The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease

AbstractChronic lung disease remains the major cause of morbidity and mortality of cystic fibrosis (CF) patients. Cftr mutant mice developed severe intestinal obstruction, but did not exhibit the characteristic CF ion transport defects (i.e. deficient cAMP-dependent Cl− secretion and increased Na+ absorption) in the lower airways, and failed to develop CF-like lung disease. These observations led to the generation of transgenic mice with airway-specific overexpression of the epithelial Na+ channel (ENaC) as an alternative approach to mimic CF ion transport pathophysiology in the lung. Studies of the phenotype of βENaC-transgenic mice demonstrated that increased airway Na+ absorption causes airway surface liquid (ASL) depletion, reduced mucus transport and a spontaneous CF-like lung disease with airway mucus obstruction and chronic airway inflammation. Here, we summarize approaches that can be applied for studies of the complex in vivo pathogenesis and preclinical evaluation of novel therapeutic strategies in this model of CF lung disease

Insulin‐like growth factor‐1 stimulates regulatory T cells and suppresses autoimmune disease

The recent precipitous rise in autoimmune diseases is placing an increasing clinical and economic burden on health systems worldwide. Current therapies are only moderately efficacious, often coupled with adverse side effects. Here, we show that recombinant human insulin‐like growth factor‐1 (rhIGF‐1) stimulates proliferation of both human and mouse regulatory T (Treg) cells in vitro and when delivered systemically via continuous minipump, it halts autoimmune disease progression in mouse models of type 1 diabetes (STZ and NOD) and multiple sclerosis (EAE) in vivo. rhIGF‐1 administration increased Treg cells in affected tissues, maintaining their suppressive properties. Genetically, ablation of the IGF‐1 receptor specifically on Treg cell populations abrogated the beneficial effects of rhIGF‐1 administration on the progression of multiple sclerotic symptoms in the EAE model, establishing a direct effect of IGF‐1 on Treg cell proliferation. These results establish systemically delivered rhIGF‐1 as a specific, effective stimulator of Treg cell action, underscoring the clinical feasibility of manipulating natural tolerance mechanisms to suppress autoimmune disease

CFTR Regulates Early Pathogenesis of Chronic Obstructive Lung Disease in βENaC-Overexpressing Mice

<div><h3>Background</h3><p>Factors determining the onset and severity of chronic obstructive pulmonary disease remain poorly understood. Previous studies demonstrated that airway surface dehydration in βENaC-overexpressing (βENaC-Tg) mice on a mixed genetic background caused either neonatal mortality or chronic obstructive lung disease suggesting that the onset of lung disease was modulated by the genetic background.</p> <h3>Methods</h3><p>To test this hypothesis, we backcrossed βENaC-Tg mice onto two inbred strains (C57BL/6 and BALB/c) and studied effects of the genetic background on neonatal mortality, airway ion transport and airway morphology. Further, we crossed βENaC-Tg mice with CFTR-deficient mice to validate the role of CFTR in early lung disease.</p> <h3>Results</h3><p>We demonstrate that the C57BL/6 background conferred increased CFTR-mediated Cl⁻ secretion, which was associated with decreased mucus plugging and mortality in neonatal βENaC-Tg C57BL/6 compared to βENaC-Tg BALB/c mice. Conversely, genetic deletion of CFTR increased early mucus obstruction and mortality in βENaC-Tg mice.</p> <h3>Conclusions</h3><p>We conclude that a decrease or absence of CFTR function in airway epithelia aggravates the severity of early airway mucus obstruction and related mortality in βENaC-Tg mice. These results suggest that genetic or environmental factors that reduce CFTR activity may contribute to the onset and severity of chronic obstructive pulmonary disease and that CFTR may serve as a novel therapeutic target.</p> </div

Genetic background modulates airway ion transport in wild-type and βENaC-Tg mice.

<p>A) Representative original recordings of the effects of amiloride and cAMP-dependent activation (IBMX/forskolin) on transepithelial voltage (V_te) and transepithelial resistance (R_te) across freshly excised tracheal tissues from neonatal (3-day-old) wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. R_te was determined from V_te deflections obtained by pulsed current injection. (B–F) Summary of basal equivalent short-circuit current (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 freshly excised tracheal tissues from neonatal WT and βENaC-Tg mice on the C57BL/6 and BALB/c background. Data are presented as mean ± SEM (<i>n</i> = 8–20 mice per group). *<i>P</i><0.05 and **<i>P</i><0.001 compared with WT mice on same strain background, ^†<i>P</i><0.05 and ^††<i>P</i><0.01 compared with mice of same genotype on C57BL/6 background.</p

Survival of βENaC-Tg mice is modified by genetic background.

<p>Survival curves of βENaC-Tg mice and wild-type (WT) littermates on mixed (C3H/He x C57BL/6), C57BL/6 and BALB/c backgrounds (n = 36–59 mice per group). *<i>P</i><0.05 and ^†<i>P</i><0.01 (Kaplan-Meier survival analysis).</p

Genetic background modulates CFTR-mediated Cl⁻ secretion in airways of wild-type and βENaC-Tg mice.

<p>A–C) Effects of genetic background on transepithelial Cl- secretion were determined by adding bumetanide or CFTR_inh-172 to amiloride-pretreated tracheal tissues from neonatal wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. A,B) Summary of bumetanide-sensitive I_sc (A) and CFTR_inh-172-sensitive I_sc (B) in the presence of amiloride (<i>n</i> = 12–23 mice per group). C) Summary of CFTR_inh-172-sensitive I_sc in the presence of amiloride and cAMP-dependent activation (IBMX/forskolin) (<i>n</i> = 5–11 mice per group). Data are presented as mean ± SEM. ^†<i>P</i><0.05 and ^††<i>P</i><0.01 compared with mice of same genotype on C57BL/6 background.</p

Genetic background modifies early airway mucus obstruction and epithelial necrosis in neonatal βENaC-Tg mice.

<p>A) Longitudinal sections of tracheae from neonatal (3-day-old) wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. Sections were stained with Alcian blue–periodic acid Schiff (AB-PAS) to determine the presence of mucus and goblet cells. Scale bars  = 200 µm. B–D) Summary of mucus content, as determined from measuring volume density of AB-PAS-positive material in the tracheal lumen (B), goblet cell numbers (C) and Transcript levels of Muc5b (D) (<i>n</i> = 4–13 mice per group). E) Airway histology from neonatal (3-day-old) WT and βENaC-Tg mice on the C57BL/6 and BALB/c background. Sections were stained with hematoxylin and eosin (H&E) and evaluated for degenerative airway epithelial cells (arrows). Scale bars  = 40 µm (upper panels) and 20 µm (lower panels). F) Summary of airway epithelial necrosis as determined from the number of degenerative epithelial cells per mm of the basement membrane (<i>n</i> = 9–11 mice per group). *<i>P</i><0.001 compared with WT mice on same strain background, ^†<i>P</i><0.05 compared with mice of same genotype on C57BL/6 background.</p

Airway Surface Liquid Volume Regulation Determines Different Airway Phenotypes in Liddle Compared with βENaC-overexpressing Mice*

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na+ channel β-subunit (βENaC-Tg) suggest that raised airway Na+ transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function βENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, βENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na+ transport measured in Ussing chambers (“flooded” conditions) was raised in both Liddle and βENaC-Tg mice. Because enhanced Na+ transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic “thin film” conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na+ absorption were intact in Liddle but defective in βENaC-Tg mice. We conclude that the capacity to regulate Na+ transport and ASL volume, not absolute Na+ transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease