Loss of Cftr function exacerbates the phenotype of Na hyperabsorption in murine airways

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1 Am J Physiol Lung Cell Mol Physiol 304: L469 L480, First published February 1, 2013; doi: /ajplung Loss of Cftr function exacerbates the phenotype of Na hyperabsorption in murine airways Alessandra Livraghi-Butrico, 1 Elizabeth J. Kelly, 1 Kristen J. Wilkinson, 1 Troy D. Rogers, 1 Rodney C. Gilmore, 1 Jack R. Harkema, 2 Scott H. Randell, 1 Richard C. Boucher, 1 Wanda K. O Neal, 1 and Barbara R. Grubb 1 1 Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and 2 Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan Submitted 7 May 2012; accepted in final form 28 January 2013 Livraghi-Butrico A, Kelly EJ, Wilkinson KJ, Rogers TD, Gilmore RC, Harkema JR, Randell SH, Boucher RC, O Neal WK, Grubb BR. Loss of Cftr function exacerbates the phenotype of Na hyperabsorption in murine airways. Am J Physiol Lung Cell Mol Physiol 304: L469 L480, First published February 1, 2013; doi: /ajplung 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 subunit in transgenic mice ( ENaC-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 doublemutant F508 CF/ ENaC-Tg mice. Survival of F508 CF/ ENaC-Tg mice was reduced compared with ENaC-Tg or F508 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 F508 CF and F508 CF/ ENaC-Tg mice. Neonatal F508 CF/ ENaC-Tg mice exhibited higher neutrophilic pulmonary inflammation and club cell (Clara cell) necrosis compared with ENaC-Tg littermates. Neonatal F508 CF/ ENaC-Tg mice also exhibited spontaneous bacterial infections, but the bacterial burden was similar to that of ENaC-Tg littermates. Adult F508 CF/ ENaC-Tg mice exhibited pathological changes associated with eosinophilic crystalline pneumonia, a phenotype not observed in age-matched ENaC-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 ENaC overexpression and absent CFTR-mediated Cl secretion to produce the increased neonatal mortality observed in F508 CF/ ENaC-Tg mice. genetically modified mice; cystic fibrosis; F508 CFTR; ENaC; airway inflammation; airway epithelia necrosis HYDRATION OF AIRWAY SURFACES, which is critically important for mucociliary clearance and lung health, is achieved by balancing transepithelial Cl secretion and Na absorption. Defective camp-mediated Cl secretion due to loss of cystic fibrosis Address for reprint requests and other correspondence: A. Livraghi-Butrico, Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, The Univ. of North Carolina at Chapel Hill, 6029 Thurston Bowles Bldg., Chapel Hill, NC ( alessandra_livraghi@med.unc.edu). transmembrane conductance regulator (CFTR) function and poorly regulated Na absorption through the epithelial Na channel (ENaC) both contribute to impaired airway surface hydration in cystic fibrosis (CF) (34, 42). Several Cftr-targeted murine models have been developed to study CF lung disease pathogenesis and test therapeutic strategies (reviewed in Refs. 14, 30, 44). Although Cftr-deficient mice exhibit intestinal and nasal phenotypes consistent with human CF (11), the lower airways of Cftr-deficient mice do not exhibit CF-like obstructive lung disease. Tracheas from adult CF mice also do not exhibit defects in either camp-mediated Cl secretion nor Na hyperabsorption (39), likely due to low endogenous Cftr expression in adult tracheal tissue combined with expression of alternative Cl channels, e.g., Tmem16a (39). In contrast, neonatal murine tracheas have a more robust Cftr mrna expression ( 2-fold more than adult tracheas; W. K. O Neal, unpublished observations), and CF neonatal mice exhibit the classic defect in camp-mediated Cl secretion, with a forskolin response reduced approximately fourfold compared with wild-type (WT) mice (38, 39). Nonetheless, neonatal CF mice have no detectable lung disease. In contrast to nasal epithelia, the lower airways of CF mice do not exhibit dysregulated Na absorption at any age (38). The importance of dyrsegulated Na absorption in eliciting a CF-like lung disease in the mouse lower airways is supported by the phenotype of ENaC-transgenic ( ENaC-Tg) mice, in which club cell (Clara cell)-targeted overexpression of the epithelial Na channel subunit ( ENaC, encoded by the Scnn1b gene) produces dysregulated Na absorption and causes defective airway mucus clearance, susceptibility to spontaneous bacterial infection, and airway inflammation that resembles human CF lung disease (27, 31, 33). ENaC-Tg mice exhibit forskolin- and UTP-regulated Cl secretory responses similar to WT mice (31), indicating that lung disease occurs in the presence of normal Cftr and Tmem16a function. To test the hypothesis that the combination of defective Cl secretion with abnormally regulated Na absorption would produce lung disease more severe than defects in either pathway alone, particularly in the neonatal period when Cftr is most highly expressed in mouse lower airways, we crossed ENaC-Tg mice with mice carrying the F508 Cftr mutation [Cftr tm1kth (46), hereafter referred to as F508 CF]. We studied the airway bioelectric properties of neonatal and adult F508 CF/ ENaC-Tg mice in Ussing chambers and evaluated the severity of lung disease by measuring histopathology, inflammatory infiltrate, and lung bacterial burden /13 Copyright 2013 the American Physiological Society L469

