CFTR modulator therapy impacts neutrophil CD39 expression in cystic fibrosis - Supplemental Data and Methodology

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Levels of eATP are tightly regulated through direct hydrolysis via the action of cell surface ectonucleotidases including CD39, however, a role for CD39 as an influencer of inflammation in CF is underexplored.
In the present study we sought to evaluate the impact of CFTR modulator therapy on eATP levels and CD39 expression. Detailed protocols of this study are outlined in the online Supplementary Methodology File. A flow chart of patients' samples processed and experiments analysed is presented in Supplementary Data File (Supplemental Figure 1).
To understand the basis for increased eATP release by CF neutrophils, we assessed membrane CD39 levels.
Prompted by results suggesting cleavage of membrane CD39 from CF neutrophils, we investigated the protease involved in CD39 release. In light of previous published findings of increased release of neutrophil elastase (NE) by CF neutrophils, we hypothesized that NE may cleave CD39, an effect potentially abated by inclusion of the NE inhibitor, alpha-1 antitrypsin (AAT). In support of this theory, recombinant NE cleaved CD39 from healthy control cells in vitro (Supplemental Figure 2).
This study concludes by stating that extracellular nucleotides were previously detected in sputum and BAL fluid of patients with CF, and in the current study correlated with sputum neutrophil counts. In the current study apparent proteolytic cleavage decreased membrane expression of CD39. Accordingly, sCD39 was detected in CF airway and plasma samples.