602 Optimization of cystic fibrosis transmembrane conductance regulatorG551D ferret model for early synchronized lung microbial colonization

425 Development of an AICDA-CreERT2 ferret model to study adaptive humoral immunity

258 Impact of temporal changes in cystic fibrosis transmembrane conductance regulator activity on fertility, conception, pregnancy, and lactation in ferrets with cystic fibrosis

episode of SARS-COV2 infection, which required adjustment of the dose administered. Conclusions: Our data confirm that ELX/TEZ/IVA treatment is safe, well tolerated, and effective in PwCF. ELX/TEZ/IVA improved pulmonary function and nutritional status and remarkably reduced hospitalization rate. Our data indicate that introduction of ELX/TEZ/IVA in CF care will radically change the natural history of and management approach to the disease. Background: Pulmonary ionocytes (PIs) are distinguished by high expression of the transcription factor FOXI1, V-ATPases, and other ion channels such as cystic fibrosis (CF)transmembrane conductance regulator (CFTR). Consistent with this expression pattern, ionocytes in fish and frogs have confirmed roles in regulating salt and fluid movement, but in vivo functions of ionocytes in mammalian respiratorysystems are 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 wild-type (WT) (n=4), FOXI1-knockout (KO) (n =4), and FOXI1 -Cre ERT2 ::ROSA-TG (n =3) ferrets using 10x droplet-based 3 ′ small conditional ribonucleic acid sequencing. Methods for enrichment of the population of rare PIs in our dataset involve upstream isolation of traced (enhanced green fluorescent protein-positive [EGFP+]) ionocytes by fluorescence-activated cell sorting (FACS). Results: We partitioned ferret airway epithelial cells (FAECs) into seven distinct clusters that we annotated based on known gene expression signatures for basal, secretory, ciliated, hillock, neuroendocrine, tuft, and ionocyte cells. 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 sixATP6V1familyprotonpumps,chloride channels (CFTR, CLCNKB, BSND), four family solute carriers (SLCs), sodium channels (SCNN1G, SCNN1A), potassium channels (KCNK5, KCNK1), consistent with unique functions in ion transport. Consistent with other species, CFTR was highlyexpressed in ferret PIs. We also observed low levels of CFTR in other cell types. Re-clusteringof PI transcriptional signatures suggested three subtypes. Type A ionocytes contained 58 differentiallyexpressed genes,manyof which were associated with oxidative phosphorylation, oxidative stress response, and tricarboxylic acid (TCA) cycle signaling pathway using ingenuity pathway analysis. Type-A ionocytes were also associated withcell cycle activation of chromosomal replication,suggesting that this subtype mayproliferate,and a subsetof FOXI1 -Cre ERT2 ::ROSA-TG – traced ionocytes in air-liquid interface cultures were EGFP+Ki67+. Type B ionocytes contained 228 differentially expressed genes, including the cation transporter ATP13A5, solute carrier genes SLC38A2 and SLC20A1, potassium channel KCNK5, and major histocompatibility complex II antigen presentation genes (CD74, H2-Aa, H2-Eb1). The Type B ionocytes were also enriched genes relevant to Type C ionocytes showed 35 differentially expressed genes having biological functions closelyassociated 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 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, B, and C, that indicate further cellular specializations in mitochondrial function and replication, antigen presentation, and cell motility, respectively. Background: Cystic fibrosis (CF) transmembrane-conductance regulator (CFTR) is a multi-transmembrane protein that functions as a chloride/ bicarbonate channel and plays an important role in maintaining water homeostasis on the apical surface of epithelial cells throughout the whole body. Mutations in the cftr gene leading to loss of or defective CFTR function causes CF. Among the efforts to enhance mutant CFTR expression and function on the plasma membrane, intensive studies have focused on the cellular mechanisms mediating CFTR degradation, but CFTR biogenesis machineries are not well understood, and better understanding of these mechanisms could lead to a new strategy to enhance mutant CFTR expression.Endoplasmicreticulum(ER)membraneproteincomplex(EMC) is a well-conserved ubiquitous protein complex expressed on the ER Methods: Inthisstudy,wegeneratedintestinalepithelialcell – specificEMC subunit 3 (EMC3) knockout (KO) mice by crossing EMC3 flox mice with Villin-Cre mice. Intestinal crypts were harvested and analyzed by reverse transcription polymerase chain reaction, mass spectrometry and western blotting. Intestinal organoids were used to study CFTR function through cyclic adenosine monophosphate (cAMP)- and calcium-dependent path-ways and to monitor calcium flux between wild-type (WT) and KO mice. Results: Although EMC3 KO micewere viable afterbirth, they were smaller than their WT littermates, indicating a functional impairment of intestinal epithelium in the EMC3 KO mice. Molecular analysis of the protein profile collected by mass spectrometry of intestinal crypts from these mice revealed downregulation of the EMC complex, CFTR, and many other ion transporters. Western blot confirmed significant lower CFTR protein in EMC3 KO villi, whereas CFTR transcription was not altered. Forskolin- and cpt-cAMP-stimulated intestinal organoid fluid secretion was greatly reduced and delayed (Figure 1A). We found that EMC3 deficiency completely inhibited carbachol-mediated intestinal organoid fluid secre- tion (Figure 1B), whereas muscarinic receptors (carbachol receptors) were upregulated at transcriptional and translational levels in EMC3 KO villi. These dataindicatethatdownstream signalingofmuscarinic receptorswas in EMC3 KO epithelial cells, and muscarinic receptor – specific agonist Oxotremorine M failed to induce intracellular calcium flux fluid secretion in EMC3 KO organoids. Pathway analysis of the above mass spectrometry data supported these