2 L470 MATERIALS AND METHODS Animals. Animals were maintained and studied under protocols approved by the University of North Carolina Institutional Animal Care and Use Committee, and experiments were performed according to the principles outlined by the Animal Welfare and the National Institutes of Health guidelines for the care and use of animals in biomedical research. All mice were housed in individually ventilated microisolator cages in a specific pathogen-free facility maintained at the University of North Carolina at Chapel Hill, on a 12-h day-night cycle. Mice were fed a regular chow diet and given water ad libitum. Weaned mice and breeders homozygous for the murine Cftr F508 mutation were given water supplemented with Colyte to reduce complications due to intestinal obstruction (7). To generate CF/ ENaC-Tg mice, we sequentially bred congenic C57BL/6N ENaC-Tg mice (line 6608) (28), with congenic C57BL/6J male Cftr F508 Cftr tm1kth mice (46) and obtained mice of four different genotypes with respect to murine Cftr F508 and ENaC-Tg expression: 1) F508/wt, wt (referred to as WT); 2) F508/wt, ENaC-Tg (referred to as ENaC-Tg); 3) F508/ F508, wt (referred to as F508 CF); 4) F508/ F508, ENaC-Tg (referred to as F508 CF/ ENaC-Tg). At birth all pups were toe clipped for identification and genotyping, as previously described (31, 46). Mice studied were littermates, age matched, and of both sexes. When used for Ussing chamber experiments, all mouse pups were euthanized by an overdose of ketamine/xylazine and adult mice were euthanized by CO 2 inhalation. When used for bronchoalveolar lavage and histopathology, mice were euthanized by exsanguination under deep 2,2,2-tribromoethanol (Avertin) anesthesia. Bioelectric studies. Mouse pups remained with the mother until time of study. Adult mice were studied at the ages indicated in figure legends. All studies were done blinded with respect to genotype. Details of the Ussing chamber preparations have been previously published (13). Amiloride (10 4 M apical addition) was used to block electrogenic Na absorption. Forskolin (10 5 M apical) and UTP (10 4 M apical) were used to induce anion secretion via an increase in intracellular camp and intracellular Ca 2 concentrations, respectively. Bumetanide (10 4 M) was added to the basolateral bath to inhibit Cl entry through the Na-K-2Cl cotransporter (NKCC1). All drugs were purchased from Sigma Aldrich with the exception of UTP (Amersham Pharmacia Biotech). BAL. For 10-day-old or older mice, bronchoalveolar lavage (BAL) cell counts and lung histology were obtained from the same animal, as described in Ref. 28. Because of their small size, 5-day-old pups were subject to either whole lung lavage or fixation for histology, but not both. Routinely, lungs were immersion-fixed in 10% neutral-buffered formalin (NBF) to prevent dislodging of airway luminal contents. For lung volume measurements, lungs were inflated with 10% NBF at a constant pressure (25 cmh 2O) and fixed in situ before removal en bloc. After wet trimming of the heart, lung volume was measured by water displacement, as previously described (33). Measurement of cytokines and chemokines in BALF. Aliquots of cell-free BAL fluid (BALF), stored at 80 C, were used to measure mouse TNF- (tumor necrosis factor-, Tnf), KC (keratinocytederived cytokine, Cxcl1), MIP-2 (macrophage inflammatory protein, Cxcl2), MIP-1 (macrophage inflammatory protein-1, Ccl3), M-CSF (macrophage colony-stimulating factor, Csf1), MCP-1 (monocyte chemoattractant protein-1, Ccl2), LIX (lipopolysaccharide-induced CXC chemokine, Cxcl5), IL-6 (interleukin-6, Il6), IL-1 (interleukin- 1, Il1a), IL-1 (interleukin-1, Il1b), GM-CSF (granulocyte-macrophage colony-stimulating factor, Csf2), and G-CSF (granulocyte colony-stimulating factor, Csf3) via a bead-based assay (Upstate- Millipore Beadlyte multiplex assays/luminex, Billerica, MA) according to the manufacturer instructions. Bacteriology. BAL was performed aseptically and plated, and colony-forming units (CFUs) were enumerated, classified on the basis of their morphology, and processed for molecular identification through ribosomal 16S gene PCR amplification and sequencing, as previously described (27). Lung histology. Fixed lungs were embedded, sectioned, and stained with hematoxylin and eosin (H&E) and Alcian blue-periodic acid Schiff staining (AB-PAS), and lung pathology was graded as previously described (28). Bronchiolar epithelial cell necrotic degeneration was quantified morphometrically by counting epithelial necrotic cells lining the bronchiolar airways and expressing the data per millimeter of the basement membrane, as described in Ref. 33. Tissue blocks received a numerical code at time of embedding, and scoring of the slides was performed by an investigator blinded to specimen genotype. Immunohistochemistry. Immunohistochemical staining for Ym1/2 and club cell secretory protein (CCSP) were performed on formalinfixed, paraffin-embedded lung sections by using rabbit polyclonal anti-ym1 antibody and anti-club cell secretory protein (anti-ccsp; Seven Hills Bioreagents, Cincinnati, OH) as described in Refs. 33 and 8, respectively Statistics. Data are shown as means SE, with the number of preparations or mice (n). Survival curves were compared by Kaplan- Meier log rank analysis and Holm-Sidak multiple comparison with the overall significance level For bioelectric studies, a one-way ANOVA was used to determine whether there was an overall significant difference among the groups, in which case the Holm-Sidak test was used for all pairwise comparisons. The 2 test was used when comparing frequencies of observed events. ANOVA followed by Tukey s post hoc test for multiple comparisons were used to determine significant differences among groups