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Ionenbewegungen über das Atemwegsepithel bilden die Grundlage einer definierten Zusammensetzung intraluminaler pulmonaler Flüssigkeit. Ein perinataler Anstieg maternaler und damit einhergehend fetaler Glukokortikoid (GC)-Serumlevel ist vergesellschaftet mit einer Veränderung im epithelialen Ionentransport der Lunge. So erfährt das respiratorische Epithel in einer perinatalen Anpassungsreaktion eine Transition fetaler Fruchtwasserproduktion hin zu überwiegend flüssigkeitsabsorbierenden Vorgängen. In den distalen Abschnitten spiegelt sich dies in einer Aktivierung flüssigkeitsabsorptiv wirkender epithelialer Natriumkanäle (ENaC) wider. Vor Beginn der Transition überwiegt ein sekretorisch aktiver, apikaler Chloridkanal, der cystic fibrosis transmembrane conductance regulator (CFTR). Als Gegenspieler zum ENaC gewährleistet der CFTR einen transepithelialen Flüssigkeitsstrom in das Lumen des respiratorischen Systems. Diese Prozesse ermöglichen den pulmonalen Gastaustausch sowie die Bereitstellung eines periziliären Flüssigkeitsfilms, welcher die postnatale mukoziliäre Clearance (MCC) gewährleistet. Während bereits gezeigt werden konnte, dass GC den ENaC vor allem in distalen Atemwegsepithelien in Expression und Aktivität steigern (Thome et al. 2003; Venkatesh und Katzberg 1997), gibt es bisher nur wenige Daten zum Einfluss von GC auf den CFTR. Zusammenfassend kann durch die vorliegende Arbeit gezeigt werden, dass es durch GC zu einer Zunahme des CFTR-abhängigen Ionentransportes kommt und dass dieser aktivitätssteigernde Einfluss von der PI3K sowie der AKT abhängig ist, während die SGK1 nicht wesentlich in diesen Wirkmechanismus involviert zu sein scheint. Es ließ sich nachweisen, dass es durch GC zu einer gesteigerten Phosphorylierung von AKT, SGK1 sowie NEDD4L kommt, woraus eine Aktivitätssteigerung der AKT und der SGK1 sowie ein Rückgang in der NEDD4L-Aktivität resultieren. Eine AKT-Inhibition verhindert die zunehmende NEDD4L Phosphorylierung, während eine Inhibition der SGK1 keinen Einfluss auf den NEDD4L-Phosphorylierungsstatus zeigt. Des Weiteren ergibt sich ein stimulierender Einfluss von GC auf die CFTR-Aktivität innerhalb von 30 Minuten. Im Gegensatz zum Ionentransport zeigten GC keinen Einfluss auf den Epithelwiderstand und damit die Barrierefunktion der Zellkultur.:1. Abkürzungsverzeichnis S. 3 2. Abbildungsverzeichnis S. 5 3. Bibliographische Beschreibung S. 6 4. Einleitung S. 7 4.1. Der CFTR und seine Rolle im epithelialen Chloridtransport S. 7 4.2.Die Struktur und Regulation des CFTR S. 8 4.3.Die (Patho-)Physiologie des CFTR S. 10 4.4.Alternative Chloridkanäle S. 11 4.5.Methodik – Etablierung der Methode S. 12 5. Originalpublikation 5.1. “Glucocorticoids Distinctively Modulate the CFTR Channel with Possible Implications in Lung Development and Transition into Extrauterine Life” S. 14 5.2. “Signaling Cascade involved in Rapid Stimulation of Cystic Fibrosis Conductance Regulator (CFTR) by Dexamethasone” S. 36 6. Zusammenfassung der Ergebnisse S. 52 6.1 Hintergrund und Ziel S. 52 6.2 Ergebnisse S. 53 7. Diskussion der Ergebnisse S. 54 8. Literaturverzeichnis S. 57 9. Anmerkungen zur Originalpublikation – Darstellung des eigenen Beitrags S. 64 10. Erklärung über die eigenständige Abfassung der Arbeit S. 65 11. Curriculum vitae S. 66 12. Danksagung S. 68

The genetic disease cystic fibrosis is the most common lethal genetic disease in Western countries. People born with cystic fibrosis suffer from many health issues including severe respiratory problems, inflammation and recurrent lung infections that can become fatal. The disease is caused by the loss of function of a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is an chloride ion channel and, in healthy people, its activity assures correct water and salt transport across the cell membrane. Most cases of cystic fibrosis are caused by a genetic defect that leads to the deletion of phenylalanine 508 (F508del) in the amino acid sequence of the protein. The molecular mechanism by which F508del leads to loss of function of the CFTR channel is still poorly understood. The mutation is found in the first nucleotide binding domain (NBD1) and studies have shown that it causes misfolding of CFTR and subsequent degradation of the protein by the cellular quality control system. It is established that the mutation affects stability and dynamics of NBD1 but does not alter its structure significantly. This destabilizing effect of F508del can be compensated by specific mutations distributed over different regions of NBD1, leading to recovery of membrane expression of a functional channel. A surprising example involves the regulatory insertion (RI), a 32-residue long segment found in all CFTR orthologs but not in related channels or transporters. The RI is not resolved in crystal structures of NBD1 nor cryo-EM structures of CFTR and has been described as intrinsically disordered. Its functional role