Rationale: CFTR (
cystic fibrosis transmembrane conductance regulator) modulator drugs restore function to mutant channels in patients with
cystic fibrosis (CF) and lead to improvements in body mass index and lung function. Although it is anticipated that early childhood treatment with CFTR modulators will significantly delay or even prevent the onset of advanced
lung disease, lung neutrophils and inflammatory
cytokines remain high in patients with CF with established
lung disease despite modulator
therapy, underscoring the need to identify and ultimately target the sources of this
inflammation in CF lungs. Objectives: To determine whether CF lungs, like
chronic obstructive pulmonary disease (
COPD) lungs, harbor potentially pathogenic stem cell "variants" distinct from the normal p63/Krt5 lung stem cells devoted to alveolar fates, to identify specific variants that might contribute to the inflammatory state of CF lungs, and to assess the impact of CFTR genetic complementation or CFTR modulators on the inflammatory variants identified herein. Methods: Stem cell cloning technology developed to resolve pathogenic stem cell heterogeneity in
COPD and
idiopathic pulmonary fibrosis lungs was applied to end-stage lungs of patients with CF (three homozygous CFTR:F508D, one CFTR F508D/L1254X; FEV1, 14-30%) undergoing therapeutic
lung transplantation. Single-cell-derived clones corresponding to the six stem cell clusters resolved by single-cell
RNA sequencing of these libraries were assessed by
RNA sequencing and
xenografting to monitor
inflammation,
fibrosis, and
mucin secretion. The impact of CFTR activity on these variants after CFTR gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies. Measurements and Main Results: End-stage CF lungs display a stem cell heterogeneity marked by five predominant variants in addition to the normal lung stem cell, of which three are proinflammatory both at the level of gene expression and their ability to drive neutrophilic
inflammation in xenografts in immunodeficient mice. The proinflammatory functions of these three variants were unallayed by genetic or pharmacological restoration of CFTR activity. Conclusions: The emergence of three proinflammatory stem cell variants in CF lungs may contribute to the persistence of
lung inflammation in patients with CF with advanced disease undergoing CFTR modulator
therapy.