Background: The adaptive immune system is increasingly appreciated for its contributions to the exuberant and maladaptive immune response in CF.We hypothesized that the CFTR functions as a negative regulator of T helper 2 (Th2) cell function, CFTR deficiency in mouse Th2 cells increases Th2 cytokine production, therapeutic CFTR modulation can decrease Th2 effector function, allergic inflammation increases in CF mice when challenged with inhaled allergen, and CFTR expression is regulated by a novel GATA binding site in Th2 cells.Methods: Naïve CD4+ T-cells from Cftr +/+ , Cftr -/-, and Cftr -/-murine spleens expressing genomic human CFTR (B6-Tg(CFTR)1Lcl/Cwr [hCFTR]) were isolated and cultured in Th2 polarizing conditions using plate-bound anti-CD3e and anti-CD28 in the presence of soluble anti-IFNγ and IL-4 for 5 days.Naïve hCFTR expressing murine CD4+ T-cells was treated with elexacaftortezacaftor-ivacaftor (ETI) or vehicle control at activation.On Day 3, CD4+ Tcells were restimulated with IL-4, and cytokine measurements were taken 48 hours later (Day 5).Flow cytometry was performed on Th2-polarized Cftr +/+ and Cftr -/-CD4+ T-cells using antibodies to CD3, CD4, GATA3, IL-5, and IL-13.Cftr +/+ and Cftr -/-mice were sensitized and challenged using Alternaria alternata to elicit an adaptive immune response.Bronchoalveolar lavage (BAL), protein was quantified from whole-lung homogenate, and histopathological analysis was performed.Chromatin immunoprecipitation (ChIP) of GATA-3-bound chromatin fragments was performed.Results: In Cftr -/-CD4+ T-cells, Cftr expression is tightly associated with Tcell activation and subsequent GATA-3 expression.ChIP analysis of a putative Gata binding motif at site -607 respective to the Cftr transcription start site demonstrated that GATA-3 binds to nucleosomal-bound DNA within the Cftr promoter to induce local chromatin rearrangement.Th2polarized CD4+ T-cells from Cftr -/-mice produce higher levels of IL-4, IL-5, and IL-13 in response to activation than Cftr +/+ CD4+ T-cells.ETI-treated hCFTR murine CD4+ T-cells secrete less IL-5 and IL-13 than vehicle controltreated hCFTR murine CD4+ T-cells.In vivo, Cftr -/-mice display greater inflammation by histology and greater Th2 cytokine production in response to Alternaria challenge than Cftr -/-mice.Conclusions: These findings demonstrate a previously undefined role for GATA-3 regulation of CFTR expression in CD4+ T-cells.These data suggest a role for CFTR as a negative regulator of Th2 effector function in CD4+ T-cells and resultant allergic inflammation, an effect reversed by current CFTR modulator therapies.These studies highlight a potential role for CFTR modulator therapies in decreasing allergic inflammation in people with CF.

Background: The fecal microbiota is reproducibly altered (dysbiosis is present) in people with CF, despite numerous and diverse medical interventions in this population.CF fecal dysbiosis is characterized by a decrease in the bacterial community complexity and conserved decreases and increases in several taxa, such as a marked expansion of Escherichia coli, which correlates with worse inflammation and, in infants, shorter stature, but we cannot infer cause and effect from these correlations.Therefore, to enable mechanistic studies with precise control of the microbiota in CF, we have generated a germ-free Cftr-null mouse model.Our published results show that this model replicates several features of fecal dysbiosis observed in humans, including a decrease in community complexity and an increase in E. coli.Methods: To limit the complexity of the microbial component, we colonized germfree CF and control non-CF mice with the defined, wellcharacterized, altered Schaedler flora (ASF) community that consists of eight bacteria.This consortium does not mediate colonization resistance.allowing us to further colonize the mice with E. coli.We then used the welldefined fluorescein isothiocyanate conjugated dextran (FITC-D) permeability assay to examine barrier function in this model.Results: We found that neither germfree nor ASF-colonized CF mice had higher serum FITC-D levels than control mice, although ASF + E. colicolonized CF mice had significantly higher serum FITC-D than control non-CF mice (median 7.2 vs. 1.0 μg/mL serum, P = 0.008), similar to what was seen with specific pathogen-free mice.We also found a higher load of E. coli in the contents of the large intestine (4.9 × 10 9 vs. 1.9 × 10 8 CFU/g content, P = 0.008).Conclusions: Expansion of E. coli in the CF gut is associated with pathology as measured by intestinal permeability.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, 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 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 with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis 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.

needed before phage therapy becomes the standard of care for treatment of multidrug-resistant infections in CF.

