The Coxsackievirus and Adenovirus Receptor Has a Short Half-Life in Epithelial Cells

The coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell adhesion, cell signaling, and viral infection. The 8-exon encoded isoform (CAREx8) resides at the apical surface of polarized epithelia, where it is accessible as a receptor for adenovirus entering the airway lumen. Given its pivotal role in viral infection, it is a target for antiviral strategies. To understand the regulation of CAREx8 and determine the feasibility of receptor down regulation, the half-life of total and apical localized CAREx8 was determined and correlated with adenovirus transduction. Total and apical CAREx8 has a relatively short half-life of approximately 2 h. The half-life of apical CAREx8 correlates well with adenovirus transduction. These results suggest that antiviral strategies that aim to degrade the primary receptor for apical adenovirus infection will be effective within a relatively short time frame after application

Neutrophils adhered to the apical surface of polarized-MDCK cells augment AdV entry without decreasing the TER.

<p>A) MDCK-CAR^Ex8 cells were either mock- or DOX-induced. A neutrophil adhesion assay was performed with increasing numbers of neutrophils, as indicated. Immediately post-neutrophil adhesion, MDCK-CAR^Ex8 epithelia were infected with AdV5-β-gal for 1 h from the apical surface. 24 h later, viral entry was determined by qPCR analysis. Fold change in viral genomes, relative to AdV5-βGal entry in the absence of DOX and neutrophils, is shown. AdV entry from the apical surface was quantitated by qPCR analysis of polarized B) MDCK-CAR^Ex8 C) MDCK-mCherry and D) MDCK-CAR^Ex7 cells that were uninduced (circles), uninduced with adhered neutrophils (squares), or induced with DOX for 24 h prior to neutrophil adhesion (triangles). E) AdV5-β-gal entry from the apical surface of MDCK-CAR^Ex8 epithelia in the presence or absence of neutrophils and AdV5 FK or AdV3 FK. F) TER of mock- or Dox-induced MDCK-CAR^Ex8 epithelia was measured in the presence or absence of neutrophils. Error bars represent standard error of the mean (SEM) from three independent experiments. No significant difference was detected by one-way ANOVA. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.001 by one-way ANOVA and Bonferroni post hoc test.</p

Apical CAR^Ex8 protein expression increases apical adhesion of infiltrating neutrophils.

<p>Neutrophil transmigration assays were performed in the basal-to-apical direction in MDCK stable cells exposed to the neutrophil chemoattractive peptide fMLP on the apical surface. A) % neutrophil adhesion and B) % neutrophil transmigration were quantitated by measuring the fluorescence intensity of fluorescently-labeled neutrophils imaged by fluorescence microscopy. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.01 by one-way ANOVA.</p

Schematic of IL-8-mediated enhancement of AdV entry into polarized epithelia.

<p>1) Pathogenic microbes that invade the airway 2) cause both the resident macrophages and the epithelial cells to secrete IL-8. 3) IL-8 exposure causes intracellular signaling within the epithelial cells that augments <i>de novo</i> protein synthesis and apical localization of CAR^Ex8. 4) IL-8 simultaneously recruits neutrophils that transmigrate through the epithelium from the basal surface to the apical surface and 5) bind to CAR^Ex8 at the apical surface of the epithelium. 6) AdV entering the airway hijacks the host innate immune response and apical CAR^Ex8 to gain entry into the host cell.</p

IL-8 activates AKT/S6K and inactivates GSK3β to increase CAR^Ex8 protein synthesis and AdV entry.

