Prolonged airway explant culture enables study of health, disease, and viral pathogenesis.

In vitro models play a major role in studying airway physiology and disease. However, the native lung's complex tissue architecture and non-epithelial cell lineages are not preserved in these models. Ex vivo tissue models could overcome in vitro limitations, but methods for long-term maintenance of ex vivo tissue has not been established. We describe methods to culture human large airway explants, small airway explants, and precision-cut lung slices for at least 14 days. Human airway explants recapitulate genotype-specific electrophysiology, characteristic epithelial, endothelial, stromal and immune cell populations, and model viral infection after 14 days in culture. These methods also maintain mouse, rabbit, and pig tracheal explants. Notably, intact airway tissue can be cryopreserved, thawed, and used to generate explants with recovery of function 14 days post-thaw. These studies highlight the broad applications of airway tissue explants and their use as translational intermediates between in vitro and in vivo studies.

BPIFB1 loss alters airway mucus properties and diminishes mucociliary clearance.

Airway mucociliary clearance (MCC) is required for host defense and is often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B protein levels using genetic approaches. Here, we show that BPIFB1 is required for effective MCC in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in epithelial ion transport or reduced ciliary beat frequency. Loss of BPIFB1 in vivo and in vitro altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and the protein mesh of secreted mucus in human airway epithelia cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.NEW & NOTEWORTHY BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC.

Mucolytic treatment of chronic rhinosinusitis in a murine model of primary ciliary dyskinesia.

Background: Genetic defects in motile cilia cause primary ciliary dyskinesia (PCD), a rare disease with no specific therapeutics. Individuals with PCD often have impaired fertility and laterality defects and universally suffer from upper and lower airway diseases. Chronic rhinosinusitis is a universal feature of PCD, and mucus accumulation and subsequent infections of the sinonasal cavity cause significant morbidity in individuals with PCD. Despite this, there are no approved treatments that specifically target mucus. Objective: The goals of this study were to determine whether computed tomography (CT) imaging could be used to quantify mucus accumulation and whether the use of a mucolytic agent to reduce disulfide cross-links present in mucins would improve the effectiveness of nasal lavage at removing mucus in a murine model of PCD. Methods: Adult mice with a deletion of the axonemal dynein Dnaic1 were imaged using CT scanning to characterize mucus accumulation. The animals were then treated by nasal lavage with saline, with/without the disulfide-reducing agent tris(2-carboxyethyl)phosphine. Post-treatment CT scans were used to quantify improvement in the sinonasal cavity. Results: Mucus accumulation in the nasal cavity was readily quantified by CT. Compared to sham-treated control animals, nasal lavage with/without a mucolytic agent resulted in a significant reduction of accumulated mucus (p < 0.01). Treatment with the mucolytic agent showed a greater reduction of accumulated mucus than treatment with saline alone. Conclusion: The results suggest that inclusion of a mucolytic agent may increase the effectiveness of nasal lavage at reducing mucus burden in PCD.

Chronic airway epithelial hypoxia exacerbates injury in muco-obstructive lung disease through mucus hyperconcentration.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O2, airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na+ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of ß and γENaC (epithelial Na+ channel) subunit expression. The combination of increased Na+ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Regional Differences in Mucociliary Clearance in the Upper and Lower Airways.