Background: The recently discovered pulmonary ionocyte is a rare cell type in the airway epithelium, comprising just 1% of all epithelial cells.Pulmonary ionocytes express the highest CFTR mRNA level of all airway epithelial cell types, and ionocyte dysfunction has been associated with development of phenotypes resembling CF lung disease [1, 2], but the function of pulmonary ionocytes is poorly understood, and their role in CF pathobiology is debated.Methods: Studying pulmonary ionocytes is complicated by how uncommon they are in the airway epithelia, and because they represent only a small fraction of the airway epithelia, other epithelial cell types can mask ionocyte function.Thus, we proposed studying ionocyte ion transport at the single-cell level.We used a high-resolution self-referencing ionselective microelectrode technique [3] to measure the ion transport (Na + , Cl -, H + ) properties of single ionocytes in primary human bronchial epithelial cell (hBEC) cultures.Results: Using microelectrode electrophysiology measurements and immunofluorescence staining, we investigated the ion flux properties and membrane transport systems involved in CFTR-mediated transport across ionocytes in CF and non-CF primary hBEC culture.We showed that V-type ATPase, CFTR, and anion exchanger 2 (SLC4A2) were expressed on the apical membrane of pulmonary ionocytes, but V-type ATPase did not contribute to the forskolin+IBMX-triggered H + flux across ionocytes.On the other hand, SLC4A2 drove the secretion of bicarbonate (HCO 3 -) in exchange

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.

nonsense mutation along the gene sequence enables investigation of a longer truncated protein.Methods: We created a germline mutation at p.W1093X that corresponds to human p.W1098X CFTR in Sprague Dawley rats using CRISPR-Cas9 tools.CFTR W1098X /+ rats were inbred to generate homozygous knock-in animals.These rats and littermates received ground chow mixed with laxative-filled water to maintain growth and minimize mortality.Newborn rats were genotyped and characterized for CFTR activity by Ussing chamber electrophysiology of tracheal explants.Airway surface liquid (ASL) depth, periciliary layer (PCL) height, and mucus transport (MCT) on excised trachea were quantified with micro-optical coherence tomography (μOCT) imaging, and lung function was assessed by flexivent oscillometry in anesthetized animals.Results: Newborn W1098X CFTR rats exhibited intestinal obstruction that was partially relieved with a specialized diet and osmotic laxatives.These rats displayed phenotypic anomalies characteristic of CF, such as slow growth and tooth enamel defects, but the histology of the intestine, vas deferens, pancreas, liver, and lungs in these animals was normal.In contrast to G542X and CFTR KO rats, they displayed significant CFTR activation in response to forskolin-nearly one-third that of wt counterparts.There was no response to VX-770 potentiator because wt rat CFTR does not respond to this drug, but unlike wt rat CFTR, there was no response to two commonly used CFTR-specific inhibitors (GlyH101 and Inh-172), suggesting an absence of adequate binding sites in this truncated CFTR channel.Consistent with other premature termination codon (PTC) mutations, mRNA abundance for W1098X CFTR was substantially reduced, yet the protein level was detectable.μOCT image analysis of excised trachea from 2-month-old rats indicated physiologic levels of ASL and PCL depths and MCT matching that of wt counterparts.At 6 months of age, these rats exhibited greater airway resistance and less inspiratory capacity than wt littermate controls.Conclusions: W1098X rats partially reproduce the phenotypes observed in CFTR KO and G542X animals and represent a novel animal model with likely truncated yet partially functional CFTR.Thus, this model is uniquely suited to offer new insights into disease biology from rare PTC mutations and is a valuable tool for testing the efficacy of emerging treatments, including genetic therapies, in a model that reflects residual CFTR function or partial CFTR restoration from ongoing treatment with currently approved CFTR modulators.

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: Cystic fibrosis (CF) is a monogenic lung disease caused by dysfunction of the CF transmembrane conductance regulator (CFTR) anion channel, resulting in significant morbidity and mortality.The progress of elucidating the role of CFTR using established animal and cell-based models led to the recent discovery of effective modulators for most individuals with CF, but a subset of individuals with CF do not respond to these modulators, and there is an urgent need to develop novel therapeutic strategies.Methods: Here, we have assembled a panel of induced pluripotent stem cells (iPSCs) derived from individuals with common or rare CFTR variants representative of three distinct classes of CFTR dysfunction.Using new advances in iPSC-directed differentiation protocols, we have generated airway epithelial cells from 12 iPSC lines encompassing unaffected (healthy) donors and donors with Class 1 to 3 CFTR mutations.We have adapted two functional assays for detection of baseline and rescued function of the CFTR channel.Results: In a three-dimensional spheroid assay (Figure 1a) using forskolininduced swelling and planar cultures composed of polarized mucociliary airway epithelial cells (Figure 1b), we quantified CFTR baseline function and response to CFTR modulators and detected genotype-specific differences.We detected CFTR genotype-specific responses that show negligible function at baseline for Phe508del, G551D, W1282X, and G542X samples, but after treatment with known therapeutics, we detected functional rescue of Phe508del and G551D samples.Treatment of premature termination codon (PTC) variants (W1282X, G542X) with experimental CFTR therapeutics (such as nonsense-mediated decay inhibitors and PTC readthrough agents) partially restores function in iPSC-derived airway epithelial cells.Comparison with primary W1282X human bronchial epithelial cells showed a remarkable similarity in amplitude and pattern of response to combinatorial therapeutics (Figure 1c).Conclusions: Our results demonstrate the potential of the human iPSC platform as a research tool to study CF and to accelerate therapeutic development for CF caused by rare variants.