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Airway acidification initiates host defense abnormalities in cystic fibrosis mice

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Science  29 Jan 2016:
Vol. 351, Issue 6272, pp. 503-507
DOI: 10.1126/science.aad5589
  • Fig. 1 ASL pH is abnormally acidic in CF pigs and humans, but not in CF mice.

    (A) ASL pH measured in differentiated primary airway epithelial cultures from humans, pigs, and mice using SNARF-dextran (n = 6). The basolateral solution contained 25 mM HCO3, and the atmosphere contained 5% CO2. (B) ASL pH measured in vivo on tracheal surfaces of newborn pigs and mature mice using a pH-sensitive optode (n = 6 for non-CF and CF pigs, 18 for non-CF mice, and 9 for CF mice). (C) TMEM16a mRNA expression in CF human, pig, and mouse cultured airway epithelia (n = 6). (D) Change in the short-circuit current (ΔIsc) after adding 1 μM ionomycin basolaterally to cultured CF airway epithelia studied in Ussing chambers (n = 5). The solution contained 140 mM Cl bilaterally without HCO3 and CO2. (E) ASL pH before and after adding the Ca2+-mediated secretagogue carbachol (CCH, 200 μM) to basolateral surfaces of CF epithelia in the presence of HCO3 and CO2 (n = 6 pigs and humans and 5 mice. (F) ΔIsc induced by adding 10 μM forskolin and 100 μM IBMX to increase intracellular cAMP (n = 5 for CF pigs and mice, 6 for non-CF humans and mice, and 7 for non-CF pigs and CF humans). The solution was free of Cl and contained 25 mM HCO3 with a 5% CO2 atmosphere. (G and H) ASL pH in non-CF and CF pig, human, and mouse cultured airway epithelia before and after basolateral addition of 10 μM forskolin and 100 μM IBMX to increase cAMP (n = 6). In all panels, each data point or pair of data points is from a different animal or human, and bars indicate means ± SEM. Analysis of variance (ANOVA) with Tukey's multiple comparison test [(C) and (D)] or paired [(E), (G), and (H)] or unpaired [(A), (B), and (F)] Student’s t tests were used to assess statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

  • Fig. 2 ATP12A secretes H+ in pig and human but not mouse airway epithelia.

    (A and B) The rate of apical acidification in cultured non-CF and CF airway epithelia (n = 6 for non-CF epithelia and 6 for CF human, 5 for CF pig, and 4 for CF mouse epithelia). 30 μl of a pH 7.0 unbuffered solution was placed on the apical surface, and pH was measured immediately and after 5 min. The basolateral solution was free of HCO3 and CO2 and buffered with 25 mM Hepes. (C) ASL pH of non-CF and CF pig cultured airway epithelia in media free of HCO3 and CO2 (n = 6 or 7). Epithelia were treated with the following agents dissolved in solvent and added apically in 20 μl of perfluorocarbon (target in parentheses): 10 μM ouabain (nongastric H+/K+ ATPase), 100 μM SCH28080 (gastric H+/K+ ATPase), 100 μM bafilomycin (V-type H+-ATPase), 100 μM dimethyl amiloride, dimethylamine (NHE3), 1 mM ZnCl2 (HVCN1), or dimethyl sulfoxide (DMSO) vehicle. (D) The rate of apical acidification in the presence and absence of K+ (n = 4 to 6). 30 mM Na+ replaced K+. Conditions were same as described for (A) and (B). (E) Effect of siRNA directed against pig ATP12A or scrambled control (Scr) on ASL pH in cultured non-CF and CF airway epithelia (n = 4). Solutions contained HCO3 and CO2. (F) ATP12A mRNA levels in non-CF and CF human, pig, and mouse airway epithelia (n = 5 for non-CF pig and non-CF mouse and 6 for all other epithelia). Values for mouse epithelia were less than for human and pig epithelia for both genotypes. (G) ATP12A immunostaining of human, pig, and mouse tracheal epithelium. The surface epithelium showed immunostaining along the apical surface in humans and pigs, but this was absent in mice. Scale bar, 18 μm. Figure S7 shows a positive control. In all panels, data points in each group are from epithelia from a different animal or human. Bars indicate means ± SEM. Data were analyzed using ANOVA with Tukey's multiple comparison test [(A) to (C) and (F)] or unpaired Student’s t tests [(D) and (E)].