As the nasal cavity is the portal of entry for inspired air in mammals, this region is exposed to the highest concentration of inhaled particulate matter and pathogens, which must be removed to keep the lower airways sterile. Thus, one might expect vigorous removal of these substances via mucociliary clearance (MCC) in this region. We have investigated the rate of MCC in the murine nasal cavity compared to the more distal airways (trachea). The rate of MCC in the nasal cavity (posterior nasopharynx, PNP) was ∼3-4× greater than on the tracheal wall. This appeared to be due to a more abundant population of ciliated cells in the nasal cavity (∼80%) compared to the more sparsely ciliated trachea (∼40%). Interestingly, the tracheal ventral wall exhibited a significantly lower rate of MCC than the tracheal posterior membrane. The trachealis muscle underlying the ciliated epithelium on the posterior membrane appeared to control the surface architecture and likely in part the rate of MCC in this tracheal region. In one of our mouse models (Bpifb1 KO) exhibiting a 3-fold increase in MUC5B protein in lavage fluid, MCC particle transport on the tracheal walls was severely compromised, yet normal MCC occurred on the tracheal posterior membrane. While a blanket of mucus covered the surface of both the PNP and trachea, this mucus appeared to be transported as a blanket by MCC only in the PNP. In contrast, particles appeared to be transported as discrete patches or streams of mucus in the trachea. In addition, particle transport in the PNP was fairly linear, in contrast transport of particles in the trachea often followed a more non-linear route. The thick, viscoelastic mucus blanket that covered the PNP, which exhibited ∼10-fold greater mass of mucus than did the blanket covering the surface of the trachea, could be transported over large areas completely devoid of cells (made by a breach in the epithelial layer). In contrast, particles could not be transported over even a small epithelial breach in the trachea. The thick mucus blanket in the PNP likely aids in particle transport over the non-ciliated olfactory cells in the nasal cavity and likely contributes to humidification and more efficient particle trapping in this upper airway region.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

Mice with a Deletion of Rsph1 Exhibit a Low Level of Mucociliary Clearance and Develop a Primary Ciliary Dyskinesia Phenotype.

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype-phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1-/- cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1-/- cells was significantly lower than in control cells (20.2 ± 0.8 vs. 25.0 ± 0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1-/- animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1-/- animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1-/-). Thus, Rsph1-/- mice display a PCD phenotype similar to but less severe than that observed in Dnaic1-/- mice, similar to what has been observed in humans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may be important to maintain this low amount of clearance.

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance.

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.

Mucociliary Clearance in Mice Measured by Tracking Trans-tracheal Fluorescence of Nasally Aerosolized Beads.

Mucociliary clearance (MCC) is the first line of defense in clearing airways. In genetically engineered mice, each component of this system (ciliary beat, mucus, airway surface hydration) can be studied separately to determine its contribution to MCC. Because MCC is difficult to measure in mice, MCC measurements are often omitted from these studies. We report a simple method to measure MCC in mice involving nasal inhalation of aerosolized fluorescent beads and trans-tracheal bead tracking. This method has a number of advantages over existing methods: (1) a small volume of liquid is deposited thus minimally disturbing the airway surface; (2) bead behavior on airways can be visualized; (3) useful for adult or neonatal mice; (4) the equipment is relatively inexpensive and easily obtainable. The type of anesthetic had no significant effect on the rate of MCC, but overloading the airways with beads significantly decreased MCC. In addition, the rate of bead transport was not different in alive (3.11 mm/min) vs recently euthanized mice (3.10 mm/min). A 5-min aerosolization of beads in a solution containing UTP significantly increased the rate of MCC, demonstrating that our method would be of value in testing the role of various pharmacological agents on MCC.

Lung disease phenotypes caused by overexpression of combinations of α-, ß-, and γ-subunits of the epithelial sodium channel in mouse airways.

