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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

Induction of CAREx8 expression increases the susceptibility of polarized epithelia to AdV entry and transduction.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-CAREx8, Flag-CAREx7 protein expression was analyzed in lysates from MDCK-CAREx8 and-CAREx7 cells, respectively, after mock (0) or DOX induction. C) Apical surface-specific biotinylation of mock- (0) or DOX-induced polarized-MDCK-CAREx8 or-CAREx7 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.
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ppat.1004696.g002: Induction of CAREx8 expression increases the susceptibility of polarized epithelia to AdV entry and transduction.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-CAREx8, Flag-CAREx7 protein expression was analyzed in lysates from MDCK-CAREx8 and-CAREx7 cells, respectively, after mock (0) or DOX induction. C) Apical surface-specific biotinylation of mock- (0) or DOX-induced polarized-MDCK-CAREx8 or-CAREx7 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.

Mentions: To confirm that CAREx8 is responsible for increased AdV5 transduction and that CAREx8 tethers neutrophils at the apical surface of polarized epithelia, model epithelial cells stably expressing CAREx8 under a Doxycycline (DOX) inducible promoter were generated. Control cells stably expressing CAREx7 or mCherry were also generated from the same parental Tet-on MDCK cell line. MDCK cells were chosen because these cells are well characterized, grow quickly, and polarize rapidly into an epithelium with an expected distribution of cellular proteins [21–23]. Polarized epithelia from cell lines derived from single-cell clones with stable integration of FLAG-tagged CAREx8, FLAG-tagged CAREx7, or mCherry, under the DOX sensitive PTight promoter were characterized and compared in the absence of DOX. Clones were selected that had similar growth and polarization characteristics, including the ability to form tight junctions, distribution of apical, basolateral, and tight junction proteins, and polarity of baseline AdV5 transduction. MDCK cells stably expressing mCherry, FLAG-tagged CAREx8, or FLAG-tagged CAREx7, demonstrated a DOX-dose dependent increase of mCherry fluorescence (Fig. 2A), or CAREx8 or CAREx7 protein levels relative to actin (Fig. 2B). To confirm the polarity of protein expression with the polarized MDCK epithelium, apical surface-specific biotinylation was performed. In contrast to CAREx7 in MDCK-CAREx7 epithelia, CAREx8 protein was detected at the apical surface of MDCK-CAREx8 epithelia at low doses of DOX and expression was saturated above 100 ng/ml of DOX (Fig. 2C).


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)

Induction of CAREx8 expression increases the susceptibility of polarized epithelia to AdV entry and transduction.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-CAREx8, Flag-CAREx7 protein expression was analyzed in lysates from MDCK-CAREx8 and-CAREx7 cells, respectively, after mock (0) or DOX induction. C) Apical surface-specific biotinylation of mock- (0) or DOX-induced polarized-MDCK-CAREx8 or-CAREx7 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.
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Related In: Results  -  Collection

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ppat.1004696.g002: Induction of CAREx8 expression increases the susceptibility of polarized epithelia to AdV entry and transduction.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-CAREx8, Flag-CAREx7 protein expression was analyzed in lysates from MDCK-CAREx8 and-CAREx7 cells, respectively, after mock (0) or DOX induction. C) Apical surface-specific biotinylation of mock- (0) or DOX-induced polarized-MDCK-CAREx8 or-CAREx7 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.
Mentions: To confirm that CAREx8 is responsible for increased AdV5 transduction and that CAREx8 tethers neutrophils at the apical surface of polarized epithelia, model epithelial cells stably expressing CAREx8 under a Doxycycline (DOX) inducible promoter were generated. Control cells stably expressing CAREx7 or mCherry were also generated from the same parental Tet-on MDCK cell line. MDCK cells were chosen because these cells are well characterized, grow quickly, and polarize rapidly into an epithelium with an expected distribution of cellular proteins [21–23]. Polarized epithelia from cell lines derived from single-cell clones with stable integration of FLAG-tagged CAREx8, FLAG-tagged CAREx7, or mCherry, under the DOX sensitive PTight promoter were characterized and compared in the absence of DOX. Clones were selected that had similar growth and polarization characteristics, including the ability to form tight junctions, distribution of apical, basolateral, and tight junction proteins, and polarity of baseline AdV5 transduction. MDCK cells stably expressing mCherry, FLAG-tagged CAREx8, or FLAG-tagged CAREx7, demonstrated a DOX-dose dependent increase of mCherry fluorescence (Fig. 2A), or CAREx8 or CAREx7 protein levels relative to actin (Fig. 2B). To confirm the polarity of protein expression with the polarized MDCK epithelium, apical surface-specific biotinylation was performed. In contrast to CAREx7 in MDCK-CAREx7 epithelia, CAREx8 protein was detected at the apical surface of MDCK-CAREx8 epithelia at low doses of DOX and expression was saturated above 100 ng/ml of DOX (Fig. 2C).

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