Influenza virus infection alters ion channel function of airway and alveolar cells: mechanisms and physiological sequelae

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) and the amiloride-sensitive epithelial sodium channels (ENaC) are located in the apical membranes of airway and alveolar epithelial cells. These transporters play an important role in the regulation of lung fluid balance across airway and alveolar epithelia by being the conduits for chloride (Cl^-) and bicarbonate ([Formula: see text]) secretion and sodium (Na⁺) ion absorption, respectively. The functional role of these channels in the respiratory tract is to maintain the optimum volume and ionic composition of the bronchial periciliary fluid (PCL) and alveolar lining fluid (ALF) layers. The PCL is required for proper mucociliary clearance of pathogens and debris, and the ALF is necessary for surfactant homeostasis and optimum gas exchange. Dysregulation of ion transport may lead to mucus accumulation, bacterial infections, inflammation, pulmonary edema, and compromised respiratory function. Influenza (or flu) in mammals is caused by influenza A and B viruses. Symptoms include dry cough, sore throat, and is often followed by secondary bacterial infections, accumulation of fluid in the alveolar spaces and acute lung injury. The underlying mechanisms of flu symptoms are not fully understood. This review summarizes our present knowledge of how influenza virus infections alter airway and alveolar epithelial cell CFTR and ENaC function in vivo and in vitro and the role of these changes in influenza pathogenesis.

Keywords: M2 protein; Na+/K+-ATPase; calcium-activated Cl− channels; cystic fibrosis transmembrane conductance regulator; epithelial sodium channels.

Fig. 1.

The respiratory tract: segments of the airways, cell types, and ion transport in epithelial cells. Na⁺ ions enter the cytoplasm of ciliated airway cells and alveolar type I (ATI) and type II (ATII) cells, through channels, down an electrochemical gradient, created by the energy-consuming, ouabain-sensitive Na⁺/K⁺-ATPase located at the basolateral surface. Two different types of Na⁺ channels have been identified. Highly selective Na⁺ channels, consisting of at least 3 subunits (α, β, and γ), which are inhibited by micromolar concentrations of amiloride (epithelial Na channels, ENaC) and nonselective cation channels, consisting of the α-subunit. K⁺ ions exit the cells via basolateral K⁺ channels (KC). Ciliated cells secrete Cl⁻ ions through cystic fibrosis transmembrane conductance regulator (CFTR), as well as Ca²⁺- activated Cl⁻ channels (CaCC) and SCL26A9. Cl⁻ ions enter the cells via the basolateral Na⁺/K⁺/2Cl⁻ (NKCC) symporter, down an electrochemical gradient created by the Na⁺/K⁺-ATPase. In alveolar cells, Cl⁻ transport across CFTR is likely bidirectional and depends on the concentration gradient. In ATII and ATI cells, Cl⁻ enters cells through CFTR or crosses across the paracellular junctions to maintain electroneutrality. PCL, pericilary fluid; ASL, airway surface fluid; ALF, alveolar lining fluid; AEC, airway epithelial cell.

Fig. 2.

Influenza virus replication cycle (1). Hemagglutinin binds to sialic acid residues at the plasma membrane (2). Influenza virus is internalized and tracked to the endosomes. Low pH in the endosome activates M2, which transports protons to the interior of the virion, causing viral ribonucleoprotein (RNP) to dissociate from the virion. Simultaneously, fusion of the hemagglutinin to the endosomal membrane, also initiated by low pH, allows the virions to track into the cytosol (3). Viral RNPs are released (4). Viral RNP is tracked to the nucleus (5). Viral RNA produces cRNA, which encodes new viral genome (vRNA) and mRNA (6). Viral mRNA is translated in the cytosol using host machinery (7). vRNA and viral proteins are tracked to the cell surface (8). Viral assembly and budding from the cell surface occurs (9). Neuraminidase cleaves sialic acid residues from the surface of the cell to allow the virus to detach and infect new host cells.

Fig. 3.

Inhibition of epithelial sodium channels (ENaC) by virus binding. Influenza virus hemagglutinin binding to sialic acid residues at the cell surface and activates Src kinase. Src activates PLC, which activates PKC. PKC downregulates ENaC expression and activity. Neuraminidase cleaves sialic acid residues, preventing virus binding and ENaC inhibition. Cytochalasin D allows the virus to bind but prevents virus internalization. Blocking internalization with cytochalasin D does not prevent ENaC inhibition. This is based on conclusions published previously (12, 51).

Fig. 4.

Expression of influenza virus M2 inhibits epithelial sodium channel (ENaC) activity. Influenza M2 expression increases cellular reactive oxygen species (ROS) either through mitochondrial dysfunction or through activation of the NADPH oxidases. ROS activates PKC, which increases ENaC ubiquitination via the E3 ubiquitin ligase Nedd4-2. Ubiquitinated ENaC is proteasomally degraded, leading to decreased ENaC-mediated sodium transport. This is based on conclusions published previously (54).

Fig. 5.

Influenza virus alteration of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) activity through the release of ATP/UTP. After influenza infection, both ATP and UTP were significantly increased 1 and 2 days postinfection due to viral infection of the lung epithelium. UTP is hydrolyzed to UDP and acts on the P2Y₆R receptor, leading to decreased amiloride-sensitive ion transport. Simultaneously, ATP is hydrolyzed to adenosine, which acts on the A₁-AR. Activation of the A₁-AR increases Cl⁻ secretion through CFTR. The combination of decreased Na⁺ absorption and increased Cl⁻ secretion leads to increased airway surface fluid and pulmonary edema. This is based on conclusions previously published (109). NKCC, Na⁺/K⁺/2Cl⁻; KC, K⁺ channels.

Fig. 6.

Modification of ion transport by influenza virus at cellular and tissue level. Mechanisms underlie ion transport regulation by influenza virus infection. 1: Influenza virus hemagglutinin binds sialic acid residues, leading to PKC activation and epithelial sodium channel (ENaC) inhibition. 2: After internalization, influenza produces viral proteins. Influenza M2 leads to enhanced ubiquitination and degradation of both ENaC and cystic fibrosis transmembrane conductance regulator (CFTR). 3: Influenza replication assembly and budding result in the release of cytokines and UTP/ATP. Reinfection propagates earlier signaling pathways.