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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.

The skin of many fishes contains large epidermal club cells (ECCs) that are known to release chemicals (alarm cues) that warn other fishes of danger. Initial research on ECCs focussed on their role in predator avoidance behaviour, however later research revealed that these cells might also have immune functions. Anthropogenic activities have dramatically increased over the past decades, with the consequence that many organisms simultaneously get exposed to multiple environmental stressors. We have seen considerable reductions in stratospheric ozone with a concomitant increase in global ultraviolet radiation (UVR). Metal pollution associated with industrial activity is also increasing on a global scale. Cadmium (Cd) is one such ubiquitous pollutant which is known to be toxic to organisms at extremely low concentrations. The main goal of my PhD research was to understand how multiple environmental stressors play a role in altering ECC investment and chemically-mediated predator-prey interactions by indirectly elucidating the evolutionary role of ECCs. The first experiment investigated the effects of in vivo ultraviolet radiation (UVR) exposure on ECC investment, physiological stress responses and potency of alarm cues in fathead minnows (Pimephales promelas). Subsequently, I investigated the interactive effects of UVR and/or waterborne cadmium (Cd) exposure using the same end points. I found that minnows exposed to UVR, either in the presence or absence of Cd, showed consistent decrease in ECC investment compared to non-exposed controls. There was a significant increase in cortisol levels of UVR exposed minnows compared to unexposed minnows. However, the combined exposure of UVR and Cd reduced cortisol levels relative to that in UVR only exposure. Surprisingly, there was no difference in the potency of the cues prepared from the skin of UVR and/or Cd exposed or non-exposed fish indicating that UVR and/or Cd exposure combined may have little influence on chemically-mediated predator-prey interactions. In aquatic systems, much of the negative effects of UVR are minimized by dissolved organic carbon (DOC) which is known to attenuate rates of UVR across the water column. In my third study, I investigated if DOC played a role in ameliorating the effects of in vivo UVR exposure on physiological stress and ECC investment in fathead minnows. I used two sources of DOC, a commercial soil based DOC (Sigma Aldrich Humic Acid) and a terrigenous source of DOC (Luther Marsh Natural Organic Matter). I found that fish exposed to UVR, in the presence of either source of DOC, in the presence and absence of UV blocking filter, maintained high ECC investment and reduced cortisol levels compared to fish exposed to UVR only. Studies that have examined factors that influence ECC investment have often been hampered by large variation in baseline levels of ECC. The larger the baseline variation in ECC number, the more difficult it is to elucidate factors responsible for changes in ECC investment. While I did not find this problematic in my work with UVR and Cd, others have failed to find effects in manipulative experiments. Consequently, my fourth study examined between and within variation in ECC investment across multiple sites in Saskatchewan and tried to investigate if holding fish under controlled laboratory conditions for up to 28 days would help reduce variation in ECC investment between and within populations. I found some evidence that I could reduce within population variation in ECC investment through time, but could not reduce among-population variation in mean ECC investment.