSP supplementation dampened CSE-induced IL-8-release in patients with COPD and returned to levels similar to those of smokers and control subjects (P = .0001). A negative correlation was found between IL-8-release in ALI and CCSP+ cell density in initial biopsy specimens (P = .0073). CONCLUSIONS In vitro, rhCCSP exogenous supplementation can reverse CSE-induced IL-8 release in biopsy specimens from patients with COPD, indicating a potential use of this strategy in vivo

Supervising differentiation for tailoring the airway epithelial cell phenotype

28th International Congress of the European-Respiratory-Society (ERS), Paris, FRANCE, SEP 15-19, 201

Club cell secretory protein serum concentration is a surrogate marker of small-airway involvement in asthmatic patients

International audiencePoor asthma control and recurrent exacerbations have been shown to be a phenotypic counterpart of asthma with predominantly small-airway involvement.1 Biomarkers are not always accurate in asthmatic patients, especially in serum, because compartmentalization can occur between the blood and airways. Blood eosinophil counts do not represent an overall view of airway inflammation, and exhaled nitric oxide measurements at different flow rates (fraction of exhaled nitric oxide [Feno] and alveolar nitric oxide [Calvno]) have been developed and validated to reflect more accurately proximal and distal airway inflammation.2Club cell secretory protein (CCSP) serum concentration has been shown to be associated with chronic obstructive pulmonary disease, bronchiolitis obliterans syndrome, and sarcoidosis, which are all predominantly diseases involving the small airways. Ranges of CCSP concentrations in healthy subjects, reproducibility, and relationships between serum and airway levels are known and can be used as potential surrogate markers. Our aim was to assess small-airway disease in asthmatic patients and to find a related biomarker. We used a dynamic assessment of gas trapping using computed tomographic (CT) imaging of the chest during methacholine challenge as a marker of small-airway disease

Epithelial ciliated beating cells essential for ex vivo ALI culture growth

7 pagesInternational audienceAbstractBackgroundBronchial epithelium plays a key role in orchestrating innate and adaptive immunity. The fate of ex vivo airway epithelial cultures growing at the air liquid interface (ALI) derived from human endobronchial biopsies or brushings is not easy to predict. Calibrating and differentiating these cells is a long and expensive process requiring rigorous expertise. Pinpointing factors associated with ALI culture success would help researchers gain further insight into epithelial progenitor behavior.MethodsA successful ALI culture was defined as one in which a pseudostratified epithelium has formed after 28 days in the presence of all differentiated epithelial cell types. A 4-year prospective bi-center study was conducted with adult subjects enrolled in different approved research protocols.Results463 consecutive endobronchial biopsies were obtained from normal healthy volunteers, healthy smokers, asthmatic patients and smokers with COPD. All demographic variables, the different fiber optic centers and culture operators, numbers of endo-bronchial biopsies and the presence of ciliated cells were carefully recorded. Univariate and multivariate models were developed. A stepwise procedure was used to select the final logistic regression model. ALI culture success was independently associated with the presence of living ciliated cells within the initial biopsy (OR = 2.18 [1.50–3.16], p < 0.001).ConclusionThis finding highlights the properties of the cells derived from the epithelium dedifferentiation process. The preferential selection of samples with ciliated beating cells would probably save time and money. It is still unknown whether successful ALI culture is related to indicators of general cell viability or a purported stem cell state specifically associated with ciliated beating cells