for body weight, lung volume, cytokine levels, necrotic epithelial cells, BAL cells, and CFU counts. P 0.05 was considered statistically significant. RESULTS F508 CF/ ENaC-Tg mice exhibited reduced survival compared with both F508 CF and ENaC-Tg littermates. As previously reported, both genetic modifications, i.e., ENaC overexpression and functional deletion of Cftr, reduced survival compared with WT littermates [Fig. 1A, 72 5%, for ENaC-Tg (26) and 55 7% for F508 CF (46) mice at postnatal day (PND) 20]. Notably, F508 CF/ ENaC-Tg mice survival was lower than either F508 CF or ENaC-Tg mice. In particular, survival of F508 CF/ ENaC-Tg mice resembled that of F508 CF mice at PND 1 10, but after PND 10 the survival of F508 CF/ ENaC-Tg mice significantly decreased and only 30 6% survived to PND 20. F508 CF, ENaC-Tg, and F508 CF/ ENaC-Tg mice all exhibited reduced body weight at PND 5 compared with WT littermates (Fig. 1B). F508 CF/ ENaC-Tg mice weighed less than ENaC-Tg mice, but their body weight was not different from that of F508 CF mice. F508 CF/ ENaC-Tg mice airway bioelectric properties reflect the combined expression of defective Cftr-dependent Cl secretion and excessive ENaC-dependent Na absorption. The tracheal bioelectric properties of 3-day-old mice for the four genotypes are shown in Fig. 2A. F508 CF mice exhibited a trend toward reduction of the basal short-circuit current (I sc ) but no change in amiloride-sensitive ENaC-mediated current compared with WT mice. The postamiloride residual I sc, the forskolin-stimulated I sc, and the bumetanide-inhibitable I sc were reduced in F508 CF compared with WT mice, consistent with reduced Cftr-mediated Cl secretion. As expected, the basal I sc and the amiloride-sensitive I sc were significantly elevated in ENaC-Tg compared with WT mice, and the fold increase in amiloride-sensitive I sc, i.e., Na absorption, with-

3 L471 significant difference in the magnitude of the bumetanide responses between groups at PND 10. Tracheas of 30-day-old mice maintained most bioelectric features observed for the different genotypes at earlier ages (Fig. 2C). The basal I sc was again significantly reduced in F508 CF tracheas compared with WT and F508 CF/ ENaC-Tg tracheas and remained elevated in ENaC-Tg compared with WT, F508 CF, and F508 CF/ ENaC-Tg tracheas. Again, the amiloride response was significantly greater in the two groups expressing the ENaC transgene, compared with WT and F508 CF mice. The residual I sc was still significantly attenuated in both groups of F508 CF mice, but the response to forskolin did not differ among the four groups. In general, the UTP responses increased with age and the Fig. 1. Gross findings in double-mutant F508 CF/ ENaC-Tg mice compared with wild-type (WT), F508 CF (mice carrying the F508 Cftr mutation), and ENaC-Tg (transgenic mice with overexpression of the epithelial Na channel subunit) littermates. A: survival curves for mice from the F508 CF ENaC-Tg cross indicate reduced survival in F508 CF/ ENaC-Tg mice ( F/ ENaC) compared with F508 CF ( F) and ENaC-Tg ( ENaC) littermates. *P 0.05 vs. WT mice, #P 0.05 vs. ENaC-Tg mice. B: comparison of body weights at 5 days of age shows decreased body weight in F508 CF, ENaC-Tg, and F508 CF/ ENaC-Tg mice. *P 0.05 vs. WT mice, #P 0.05 vs. ENaC-Tg mice. out changes in forskolin- or UTP-stimulated I sc, was similar to that previously reported (31). F508 CF/ ENaC-Tg tracheas exhibit bioelectric properties of both F508 CF mice, with decreased postamiloride residual I sc, forskolin-stimulated I sc, and bumetanide-sensitive I sc, and ENaC-Tg mice, with elevated amiloride-sensitive I sc. However, F508 CF/ ENaC-Tg tracheas did not exhibit the elevated residual I sc typical of neonatal ENaC-Tg tracheas, indicating that Cftr contributes to the basal Cl secretory current. In addition, the basal I sc of F508 CF/ ENaC-Tg tracheas was significantly lower than that of ENaC-Tg tracheas and not different from WT, suggesting that the raised Na currents due to ENaC overexpression were offset by reduced basal Cftr-mediated Cl secretion. Thus the 3-day-old F508 CF/ ENaC-Tg tracheas exhibited properties of both accelerated Na absorption and decreased Cl secretion. The bioelectric properties of tracheas from pups that reached 10 days of age were similar to those at 3 days (Fig. 2B). The basal I sc of each group was significantly different from each of the other groups. The basal I sc of both groups carrying the ENaC transgene were significantly elevated compared with the other two groups, and the F508 CF/ ENaC-Tg mice exhibited a trend toward reduced basal I sc compared with the ENaC-Tg mice. As noted above, this trend likely reflects decreased Cftr-mediated Cl secretion, as the basal I sc of CF mice was also reduced compared with WT mice. Unlike 3-day-old tracheas, the UTP response was significantly enhanced in 10-day-old F508 CF tracheas compared with WT (P 0.01), with all other groups being similar. There was no Fig. 2. F508 CF/ ENaC-Tg mice exhibit increased Na absorption and defective Cl secretion. Ion transport properties of freshly excised tracheal tissues of 3-day-old mice (A), 10-day-old mice (B), and adult mice (C) from the F508 CF ENaC-Tg cross. Basal indicates the short-circuit current (I sc) before drug application. The change in I sc ( ) after sequential drug addition is shown. Residual I sc is the I sc remaining after amiloride application. *P 0.05 vs. WT mice, #P 0.05 vs. ENaC-Tg mice, P 0.05 vs. ENaC-Tg mice in B, and P 0.05 vs. F508 CF ( F) and F508 CF/ ENaC-Tg ( F/ ENaC) mice in C.

