Select Amino Acid Formulation Influenced Anion Secretion Induced by Th2 Cytokines in Human Bronchial Epithelial Cells (HBECs)
Authors: Grosche Astrid , Damiano Angoli , Sasidharan Anusree , Xu Xiaodong , Vidyasagar Sadasivan 
Published: May 2022

Introduction

Airway surface liquid is tightly regulated by ion channels and/or transporters, and plays a critical role in inflammatory airway diseases, such as ARDS, and in edema fluid clearance during lung injury. Although beta‐adrenergic agonists have been shown to improve alveolar fluid clearance, they have limited effect in late stages of ARDS due to reduced epithelial beta‐adrenergic receptor function or agonist delivery to target regions. CFTR, CLCA2, TMEM16A, and alternative Cl‐/HCO3‐ exchangers (e.g., SLC4 family) are known to be expressed in lung epithelia. Their relative contribution during late stages of lung injury where TH2 cytokines such as IL13 are elevated is not fully understood. The expression pattern of these anion channels are dysregulated in ARDS, and restoration of channel activities could improve fluid accumulation within the lung. In the present study, various anion channel and/or exchanger activities were measured in HBECs exposed to IL13. We have shown that select amino acids influence anion channel activity. Therefore, the effect of a proprietary amino acid formulation on specific anion channels were studied in IL13‐treated HBECs.

Methods

Transepithelial short‐circuit current (Isc) was sequentially blocked using inhibitors for ENaC (benzamil), CFTR CFTRInh172), TMEM16A (CaCCInhA01) and Na+/K+/2Cl‐ co‐transporter (bumetanide) in differentiated HBECs mounted in Ussing chambers. Patch‐clamp whole‐cell recordings were performed to determine anion channel activity after IL13 treatment, and channel and/or exchanger expression was analyzed by immunofluorescence.

Results

IL13 increased benzamil‐insensitive (anion) Isc (87.5 ± 4.7 µA.cm‐2 vs 21.9 ± 1.8 µA.cm‐2; P <0.001) and decreased CFTR Isc (1.4 ± 0.52 µA.cm‐2 vs 12.2 ± 0.83 µA.cm‐2; P <0.001) compared to controls. Amino acids reduced benzamil‐insensitive Isc (55.9 ± 4.8 µA.cm‐2; P <0.001) and increased CFTR Isc (6.7 ± 0.75 µA.cm‐2; P <0.001). There was no change in TMEM16A Isc with IL13 treatment. Bumetanide‐sensitive Isc was higher in IL13‐treated cells compared to controls (52.5 ± 3.6 µA.cm‐2 vs 5.5 ± 0.62 µA.cm‐2; P <0.001) suggesting anion secretion via alternate mechanisms, and amino acids decreased bumetanide‐sensitive anion secretion (20.5 ± 2.9 µA.cm‐2; P <0.001). Similarly, whole‐cell patch clamp recordings showed that amino acids restored the CFTR response in IL13‐treated cells. Immunofluorescence imaging demonstrated decreased CFTR expression with IL13, while amino acids recovered CFTR at the apical membrane.

Conclusion

Treatment with IL13 increased anion secretion while reducing CFTR channel activity in HBECs. Amino acid formulation restored CFTR activity and decreased total anion current in the presence of IL13. These studies suggest anion secretion via alternate anion secretory pathways. Future studies should be directed at understanding the link between CFTR and alternate anion channels in conditions associated with elevated IL13, and how amino acids regulate these channel and/or exchanger functions