665 Single-cell ribonucleic acid sequencing reveals pulmonary ionocytes subtypes in proximal ferret airway epithelium

Background: Osmoregulation, which maintains the osmolarity of fluid surrounding cells, is a key feature of ionocytes across species and has been studied extensively in fish adaptation to environmental changes in salinity.In the mammalian kidney, osmoregulation is coordinated by principal cells and intercalated cells of the collecting ducts, which control water movement; acid-base regulation; and Na + , Cl -, K + , and Ca 2+ homeostasis.Given the overlap in expressed channels between pulmonary ionocytes and kidney principal cells and intercalated cells, we hypothesized that pulmonary ionocytes may also participate in osmoregulation by airway epithelia.Methods: To mimic fish gill ionocytes in freshwater and seawater environments, we created hypertonic and hypotonic air-liquid interface (ALI) culture media.FOXI1-Cre ERT2 lineage-traced proximal airway stem cells were differentiated under hypertonic or hypotonic conditions in ALI culture.At full differentiation (21 days), we quantified ionocyte numbers and ionocyte subtype gene expression patterns.Results: We exposed actively differentiating ferret basal cells to slightly hypertonic media (+77 mOsm/L NaCl) and observed a marked increase (7.32-fold; p < 0.0015) in the number of lineage-traced pulmonary ionocytes at full differentiation (21 days).Under hyperosmotic stress, greater numbers of ionocytes was correlated with high mRNA expression of key ionocyte markers FOXI1 and ASCL3.We have previously shown that three ionocyte subtypes exist (Type-A, Type-B, Type-C) in ferret airway ALI culture.We found that hyperosmotic stress increased expression of Type-A (BSND) and Type-C (CXCL17) ionocyte marker genes but decreased expression of the Type-B ionocyte marker ID3.The observed downregulation in CFTR expression under hyperosmotic stress is consistent with the expansion of Type-C ionocytes, which express significantly ( p = 2 × 10 -11 ) less CFTR than other ionocyte subtypes.Hyperosmotic stress imposed on fully differentiated ALI cultures did not alter the frequency of lineage-traced pulmonary ionocytes, suggesting that a hyperosmotic environment affects progenitor cell specification of Type-A and Type-C ionocytes.Exposure of differentiating ferret basal cells to hypotonic media (-77 mOsm/L) also led to expansion of ATP6V1G3 + ionocytes when fully differentiated at the ALI.Ongoing gene expression profiling is defining the dominant ionocyte subtype under hypotonic conditions.Conclusions: These findings indicate that osmolarity affects basal cell specification of pulmonary ionocytes and suggests that there are specialized ionocyte functions that adapt to environmental changes in airway osmolarity.Hyperosmotic fluid at the basolateral surface of the airway would be expected to be extracted water from cells and the airway surface liquid (ASL), leading to cell shrinkage and ASL dehydration.Our data suggest that apical membrane Cl -permeability is constrained by CFTR-expressing ionocytes and that the observed hyperosmotic downregulation of CFTR may be a compensatory mechanism to limit ASL dehydration.This finding may be analogous to the inactivation of ionocyte CFTR expression in fish gills during seawater to freshwater transition. 

163 Ferret pulmonary ionocytes abundance is regulated by extracellular osmolarity in air-liquid interface culture

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