4 L472 individual responses in each group differed significantly from each other, with the F508 CF tracheas exhibiting a significantly greater UTP response than the other groups. The response to bumetanide did not differ among the four groups at this age. F508 CF/ ENaC-Tg mice exhibit tracheal abnormalities similar to F508 CF mice and more severe lung pathology compared with ENaC-Tg littermates. At dissection for Ussing chamber studies, we observed mucus plugs in 14 20% of 3-day-old F508 CF/ ENaC-Tg and ENaC-Tg, whereas no mucus plug was found in the tracheas of F508 CF or WT pups. The morphology of tracheal tissues was examined and gross defects that resulted in narrowing of the trachea were observed in the cartilaginous tracheas of some F508 CF and F508 CF/ ENaC-Tg mice (Fig. 3, A and B, comparing WT and F508 CF/ ENaC-Tg mice, respectively). In both F508 CF and F508 CF/ ENaC-Tg mice exhibiting this phenotype, the cartilaginous rings in the upper trachea were discontinuous, resulting in a significantly decreased upper tracheal diameter (0.045 vs cm for WT, n 3 and CF, n 4, respectively, P 0.007). This defect was confined to the upper third of the trachea, and there was no difference in lower tracheal or bronchial diameter between WT and Cftr-deficient pups. Although there was a trend for the F508 CF/ ENaC-Tg tracheas to exhibit this defect more frequently (7/9 pups; 77%) than F508 CF neonatal tracheas (3/9 pups; 33%), the difference was not significant ( 2 ). In contrast, tracheas of WT or ENaC-Tg pups did not show tracheal defects. Histologically, the lungs of 5-day-old WT and F508 CF pups exhibited no phenotypic abnormalities, whereas lungs of both ENaC-Tg and F508 CF/ ENaC-Tg pups exhibited air space enlargement, bronchial airway inflammation, and airway epithelial necrotic degeneration, which are all features of the lung phenotype originally described for neonatal ENaC-Tg mice (33). In particular, the degree of air space enlargement, observed histologically (Fig. 3C, compare WT and F508 CF top with ENaC and F508 CF/ ENaC bottom) and measured as a function of lung volume (Fig. 3D), was similar between ENaC-Tg and F508 CF/ ENaC-Tg mice. In contrast, the airways of neonatal F508 CF/ ENaC-Tg mice were characterized by focal areas of massive intraluminal neutrophil aggregation (Fig. 3C, arrows and high magnification inset), which were never observed in ENaC-Tg, WT or F508 CF mice. Moreover, F508 CF/ ENaC-Tg mice exhibited an increased incidence of necrotic airway epithelial cells compared with ENaC-Tg littermates (Fig. 4, A D). Consistent with the previously described developmental pattern of airway mucus obstruction in ENaC-Tg mice (28, 33, 43), which features a progression from the trachea in the early neonatal period (PND 5 10) to the intrapulmonary bronchi as the mice age, no AB-PAS-positive material was found in the lungs of 5-day-old ENaC-Tg or F508 CF/ ENaC-Tg mice. Because of the extremely poor survival of F508 CF/ ENaC-Tg mice (Fig. 1A), it was difficult to obtain mice at later time points. However, since Cftr-deficient mice on the C57BL/6 background have been reported to develop some lung pathology later in life (10, 21), we studied the few F508 CF/ ENaC-Tg mice that survived until 10 mo of age. Compared with the phenotype of age-matched F508 CF and ENaC-Tg mice (Fig. 5, A and B, respectively), the lesions associated with the F508 CF/ ENaC-Tg genotype were much more severe. All 10-mo-old F508 CF/ ENaC-Tg mice exhibited multiple lung lesions (Fig. 5, C F), strikingly resembling the pathology of eosinophilic crystalline pneumonia (18). In particular, lung histology revealed major areas of lobar consolidation (Fig. 5C). Airways and alveoli were filled with mucus and a mixed inflammatory cell exudate containing hypertrophic macrophages, neutrophils, eosinophils, and eosinophilic crystals (Fig. 5, D F), which have been previously identified as consisting of chitinases YM1/2 (33). Strong YM1/2 immunoreactivity was found in hypertrophic macrophages and epithelial cells (Fig. 5G). Club cell secretory protein accumulated in the lumen of F508 CF/ ENaC-Tg mice airways and was entrapped in mucus (Fig. 5H). F508 CF/ ENaC-Tg mice also had significant proliferation of bronchial associated lymphoid tissue (BALT, Fig. 5, C and I) with Russell bodies, i.e., inclusions found in plasma cells undergoing excessive synthesis of immunoglobulins [these inclusions appear eosinophilic if stained with H&E (Fig. 5J) or glycoprotein-rich if stained with AB-PAS (Fig. 5K)]. These findings suggest a robust immune response in F508 CF/ ENaC-Tg mice. F508 CF/ ENaC-Tg mice exhibit increased pulmonary inflammation compared with F508 CF and ENaC-Tg littermates. Evaluation of the inflammatory infiltrate was performed by BAL cell counts in 5-day-old pups and 10-mo-old mice from the F508 CF ENaC-Tg cross. As shown in Fig. 6A, ENaC-Tg and F508 CF/ ENaC-Tg pups had significantly more neutrophils than WT and F508 CF mice, and F508 CF/ ENaC-Tg pups had more neutrophils than ENaC-Tg littermates. Note that the degree of neutrophilia shown in Fig. 6A for F508 CF/ ENaC-Tg mice is likely underestimated, because the luminal neutrophil