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Transgenic Ferret Models Define Pulmonary Ionocyte Diversity and Function

ABSTRACT: Speciation leads to adaptive changes in organ cellular anatomy and physiology. These evolutionary changes create challenges for studying rare cell type functions that diverge between human and mice. Rare CFTR-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of novel conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO), and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models to cystic fibrosis (CF) ferrets, we demonstrate that ionocytes control airway surface liquid (ASL) absorption, secretion, pH, and mucus viscosity—leading to reduced ASL volume and impaired mucociliary clearance in CF, FOXI1-KO, and FOXI1-CreERT2::CFTRL/L ferrets. These processes were regulated by CFTR- dependent ionocyte transport of Cl– and HCO3–, as determined electrophysiologically and by single-cell imaging with a conditionally activated halide fluorescent sensor. Single-cell transcriptomics, using pulse-seq of lineage-traced ionocyte-enriched cultures, revealed three subtypes of pulmonary ionocytes with unique biologic functions and a common rare cell progenitor of ionocytes, tuft, and neuroendocrine cells. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of CF airway disease. These studies provide a road map for using conditional genetics in the first non- rodent mammal to address gene function, cell biology, and disease processes that have greater evolutionary conservation between humans and ferrets.

ORGANISM(S): Mustela putorius furo

PROVIDER: GSE233654 | GEO | 2023/08/01

REPOSITORIES: GEO

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Project description:Background: CFTR modulators will decrease some etiologies of inflammation in the CF airway; however, current data indicate that non-resolving airway infection and inflammation will persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish CF airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation require a better understanding of the cellular contributions to chronic CF airway disease, and how cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, and represent intriguing targets for focused therapies to dampen CF airway inflammation. Material and Methods: In order to characterize the inflammatory phenotype of CF blood monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq). Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. Results: RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional pathways were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including transcriptional modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. These results demonstrate that ivacaftor causes acute changes in blood monocytes and plasma chemokines, and suggest that increased monocyte inflammatory signals and potentially improved trafficking to the lung contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of isolated blood monocytes in CF subjects.

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Transgenic Ferret Models Define Pulmonary Ionocyte Diversity and Function

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