(665) Single-cell RNA sequencing reveals pulmonary ionocytes subtypes in proximal ferret airway epithelium

Background: Pulmonary ionocytes (PIs) are distinguished by high expression of the transcription factor FOXI1, V-ATPases, and other ion channels such as CFTR. Consistent with this expression pattern, ionocytes in fish and frogs have confirmed roles in regulating salt and fluid movement; however, in vivo functions of ionocytes in mammalian respiratory systems remains unclear. Here we transcriptionally profiled ferret airway epithelial cells together with FOXI1-Cre^ERT2 lineage traced PIs to infer potential roles in mammalian airway function.

Methods: We initially profiled 70,000 proximal tracheal basal cell derived epithelial cells from WT (n=4), FOXI1-KO (n=4) and FOXI1-Cre^ERT2::ROSA-TG ferrets (n=3) using 10x droplet-based 3’ scRNA-seq. Methods for enrichment of the population of rare PIs in our dataset involve upstream isolation of traced (EGFP+) ionocytes by FACS.

Results: We partitioned ferret airway epithelial cells (FAECs) into 7 distinct clusters that we annotated based on known gene expression signatures for basal, secretory, ciliated, hillock, neuroendocrine, tuft, and ionocyte. PIs were not captured from FOXI1-KO airway cultures, consistent with FOXI1 being required for PI specification. We profiled 256 PIs from WT and FOXI1-Cre^ERT2::ROSA-TG ferret airway cultures and defined their consensus expression signature. Ferret PIs express genes for 6 ATP6V1 family proton pumps, chloride channels (CFTR, CLCNKB and BSND), 4 SLC family solute carriers, sodium channels (SCNN1G and SCNN1A), potassium channels (KCNK5 and KCNK1), consistent with unique functions in ion transport. Consistent with other species, CFTR was highly expressed in ferret PIs. We also observed low levels of CFTR in other cell types. Re-clustering of PI transcriptional signatures suggested three subtypes: Type-A, Type-B and Type-C. Type-A ionocytes contained 58 genes differentially expressed (DE) genes, many of which were associated with oxidative phosphorylation, oxidative stress response and tricarboxylic acid (TCA) cycle signaling pathway using ingenuity pathway analysis (IPA). Type-A ionocytes were also associated with “cell cycle activation of chromosomal replication”, suggested this subtype may proliferate. Indeed, a subset of FOXI1-Cre^ERT2::ROSA-TG traced ionocytes in ALI cultures were EGFP+Ki67+. Type-B ionocytes contained 228 DE genes including the cation transporter ATP13A5, solute carrier genes SLC38A2 and SLC20A1, potassium channel KCNK5, and MHC II antigen presentation genes (CD74, H2-Aa, H2-Eb1). The Type-B ionocytes were also were enriched genes relevant to immune processing. Type-C ionocytes showed 35 DE genes having biological functions closely associated with cell movement, a finding consistent with dynamic movement of appendages in time-lapse live ionocyte imaging. The highly expressed SLC34A2 channel in Type-C ionocytes also suggests unique function in phosphate homeostasis.

Conclusions: Transcriptional profiling of PIs in ferrets indicates that they are enriched for the expression of ion channels that is consistent with a specialized functional role in ion transport in the proximal lung epithelium. We identify putative subtypes of ionocytes, Types-A-C, that indicate further cellular specializations in mitochondrial function and replication, antigen presentation, and cell motility, respectively.

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