The epithelial Na+ channel (ENaC) regulates airway surface hydration. In mouse airways, ENaC is composed of three subunits, α, ß, and γ, which are differentially expressed (α > ß > γ). Airway-targeted overexpression of the ß subunit results in Na+ hyperabsorption, causing airway surface dehydration, hyperconcentrated mucus with delayed clearance, lung inflammation, and perinatal mortality. Notably, mice overexpressing the α- or γ-subunit do not exhibit airway Na+ hyperabsorption or lung pathology. To test whether overexpression of multiple ENaC subunits produced Na+ transport and disease severity exceeding that of ßENaC-Tg mice, we generated double (αß, αγ, ßγ) and triple (αßγ) transgenic mice and characterized their lung phenotypes. Double αγENaC-Tg mice were indistinguishable from WT littermates. In contrast, double ßγENaC-Tg mice exhibited airway Na+ absorption greater than that of ßENaC-Tg mice, which was paralleled by worse survival, decreased mucociliary clearance, and more severe lung pathology. Double αßENaC-Tg mice exhibited Na+ transport rates comparable to those of ßENaC-Tg littermates. However, αßENaC-Tg mice had poorer survival and developed severe parenchymal consolidation. In situ hybridization (RNAscope) analysis revealed both alveolar and airway αENaC-Tg overexpression. Triple αßγENaC-Tg mice were born in Mendelian proportions but died within the first day of life, and the small sample size prevented analyses of cause(s) of death. Cumulatively, these results indicate that overexpression of ßENaC is rate limiting for generation of pathological airway surface dehydration. Notably, airway co-overexpression of ß- and γENaC had additive effects on Na+ transport and disease severity, suggesting dose dependency of these two variables.

Dietary Salt Exacerbates Experimental Colitis.

The Western diet is characterized by high protein, sugar, fat, and low fiber intake, and is widely believed to contribute to the incidence and pathogenesis of inflammatory bowel disease (IBD). However, high sodium chloride salt content, a defining feature of processed foods, has not been considered as a possible environmental factor that might drive IBD. We set out to bridge this gap. We examined murine models of colitis on either a high salt diet (HSD) or a low salt diet. We demonstrate that an HSD exacerbates inflammatory pathology in the IL-10-deficient murine model of colitis relative to mice fed a low salt diet. This was correlated with enhanced expression of numerous proinflammatory cytokines. Surprisingly, sodium accumulated in the colons of mice on an HSD, suggesting a direct effect of salt within the colon. Similar to the IL-10-deficient model, an HSD also enhanced cytokine expression during infection by Salmonella typhimurium This occurred in the first 3 d of infection, suggesting that an HSD potentiates an innate immune response. Indeed, in cultured dendritic cells we found that high salt media potentiates cytokine expression downstream of TLR4 activation via p38 MAPK and SGK1. A third common colitis model, administration of dextran sodium sulfate, was hopelessly confounded by the high sodium content of the dextran sodium sulfate. Our results raise the possibility that high dietary salt is an environmental factor that drives increased inflammation in IBD.

Reduced mucociliary clearance in old mice is associated with a decrease in Muc5b mucin.

Respiratory infections are a major cause of morbidity and mortality in the elderly. Previous reports have suggested that mucociliary clearance (MCC) is impaired in older individuals, but the cause is unclear. To unravel the mechanisms responsible for the age-associated decline in MCC, we investigated the MCC system in young (3 mo) and old (2 yr) C57BL/6 mice. We found that old mice had significantly reduced MCC function in both the upper and lower airways compared with young mice. Measurement of bioelectric properties of isolated tracheal and bronchial tissue revealed a significant decrease in Cl(-) secretion, suggesting that the older mice may have a reduced ability to maintain a sufficiently hydrated airway surface for efficient MCC. Ciliary beat frequency was also observed to be reduced in the older animals; however, this reduction was small relative to the reduction in MCC. Interestingly, the level of the major secreted mucin, Muc5b, was found to be reduced in both bronchioalveolar lavage and isolated tracheal tissue. Our previous studies of Muc5b(-/-) mice have demonstrated that Muc5b is essential for normal MCC in the mouse. Furthermore, examination of Muc5b(+/-) and wild-type animals revealed that heterozygous animals, which secrete ∼50% of the wild-type level of Muc5b, also demonstrate a markedly reduced level of MCC, confirming the importance of Muc5b levels to MCC. These results demonstrate that aged mice exhibit a decrease in MCC and suggest that a reduced level of secretion of both Cl(-) and Muc5b may be responsible.

Loss of Cftr function exacerbates the phenotype of Na(+) hyperabsorption in murine airways.

