SLC26A9-mediated chloride secretion prevents mucus obstruction in airway inflammation

Abstract

Asthma is a chronic condition with unknown pathogenesis, and recent evidence suggests that enhanced airway epithelial chloride (Cl-) secretion plays a role in the disease. However, the molecular mechanism underlying Cl- secretion and its relevance in asthma pathophysiology remain unknown. To determine the role of the solute carrier family 26, member 9 (SLC26A9) Cl- channel in asthma, we induced Th2-mediated inflammation via IL-13 treatment in wild-type and Slc26a9-deficient mice and compared the effects on airway ion transport, morphology, and mucus content. We found that IL-13 treatment increased Cl- secretion in the airways of wild-type but not Slc26a9-deficient mice. While IL-13-induced mucus overproduction was similar in both strains, treated Slc26a9-deficient mice exhibited airway mucus obstruction, which did not occur in wild-type controls. In a study involving healthy children and asthmatics, a polymorphism in the 3' UTR of SLC26A9 that reduced protein expression in vitro was associated with asthma. Our data demonstrate that the SLC26A9 Cl- channel is activated in airway inflammation and suggest that SLC26A9-mediated Cl- secretion is essential for preventing airway obstruction in allergic airway disease. These results indicate that SLC26A9 may serve as a therapeutic target for airway diseases associated with mucus plugging.

Figure 1. Lung morphology and airway ion transport are normal in naive Slc26a9^–/– mice.

(A and B) Representative morphology of conducting airways (A) and lung parenchyma (B) in naive wild-type and Slc26a9^–/– mice. Sections were stained with H&E. Scale bars: 100 εm. (C–F) Summary of V_te (C), R_te (D), and I_sc (E) under basal conditions, and amiloride-sensitive I_sc (F) in freshly excised bronchial tissues from wild-type and Slc26a9^–/– mice. (G and H) Amiloride-insensitive I_sc and effects of CFTR_inh-172 and bumetanide in the absence (–cAMP) (G) and presence (+cAMP) (H) of cAMP-mediated stimulation. (I) UTP-induced I_sc in the presence of amiloride and cAMP-dependent stimulation. n = 5–13 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with mice of the same genotype.

Figure 2. SLC26A9 determines constitutive Cl^– secretion induced in Th2-mediated airway inflammation.

(A) Original recordings of effects of amiloride, cAMP-mediated (IBMX/forskolin [Fors]) activation, and UTP on V_te and R_te across freshly excised bronchial tissues from vehicle- and IL-13–treated wild-type and Slc26a9^–/– mice. Ctrl, control. (B–F) Summary of basal I_sc (B), amiloride-sensitive I_sc (C), amiloride-insensitive I_sc (D), cAMP-induced I_sc (E), and UTP-induced I_sc (F) in bronchi from vehicle-treated and IL-13–treated wild-type and Slc26a9^–/– mice. (G) Summary of bumetanide-sensitive I_sc in amiloride-pretreated tissues from vehicle- and IL-13–treated wild-type and Slc26a9^–/– mice (G). n = 8–11 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle-treated mice of the same genotype. ^†P < 0.05, ^‡P < 0.01 compared with IL-13–treated wild-type mice.

Figure 3. SLC26A9 prevents airway mucus obstruction in the presence of mucin overproduction in Th2-mediated airway disease.

(A) Representative airway morphology in vehicle- and IL-13–treated wild-type and Slc26a9^–/– mice. Sections were stained with Alcian blue–PAS to determine the presence of goblet cells and airway mucus. Scale bars: 100 εm. (B–E) Summary of goblet cell densities (B) and epithelial volume density (C) in main axial airways; fraction (%) of mice showing accumulation of airway mucus (D); and mucus volume density in the airway lumen (E) from vehicle-treated and IL-13–treated wild-type and Slc26a9^–/– mice. n = 7–11 mice for each group. **P < 0.01, ***P < 0.001 compared with vehicle-treated mice of the same genotype. ^‡P < 0.01, ^§P < 0.001 compared with IL-13–treated wild-type mice.

Figure 4. A SNP in the 3′ UTR of SLC26A9 reduces reporter gene activity in luciferase reporter assays.

(A) Alignment of the partial wild-type 3′ UTR sequence of SLC26A9 (nucleotides 4511–4529) with the SNP rs2282430 (indicated by arrowhead) and hsa-miR-632. (B) Summary of relative luciferase activity of reporter constructs containing the wild-type versus polymorphic (SNP) 3′ UTR of SLC26A9 transfected into A549 cells. (C) Effects of co-transfection with precursor hsa-miR-632 or a pre-miR negative control (miR-Ctrl) on luciferase activity of wild-type versus SNP SLC26A9 3′ UTR constructs in HEK293 cells. n = 10–11 experiments per group. *P < 0.05.