in CFTR is unknown. Removal of the RI increases the stability of the NBD1 domain and, in the context of F508del-CFTR, this deletion restores maturation, cell surface expression and activity of the mutant channel. We probed the effect of the RI on NBD1 structure, dynamics and allostery using X-ray crystallography, single molecule FRET and hydrogen-deuterium exchange. We discovered that the RI enables an alternative NBD1 fold which departs markedly from the canonical fold previously observed for this domain and the NBDs of other ABC transporters. The conformational equilibrium between these states is regulated by ATP binding and affected by disease-associated conditions. Aside from clear alterations to structure and dynamics of NBD1, the RI also affects allostery, i.e. how NBD1 structure and dynamics respond to perturbations such as ligand binding. Finally, we show that the RI-enabled conformation is adopted in full-length CFTR and associated with increased channel activity in electrophysiological assays. We then identify an allosteric network that links the structural hotspots of the conformational changes to F508 and its surroundings. Lastly, we argue that these conformational changes lead to unfolding of NBD1 in the context of F508del, providing a new model for the molecular mechanism leading to pathogenesis. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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In cystic fibrosis (CF) therapy, the recent approval of CF-transmembrane conductance regulator (CFTR) channel modulators is considered to be the major breakthrough. However, the current first-line approach based mainly on pulmonary function to measure effects of the novel therapy, tested by forced expiratory volumes in one second (FEV1), provides restricted sensitivity to detect early structural damages. Accordingly, there is a need for new sensitive surrogate parameters. Most interestingly, these should quantify inflammation that precedes a decline of pulmonary function. We present a novel method assessing inflammatory markers in the upper airways’ epithelial lining fluid (ELF) obtained by nasal lavage (NL). In contrast to broncho-alveolar lavage, ELF sampling by NL is an attractive method due to its limited invasiveness which allows repeated analyses, even performed in a home-based setting. In a longitudinal cohort study (ClinicalTrials.gov, Identifier: NCT02311140), we assessed changes of inflammatory mediators in 259 serially obtained nasal lavages taken up to every second day before and during therapy with ivacaftor from ten CF patients carrying a G551D mutation. Patients were trained to sample NL-fluid at home, to immediately freeze and transfer chilled secretions to centers. Neutrophil Elastase, Interleukins IL-1b, IL-6 and IL-8 in NL were quantified. During 8-12 weeks of ivacaftor-treatment, median values of IL-1b and IL-6 significantly declined 2.29-fold (2.97!1.30 pg/mL), and 1.13-fold (6.48!5.72 pg/mL), respectively. In parallel, sweat tests and pulmonary function improved considerably. This is the first study assessing changes of airway inflammation on a day-to-day basis in CF patients receiving a newly administered CFTR-modulator therapy. It proves a decline of airway inflammation during ivacaftor-therapy.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder involving a mutation in the CF transmembrane conductance regulator protein (CFTR), which causes dysfunctional transport of chloride ions across cell membranes. CF affects multiple body systems and a few of its symptoms include chronic cough, difficulty breathing, obstructive airway disease, bacterial pulmonary infections, maldigestion, malabsorption, pancreatitis, and male infertility. Until recently, treatment options have been limited to alleviating symptoms, but a new classification of drugs, CFTR modulators, provide an opportunity to slow the progression of the disease and improve clinical outcomes. The effect of CFTR modulators may be attributed to the reduction of persistently colonizing bacteria in CF lungs. Though, the effects of modulators on microbial communities colonizing the CF lung remains unknown, specifically with common respiratory pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. Particularly, previous CF studies have been limited in scope due to focusing on only one type of modulator and by using low-yield sequencing techniques. To address this gap, we seek to study the changes in CF respiratory pathogens of patients initiating CFTR modulator therapy at Nemours Hospital using long-read metagenomic sequencing (Oxford Nanopore) of longitudinally collected respiratory samples. We have optimized a protocol for host DNA depletion and microbial metagenomic sequencing to characterize the respiratory microbiome. This study focuses on utilizing these sequencing data to compare the microbiome among two healthy controls to pre-CFTR-treatment microbial communities of two recruited pediatric CF patients.

