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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

Neutrophils adhered to the apical surface of polarized-MDCK cells augment AdV entry without decreasing the TER.A) MDCK-CAREx8 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-CAREx8 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-CAREx8 C) MDCK-mCherry and D) MDCK-CAREx7 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-CAREx8 epithelia in the presence or absence of neutrophils and AdV5 FK or AdV3 FK. F) TER of mock- or Dox-induced MDCK-CAREx8 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.
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ppat.1004696.g005: Neutrophils adhered to the apical surface of polarized-MDCK cells augment AdV entry without decreasing the TER.A) MDCK-CAREx8 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-CAREx8 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-CAREx8 C) MDCK-mCherry and D) MDCK-CAREx7 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-CAREx8 epithelia in the presence or absence of neutrophils and AdV5 FK or AdV3 FK. F) TER of mock- or Dox-induced MDCK-CAREx8 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.

Mentions: Neutrophils are part of the innate immune system and the first cells recruited to sites of injury or pathogenic invasion. In order to understand the contribution of neutrophils bound to the apical surface to AdV infection, increasing amounts of primary human neutrophils (0–1 x 107 cells) were allowed to bind to the apical surface of mock-induced or DOX-induced polarized MDCK-CAREx8 epithelia. Unbound neutrophils were removed by washing and AdV5-β-Gal was added to the apical surface for 1 h at 37°C. Viral entry was quantified 24 h later by qPCR. Neutrophils enhanced AdV entry by approximately 2–3 fold (Fig. 5A, white bars) and, consistent with a significant increase in neutrophil binding, AdV entry was increased by an additional 2-fold when CAREx8 expression was induced with DOX (Fig. 5A, grey bars). To determine whether neutrophil-enhanced AdV entry was dependent on viral dose, 2 X 106 neutrophils were allowed to bind CAREx8, or control CAREx7 and mCherry, mock-induced or DOX-induced epithelia followed by apical transduction with increasing MOI of AdV5-β-Gal (Fig. 5B-D). To control for baseline infection, mock-induced epithelia having no neutrophils were also similarly infected with AdV5-β-Gal. Neutrophils increased apical AdV entry by 3–10-fold in MDCK-CAREx8 epithelia at all MOI (Fig. 5B). Except at MOI 1, this increase was further amplified by at least 3 fold in the presence of DOX, indicating that both neutrophils and the level of apical CAREx8 play a major role in AdV entry. In the case of uninduced mCherry epithelia (Fig. 5C), neutrophils significantly increased AdV entry in a similar manner as uninduced MDCK-CAREx8 cells, while MDCK-CAREx7 epithelia followed this trend (Fig. 5D). However, no significant change in AdV entry occurred in the presence of DOX indicating the importance of CAREx8 expression. Taken together, these data show that neutrophils facilitate viral entry into the polarized MDCK epithelium, particularly upon induction of apical CAREx8 expression. To confirm that the effect of neutrophils on AdV entry depends on CAR, polarized MDCK-CAREx8 cells were treated with either AdV5 FK or AdV3 FK, followed by neutrophil adhesion and infection with AdV5-β-Gal. We observed that AdV5 FK blocked AdV5-β-Gal entry by ∼7-fold in the absence of adhered neutrophils (p<0.0001, Fig. 5E). In the presence of adhered neutrophils, AdV5 FK, but not AdV3 FK, blocked AdV5-β-Gal entry by ∼25-fold (p<0.0001). The difference in fold change reflects the increased AdV-β-Gal entry in the presence of neutrophils. These results indicate that neutrophils promote adenoviral entry via CAREx8. To further confirm that neutrophils were not simply disrupting the epithelial tight junction, TER was measured in the presence or absence of apically adhered neutrophils. Interestingly, a trend towards increased transepithelial resistance was observed when compared to MDCK-CAREx8 cells without neutrophils (Fig. 5F). A lack of tight junction disruption is consistent with the evidence that increasing the basolateral CAREx7 isoform does not further augment viral infection in the presence of neutrophils (Figs. 2B and 5D).


