Evaluation of a novel CFTR potentiator in COPD ferrets with acquired CFTR dysfunction.

RATIONALE: The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure. OBJECTIVES: To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis. METHODS: Ferrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes. MEASUREMENTS AND MAIN RESULTS: Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy. CONCLUSIONS: The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.

Influenza-Induced Oxidative Stress Sensitizes Lung Cells to Bacterial-Toxin-Mediated Necroptosis.

Pneumonias caused by influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity and high mortality rate. Previously, we have shown that bacterial pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondary Streptococcus pneumoniae (Spn) infection. We observe that IAV synergistically sensitizes lung epithelial cells for PFT-mediated necroptosis in vitro and in murine models of Spn co-infection and secondary infection. Pharmacoelogical induction of oxidative stress without virus sensitizes cells for PFT-mediated necroptosis. Antioxidant treatment or inhibition of necroptosis reduces disease severity during secondary bacterial infection. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identify necroptosis inhibition and antioxidant therapy as potential intervention strategies.

Pulmonary surfactant lipids inhibit infections with the pandemic H1N1 influenza virus in several animal models.

The influenza A (H1N1)pdm09 outbreak in 2009 exemplified the problems accompanying the emergence of novel influenza A virus (IAV) strains and their unanticipated virulence in populations with no pre-existing immunity. Neuraminidase inhibitors (NAIs) are currently the drugs of choice for intervention against IAV outbreaks, but there are concerns that NAI-resistant viruses can transmit to high-risk populations. These issues highlight the need for new approaches that address the annual influenza burden. In this study, we examined whether palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI) effectively antagonize (H1N1)pdm09 infection. POPG and PI markedly suppressed cytopathic effects and attenuated viral gene expression in (H1N1)pdm09-infected Madin-Darby canine kidney cells. POPG and PI bound to (H1N1)pdm09 with high affinity and disrupted viral spread from infected to noninfected cells in tissue culture and also reduced (H1N1)pdm09 propagation by a factor of 102 after viral infection was established in vitro In a mouse infection model of (H1N1)pdm09, POPG and PI significantly reduced lung inflammation and viral burden. Of note, when mice were challenged with a typically lethal dose of 1000 plaque-forming units of (H1N1)pdm09, survival after 10 days was 100% (14 of 14 mice) with the POPG treatment compared with 0% (0 of 14 mice) without this treatment. POPG also significantly reduced inflammatory infiltrates and the viral burden induced by (H1N1)pdm09 infection in a ferret model. These findings indicate that anionic phospholipids potently and efficiently disrupt influenza infections in animal models.

Influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis.

Severe influenza (IAV) infection can develop into bronchopneumonia and edema, leading to acquired respiratory distress syndrome (ARDS) and pathophysiology. Underlying causes for pulmonary edema and aberrant fluid regulation largely remain unknown, particularly regarding the role of viral-mediated mechanisms. Herein, we show that distinct IAV strains reduced the functions of the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane regulator (CFTR) in murine respiratory and alveolar epithelia in vivo, as assessed by measurements of nasal potential differences and single-cell electrophysiology. Reduced ion channel activity was distinctly limited to virally infected cells in vivo and not bystander uninfected lung epithelium. Multiple lines of evidence indicated ENaC and CFTR dysfunction during the acute infection period; however, only CFTR dysfunction persisted beyond the infection period. ENaC, CFTR, and Na,K-ATPase activities and protein levels were also reduced in virally infected human airway epithelial cells. Reduced ENaC and CFTR led to changes in airway surface liquid morphology of human tracheobronchial cultures and airways of IAV-infected mice. Pharmacologic correction of CFTR function ameliorated IAV-induced physiologic changes. These changes are consistent with mucous stasis and pulmonary edema; furthermore, they indicate that repurposing therapeutic interventions correcting CFTR dysfunction may be efficacious for treatment of IAV lung pathophysiology.

Assessment of ciliary phenotype in primary ciliary dyskinesia by micro-optical coherence tomography.