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Dans mon projet de doctorat, j’ai étudié des fonctions primordiales de l’épithélium respiratoire telles que la régulation du transport ionique, la clairance liquidienne et la réparation épithéliale. J’ai particulièrement mis l’emphase sur le rôle des canaux potassiques qui interviennent dans ces trois fonctions de l’épithélium respiratoire. J’ai tout d’abord prouvé que la modulation des canaux potassiques régulait l’activité du promoteur de αENaC, en partie via la voie de signalisation ERK1/2, dans des cellules alvéolaires. Cette régulation entraîne une variation de l’expression génique et protéique du canal ENaC. Physiologiquement, il en résulte une augmentation du phénomène de clairance liquidienne suite à l’activation des canaux K+, tandis que l’inhibition de ces canaux la diminue sévèrement. J’ai aussi pu démontrer que l’absence de canal KvLQT1 entraînait une diminution du courant (ENaC) sensible à l’amiloride, dans les cellules de trachée en culture primaire, isolées de souris KO pour kcnq1. Dans la seconde partie de mon étude, j’ai évalué l’impact de l’hyperglycémie sur la capacité de transport ionique et de réparation de cellules épithéliales bronchiques saines ou Fibrose Kystique. Mes résultats montrent que l’hyperglycémie diminue le transport transépithélial de chlore et le transport basolatéral de potassium. Des études préalables du laboratoire ayant montré que les canaux K+ et Cl- contrôlent les processus de réparation, j’ai donc évalué si ceux-ci étaient modifiés par l’hyperglycémie. Et en effet, l’hyperglycémie ralentit la vitesse de réparation des cellules issues des voies aériennes (CFBE-wt et CFBE-ΔF508). J’ai donc démontré que le transport de potassium intervenait dans des fonctions clés de l’épithélium respiratoire, comme dans la régulation génique de canaux ioniques, le contrôle de la clairance liquidienne alvéolaire, et que l’hyperglycémie diminuait le transport ionique (K+ et Cl-) et la réparation épithéliale. / During my Ph.D. training, I studied 3 important functions of respiratory epithelium : regulation of ion transport, liquid clearance and epithelial repair. I focused on potassium channels, because they control these three respiratory epithelial functions. First, I proved that αENaC promoter activity was regulated following K+ channel modulation, in alveolar cells. This regulation of αENaC promoter which might be through a modification of ERK1/2 phosphorylation, was followed by ENaC mRNA and protein expression regulation. I then showed that activation of KvLQT1 and KATP channels increased alveolar liquid clearance, whereas inhibition of these K+ channels decreased the alveolar clearance. I showed that the absence of KvLQT1 channel inhibited the amiloride-sensitive current (ENaC), in tracheal epithelial cells isolated from KvLQT1-KO mice. In the second part of my Ph.D. project, I studied the impact of hyperglycemia on Cystic Fibrosis (CF) and non-CF epithelial cells. I first observed that K+ and Cl- currents were reduced by hyperglycemia. Because we have previously shown that wound-healing process was dependant on K+ and Cl- channels, I then evaluated the impact of hyperglycemia on wound-healing. As expected, hyperglycemia slowed the repair rate of non-CF (CFBE-wt) and CF (CFBE-ΔF508) cell monolayers.