aggregates present in F508 CF/ ENaC-Tg airways (Fig. 3C) were not readily displaced by the BAL procedure, as assessed histologically in lungs subjected to BAL (data not shown). Multiplex profiling of inflammatory mediators in BAL fluid harvested from 5-day-old mice indicated that TNF-, KC, MIP-2, LIX, G-CSF, M-CSF, and MIP-1 were similarly elevated in ENaC-Tg and F508 CF/ ENaC-Tg mice compared with either F508 CF or WT mice (Fig. 6B). The only exception was IL-6, which was significantly elevated in F508 CF/ ENaC-Tg mice compared with WT, F508 CF, or ENaC-Tg mice. F508 CF/ ENaC-Tg mice had higher MCP-1 compared with WT mice and, although not significant, a slight increase in IL-1 was also noted. BAL analysis confirmed that 10-mo-old F508 CF/ ENaC-Tg mice had significantly more severe lung inflammation than age-matched ENaC-Tg, F508 CF, or WT mice (Fig. 6C). In particular, the number of macrophages and neutrophils was significantly higher than F508 CF or ENaC-Tg mice, and eosinophils were still detectable in F508 CF/ ENaC-Tg mice, an unusual finding, since in congenic C57BL/6N ENaC-Tg mice early postnatal eosinophilia is transient and usually wanes after 6 wk of age (26). Interestingly, F508 CF mice exhibited an increase in macrophage number compared with WT or ENaC-Tg mice, in agreement with data previously reported for a different strain of Cftr F508 mice (23). However, in this study we did not observe any appreciable pathological change in the lungs of F508 CF mice (Fig. 5J).

5 DOUBLE-MUTANT F508 CF/ ENAC-TRANSGENIC MICE L473 Fig. 3. F508 CF/ ENaC-Tg mice exhibited airway and lung pathology distinct from F508 CF and ENaC-Tg mice. A and B: representative photomicrographs of tracheas from 3-day-old WT (A) and F508 CF/ ENaC-Tg (B) mice, illustrating tracheal abnormalities in the latter. Tracheas from F508 CF mice exhibited the same tracheal defect as in B. Scale bar 100 m. C: representative photomicrographs of lung histology in 5-day-old WT, F508 CF ( F), ENaC-Tg ( ENaC), and F508 CF/ ENaC-Tg ( F/ ENaC) mice. Hematoxylin and eosin (H&E) stain, low magnification, scale bar 200 m. Rectangles indicate areas shown at higher magnification in the insets, scale bar 20 m. Arrows indicate dense neutrophil aggregates, commonly found in F508 CF/ ENaC-Tg mice airway lumen. D: comparison of lung volumes at 5 days of age shows equivalent air space enlargement in ENaC-Tg and F508 CF/ ENaC-Tg mice. *P 0.05 vs. WT mice.

6 L474 Fig. 4. Prominent airway epithelial cell necrosis in F508 CF/ ENaC-Tg mice. A C: representative photomicrographs of airway epithelia from 5-day-old F508 CF ( F; A), ENaC-Tg ( ENaC; B), and F508 CF/ ENaC-Tg ( F/ ENaC; C) mice. H&E stain, scale bar 20 m. D: morphometric quantification of necrotic cells in 5-day-old mice. *P 0.05 vs. WT mice, #P 0.05 vs. ENaC-Tg mice. F508 CF/ ENaC-Tg mice exhibit spontaneous lung bacterial infection similar to ENaC-Tg littermates. As recently reported, neonatal ENaC-Tg mice routinely exhibit spontaneous bacterial infection with a predominance of Streptococcus, Pasteurella, and Actinobacillus species (27). Quantification of the bacterial load among genotypes, using culture conditions that permitted detection of nonfastidious aerobes and microaerophilic bacteria, revealed that WT and F508 CF mice exhibited little or no evidence of infection. In contrast, F508 CF/ ENaC-Tg mice exhibited infection, with both bacterial counts (Fig. 7A) and lung microflora composition (Fig. 7B) similar to ENaC-Tg littermates. DISCUSSION Effective airway mucus clearance is dependent on adequate airway surface hydration. In CF, the pathogenic failure of mucus clearance has been attributed to inadequate airway hydration caused by both defective Cl secretion, due to the absence of functional CFTR, and, although still controversial (6), enhanced Na absorption, due to unregulated ENaC activity (9). Models of inadequate airway hydration are thus needed to better understand the pathophysiology of CF and to aid the design of effective therapies. The various CF mouse models generated thus far have not exhibited, as adults, either defective camp-mediated Cl secretion nor Na hyperabsorption in the lower airways. We have reported, however, that neonatal CF pups do exhibit a defect in camp-mediated Cl secretion (38, 39). The availability of ENaC-Tg mice, which exhibit constitutive airway ENaC hyperactivity and CF-like lung pathology (31, 33), allowed us to generate a mouse model, i.e., the F508 CF/ ENaC-Tg mouse, that could, at least during the early postnatal period, recapitulate both ion transport defects that likely initiate CF lung disease. The F508 CF mouse model was chosen for these studies because of its availability on the C57BL/6 background and its potential usefulness in pharmacological studies with F508 CFTR correctors/potentiators. Survival analysis of mice generated from F508 CF ENaC-Tg crosses revealed distinct differences among the resulting genotypes (Fig. 1A). ENaC-Tg mice exhibited a small but significant drop in neonatal survival, which is typical for the congenic C57BL/6N strain (26). F508 CF mice exhibited survival consistent with that reported for this line at 20 