  • Fig. 3 Inhibiting ATP12A increases pig and human ASL pH and enhances host defense properties of ASL.

    (A to C) Non-CF and CF cultured pig airway epithelia were treated with 10 μM apical ouabain or DMSO vehicle for 2 hours (n = 6). The data shown are ASL pH (A), bacterial killing by ASL (B), and the time constant (τ) for fluorescence recovery after photobleaching of ASL relative to saline (C). For testing antibacterial activity, S. aureus–coated grids were placed on ASL for 1 min and then evaluated with a live/dead stain (fig. S9 shows a negative control). A higher τASLsaline indicates a greater viscosity (fig. S10 shows that ASL depth did not differ). (D and E) Effect of ouabain (dissolved in DMSO suspended in 100 μl of perfluorocarbon; estimated final concentration, 10 μM) applied to tracheal surfaces of non-CF and CF newborn pigs (n = 6). Thirty min later, we measured ASL pH (D) and bacterial killing [S. aureus-coated grids placed on the airway surface for 1 min (E)]. Figure S11 shows that aerosolized DMSO vehicle did not alter ASL pH, and fig. S12 shows a lack of histopathological changes after ouabain delivery. (F to H) Effect of 1 mM apical ouabain or vehicle on ASL pH (F), antibacterial activity (G), and ASL viscosity (H) in human non-CF and CF epithelia (n = 6 or 8). In all panels, each data point or pair of data points is from epithelia from a different pig [(A) to (E)] or human [(F) to (H)]. Bars indicate means ± SEM. Data were analyzed using unpaired [(A) to (C) and (F) to (H)] or paired [(D) and (E)] Student’s t tests.

  • Fig. 4 Expressing ATP12A in CF mouse airways acidifies ASL, induces abnormalities in host defense processes, and increases the number of bacteria in lungs.

    (A) Effect of treating cultured mouse airway epithelia with adenovirus expressing ATP12A (Ad-ATP12A) or vehicle (control) on ASL pH, measured Three days later (n = 7). (B and C) Cultured mouse airway epithelia were treated with Ad-ATP12A or vehicle (control). Three days later, antimicrobial activity was measured by placing a S. aureus–coated grid on the apical surface for 1 min (B), and ASL viscosity was assessed by measuring the time constant (τ) for fluorescence recovery after photobleaching of ASL relative to saline (C) (n = 5). (D and E) Ad-ATP12A or Ad-GFP (control; GFP, green fluorescent protein) was administered to CF mice. ASL pH (D) and antimicrobial activity [S. aureus–coated grid placed on the apical surface for 1 min (E)] were measured in vivo 3 days later (n = 6 or 7). (F) Ad-ATP12A or Ad-GFP (control) were administered to CF mice. Three days later, lungs were removed, homogenized, and cultured. The data shown are colony-forming units (CFU) (n = 8; see also figs. S17H and S18). (G) Total cells/ml in bronchoalveolar lavage liquid from CF mice treated with Ad-ATP12A or Ad-GFP (control) 3 days earlier (n = 5 to 7). (H and I) Relationships between ATP12A mRNA and bacterial CFU from a partial lung homogenate (H) and composite macrophage score [foamy macrophage plus Iba1 (ionized calcium binding adapter molecule 1) staining (I)]. (J) Histopathological sections from CF mice 3 days after administering Ad-ATP12A. The left panel is from a mouse that had a low level of ATP12A mRNA, and the right panel is from a mouse with greater ATP12A mRNA (log10 relative to Ad-GFP control). Arrows identify macrophages admixed with cell debris and inflammation [see (I)]. In all panels, each data point or pair of data points is from a different mouse, and bars indicate means ± SEM. Data were analyzed using unpaired Student’s t tests [(A) to (E) and (G)] Mann-Whitney U rank sum test (F), or linear correlation [(H) and (I)].

Supplementary Materials

  • Airway acidification initiates host defense abnormalities in cystic fibrosis mice

    Viral S. Shah, David K. Meyerholz, Xiao Xiao Tang, Leah Reznikov, Mahmoud Abou Alaiwa, Sarah E. Ernst, Philip H. Karp, Christine L. Wohlford-Lenane, Kristopher P. Heilmann, Mariah R. Leidinger, Patrick D. Allen, Joseph Zabner, Paul B. McCray Jr., Lynda S. Ostedgaard, David A. Stoltz, Christoph O. Randak, Michael J. Welsh

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Figs. S1 to S20
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