Alcohol potentiates RSV-mediated injury to ciliated airway epithelium.

Alcohol impairs resolution of respiratory viral infections. Numerous immune response pathways are altered in response to alcohol misuse, including alcohol-induced ciliary dysfunction in the lung. We hypothesized that mucociliary clearance-mediated innate immunity to respiratory syncytial virus (RSV) would be compromised by alcohol exposure. Cilia were assayed using Sisson-Ammons Video Analysis by quantitating the average number of motile points in multiple whole field measurements of mouse tracheal epithelial cells grown on an air-liquid interface. Pretreatment with ethanol alone (100 mM for 24 hours) had no effect on the number of motile cilia. A single dose (TCID50 1 × 105) of RSV resulted in a significant (p < 0.05) decrease in motile cilia after 2 days. Ethanol pretreatment significantly (p < 0.05) potentiated RSV-induced cilia loss by 2 days. Combined RSV and ethanol treatment led to a sustained activation-induced auto-downregulation of PKC epsilon (PKCε). Ethanol-induced enhancement of ciliated cell detachment was confirmed by dynein ELISA and LDH activity from the supernates. RSV-induced cilia loss was evident until 7 days, when RSV-only infected cells demonstrated no significant cilia loss vs. control cells. However, cells pretreated with ethanol showed significant cilia loss until 10 days post-RSV infection. To address the functional significance of ethanol-enhanced cilia detachment, mice fed alcohol ad libitum (20% for 12 weeks) were infected once with RSV, and clearance was measured by plaque-forming assay from lung homogenates for up to 7 days. After 3 days, RSV plaque formation was no longer detected from the lungs of control mice, while significant (p < 0.01) RSV plaque-forming units were detected at 7 days in alcohol-fed mice. Alcohol-fed mice demonstrated enhanced cilia loss and delayed cilia recovery from tracheal measurements in wild-type C57BL/6 mice, but not PKCε KO mice. These data suggest that alcohol worsens RSV-mediated injury to ciliated epithelium in a PKCε-dependent manner.

Non-typeable Haemophilus influenzae decreases cilia beating via protein kinase Cε.

BACKGROUND: Haemophilus influenzae infection of the nasal epithelium has long been associated with observations of decreased nasal ciliary beat frequency (CBF) and injury to the ciliated epithelium. Previously, we have reported that several agents that slow CBF also have the effect of activating protein kinase C epsilon (PKCε) activity in bronchial epithelial cells. The subsequent auto-downregulation of PKCε or the direct inhibition of PKCε leads to the specific detachment of the ciliated cells. METHODS: Primary cultures of ciliated bovine bronchial epithelial cells were exposed to filtered conditioned media supernatants from non-typeable H. influenzae (NTHi) cultures. CBF and motile points were measured and PKCε activity assayed. RESULTS: NTHi supernatant exposure significantly and rapidly decreased CBF in a dose-dependent manner within 10 minutes of exposure. After 3 hours of exposure, the number of motile ciliated cells significantly decreased. Direct measurement of PKCε activity revealed a dose-dependent activation of PKCε in response to NTHi supernatant exposure. Both CBF and PKCε activity changes were only observed in fresh NTHi culture supernatant and not observed in exposures to heat-inactivated or frozen supernatants. CONCLUSIONS: Our results suggest that CBF slowing observed in response to NTHi is consistent with the stimulated activation of PKCε. Ciliated cell detachment is associated with PKCε autodownregulation.

RACK1, a PKC targeting protein, is exclusively localized to basal airway epithelial cells.

The novel isoform of protein kinase C (PKC), PKCepsilon, is an important regulator of ciliated cell function in airway epithelial cells, including cilia motility and detachment of ciliated cells after environmental insult. However, the mechanism of PKCepsilon signaling in the airways and the potential role of the PKCepsilon-interacting protein, receptor for activated C kinase 1 (RACK1), has not been widely explored. We used immunohistochemistry and Western blot analysis to show that RACK1 is localized exclusively to basal, non-ciliated (and non-goblet) bovine and human bronchial epithelial cells. Our immunohistochemistry experiments used the basal body marker pericentrin, a marker for cilia, beta-tubulin, and an airway goblet cell marker, MUC5AC, to confirm that RACK1 was excluded from differentiated airway cell subtypes and is only expressed in the basal cells. These results suggest that PKCepsilon signaling in the basal airway cell may involve RACK1; however, PKCepsilon regulation in ciliated cells uses RACK1-independent pathways.

Inhibition of protein kinase C epsilon causes ciliated bovine bronchial cell detachment.

This study defines the in vitro phenomenon of ciliated bovine bronchial epithelial cell (BBEC) detachment from the basal epithelium and regulation of cilia motility mediated through protein kinase C epsilon (PKCepsilon). The authors determined the time course of activation and downregulation of PKCepsilon by the known PKC activator phorbol 12-myristate 13-acetate (PMA) and demonstrate that chemical inhibition of PKC by calphostin C or the novel PKC isoform inhibitor Ro 31-8220 induced striking detachment of ciliated BBECs from the basal cell monolayer within 1 hour, independent of apoptosis or necrotic cell death. The results of this study support a possible novel PKCepsilon-mediated signaling pathway through which ciliated cell attachment is maintained.