<p>A) The apical surfaces of polarized primary airway epithelial cells were either mock (0, white bars) or IL-8 (30 ng/ml, gray bars) treated for the indicated time and analyzed for CAR^Ex8, CAR^Ex7, or E-cadherin (E-cad) gene expression by qPCR, relative to GAPDH. B) The apical surfaces of polarized primary airway epithelial cells were mock (0) or IL-8 treated in the presence or absence of cycloheximide (CHX) and lysates were analyzed for CAR^Ex8 and actin protein expression. Activation state of C) AKT, D) S6K and H) GSK3β was analyzed after IL-8 treatment by probing for the pAKT T308, pS6K T389, and pGSK3β S9 respectively. Lysates from polarized cells treated with IL-8 in the presence or absence of chemical inhibitors for E) AKT (Ly294002, 30 μM), F) S6K (RO3118220, 300 nM), I) GSK3β (SB415286, 45 μM, or LiCl, 10 mM), or J) a combination of S6K (RO3118220, 300 nM) and GSK3β (SB415286, 45 μM) were investigated for CAR^Ex8 and actin protein expression. G) Polarized cells were either transfected or not with myc-tagged S6K plasmid prior to mock (0) or IL-8 treatment followed by the analysis of CAR^Ex8 and actin protein expression from cell lysates. K) Polarized cells exposed to IL-8 in the presence or absence of the indicated chemical inhibitors for 4 h were washed and transduced with AdV5-βGal for 1 h. Genomic DNA was isolated 24 h post-transduction and analyzed for the fold change in Vg normalized to GAPDH and relative to mock. Error bars represent the SEM from three independent experiments: **p < 0.001 by one way ANOVA and Bonferroni post hoc test. L) A schematic of a predicted model showing that 1) IL-8 binds to the IL-8 receptor (CXCR1/2) and 2) activates AKT. 3) Activated AKT (pAKT T308) further activates S6K (pS6K T389) and 4) activated AKT directly and/or via inhibition of GSK3β (pGSK3β S9) stimulates CAR^Ex8 protein synthesis. 5) Newly synthesized CAR^Ex8 traffics to the apical surface and 6) can mediate apical AdV infection.</p

Induction of CAR^Ex8 expression increases the susceptibility of polarized epithelia to AdV entry and transduction.

<p>A) MDCK-mCherry cells either mock (0) or DOX treated for 24 h were imaged using fluorescence microscopy (20X, white bar = 30 μm). Hoechst 33342 staining (blue) indicates cellular nuclei. B) Flag-CAR^Ex8, Flag-CAR^Ex7 protein expression was analyzed in lysates from MDCK-CAR^Ex8 and-CAR^Ex7 cells, respectively, after mock (0) or DOX induction. C) Apical surface-specific biotinylation of mock- (0) or DOX-induced polarized-MDCK-CAR^Ex8 or-CAR^Ex7 cells analyzed by Western blot using an anti-FLAG-tag Ab. D) Polarized MDCK-stable cells were treated with increasing concentrations of DOX for 24 h, transduced with AdV5-βGal from the apical surface for 1 h, and analyzed 24 h post-infection for viral entry by qPCR (viral genomes, Vg) or E) viral transduction via β-gal activity. Error bars represent the SEM from three independent experiments; *p < 0.05 by two-way ANOVA.</p

IL-8 increases the susceptibility of polarized airway epithelia to AdV entry, apical CAR^Ex8 protein expression, and neutrophil adhesion at the apical surface.

<p>The apical surfaces of polarized A-D) Calu-3 cells or F-I) primary human airway epithelia were exposed to IL-8 for 4 h. A) Mock (0) or IL-8-exposed Calu-3 epithelia were transduced with AdV5 from the apical surface and analyzed 24 h later for the fold change in viral genomes (Vg) relative to GAPDH by qPCR. B) TER before or after IL-8 (30 ng/ml) exposure. C) Western blots for CAR^Ex8, total CAR, actin, and E-cadherin protein expression in lysates or D) CAR^EX8 and actin after apical surface-specific biotinylation. E) CAR^Ex8 and actin protein expression in lysates from Calu-3 cells exposed to IL-8 for different lengths of time. The apical surface of polarized primary airway epithelial cells were exposed to IL-8 and F) CAR^Ex8, actin, and E-cadherin protein expression in lysates or G) after apical surface-specific biotinylation. H) Polarized primary human airway epithelia were either mock or IL-8 treated for 4 h. Cells were then either untreated or treated with purified AdV5 FK, as indicated, followed by an adhesion assay with primary neutrophils stained with calcein green. Bound neutrophils were imaged using fluorescence microscopy (10X lens, white bar = 150 μm) and I) quantified using Metamorph software. Error bars represent the SEM from three independent experiments: *p < 0.05, A and B by one-way ANOVA or I, IL-8 treatment versus untreated or FK treated.</p

Adenovirus Entry From the Apical Surface of Polarized Epithelia Is Facilitated by the Host Innate Immune Response

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apicalepithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection