Inflammatory Activity of Epithelial Stem Cell Variants from Cystic Fibrosis Lungs Is Not Resolved by CFTR Modulators

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

Lung Stem Cell Heterogeneity in Advanced Cystic Fibrosis

Despite congenital CFTR mutations and the severity of advanced cystic fibrosis (CF), the onset and progression of lung disease in these patients is not uniform. This phenomenon raises the possibility that symptomatic CF involves contributions to the disease process of an epigenetic nature beyond those conferred by CFTR mutations themselves. As the first test of this hypothesis, I asked in my studies how lung stem cells from patients with end-stage CF differ from those of normal patients without CFTR mutations. Using technology that enables single stem cell cloning, we found that in addition to the normal lung stem cell (NM) of control patients that can differentiate to cells of terminal bronchiolar and alveolar lineages, end-stage CF patients are dominated by five additional stem cell variants including ones committed to goblet cell metaplasia (GCM), GCM marked by constitutive inflammation (iGCM), squamous cell metaplasia (SCM), SCM marked by inflammatory gene expression (iSCM), and finally, one that is apparently normal but with constitutive inflammation (iNM). Importantly, stem cell libraries generated from all four of the CF patients express each of these stem cell types, and xenografts of these libraries yield epithelial structures marked by massive intra-luminal leukocyte infiltration and extensive submucosal fibrosis. Xenografting of individual NM, iNM, GCM, iGCM, SCM, and iSCM clones has revealed that fibrosis is primarily mediated by SCM and iSCM while the leukocyte attraction is triggered by iNM, iGCM, and iSCM. Long-term passaging of these clonal types revealed that these phenotypes were absolute and likely epigenetically committed. Remarkably, similar variants pre-exist as minor constituents in control lung and fetal lung that conceivably act as sentinels for pathogen incursions. And while these individual clones and the collective library of such clones recapitulate some of the major pathological features of the CF lung including inflammation, correction of CFTR deficiency via lentiviral transduction has no impact on the proinflammatory phenotype of these variant epithelial clones. Our conclusions from these studies are that end-stage CF patients display a heterogeneity of lung stem cells that is epigenetic in origin and includes variants that conceivably contribute to the progression of CF disease presentation