Characterization and Pharmacological Modulation of Ion Transport Mechanisms Involved in the Innate Defence of Human Airway Epithelium

Genovese, Michele (2024). Characterization and Pharmacological Modulation of Ion Transport Mechanisms Involved in the Innate Defence of Human Airway Epithelium. PhD thesis The Open University.



The airway epithelium plays a protective role against microbial pathogens delivered by inhaled air. We used cultured bronchial epithelia, differentiated in vitro under air-liquid interface condition, to study the defence mechanisms of the airway epithelium against pathogens, with a particular focus on those regulated by intracellular Ca2+. As a first step, we investigated the response to inflammatory stimuli, in particular IL-4 and IL-17/TNF-α,which both elicited profound changes in transepithelial ion transport mechanisms but with some important differences. IL-4 strongly increased the expression and function of the Ca2+-activated Cl- channel TMEM16A, and decreased the Na+ absorption mediated by ENaC. In contrast, the IL-17/TNF-α treatment had no effect on TMEM16A and markedly increased ENaC function. This condition could promote dehydration of the airway surface, and impairment of mucociliary clearance, particularly in patients affected by cystic fibrosis (CF) which suffer from defective function of CFTR, a second type of Cl- channel. We thought that potentiators of TMEM16A-mediated Cl- secretion could be useful to improve airway surface hydration and hence mucociliary clearance, in particular under inflammatory conditions associated with IL-17/TNF-α and in CF patients. To find TMEM16A potentiators, we screened a large (11,300 compounds) library with a functional assay. We found three active molecules, ARN7149, ARN4550, and ARN11391. Analysis of mechanism of action of these compounds indicate they act upstream TMEM16A. In particular, ARN7149 is a potentiator of the purinergic P2RY2 receptor, whereas ARN11391 is a potentiator of the inositol triphosphate receptor ITPR1. Interestingly, ARN7149 and ARN4550 also caused inhibition of ENaC, probably by causing breakdown of PIP2, a known regulator of ENaC function. Combined effects on TMEM16A (potentiation) and ENaC (inhibition) could have beneficial effects on mucociliary clearance.We also evaluated the specificity of known pharmacological inhibitors of TMEM16A, in particular niclosamide, which has been used to assign physiological roles to TMEM16A. Surprisingly, most compounds, except Ani9, showed an indirect mechanism of action involving alteration of mechanisms that control intracellular Ca2+ mobilization. Therefore, conclusions based on many TMEM16A inhibitors need to be reconsidered. Finally, we investigated the role of TRPV4, a Ca2+ channel that acts as a sensor of mechanical and chemical stimuli. It has been shown that TRPV4 activation enhances the beating of cilia in the airway epithelium. We found that TRPV4 has an additional role in innate defence mechanisms. Indeed, stimulation of TRPV4-dependent Ca2+ influx resulted in H2O2 release, a potential bactericidal molecule.

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