days, i.e., a modest decrease that precedes the more significant drop (40%) occurring after weaning (46). Conversely, the survival decline in F508 CF/ ENaC-Tg mice appeared to be biphasic. During the first 10 days of life, both F508 CF and F508 CF/ ENaC-Tg pups exhibited similar declines in survival (Fig. 1A), likely reflecting a survival disadvantage due to a common dominant effect, i.e., failure to thrive imposed by the Cftr deletion (17). This notion is supported by the similar reduced weight for the two genotypes (Fig. 1B). However, after 10 days, F508 CF pups survival stabilized, whereas F508 CF/ ENaC-Tg pups suffered a further drop in survival. Histopathological and BAL data showed that, by PND 5, F508 CF/ ENaC-Tg pups had an inflammatory burden and severity of lung disease that exceeded that of ENaC-Tg pups (Fig. 3C, 4C, and 6A). Moreover, F508 CF/ ENaC-Tg pups exhibited the CF-related tracheal anatomical defect (Fig. 3B) previously described in CF mice (1) and pigs (35, 37). This defect may produce increased airways compliance and enhance airway closure, reducing the ability of F508 CF/ ENaC-Tg pups to clear lethal mucus plugs. Collectively, these data suggest that the second component of the decline in survival of F508 CF/ ENaC-Tg mice reflected more severe respiratory disease. The bioelectric properties of the genetically modified mice utilized in this study revealed three distinct patterns of ion transport defects with reference to WT. First, ENaC-Tg mice exhibited consistently elevated Na transport rates, slightly waning with time (from 3- to 4-fold of WT mice at PND 3 10 to 1.5-fold

7 DOUBLE-MUTANT F508 CF/ ENAC-TRANSGENIC MICE L475 Fig. 5. Surviving F508 CF/ ENaC-Tg mice exhibit severe lung pathology reminiscent of eosinophilic crystalline pneumonia. A and B: representative micrograph of lung histopathology in 10-mo-old F508 CF (A), ENaC-Tg (B), and F508 CF/ ENaC-Tg (C) mice. H&E stain, scale bar 100 m. C: lung pathology in adult F508 CF/ ENaC-Tg mice comprised airway mucus obstruction (arrow), bronchial-associated lymphoid tissue (BALT) (arrowhead), and extensive lobar consolidation. D: intraluminal mucus plugs (MP), granulocytes (G), hypertrophic macrophages (M), and eosinophilic crystals (C), scale bar 10 m. E and F: dense aggregates of hypertrophic and vacuolated macrophages in the parenchyma and airway lumen, respectively. G: substantial immunoreactivity for YM1/2 in alveolar macrophages (solid arrows) and epithelial cells (dotted arrows). H: immunoreactivity for CCSP in the lumen (solid arrows) and epithelial cells (dotted arrows). ep, Epithelium; aa, airway lumen; ap, alveolar parenchyma. I: BALT, adjacent a small bronchiole filled with hypertrophic macrophages. J and K: Russell bodies, large cytoplasmic inclusions identifiable as eosinophilic if stained with H&E (J) or glycoprotein-rich if stained with Alcian blueperiodic acid Schiff staining (AB-PAS; K). of WT mice at PND 30 40), and normal capacity for regulated Cl secretion. Second, F508 CF mice exhibited persistent defects in basal Cl secretion, abnormal forskolin-regulated secretion in neonates but not adults, persistent upregulation of UTPregulated Cl secretion and normal Na transport. Third, F508 CF/ ENaC-Tg exhibited the combined bioelectric hallmarks of ENaC-Tg and F508 CF mouse airways (Fig. 2A), i.e., increased amiloride-sensitive Isc, an index of raised Na transport, and reduced postamiloride residual current, an index of decreased Cl secretion. This pattern was observed in mice at PND 3 and

8 L476 Fig. 6. Quantification of airway inflammatory infiltrate and inflammatory mediators profile indicate more severe lung pathology in F508 CF/ ENaC-Tg mice. A: differential bronchoalveolar lavage (BAL) cell counts in 5-day-old WT, F508 CF ( F), ENaC-Tg ( ENaC), and F508 CF/ ENaC-Tg ( F/ ENaC) mice. *P 0.05 vs. WT mice, #P 0.05 vs. ENaC-Tg mice. B: BAL fluid (BALF) cytokines in 5-day-old mice. Dotted line represents the assay lower detection limit (LOD). *P 0.05 vs. WT mice, #P 0.05 vs. ENaC-Tg mice. TNF-, tumor necrosis factor- ; KC, keratinocyte-derived cytokine; MIP-2, macrophage inflammatory protein; LIX, lipopolysaccharide-induced CXC chemokine; MIP-1, macrophage inflammatory protein-1; G-CSF, granulocyte colonystimulating factor; M-CSF, macrophage colonystimulating factor; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; IL-1, interleukin-1 ; IL-1, interleukin-1 ; GM-CSF, granulocyte-macrophage colony-stimulating factor. C: differential BAL cell counts in 10-mo-old F508 CF and F508 CF/ ENaC-Tg mice. BAL cell counts in 12-mo-old WT and congenic C57BL/6N ENaC-Tg mice from (26) are plotted in the same graph for comparison. *P 0.05 vs. WT mice, #P 0.05 vs. ENaC-Tg mice. was similar at PND 10 and PND (Fig. 2, B and C). Notably, a selective increase in Na transport ( ENaC-Tg mice) was associated with moderate lung disease, whereas a selective defect in Cl secretion ( F508 CF mice) was not associated with disease. Importantly, despite the apparent absence of disease in F508 CF mice, the combined defects in both Na absorption and Cl secretion in F508 CF/ ENaC mice were associated with severe airways pathology.

