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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

Apical CAREx8 protein expression increases apical neutrophil adhesion that is sensitive to AdV5 FK.A neutrophil adhesion assay was performed on mock (0) or DOX-induced A) MDCK-CAREx8, B)-CAREx7, or C)-mCherry cells. Adhered neutrophils (green) on the surface of the epithelial cells were captured by fluorescence microscopy (10X; white bar = 100 μm). D) MDCK-CAREx8 either mock (0) or DOX-induced, as indicated, were treated with AdV5 FK or AdV3 FK immediately prior to performing the neutrophil adhesion assay. Adhered neutrophils were captured by using fluorescence microscopy and quantitated using Metamorph software. Images and quantitation are representative of 5–10 images from at least 3 separate experiments. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.01 by one-way ANOVA. White bar, 100 μM.
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ppat.1004696.g003: Apical CAREx8 protein expression increases apical neutrophil adhesion that is sensitive to AdV5 FK.A neutrophil adhesion assay was performed on mock (0) or DOX-induced A) MDCK-CAREx8, B)-CAREx7, or C)-mCherry cells. Adhered neutrophils (green) on the surface of the epithelial cells were captured by fluorescence microscopy (10X; white bar = 100 μm). D) MDCK-CAREx8 either mock (0) or DOX-induced, as indicated, were treated with AdV5 FK or AdV3 FK immediately prior to performing the neutrophil adhesion assay. Adhered neutrophils were captured by using fluorescence microscopy and quantitated using Metamorph software. Images and quantitation are representative of 5–10 images from at least 3 separate experiments. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.01 by one-way ANOVA. White bar, 100 μM.

Mentions: Since IL-8 induces CAREx8 protein expression and increases neutrophil retention at the apical surface of polarized epithelia (Fig. 1), we hypothesized that induction of CAREx8 protein expression in the absence of IL-8 would be sufficient to increase the binding of neutrophils at the apical surface of polarized epithelia. To test this, polarized MDCK-CAREx8,-CAREx7 and-mCherry epithelial cells were induced with increasing concentrations of DOX for 24 h and a neutrophil adhesion assay was performed. Increasing apical CAREx8 protein levels in MDCK-CAREx8 epithelia correlated directly with increased neutrophil adhesion on the epithelial cell surface (Fig. 3A). By contrast, MDCK-CAREx7 and-mCherry DOX-induced epithelia only showed baseline neutrophil adhesion (Fig. 3B, C). These data suggest that CAREx8 is able to tether neutrophils at the apical epithelial cell surface. To confirm that this was a CAREx8-mediated effect, purified AdV5 fiber knob (FK), which has a 500–1000 fold higher affinity for the overlapping CAR-JAML or CAR-CAR binding site [24–26], was used to compete with the putative interaction between epithelial apical CAREx8 and neutrophil JAML. AdV5 FK decreased neutrophil adhesion in a dose-dependent manner in both mock and DOX-induced MDCK-CAREx8 cells, including a complete block of neutrophil adhesion at the highest concentration of AdV5 FK (Fig. 3D). In contrast, FK from AdV3, a group B AdV that does not use CAR as a primary receptor [27], did not block neutrophil adhesion (Fig. 3D). Taken together, these data show that CAREx8 tethers neutrophils at the apical epithelial cell surface and that AdV may potentially be able to out-compete neutrophils to bind apical CAREx8.


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)

Apical CAREx8 protein expression increases apical neutrophil adhesion that is sensitive to AdV5 FK.A neutrophil adhesion assay was performed on mock (0) or DOX-induced A) MDCK-CAREx8, B)-CAREx7, or C)-mCherry cells. Adhered neutrophils (green) on the surface of the epithelial cells were captured by fluorescence microscopy (10X; white bar = 100 μm). D) MDCK-CAREx8 either mock (0) or DOX-induced, as indicated, were treated with AdV5 FK or AdV3 FK immediately prior to performing the neutrophil adhesion assay. Adhered neutrophils were captured by using fluorescence microscopy and quantitated using Metamorph software. Images and quantitation are representative of 5–10 images from at least 3 separate experiments. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.01 by one-way ANOVA. White bar, 100 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358923&req=5

ppat.1004696.g003: Apical CAREx8 protein expression increases apical neutrophil adhesion that is sensitive to AdV5 FK.A neutrophil adhesion assay was performed on mock (0) or DOX-induced A) MDCK-CAREx8, B)-CAREx7, or C)-mCherry cells. Adhered neutrophils (green) on the surface of the epithelial cells were captured by fluorescence microscopy (10X; white bar = 100 μm). D) MDCK-CAREx8 either mock (0) or DOX-induced, as indicated, were treated with AdV5 FK or AdV3 FK immediately prior to performing the neutrophil adhesion assay. Adhered neutrophils were captured by using fluorescence microscopy and quantitated using Metamorph software. Images and quantitation are representative of 5–10 images from at least 3 separate experiments. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.01 by one-way ANOVA. White bar, 100 μM.
Mentions: Since IL-8 induces CAREx8 protein expression and increases neutrophil retention at the apical surface of polarized epithelia (Fig. 1), we hypothesized that induction of CAREx8 protein expression in the absence of IL-8 would be sufficient to increase the binding of neutrophils at the apical surface of polarized epithelia. To test this, polarized MDCK-CAREx8,-CAREx7 and-mCherry epithelial cells were induced with increasing concentrations of DOX for 24 h and a neutrophil adhesion assay was performed. Increasing apical CAREx8 protein levels in MDCK-CAREx8 epithelia correlated directly with increased neutrophil adhesion on the epithelial cell surface (Fig. 3A). By contrast, MDCK-CAREx7 and-mCherry DOX-induced epithelia only showed baseline neutrophil adhesion (Fig. 3B, C). These data suggest that CAREx8 is able to tether neutrophils at the apical epithelial cell surface. To confirm that this was a CAREx8-mediated effect, purified AdV5 fiber knob (FK), which has a 500–1000 fold higher affinity for the overlapping CAR-JAML or CAR-CAR binding site [24–26], was used to compete with the putative interaction between epithelial apical CAREx8 and neutrophil JAML. AdV5 FK decreased neutrophil adhesion in a dose-dependent manner in both mock and DOX-induced MDCK-CAREx8 cells, including a complete block of neutrophil adhesion at the highest concentration of AdV5 FK (Fig. 3D). In contrast, FK from AdV3, a group B AdV that does not use CAR as a primary receptor [27], did not block neutrophil adhesion (Fig. 3D). Taken together, these data show that CAREx8 tethers neutrophils at the apical epithelial cell surface and that AdV may potentially be able to out-compete neutrophils to bind apical CAREx8.

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