Alveolar Responses to Influenza Lung Infection Determined by Confocal Imaging of Live Lungs

Abstract

One‐third of patients with severe lung infection by influenza A virus (IAV) develop secondary lung infection by inhaled S. aureus(SA), leading to mortality in 30% of cases. It is unclear how IAV promotes secondary SA infection in lung alveoli, where fluid barrier dysfunction causes fatal acute lung injury (ALI). Our objective is to define alveolar responses to IAV that promote SA infection, leading to SA‐induced alveolar damage and mortality. Our hypothesis is IAV lung infection blocks alveolar wall liquid (AWL) secretion, a homeostatic alveolar function that defends against alveolar stabilization of inhaled particles. AWL secretion depends on function of the alveolar epithelial cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Our methods include optical imaging of live, intact mouse lungs and mouse survival determinations. Mice were untreated or intranasally instilled with IAV (2000 PFU, A/PR/8/34). We excised, inflated, and blood‐perfused the lungs at 24h, then viewed live alveoli by confocal microscopy. Alveolar airspaces were microinstilled separately with: (A) the fluorescent cytosolic dye, calcein‐AM; (B) the fluorescent AWL tracer, tetramethylrhodamine‐conjugated dextran (70 kD); (C) the CFTR potentiator, ivacaftor; and (D) the SA toxin, alpha hemolysin (Hla). For survival studies, IAV‐infected mice were intranasally instilled with SA (USA300, 1x10^8 CFU) at 24h, then observed for 72h. Mice were given intraperitoneal injections of ivacaftor or vehicle at 4, 24, and 48h after SA instillation. Analysis was by t test (n=3) and log rank. Our results follow. Calcein fluorescence indicated the alveolar epithelium of IAV‐infected lungs was viable. In alveoli of untreated lungs, airspace dextran fluorescence decreased over 1h, indicating dextran was progressively diluted by AWL secretion. However, in IAV‐infected lungs, dextran fluorescence was steady (P<0.05), indicating IAV blocked AWL secretion. Since alveolar ivacaftor pretreatment restored dextran loss (P=NS versus untreated), we interpret IAV blocked AWL secretion in a CFTR‐dependent manner, and CFTR potentiation rescued AWL secretion. Hla microinstillation into alveoli of IAV‐infected lungs caused loss of epithelial calcein fluorescence, but alveolar pretreatment with ivacaftor blocked the loss (P<0.05). We interpret that rescue of AWL secretion protected against Hla‐induced alveolar damage, probably by promoting Hla clearance and disrupting Hla‐epithelial interactions. IAV‐infected mice treated with ivacaftor were protected from SA‐induced mortality (60% and 0% in vehicle‐ versus ivacaftor‐treated mice, respectively; P<0.05; N=10 each). We conclude that IAV disrupted AWL secretion, causing retention of airspace contents and augmenting Hla‐induced alveolar damage. Rescue of AWL secretion blocked Hla‐induced alveolar damage and increased survival in IAV‐SA coinfected mice. We propose strategies that rescue AWL secretion may represent a new therapeutic approach for ALI caused by IAV‐SA coinfection.