Sphingomyelinase decreases transepithelial anion secretion in airway epithelial cells in part by inhibiting CFTR‐mediated apical conductance

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel whose dysfunction causes cystic fibrosis (CF). The loss of CFTR function in pulmonary epithelial cells causes surface dehydration, mucus build‐up, inflammation, and bacterial infections that lead to lung failure. Little has been done to evaluate the effects of lipid perturbation on CFTR activity, despite CFTR residing in the plasma membrane. This work focuses on the acute effects of sphingomyelinase (SMase), a bacterial virulence factor secreted by CF relevant airway bacteria which degrades sphingomyelin into ceramide and phosphocholine, on the electrical circuitry of pulmonary epithelial monolayers. We report that basolateral SMase decreases CFTR‐mediated transepithelial anion secretion in both primary bronchial and tracheal epithelial cells from explant tissue, with current CFTR modulators unable to rescue this effect. Focusing on primary cells, we took a holistic ion homeostasis approach to determine a cause for reduced anion secretion following SMase treatment. Using impedance analysis, we determined that basolateral SMase inhibits apical and basolateral conductance in non‐CF primary cells without affecting paracellular permeability. In CF primary airway cells, correction with clinically relevant CFTR modulators did not prevent SMase‐mediated inhibition of CFTR currents. Furthermore, SMase was found to inhibit only apical conductance in these cells. Future work should determine the mechanism for SMase‐mediated inhibition of CFTR currents, and further explore the clinical relevance of SMase and sphingolipid imbalances.

Mechanistic analysis and significance of sphingomyelinase‐mediated decreases in transepithelial CFTR currents in nHBEs

“…Two different methods for preparing and culturing primary airway epithelial cells were used, as described previously (Cottrill et al, 2021 ). One method is based on that developed in the Randell laboratory (Fulcher et al, 2005 ).…”

“…Recombinant wild‐type (WT) and enzyme‐dead H322A S. aureus SMase were purified from BL21 (DE3) E. coli bacteria similarly to previously described (Cottrill et al, 2021 ; Stauffer et al, 2017 ). Briefly, bacteria were grown using kanamycin sulfate (Thermo Fisher) selection and IPTG induction.…”

“…This indicates that CFTR is in one activatable state under control conditions, but a different and potentially common activatable state under the other conditions. sphingomyelins and increases all ceramides of interest (Cottrill et al, 2021). Analysis of nHAEs acutely treated with ceranib-1 indicated that ceranib-1 caused no significant effect on bulk cellular sphingomyelins or ceramides (Figure 3a,b).…”

“…The amplifier was set to voltage clamp at 0 mV to record short-circuit currents. Bath buffer was prepared according to the protocol described previously (Ito et al, 2004;Cottrill et al, 2021 Cells were stabilized for 30 min prior to treatment with 1 µg/ml WT or enzyme-dead H322A (mutant) SMase basolaterally for 30 min. To inhibit ENaC-mediated sodium currents, 20 µM amiloride was added apically.…”

“…Fortunately, highly effective modulator therapies have been developed recently that target the primary CFTR defect (Guimbellot et al, 2017 ; Joshi et al, 2019 ). However, we have shown that these modulator therapies are unable to recover a loss of CFTR currents and CFTR‐mediated apical membrane conductance following exposure to sphingomyelinase (SMase), a bacterial virulence factor (Cottrill et al, 2021 ; Stauffer et al, 2017 ). SMase converts sphingomyelin into ceramide and phosphocholine.…”