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)

Neutrophils adhered to the apical surface of polarized-MDCK cells augment AdV entry without decreasing the TER.A) MDCK-CAREx8 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-CAREx8 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-CAREx8 C) MDCK-mCherry and D) MDCK-CAREx7 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-CAREx8 epithelia in the presence or absence of neutrophils and AdV5 FK or AdV3 FK. F) TER of mock- or Dox-induced MDCK-CAREx8 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.
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ppat.1004696.g005: Neutrophils adhered to the apical surface of polarized-MDCK cells augment AdV entry without decreasing the TER.A) MDCK-CAREx8 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-CAREx8 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-CAREx8 C) MDCK-mCherry and D) MDCK-CAREx7 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-CAREx8 epithelia in the presence or absence of neutrophils and AdV5 FK or AdV3 FK. F) TER of mock- or Dox-induced MDCK-CAREx8 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.
Mentions: Neutrophils are part of the innate immune system and the first cells recruited to sites of injury or pathogenic invasion. In order to understand the contribution of neutrophils bound to the apical surface to AdV infection, increasing amounts of primary human neutrophils (0–1 x 107 cells) were allowed to bind to the apical surface of mock-induced or DOX-induced polarized MDCK-CAREx8 epithelia. Unbound neutrophils were removed by washing and AdV5-β-Gal was added to the apical surface for 1 h at 37°C. Viral entry was quantified 24 h later by qPCR. Neutrophils enhanced AdV entry by approximately 2–3 fold (Fig. 5A, white bars) and, consistent with a significant increase in neutrophil binding, AdV entry was increased by an additional 2-fold when CAREx8 expression was induced with DOX (Fig. 5A, grey bars). To determine whether neutrophil-enhanced AdV entry was dependent on viral dose, 2 X 106 neutrophils were allowed to bind CAREx8, or control CAREx7 and mCherry, mock-induced or DOX-induced epithelia followed by apical transduction with increasing MOI of AdV5-β-Gal (Fig. 5B-D). To control for baseline infection, mock-induced epithelia having no neutrophils were also similarly infected with AdV5-β-Gal. Neutrophils increased apical AdV entry by 3–10-fold in MDCK-CAREx8 epithelia at all MOI (Fig. 5B). Except at MOI 1, this increase was further amplified by at least 3 fold in the presence of DOX, indicating that both neutrophils and the level of apical CAREx8 play a major role in AdV entry. In the case of uninduced mCherry epithelia (Fig. 5C), neutrophils significantly increased AdV entry in a similar manner as uninduced MDCK-CAREx8 cells, while MDCK-CAREx7 epithelia followed this trend (Fig. 5D). However, no significant change in AdV entry occurred in the presence of DOX indicating the importance of CAREx8 expression. Taken together, these data show that neutrophils facilitate viral entry into the polarized MDCK epithelium, particularly upon induction of apical CAREx8 expression. To confirm that the effect of neutrophils on AdV entry depends on CAR, polarized MDCK-CAREx8 cells were treated with either AdV5 FK or AdV3 FK, followed by neutrophil adhesion and infection with AdV5-β-Gal. We observed that AdV5 FK blocked AdV5-β-Gal entry by ∼7-fold in the absence of adhered neutrophils (p<0.0001, Fig. 5E). In the presence of adhered neutrophils, AdV5 FK, but not AdV3 FK, blocked AdV5-β-Gal entry by ∼25-fold (p<0.0001). The difference in fold change reflects the increased AdV-β-Gal entry in the presence of neutrophils. These results indicate that neutrophils promote adenoviral entry via CAREx8. To further confirm that neutrophils were not simply disrupting the epithelial tight junction, TER was measured in the presence or absence of apically adhered neutrophils. Interestingly, a trend towards increased transepithelial resistance was observed when compared to MDCK-CAREx8 cells without neutrophils (Fig. 5F). A lack of tight junction disruption is consistent with the evidence that increasing the basolateral CAREx7 isoform does not further augment viral infection in the presence of neutrophils (Figs. 2B and 5D).

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