Ciliary motion defects cause defective mucociliary transport (MCT) in primary ciliary dyskinesia (PCD). Current diagnostic tests do not assess how MCT is affected by perturbation of ciliary motion. In this study, we sought to use micro-optical coherence tomography (µOCT) to delineate the mechanistic basis of cilia motion defects of PCD genes by functional categorization of cilia motion. Tracheae from three PCD mouse models were analyzed using µOCT to characterize ciliary motion and measure MCT. We developed multiple measures of ciliary activity, integrated these measures, and quantified dyskinesia by the angular range of the cilia effective stroke (ARC). Ccdc39-/- mice, with a known severe PCD mutation of ciliary axonemal organization, had absent motile ciliary regions, resulting in abrogated MCT. In contrast, Dnah5-/- mice, with a missense mutation of the outer dynein arms, had reduced ciliary beat frequency (CBF) but preserved motile area and ciliary stroke, maintaining some MCT. Wdr69-/- PCD mice exhibited normal motile area and CBF and partially delayed MCT due to abnormalities of ciliary ARC. Visualization of ciliary motion using µOCT provides quantitative assessment of ciliary motion and MCT. Comprehensive ciliary motion investigation in situ classifies ciliary motion defects and quantifies their contribution to delayed mucociliary clearance.

A ferret model of COPD-related chronic bronchitis.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US. The majority of COPD patients have symptoms of chronic bronchitis, which lacks specific therapies. A major impediment to therapeutic development has been the absence of animal models that recapitulate key clinical and pathologic features of human disease. Ferrets are well suited for the investigation of the significance of respiratory diseases, given prior data indicating similarities to human airway physiology and submucosal gland distribution. Here, we exposed ferrets to chronic cigarette smoke and found them to approximate complex clinical features of human COPD. Unlike mice, which develop solely emphysema, smoke-exposed ferrets exhibited markedly higher numbers of early-morning spontaneous coughs and sporadic infectious exacerbations as well as a higher level of airway obstruction accompanied by goblet cell metaplasia/hyperplasia and increased mucus expression in small airways, indicative of chronic bronchitis and bronchiolitis. Overall, we demonstrate the first COPD animal model exhibiting clinical and pathologic features of chronic bronchitis to our knowledge, providing a key advance that will greatly facilitate the preclinical development of novel treatments for this disease.

Comparison of the effectiveness of antibody and cell-mediated immunity against inhaled and instilled influenza virus challenge.

BACKGROUND: To evaluate immunity against influenza, mouse challenge studies are typically performed by intranasal instillation of a virus suspension to anesthetized animals. This results in an unnatural environment in the lower respiratory tract during infection, and therefore there is some concern that immune mechanisms identified in this model may not reflect those that protect against infectious virus particles delivered directly to the lower respiratory tract as an aerosol. METHOD: To evaluate differences in protection against instilled and inhaled virus, mice were immunized with influenza antigens known to induce antibody or cell-mediated responses and then challenged with 100 LD50 A/PR/8/34 (PR8) in the form of aerosol (inhaled) or liquid suspension (instilled). RESULTS: Mice immunized with recombinant adenovirus (Ad) expressing hemagglutinin were protected against weight loss and death in both challenge models, however immunization with Ad expressing nucleoprotein of influenza A (NPA) or M2 resulted in greater protection against inhaled aerosolized virus than virus instilled in liquid suspension. Ad-M2, but not Ad-NPA-immunized mice were protected against a lower instillation challenge dose. CONCLUSIONS: These results demonstrate differences in protection that are dependent on challenge method, and suggest that cell-mediated immunity may be more accurately demonstrated in mouse inhalation studies. Furthermore, the data suggest immune mechanisms generally characterized as incomplete or weak in mouse models using liquid intranasal challenge may offer greater immunity against influenza infection than previously thought.

Development of a murine nose-only inhalation model of influenza: comparison of disease caused by instilled and inhaled A/PR/8/34.

Influenza continues to cause widespread disease and death during winter months. In preclinical studies to evaluate the potential efficacy of drugs and vaccines, influenza challenge virus is usually instilled into the noses of animals in the form of large liquid drops. Since inhalation of aerosolized influenza is commonly associated with human transmission, instillation of challenge virus raises uncertainty about the applicability of results. In order to compare the challenge methods, we established conditions to generate influenza aerosols with a mass median aerodynamic diameter (MMAD) of 1 µm that were delivered to mice in a nose-only inhalation system. In this report, we describe the system and compare the 50% lethal dose (LD(50)) of instilled and inhaled A/PR/8/34 (PR8) in BALB/c mice. The estimated LD(50) for inhaled virus was 8.7 plaque forming units (PFU) and the mean time to death was 7.7 days, whereas the estimated LD(50) for instilled virus was 51.6 PFU and the mean time to death was 8.2 days. Our results show that mice are more sensitive to inhaled virus than virus delivered by intranasal instillation. The murine nose-only inhalation model of influenza infection can be used to infect large numbers of animals simultaneously with well-characterized, homogenous PR8 bioaerosol in a controlled and reproducible manner. This model provides the means to evaluate the efficacy of drug and vaccine candidates against the relevant route of challenge, thereby providing data that may better predict clinical outcome.