Airway surface hydration depends on the balance between transepithelial Na(+) absorption and Cl(-) secretion. In adult mice, absence of functional cystic fibrosis transmembrane conductance regulator (Cftr) fails to recapitulate human cystic fibrosis (CF) lung disease. In contrast, overexpression of the epithelial Na(+) channel ß subunit in transgenic mice (ßENaC-Tg) produces unregulated Na(+) hyperabsorption and results in CF-like airway surface dehydration, mucus obstruction, inflammation, and increased neonatal mortality. To investigate whether the combination of airway Na(+) hyperabsorption and absent Cftr-mediated Cl(-) secretion resulted in more severe lung pathology, we generated double-mutant ΔF508 CF/ßENaC-Tg mice. Survival of ΔF508 CF/ßENaC-Tg mice was reduced compared with ßENaC-Tg or ΔF508 CF mice. Absence of functional Cftr did not affect endogenous or transgenic ENaC currents but produced reduced basal components of Cl(-) secretion and tracheal cartilaginous defects in both ΔF508 CF and ΔF508 CF/ßENaC-Tg mice. Neonatal ΔF508 CF/ßENaC-Tg mice exhibited higher neutrophilic pulmonary inflammation and club cell (Clara cell) necrosis compared with ßENaC-Tg littermates. Neonatal ΔF508 CF/ßENaC-Tg mice also exhibited spontaneous bacterial infections, but the bacterial burden was similar to that of ßENaC-Tg littermates. Adult ΔF508 CF/ßENaC-Tg mice exhibited pathological changes associated with eosinophilic crystalline pneumonia, a phenotype not observed in age-matched ßENaC-Tg mice. Collectively, these data suggest that the combined abnormalities in Na(+) absorption and Cl(-) secretion produce more severe lung disease than either defect alone. Airway cartilage abnormalities, airway cell necrosis, and exaggerated neutrophil infiltration likely interact with defective mucus clearance caused by ßENaC overexpression and absent CFTR-mediated Cl(-) secretion to produce the increased neonatal mortality observed in ΔF508 CF/ßENaC-Tg mice.

Regional differences in rat conjunctival ion transport activities.

Active ion transport and coupled osmotic water flow are essential to maintain ocular surface health. We investigated regional differences in the ion transport activities of the rat conjunctivas and compared these activities with those of cornea and lacrimal gland. The epithelial sodium channel (ENaC), sodium/glucose cotransporter 1 (Slc5a1), transmembrane protein 16 (Tmem16a, b, f, and g), cystic fibrosis transmembrane conductance regulator (Cftr), and mucin (Muc4, 5ac, and 5b) mRNA expression was characterized by RT-PCR. ENaC proteins were measured by Western blot. Prespecified regions (palpebral, fornical, and bulbar) of freshly isolated conjunctival tissues and cell cultures were studied electrophysiologically with Ussing chambers. The transepithelial electrical potential difference (PD) of the ocular surface was also measured in vivo. The effect of amiloride and UTP on the tear volume was evaluated in lacrimal gland excised rats. All selected genes were detected but with different expression patterns. We detected αENaC protein in all tissues, ßENaC in palpebral and fornical conjunctiva, and γENaC in all tissues except lacrimal glands. Electrophysiological studies of conjunctival tissues and cell cultures identified functional ENaC, SLC5A1, CFTR, and TMEM16. Fornical conjunctiva exhibited the most active ion transport under basal conditions amongst conjunctival regions. PD measurements confirmed functional ENaC-mediated Na(+) transport on the ocular surface. Amiloride and UTP increased tear volume in lacrimal gland excised rats. This study demonstrated that the different regions of the conjunctiva exhibited a spectrum of ion transport activities. Understanding the specific functions of distinct regions of the conjunctiva may foster a better understanding of the physiology maintaining hydration of the ocular surface.

Human alveolar type II cells secrete and absorb liquid in response to local nucleotide signaling.

