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A3839 - Influenza Infection Reduces Ion Channel Activity and Dysregulates Airway Surface Liquid Homeostasis
Author Block: J. D. Brand1, A. Lazrak1, J. Trombley1, R. Shei2, T. Adewale2, J. L. Tipper1, A. R. Ashtekar1, S. M. Rowe3, S. Matalon3, K. S. Harrod1; 1Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL, United States, 2Pulm Allergy and Criticaly Care Medicine, University of Alabama at Birmingham, Birmingham, AL, United States, 3Univ of Alabama At Birmingham, Birmingham, AL, United States.
Rationale: Influenza virus infection can result in bronchoalveolar edema and proceed to acute respiratory distress syndrome. Edema resolution and airway surface liquid (ASL) homeostasis rely on epithelial ion channels such as the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane regulator (CFTR). ASL maintenance is vital for host defense against pathogens. Previous studies indicate influenza infection may alter both the expression and function of these channels, though the mechanism underlying this phenomenon remain unresolved. We hypothesized epithelial influenza infection would dysregulate ASL homeostasis via reduced ENaC and CFTR function causing ASL imbalance.
Methods: Normal human bronchial epithelial cells (NHBEs) were grown at air-liquid interface and allowed to differentiate prior to infection with either A/California/7/2009 or GFP encoded A/Puerto Rico/8/34 H1N1 influenza strains at various multiplicities of infection. ENaC, CFTR, and Na+/K+-ATPase function were analyzed via Ussing chamber and protein expression was analyzed via Western Blot. Ten (10)-12 week old C57BL/6 mice were lightly anesthetized and intranasally infected with GFP tagged PR8 at 4000 plaque forming units per animal and sacrificed 1-14 days post-infection (p.i.). Lung slices from these mice were prepared for single cell patch clamp recordings on both fluorescent (infected) and uninfected cells. Excised trachea from infected mice were used to measure ASL depth and functional airway micro-anatomy via micro-optical coherence tomography.
Results: NHBE influenza infection reduced ENaC, CFTR, and Na+/K+-ATPase activity and transepithelial resistance 48 and 72 hours p.i. Reductions in channel activity were accompanied by reduced expression of ENaC, CFTR , and Na+/K+-ATPase at the selected time points. Ex vivo single cell patch clamp studies demonstrated ENaC activity was affected only within cells with active viral infection and not adjacent uninfected cells. ENaC function was reduced 2 days p.i. which persisted 15 days p.i. ASL height and ciliary beat frequency (CBF) was also reduced 4 days p.i.; returning to control levels by day 10. Finally, in vivo NPD analysis revealed reduced ENaC function 5 day p.i. returning to normal levels day 15 p.i. while CFTR function was also reduced at day 5 and day 15 p.i.
Conclusion: Influenza infection reduces ASL depth and CBF with coincident decreases in both ENaC and CFTR expression and function. Ion channel dysfunction is localized to influenza infected cells, but not uninfected neighboring cells. These events parallel morbidity associated with influenza infection. Influenza mediated alteration of ASL homeostasis may compromise host defense and lead to lethal lung pathophysiology.