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Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

Kotha PL, Sharma P, Kolawole AO, Yan R, Alghamri MS, Brockman TL, Gomez-Cambronero J, Excoffon KJ - PLoS Pathog. (2015)

Bottom Line: Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection.We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity.In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biological Sciences, Wright State University, Dayton, Ohio, United States of America.

ABSTRACT
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 apical-epithelial 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.

No MeSH data available.


Related in: MedlinePlus

IL-8 increases the susceptibility of polarized airway epithelia to AdV entry, apical CAREx8 protein expression, and neutrophil adhesion at the apical surface.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 CAREx8, total CAR, actin, and E-cadherin protein expression in lysates or D) CAREX8 and actin after apical surface-specific biotinylation. E) CAREx8 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) CAREx8, 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.
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ppat.1004696.g001: IL-8 increases the susceptibility of polarized airway epithelia to AdV entry, apical CAREx8 protein expression, and neutrophil adhesion at the apical surface.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 CAREx8, total CAR, actin, and E-cadherin protein expression in lysates or D) CAREX8 and actin after apical surface-specific biotinylation. E) CAREx8 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) CAREx8, 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.

Mentions: To investigate the effect of IL-8 on AdV infection in polarized epithelia, we first used polarized Calu-3 airway epithelial model cells. Polarized Calu-3 epithelia were treated with increasing concentrations of IL-8 (0–100 ng/ml (0–12.5 nM)) for 4 h, followed by apical infection with recombinant, replication-defective, AdV type 5 (AdV5). Quantitative PCR (qPCR) analysis for AdV5 genomes (Vg) was performed by determining the copy number of the AdV5 hexon gene relative to a cellular housekeeping gene after DNA extraction. QPCR showed that AdV entry was increased in response to IL-8 treatment in a dose-dependent manner (Fig. 1A). Viral entry reached its maximum and plateaued at 3, 10 and 30 ng/ml of IL-8, with ∼5-fold increase in Vg when compared to control (0 ng/ml IL-8; p<0.05), followed by a decrease at 100 ng/ml. However, there was no significant change in the transepithelial resistance (TER) indicating that the effect of IL-8 on viral entry was not due to decreased integrity of the epithelial junctions (Fig. 1B). These data suggest that the increase in epithelial susceptibility to AdV entry upon IL-8 exposure may be due to specific cellular effects, such as increased primary receptor expression at the apical surface of the polarized epithelium.


Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

Kotha PL, Sharma P, Kolawole AO, Yan R, Alghamri MS, Brockman TL, Gomez-Cambronero J, Excoffon KJ - PLoS Pathog. (2015)

IL-8 increases the susceptibility of polarized airway epithelia to AdV entry, apical CAREx8 protein expression, and neutrophil adhesion at the apical surface.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 CAREx8, total CAR, actin, and E-cadherin protein expression in lysates or D) CAREX8 and actin after apical surface-specific biotinylation. E) CAREx8 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) CAREx8, 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.
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ppat.1004696.g001: IL-8 increases the susceptibility of polarized airway epithelia to AdV entry, apical CAREx8 protein expression, and neutrophil adhesion at the apical surface.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 CAREx8, total CAR, actin, and E-cadherin protein expression in lysates or D) CAREX8 and actin after apical surface-specific biotinylation. E) CAREx8 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) CAREx8, 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.
Mentions: To investigate the effect of IL-8 on AdV infection in polarized epithelia, we first used polarized Calu-3 airway epithelial model cells. Polarized Calu-3 epithelia were treated with increasing concentrations of IL-8 (0–100 ng/ml (0–12.5 nM)) for 4 h, followed by apical infection with recombinant, replication-defective, AdV type 5 (AdV5). Quantitative PCR (qPCR) analysis for AdV5 genomes (Vg) was performed by determining the copy number of the AdV5 hexon gene relative to a cellular housekeeping gene after DNA extraction. QPCR showed that AdV entry was increased in response to IL-8 treatment in a dose-dependent manner (Fig. 1A). Viral entry reached its maximum and plateaued at 3, 10 and 30 ng/ml of IL-8, with ∼5-fold increase in Vg when compared to control (0 ng/ml IL-8; p<0.05), followed by a decrease at 100 ng/ml. However, there was no significant change in the transepithelial resistance (TER) indicating that the effect of IL-8 on viral entry was not due to decreased integrity of the epithelial junctions (Fig. 1B). These data suggest that the increase in epithelial susceptibility to AdV entry upon IL-8 exposure may be due to specific cellular effects, such as increased primary receptor expression at the apical surface of the polarized epithelium.

Bottom Line: Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection.We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity.In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biological Sciences, Wright State University, Dayton, Ohio, United States of America.

ABSTRACT
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 apical-epithelial 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.

No MeSH data available.


Related in: MedlinePlus