A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na(+) channel-mediated Na(+) absorption and cystic fibrosis transmembrane conductance regulator-mediated Cl(-) secretion, both significantly regulated by extracellular nucleotides. In addition, we observed in normal AT2 cells an absence of cystic fibrosis transmembrane conductance regulator regulation of epithelial Na(+) channel activity and an absence of expression/activity of reported calcium-activated chloride channels (TMEM16A, Bestrophin-1, ClC2, and SLC26A9), both features strikingly different from normal airway epithelial cells. Measurements of alveolar surface liquid volume revealed that normal AT2 cells: 1) achieved an extracellular nucleotide concentration-dependent steady state alveolar surface liquid height of ∼4 µm in vitro; 2) absorbed liquid when the lumen was flooded; and 3) secreted liquid when treated with UTP or forskolin or subjected to cyclic compressive stresses mimicking tidal breathing. Collectively, our studies suggest that human AT2 cells in vitro have the capacity to absorb or secrete liquid in response to local alveolar conditions.

Conditional deletion of dnaic1 in a murine model of primary ciliary dyskinesia causes chronic rhinosinusitis.

Studies of primary ciliary dyskinesia (PCD) have been hampered by the lack of a suitable animal model because disruption of essential ciliary genes in mice results in a high incidence of lethal hydrocephalus. To develop a viable mouse model for long-term studies of PCD, we have generated a transgenic mouse line in which two conserved exons of the mouse intermediate dynein chain gene, Dnaic1, are flanked by loxP sites (Dnaic1(flox/flox)). Dnaic1 is the murine homolog of human DNAI1, which is mutated in approximately 10% of human PCD cases. These mice have been crossed with mice expressing a tamoxifen-inducible Cre recombinase (CreER). Treatment of adult Dnaic1(flox/flox)/CreER(+/-) mice with tamoxifen results in an almost complete deletion of Dnaic1 with no evidence of hydrocephalus. Treated animals have reduced levels of full-length Dnaic1 mRNA, and electron micrographs of cilia demonstrate a loss of outer dynein arm structures. In treated Dnaic1(flox/flox)/CreER(+/-) animals, mucociliary clearance (MCC) was reduced over time. After approximately 3 months, no MCC was observed in the nasopharynx, whereas in the trachea, MCC was observed for up to 6 months, likely reflecting a difference in the turnover of ciliated cells in these tissues. All treated animals developed severe rhinosinusitis, demonstrating the importance of MCC to the health of the upper airways. However, no evidence of lung disease was observed up to 11 months after Dnaic1 deletion, suggesting that other mechanisms are able to compensate for the lack of MCC in the lower airways of mice. This model will be useful for the study of the pathogenesis and treatment of PCD.

Olfactory epithelia exhibit progressive functional and morphological defects in CF mice.

In normal nasal epithelium, the olfactory receptor neurons (ORNs) are continuously replaced through the differentiation of progenitor cells. The olfactory epithelium (OE) of the cystic fibrosis (CF) mouse appears normal at birth, yet by 6 mo of age, a marked dysmorphology of sustentacular cells and a dramatic reduction in olfactory receptor neurons are evident. Electroolfactograms revealed that the odor-evoked response in 30-day-old CF mice was reduced approximately 45%; in older CF mice, a approximately 70% reduction was observed compared with the wild type (WT) response. Consistent with studies of CF airway epithelia, Ussing chamber studies of OE isolated from CF mice showed a lack of forskolin-stimulated Cl(-) secretion and an approximately 12-fold increase in amiloride-sensitive sodium absorption compared with WT mice. We hypothesize that the marked hyperabsorption of Na(+), most likely by olfactory sustentacular cells, leads to desiccation of the surface layer in which the sensory cilia reside, followed by degeneration of the ORNs. The CF mouse thus provides a novel model to examine the mechanisms of disease-associated loss of olfactory function.

A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms.

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an approximately 5 x 10(3) strain PAO1 P. aeruginosa. Although bacteria grew to 10(10) cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical-chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.