9 Fig. 7. Spontaneous bacterial colonization in mice from the F508 CF ENaC-Tg cross. A: analysis of total colony forming units (CFU) in BAL samples from 5-day-old mice. (Log 10 1)-transformed data; n number of mice/group. *P 0.05 vs. WT littermates. B: individual CFUs and bacterial species isolated from 5-day-old mice. Each tick on the x-axis represents an individual mouse. L477 The combined Na and Cl transport defects could account for increased disease severity in F508 CF/ ENaC mice by multiple mechanisms. With respect to decreased survival, we speculate that mucus concentration was increased in F508 CF/ ENaC mice due to the combined airway surface liquid (ASL) volume deficits resulting from abnormalities in both Na and Cl transport. Although direct measurements of mucus concentration in neonatal airways are not yet feasible, a recent report from Johannesson et al. (20) found increased intraluminal mucus content in the tracheas of Cftr-deficient ENaC-Tg mice compared with ENaC-Tg mice (4), a finding consistent with increased mucus adhesion to tracheal surfaces due to increased mucus concentration. Because tracheal mucus plugging is the dominant cause of death in neonatal ENaC-Tg mice, increased airway obstruction from dehydrated, i.e., concentrated, mucus likely contributed to the increased mortality of F508 CF/ ENaC-Tg mice. It is also plausible that the cartilage defects characteristic of CF mice contributed to poor survival of F508 CF/ ENaC mice if tracheal occlusion occurred in the context of dehydrated, adhesive mucus lining their excessively compliant tracheal walls. The combined Na and Cl transport defects may have also determined the increased incidence of club cell necrotic degeneration observed in F508 CF/ ENaC-Tg mice (Fig. 4). Previous studies have shown that club cell necrosis in the intrapulmonary airways of neonatal ENaC mice is strictly dependent on the ENaC transgene function (26) and resembles that reported in nematode neurons expressing hyperactive degenerin Na channels (15). We speculate that the increased abundance of necrotic cells in the airways of neonatal F508 CF/ ENaC mice reflected the absence of Cftr-dependent compensation for hyperactive/dysregulated ENaC-dependent club cell swelling. For example, in response to Na -dependent cell swelling, airway epithelial cells exhibit a regulatory volume decrease response that involves release ( dumping ) of intracellular KCl (36). Genetic removal of Cftr could decrease the capacity of club cells to release intracellular Cl, limiting their compensatory responses to ENaC-mediated unrestrained Na entry and increasing the likelihood of necrotic cell death. It is also possible that the more severe proximal airways mucus plugging and local hypoxia in F508 CF/ ENaC mice could also have increased the incidence of necrotic degeneration. With respect to the increase inflammatory burden observed in F508 CF/ ENaC-Tg mice, the increased number of necrotic cells likely triggered inflammation by the IL-1R/MyD88 signaling pathway (5). Activation of this inflammatory cascade, coupled with inefficient removal of debris by mucus clearance, could produce the massive recruitment of neutrophils observed in F508 CF/ ENaC-Tg neonatal mice. Interestingly, the large intraluminal neutrophil influx in F508 CF/ ENaC-Tg mice correlated with higher BALF levels of IL-6 but was not paralleled by an increase in the levels of neutrophil chemoattractants, i.e., KC, MIP-2, or LIX, or other inflammatory mediators (Fig. 6). Increased levels of other neutrophil chemoattractants, i.e., high mobility box 1 (HMGB1) and the tripeptide proline-glycine-proline (PGP), both previously described in ENaC-Tg mice BALF (40), could have been responsible for the higher neutrophil influx exhibited by F508 CF/ ENaC-Tg mice, but their concentration was not assessed in the neonatal samples because of the small volume of BALF available. In addition to sterile inflammation driven by necrotic cell death, other variables might have also contributed to the increased inflammation observed in neonatal F508 CF/ ENaC-Tg mice, such as bacterial burden. On the basis of quantitative bacterial cultures, no significant differences were observed in bacterial load between ENaC-Tg and F508 CF/ ENaC-Tg mice. These data suggest that there are no Cftr-specific defects in bacterial clearance superimposed on the muco-obstructive phenotype exhibited by ENaC-Tg mice. However, the massive neutrophilic infiltrate found in the airways of F508 CF/ ENaC-Tg mice, possibly recruited in response to increased epithelial necrosis, might have cleared a fraction of the bacterial burden in F508 CF/ ENaC-Tg mice, reducing CFUs numbers. Alternatively, as noted above for neutrophil counts, an incomplete retrieval of intraluminal contents by BAL due to patchy inflammatory airway obstructions might have decreased the recovery of bacteria in F508 CF/ ENaC-Tg mice, again leading to an underestimation of bacterial numbers. Thus a definitive conclusion regarding the absence of differences in bacterial load or lung microflora composition in F508 CF/ ENaC-Tg vs. ENaC-Tg mice may await more complete, culture-independent, microbiome analyses. Cftr-deficient mice have been reported to have immunological defects, including altered macrophage function (3) and hyperinflammation (10, 23). However, the absence of profound differences in cytokine profiles between neonatal F508 CF/ ENaC-Tg and ENaC-Tg mice, as well as the absence of a proinflammatory signature in neonatal CF mice compared with WT mice, may argue against a dominant effect of CFTR-specific inflammatory pathways in determining the increased inflammatory response in the lungs of F508 CF/ ENaC-Tg mice (Fig. 6). The etiology of eosinophilic crystalline pneumonia, a subclinical to fatal idiopathic disease that occurs spontaneously in inbred C57BL/6 and 129/Sv mice, remains elusive. However, this condition is known to develop in conjunction with other

