(399) Dynamic Regulation of ASL pH by TMEM16A in Nasal Epithelia Derived from CF Donors with Rare Class I Mutations.

Background: Airway surface liquid (ASL) pH is more acidic in CF due to faulty CFTR, which contributes to mucus stasis and bacterial infection. ASL pH is therefore a potential factor that could be targeted for future CF therapies. However, we lack a complete understanding of the complex mechanisms underlying ASL pH regulation. In addition to CFTR, possible targets, such as Na⁺/H⁺-exchanger, K⁺H⁺-ATPase, SLC26 ion transporters and TMEM16A, a calcium activated chloride channel, have all been suggested to regulate ASL pH, as well as the paracellular ion shunt. Currently the role of TMEM16A in the ASL pH regulation is unclear, so our aim here was to investigate its potential role in acid-base homeostasis in fully differentiated nasal CF airway cultures derived from donors with class I mutations, under both normal and inflammatory conditions.

Methods: TMEM16A (T16A) knock-out (KO) cells were produced by CRISPR-Cas9 gene-editing of CF-null basal cells from 3 donors with class I mutations. Cells were differentiated at an air-liquid interface for 4 weeks. ASL pH was measured in real-time using a fluorescent plate-reader under thin film conditions [1]. Transepithelial resistance (TEER) and transepithelial ion-transport (short circuit current; Isc) was evaluated by Ussing chamber. Fluorescein flux was used to assess paracellular transport. To induce a pro-inflammatory phenotype, and increase the expression of T16A, cell cultures were treated with IL-4 [10ng/mL] for 48hrs.

Results: Baseline ASL pH in CF nasal epithelia was unaffected by IL-4 treatment. However, it was significantly increased in T16A KO cells, under both normal and IL-4 conditions. The Ca²⁺ agonist, carbachol, caused a small, but not significant, acidification of ASL pH under all the conditions. In contrast, the subsequent addition of the cAMP-agonist, forskolin (FSK), alkalinized ASL pH, a response that was significantly enhanced in IL-4 treated epithelia, but which was unaffected by T16A KO. FSK failed to increase Isc under all conditions tested. To further evaluate the role of T16A in ASL pH modulation other Ca²⁺ agonists were tested. The SERCA pump inhibitor CPA markedly alkalinized ASL pH but only in CF epithelia pre-treated with IL-4, and importantly this response was absent in T16A KO cultures. Complimentary Isc studies showed that CPA induced a T16A-dependent Isc. While the putative T16A activator E_act alkalinized ASL pH under all conditions, including in T16A KO cultures, it caused a marked, time-dependent, drop in TEER, and a corresponding increase in fluorescein flux, indicating a negative effect on epithelia integrity.

Conclusions: Our studies show that genetic KO of T16A increased baseline ASL pH under all conditions suggesting that T16A indirectly contributes to steady-state ASL pH homeostasis. T16A did not contribute to the FSK-induced alkalinization. A direct role for T16A in ASL pH regulation was only detected using agonists that generate a sustained rise in cytosolic Ca²⁺. The E_act-induced alkalinization was not due to T16A but was likely an off-target effect on tight junctions. Overall, our results show that T16A only plays a direct role in ASH pH regulation under inflammatory conditions, which suggests this channel may be an alternative target for ASL pH therapy in CF.

Acknowledgements: Supported by the CF Trust [SRC013]

References: [1] Saint-Criq V, Haq IJ, Gardner AI, et al. Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells. J Vis Exp. 2019(148). doi:10.3791/59815