Troy D. Rogers

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 ≈5 × 103 strain PAO1 P. aeruginosa. Although bacteria grew to 1010 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.

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.

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 β subunit in transgenic mice (βENaC-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/βENaC-Tg mice. Survival of ΔF508 CF/βENaC-Tg mice was reduced compared with βENaC-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/βENaC-Tg mice. Neonatal ΔF508 CF/βENaC-Tg mice exhibited higher neutrophilic pulmonary inflammation and club cell (Clara cell) necrosis compared with βENaC-Tg littermates. Neonatal ΔF508 CF/βENaC-Tg mice also exhibited spontaneous bacterial infections, but the bacterial burden was similar to that of βENaC-Tg littermates. Adult ΔF508 CF/βENaC-Tg mice exhibited pathological changes associated with eosinophilic crystalline pneumonia, a phenotype not observed in age-matched βENaC-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 βENaC overexpression and absent CFTR-mediated Cl(-) secretion to produce the increased neonatal mortality observed in ΔF508 CF/βENaC-Tg mice.

Expression of CFTR from a ciliated cell-specific promoter is ineffective at correcting nasal potential difference in CF mice.

Successful gene therapy will require that the therapeutic gene be expressed at a sufficient level in the correct cell type(s). To improve the specificity of gene transfer for cystic fibrosis (CF) and other airway diseases, we have begun to develop cell-type specific promoters to target the expression of transgenes to specific airway cell types. Using a FOXJ1 promoter construct previously shown to direct transgene expression specifically to ciliated cells, we have generated transgenic mice expressing human cystic fibrosis transmembrane conductance regulator (CFTR) in the murine tracheal and nasal epithelia. RNA analysis demonstrated levels of CFTR expression is greater than or equal to the level of endogenous mouse CFTR. Immunoprecipitation and western blotting demonstrated the production of human CFTR protein, and immunochemistry confirmed that CFTR was expressed in the apical region of ciliated cells. The transgenic animals were bred to CFTR null mice (Cftrtm1Unc) to determine if expression of CFTR from the FOXJ1 promoter is capable of correcting the airway defects in Cl− secretion and Na+ absorption that accompany CF. Isolated trachea from neonatal CF mice expressing the FOXJ1/CFTR transgene demonstrated a correction of forskolin-stimulated Cl− secretion. However, expression of human CFTR in ciliated cells of the nasal epithelia failed to significantly change the nasal bioelectrics of the CF mice.

Ion transport across CF and normal murine olfactory and ciliated epithelium

The nasal epithelium of the cystic fibrosis (CF) mouse has been used extensively in CF research because it exhibits ion transport defects similar to those of human CF airways. This tissue is composed of approximately 50% olfactory (OE) and approximately 50% ciliated epithelium (CE), and on the basis of previous observations, we hypothesized that a significant fraction of the bioelectric signals from murine nasal tissue may arise from OE rather than CE, while CE is the target tissue for CF gene therapy. We compared the bioelectric properties of isolated OE from the nasal cavity and CE from the nasopharynx in Ussing chamber studies. Hyperabsorption of Na(+) [amiloride response; CF vs. wild type (WT)] was approximately 7.5-fold greater in the OE compared with the CE. The forskolin response in native tissues did not reliably distinguish genotypes, likely due to a cyclic nucleotide-gated cation conductance in OE and a calcium-mediated Cl(-) conductance in CE. By potential difference assay, hyperabsorption of Na(+) (CF vs. WT) and the difference in response to apical 0 Cl(-) buffer (CF vs. WT) were approximately 2-fold greater in the nasal cavity compared with the nasopharynx. Our studies demonstrate that in the CF mouse, both the hyperabsorption of Na(+) and the Cl(-) transport defect are of larger magnitude in the OE than in the CE. Thus, while the murine CF nasal epithelium is a valuable model for CF studies, the bioelectrics are likely dominated by the signals from the OE, and assays of the nasopharynx may be more specific for studying the ciliated epithelium.

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.

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, βENaC 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.

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.

Restoring ciliary function to differentiated Primary Ciliary Dyskinesia cells with a lentiviral vector

Primary ciliary dyskinesia (PCD) is a genetically heterogenous autosomal recessive disease in which mutations disrupt ciliary function, leading to impaired mucociliary clearance and life-long lung disease. Mouse tracheal cells with a targeted deletion in the axonemal dynein intermediate chain 1 (Dnaic1) gene differentiate normally in culture but lack ciliary activity. Gene transfer to undifferentiated cultures of mouse Dnaic1−/− cells with a lentiviral vector pseudotyped with avian influenza hemagglutinin restored Dnaic1 expression and ciliary activity. Importantly, apical treatment of well-differentiated cultures of mouse Dnaic1−/− cells with lentiviral vector also restored ciliary activity, demonstrating successful gene transfer from the apical surface. Treatment of Dnaic1flox/flox mice expressing an estrogen-responsive Cre recombinase with different doses of tamoxifen indicated that restoration of ∼20% of ciliary activity may be sufficient to prevent the development of rhinosinusitis. However, although administration of a β-galactosidase-expressing vector into control mice demonstrated efficient gene transfer to the nasal epithelium, treatment of Dnaic1−/− mice resulted in a low level of gene transfer, demonstrating that the severe rhinitis present in these animals impedes gene transfer. The results demonstrate that gene replacement therapy may be a viable treatment option for PCD, but further improvements in the efficiency of gene transfer are necessary.