10 L478 pulmonary diseases, and its severity tends to increase with age (18). Here, we report that eosinophilic crystalline pneumonia occurred in aged (10 mo) F508 CF/ ENaC-Tg mice only. This phenotype was associated with abundant production of YM1/2, members of the chitinase family associated with Th2 type inflammation in asthma and chronic obstructive pulmonary disease (16, 19, 24), and of CCSP, a small anti-inflammatory protein recently found to be involved in the regulation of macrophage function (41). This paper represents the third report describing comparisons and/or interactions of the ENaC-Tg mouse model with other mouse models of airway ion transport processes. In the first report, the ENaC-Tg mouse was compared with mice bearing the Liddle s gain-of-function mutation in the ENaC subunit (32). Although both ENaC-Tg and Liddle s mice exhibited elevated Na transport as measured in Ussing chambers, assessment of ASL volumes by confocal microscopy under thin film conditions revealed that ASL volume-dependent regulation of Na transport was preserved in Liddle s mice but not in ENaC-Tg mice. Importantly, the absence of volume-dependent regulation on ENaC function correlated with the presence of muco-obstructive lung disease. The second report described the effects of club cell-targeted transgenic over expression of human CFTR in WT mice or in combination with the ENaC transgene (hcftr/ ENaC double transgenic mice) (12). These studies showed that expression of hcftr-tg produced increased Cl secretion as measured in Ussing chambers under voltage-clamp conditions (which can generate driving forces for Cl secretion) but failed to restore volume secretion in hcftr/ ENaC airway preparations, as assessed by thin film ASL microscopy. Again, the failure of hcftr-tg to restore ASL volume correlated with the failure to modulate the severity of ENaC-Tg mucoobstructive lung disease. An explanation for the failure of club cell-targeted hcftr to restore volume secretion is that in the context of ENaC-Tg club cells, the unregulated ENaC channel prevented the generation of driving forces for Cl secretion, so the magnitude of apical Cl conductance was irrelevant (12, 32, 45). In this study, we report that deletion of Cftr function in all cell types within the pulmonary epithelium increased the severity of the ENaC-Tg lung phenotype. On the basis of analogy to the hcftr transgenic studies mentioned above, the absence of functional Cftr in club cells would not be predicted to add to the severity of the dehydration insult, owing to the absence of Cl secretion driving forces in this cell type. However, it is likely that Cftr is also expressed in other cell types, including ciliated cells. Endogenous Cftr, coexpressed with WT ENaC in ciliated cells, is predicted to respond to ASL volume depletion with Cl secretion. This response, however, would be absent in F508 CF/ ENaC-Tg mice, adding a new component to airway mucus dehydration. In preliminary studies, we have observed that double-transgenic ENaC-Tg mice, obtained by crossing ENaC-Tg and ENaC-Tg mice, exhibited a twofold increase in Na transport compared with ENaC-Tg mice and had worse survival and lung phenotype (29), suggesting that the ENaC phenotype can indeed be worsened in response to more severe airway surface volume depletion. Of note, it is also possible that absent Cftr function in a different lung region, i.e., the alveolus, may contribute to airway dehydration in the F508 CF/ ENaC-Tg mouse. Previous studies have suggested that CFTR contributes to alveolar secretion (2, 25), and its absence enhances ENaC activity in alveolar epithelial cells of F508 and null CF mice (22). Although asymptomatic in F508 CF mice, the reduction in alveolar secretion moving onto airways surfaces might exacerbate the severity of airway mucus dehydration in ENaC-Tg mice. The presence of a striking alveolar disease in older F508 CF/ ENaC-Tg mice (Fig. 5) is consistent with small airways or alveolar secretory defect. In conclusion, generation of congenic F508 CF/ ENaC-Tg mice has produced an animal model with an increased severity of lung disease, consistent with the prediction that the combination of two airway epithelial ion transport defects would additively act to reduce ASL volume/airway surface hydration. This animal model will be useful 1) to test hypotheses identifying the spectrum of CFTR-dependent mechanisms that may be responsible for the severe lung inflammation observed in CF and 2) to develop both ENaC/CFTR-specific and nonspecific anti-inflammatory molecules as therapies for CF lung disease. ACKNOWLEDGMENTS The authors thank Kimberly Burns, Donald Joyner, Tracy Eldred, and Lori Bramble for outstanding technical assistance with histology and immunohistochemistry; Brian Brighton for performing morphometric analysis of necrotic epithelial cells; the UNC Michael Hooker Microscopy Facility, funded by an anonymous private donor, for assistance with imaging; and the Thurston Arthritis Research Center, Clinical Proteomics Laboratory for Luminex assays. GRANTS This study was funded by Cystic Fibrosis Foundation (CFF) grant RANDEL07P0 (to S. H. Randell), CFF grant LIVRAG08I0 (to A. Livraghi- Butrico), National Institute of Health (NIH) P30 DK and CFF grant R026-CR02 (to W. K. O Neal), and NIH grants P50 HL060280, P01 HL034322, P30 DK065988, and P50 HL (to R. C. Boucher). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). 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