Influenza A viruses (IAV) can cause lung injury and acute respiratory distress syndrome (ARDS), which is characterized by accumulation of excessive fluid (edema) in the alveolar airspaces and leads to hypoxemia and death if not corrected. Clearance of excess edema fluid is driven mostly by the alveolar epithelial Na,K-ATPase and is crucial for survival of patients with ARDS. We therefore investigated whether IAV infection alters Na,K-ATPase expression and function in alveolar epithelial cells (AECs) and the ability of the lung to clear edema. IAV infection reduced Na,K-ATPase in the plasma membrane of human and murine AECs and in distal lung epithelium of infected mice. Moreover, induced Na,K-ATPase improved alveolar fluid clearance (AFC) in IAV-infected mice. We identified a paracrine cell communication network between infected and noninfected AECs and alveolar macrophages that leads to decreased alveolar epithelial Na,K-ATPase function and plasma membrane abundance and inhibition of AFC. We determined that the IAV-induced reduction of Na,K-ATPase is mediated by a host signaling pathway that involves epithelial type I IFN and an IFN-dependent elevation of macrophage TNF-related apoptosis–inducing ligand (TRAIL). Our data reveal that interruption of this cellular crosstalk improves edema resolution, which is of biologic and clinical importance to patients with IAV-induced lung injury.

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1β production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R–dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1β–dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1β, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

Since being first described more than 60 years ago, Na,K-ATPase has been extensively studied, while novel concepts about its structure, physiology, and biological roles continue to be elucidated. Cardiac glycosides not only inhibit the pump function of Na,K-ATPase but also activate intracellular signal transduction pathways, which are important in many biological processes. Recently, antiviral effects have been described as a novel feature of Na,K-ATPase inhibition with the use of cardiac glycosides. Cardiac glycosides have been reported to be effective against both DNA viruses such as cytomegalovirus and herpes simplex and RNA viruses such as influenza, chikungunya, coronavirus, and respiratory syncytial virus, among others. Consequently, cardiac glycosides have emerged as potential broad-spectrum antiviral drugs, with the great advantage of targeting cell host proteins, which help to minimize resistance to antiviral treatments, making them a very promising strategy against human viral infections. Here, we review the effect of cardiac glycosides on viral biology and the mechanisms by which these drugs impair the replication of this array of different viruses.

Hypoxia impairs alveolar fluid reabsorption by promoting Na,K-ATPase endocytosis, from the plasma membrane of alveolar epithelial cells. The present study was designed to determine whether hypoxia induces Na,K-ATPase endocytosis via reactive oxygen species (ROS)-mediated RhoA activation. In A549 cells, RhoA activation occurred within 15 minutes of cells exposure to hypoxia. This activation was inhibited in cells infected with adenovirus coding for gluthatione peroxidase (an H2O2 scavenger), in mitochondria depleted (ρ0) cells or cells expressing decreased levels of the Rieske iron-sulfur protein (inhibitor of mitochondrial complex III), which suggests a role for mitochondrial ROS. Moreover, exogenous H2O2 treatment during normoxia mimicked the effects of hypoxia on RhoA, further supporting a role for ROS. Cells expressing dominant negative RhoA failed to endocytose the Na,K-ATPase during hypoxia or after H2O2 treatment. Na,K-ATPase endocytosis was also prevented in cells treated with Y-27632, a Rho-associated kinase (ROCK) inhibitor, and in cells expressing dominant negative ROCK. In summary, we provide evidence that in human alveolar epithelial cells exposed to hypoxia, RhoA/ROCK activation is necessary for Na,K-ATPase endocytosis via a mechanism that requires mitochondrial ROS.

The purpose of this study was to define the role of the Rho family of small GTPases in the β-adrenergic regulation of the Na,K-ATPase in alveolar epithelial cells (AEC). The β-adrenergic receptor agonist isoproterenol (ISO) increased the Na,K-ATPase protein abundance at the plasma membrane and activated RhoA in a time-dependent manner. AEC pretreated with mevastatin, a specific inhibitor of prenylation, or transfected with the dominant negative RhoAN19, prevented ISO-mediated Na,K-ATPase exocytosis to the plasma membrane. The ISO-mediated activation of RhoA in AEC occurred via β₂-adrenergic receptors and involved G_s-PKA as demonstrated by incubation with the protein kinase A (PKA)-specific inhibitors H89 and PKI (peptide specific inhibitor), and G_i, as incubation with pertussis toxin or cells transfected with a minigene vector for G_iinhibited the ISO-mediated RhoA activation. However, cells transfected with minigene vectors for G₁₂and G₁₃did not prevent RhoA activation by ISO. Finally, the ISO-mediated Na,K-ATPase exocytosis was regulated by the Rho-associated kinase (ROCK), as preincubation with the specific inhibitor Y-27632 or transfection with dominant negative ROCK, prevented the increase in Na,K-ATPase at the plasma membrane. Accordingly, ISO regulates Na,K-ATPase exocytosis in AEC via the activation of β₂-adrenergic receptor, G_s, PKA, G_i, RhoA, and ROCK.