Ambra Gianotti

Epithelial Sodium Channel Silencing as a Strategy to Correct the Airway Surface Fluid Deficit in Cystic Fibrosis

In the respiratory system, Na(+) absorption and Cl(-) secretion are balanced to maintain an appropriate airway surface fluid (ASF) volume and ensure efficient mucociliary clearance. In cystic fibrosis (CF), this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the absence of functional CFTR-dependent Cl(-) secretion. The consequences of defective Cl(-) transport are worsened by the persistence of Na(+) absorption, which contributes to airway surface dehydration. We asked whether normal ASF can be restored to an equal extent by recovering Cl(-) secretion from mutated CFTR or by reducing Na(+) absorption. This is highly relevant in the selection of the best strategy for the treatment of patients with CF. We analyzed the ASF thickness of primary cultured bronchial CF and non-CF epithelia after silencing the epithelial Na(+) channel (ENaC) with specific short, interfering RNAs (siRNAs) and after the pharmacological stimulation of CFTR. Our results indicate that (1) single siRNAs complementary to ENaC subunits are sufficient to reduce ENaC transcripts, Na(+) channel activity, and fluid transport, but only silencing both the α and β ENaC subunits at the same time leads to an increase of ASF (from nearly 7 µm to more than 9 µm); (2) the ASF thickness obtained in this way is about half that measured after maximal CFTR stimulation in non-CF epithelia (10-14 µm); and (3) the pharmacological rescue of mutant CFTR increases the ASF to the same extent as ENaC silencing. Our results indicate that CFTR rescue and ENaC silencing both produce a significant and long-lasting increase of airway hydration in vitro.

Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release.

Goblet cell hyperplasia, a feature of asthma and other respiratory diseases, is driven by the Th-2 cytokines IL-4 and IL-13. In human bronchial epithelial cells, we find that IL-4 induces the expression of many genes coding for ion channels and transporters, including TMEM16A, SLC26A4, SLC12A2, and ATP12A. At the functional level, we find that IL-4 enhances calcium- and cAMP-activated chloride/bicarbonate secretion, resulting in high bicarbonate concentration and alkaline pH in the fluid covering the apical surface of epithelia. Importantly, mucin release, elicited by purinergic stimulation, requires the presence of bicarbonate in the basolateral solution and is defective in cells derived from cystic fibrosis patients. In conclusion, our results suggest that Th-2 cytokines induce a profound change in expression and function in multiple ion channels and transporters that results in enhanced bicarbonate transport ability. This change is required as an important mechanism to favor release and clearance of mucus.

Genetic Inhibition of the Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated to F508del Cystic Fibrosis Mutation

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins.

Synthesis and structure-activity relationship of aminoarylthiazole derivatives as correctors of the chloride transport defect in cystic fibrosis.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in the membrane of epithelial cells. Mutations affecting the CFTR gene cause cystic fibrosis (CF), a multi-organ severe disease. The most common CF mutation, F508del, impairs the processing and activity (gating) of CFTR protein. Other mutations, like G551D, only cause a gating defect. Processing and gating defects can be targeted by small molecules called generically correctors and potentiators, respectively. Aminoarylthiazoles (AATs) represent an interesting class of compounds that includes molecules with dual activity, as correctors and potentiators. With the aim to improve the activity profile of AATs, we have now designed and synthesized a library of novel compounds in order to establish an initial SAR that may provide indications about the chemical groups that are beneficial or detrimental for rescue activity. The new compounds were tested as correctors and potentiators in CFBE41o-expressing F508del-CFTR using a functional assay. A dual active compound, AAT-4a, characterized by improved efficacy and marked synergy when combined with the corrector VX-809 has been identified. Moreover, by computational methods, a possible binding site for AATs in nucleotide binding domain NBD1 has been detected. These results will direct the synthesis of new analogues with possibly improved activity.

Upregulation of TMEM16A protein in bronchial epithelial cells by bacterial pyocyanin

Induction of mucus hypersecretion in the airway epithelium by Th2 cytokines is associated with the expression of TMEM16A, a Ca2+-activated Cl- channel. We asked whether exposure of airway epithelial cells to bacterial components, a condition that mimics the highly infected environment occurring in cystic fibrosis (CF), also results in a similar response. In cultured human bronchial epithelial cells, treatment with pyocyanin or with a P. aeruginosa culture supernatant caused a significant increase in TMEM16A function. The Ca2+-dependent Cl- secretion, triggered by stimulation with UTP, was particularly enhanced by pyocyanin in cells from CF patients. Increased expression of TMEM16A protein and of MUC5AC mucin by bacterial components was demonstrated by immunofluorescence in CF and non-CF cells. We also investigated TMEM16A expression in human bronchi by immunocytochemistry. We found increased TMEM16A staining in the airways of CF patients. The strongest signal was observed in CF submucosal glands. Our results suggest that TMEM16A expression/function is upregulated in CF lung disease, possibly as a response towards the presence of bacteria in the airways.

Pharmacological rescue of mutant CFTR protein improves the viscoelastic properties of CF mucus

BACKGROUND In CF patients, the defective ion transport causes a simultaneous reduction of fluid, Cl(-) and HCO3(-) secretion. We aimed to demonstrate that the resulting altered properties of mucus can be recovered using lumacaftor, a CFTR corrector. METHODS The micro-rheology of non-CF and CF mucus was analysed using Multiple Particle Tracking. RESULTS The diffusion coefficient of nano-beads imbedded in mucus from CF human bronchial epithelium was lower than in non-CF mucus, and the elastic and viscous moduli were higher. We found that 25% correction of F508del-CFTR mutation with lumacaftor was enough to improve significantly CF mucus properties. Surprisingly, also incubation with amiloride, a compound that reduces fluid absorption but might not change the secretion of HCO3(-) towards the airway surface fluid, improved CF mucus properties. CONCLUSION CF mucus properties can be recovered by either improving the hydration of the airways or recovering Cl(-) and HCO3(-) secretion across the mutated protein treated with a corrector compound.

Pharmacological analysis of epithelial chloride secretion mechanisms in adult murine airways

Defective epithelial chloride secretion occurs in humans with cystic fibrosis (CF), a genetic defect due to loss of function of CFTR, a cAMP-activated chloride channel. In the airways, absence of an active CFTR causes a severe lung disease. In mice, genetic ablation of CFTR function does not result in similar lung pathology. This may be due to the expression of an alternative chloride channel which is activated by calcium. The most probable protein performing this function is TMEM16A, a calcium-activated chloride channel (CaCC). Our aim was to assess the relative contribution of CFTR and TMEM16A to chloride secretion in adult mouse trachea. For this purpose we tested pharmacological inhibitors of chloride channels in normal and CF mice. The amplitude of the cAMP-activated current was similar in both types of animals and was not affected by a selective CFTR inhibitor. In contrast, a CaCC inhibitor (CaCCinh-A01) strongly blocked the cAMP-activated current as well as the calcium-activated chloride secretion triggered by apical UTP. Although control experiments revealed that CaCCinh-A01 also shows inhibitory activity on CFTR, our results indicate that transepithelial chloride secretion in adult mouse trachea is independent of CFTR and that another channel, possibly TMEM16A, performs both cAMP- and calcium-activated chloride transport. The prevalent function of a non-CFTR channel may explain the absence of a defect in chloride transport in CF mice.

Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity

TMEM16A and TMEM16B are plasma membrane proteins with Ca2+-dependent Cl− channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the “activating domain” to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca2+ concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl− transport.

In vitro Methods for the Development and Analysis of Human Primary Airway Epithelia

Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies.