Sustained Protein Kinase D Activation Mediates Respiratory Syncytial Virus-Induced Airway Barrier Disruption

Abstract

Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o-bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases. R espiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children (1) and an important cause of death in childhood (2). RSV has been identified as a source of morbidity and mortality in elderly and high-risk adults (3). RSV infects airway epithelial cells and is thought to cause tissue pathology by inducing the expression of proinflammatory mediators, leading to airway inflammation and, ultimately, an antiviral immune response (4). RSV also induces the expression of antiapoptotic genes and promotes epithelial cell survival, which is probably a strategy to ensure viral replication in infected cells (5).Emerging evidence points to a role for airway barrier dysfunction during respiratory viral infections (6), as well as in stable asthmatics (7). The airway barrier is made up of the surface mucus layer, as well as apical junction complexes (AJC) that regulate paracellular permeability (8). Previously we demonstrated that polyinosinic-polycytidylic acid [poly(I-C)], a synthetic doublestranded RNA and viral mimetic, induces potent breakdown of the airway epithelial AJC in a protein kinase D (PKD)-dependent manner (9). PKD, formerly known as PKC, is a serine/threonine protein kinase fami...

Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation

“…Previously, cortactin deficiency in mice had been demonstrated to affect mammalian zygotic development by modulating its association with actin filaments (Yu et al, 2010), but a role in cell-cell adhesion in mice had not been investigated. Different studies have attributed cortactin a role in epithelial (Rezaee et al, 2013;Zhao et al, 2012) and endothelial barrier functions (Dudek et al, 2004;Schnoor et al, 2011). However, wildtype PKD1-KI mice, in line with literature (Sinnett-Smith et al, 2011), did not display obvious physical phenotypes or alterations in tight junction proteins, such as claudin 1 (Fig.…”

Protein Kinase D Promotes Airway Epithelial Barrier Dysfunction and Permeability through Down-regulation of Claudin-1

“…In airway epithelial cells, it has been shown that the synthetic double-stranded RNA polyI:C induces PKD phosphorylation/activation and epithelial barrier disruption and that the epithelial barrier disruption by polyI:C could be attenuated by Gö6976, a selective inhibitor of PKD and classical PKC isoforms (35). Moreover, Rezaee et al (59) recently reported that airway epithelial barrier disruption induced by respiratory syncytial virus involved PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. These findings support a role of PKD in the regulation of airway epithelial barrier formation.…”

Epithelial barrier function: At the front line of asthma immunology and allergic airway inflammation

“…Examples of environmental exposures implicated in airway epithelial barrier dysfunction include respiratory viruses (e.g. coxsackie virus, rhinovirus and respiratory syncytial virus 48-51 ), air pollution components (e.g. ozone and ambient particulate matter 52-54 ), cigarette smoke 55, 56 , and allergens (considered below).…”