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Intestinal epithelia activate anti-viral signaling via intracellular sensing of rotavirus structural components.

Frias AH, Vijay-Kumar M, Gentsch JR, Crawford SE, Carvalho FA, Estes MK, Gewirtz AT - Mucosal Immunol (2010)

Bottom Line: Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV.In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication.The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Emory University, Atlanta, Georgia, USA.

ABSTRACT
Rotavirus (RV), a leading cause of severe diarrhea, primarily infects intestinal epithelial cells (IECs) causing self-limiting illness. To better understand innate immunity to RV, we sought to define the extent to which IEC activation of anti-viral responses required viral replication or could be recapitulated by inactivated RV or its components. Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV. Use of anti-interferon (IFN) neutralizing antibodies revealed that such replication-independent anti-viral gene expression required type I IFN signaling. In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication. The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC. Together, these results suggest that RV proteins mediate viral entry into epithelial cells leading to intracellular detection of RV RNA that generates an anti-viral response.

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Anti-viral protein expression exhibited in epithelia treated with UV-RV, RV VLPs and RV RNAConfluent intestinal epithelia (HT29) were grown in 6 well plates and treated with UV-RV (MOI 0.5–1), RV RNA (0.5–5 μg/ml), and VLPs (0.5–5 μg/ml). Control samples received equivalent amounts of trypsin diluted in SFM (Mock). Cell lysates and supernatants were collected at various time points (0–24 hpi). Western blot analyses were used to detect anti-viral gene expression in cell lysates (A). ELISA assays were performed to measure IFN-β (B) or IL-8 (C) secretion in supernatants at 24 hpi. qRT-PCR analyses were utilized to confirm mRNA synthesis of select anti-viral genes from microarray experiments (see Table III). Data in A are results of a single experiment and representative of 3 separate experiments that gave similar results. Data in B, C, D, and E shows the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).
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Figure 6: Anti-viral protein expression exhibited in epithelia treated with UV-RV, RV VLPs and RV RNAConfluent intestinal epithelia (HT29) were grown in 6 well plates and treated with UV-RV (MOI 0.5–1), RV RNA (0.5–5 μg/ml), and VLPs (0.5–5 μg/ml). Control samples received equivalent amounts of trypsin diluted in SFM (Mock). Cell lysates and supernatants were collected at various time points (0–24 hpi). Western blot analyses were used to detect anti-viral gene expression in cell lysates (A). ELISA assays were performed to measure IFN-β (B) or IL-8 (C) secretion in supernatants at 24 hpi. qRT-PCR analyses were utilized to confirm mRNA synthesis of select anti-viral genes from microarray experiments (see Table III). Data in A are results of a single experiment and representative of 3 separate experiments that gave similar results. Data in B, C, D, and E shows the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).

Mentions: Next, we sought to better define the structural determinant of RV that played an important role in activating IEC anti-viral signaling. Specifically, we compared epithelial responses to UV-RV, RV virus-like particles (VLPs), and purified RV RNA. The VLPs used here are protein shells comprised of 4 major RV structural proteins (VP 2/4/6/7) that lack nucleic acid (31). RV VLPs were used at protein concentrations of 0.5–5μg/ml, which is equivalent to the concentration of UV-RV that corresponds to MOIs of 1–10. Purified RV RNA was used at concentrations of 0.5–5 μg/ml, which is approximately 100 times the amount of RNA in UV-RV that corresponds to MOIs of 1–10. In contrast to UV-RV, neither RV VLPs or RNA induced detectable elevations in levels of phospho-STAT1 or IRF7 (Figure 6A). RV VLPs and RNA also failed to recapitulate the induction of IFN-β or IL-8 elicited by UV-RV (Figure 6B, C). To more broadly understand the extent to which these components of UV-RV might recapitulate its ability to activate gene expression in epithelial cells, we measured changes in epithelial gene expression via microarray analysis. A modest concentration of UV-RV (MOI 0.5) induced 401 genes by > 1.3 fold relative to mock-treated control cells including key anti-viral genes such as Mda-5, IFN-β, MHC I (Tables II and III). Only a small portion of the genes upregulated by UV-RV were similarly induced by treatment with RV VLPs or RNA (Table II). In accordance, induction of anti-viral gene expression by RV viral components was also not observed to be comparable to UV-RV (Table III), and these trends were confirmed by measuring mRNA synthesis of select anti-viral genes via qRT-PCR (Figure 6D, E). Thus, neither RV RNA nor VLPs could substantially recapitulate the changes in gene expression induced by inactive but structurally intact RV.


Intestinal epithelia activate anti-viral signaling via intracellular sensing of rotavirus structural components.

Frias AH, Vijay-Kumar M, Gentsch JR, Crawford SE, Carvalho FA, Estes MK, Gewirtz AT - Mucosal Immunol (2010)

Anti-viral protein expression exhibited in epithelia treated with UV-RV, RV VLPs and RV RNAConfluent intestinal epithelia (HT29) were grown in 6 well plates and treated with UV-RV (MOI 0.5–1), RV RNA (0.5–5 μg/ml), and VLPs (0.5–5 μg/ml). Control samples received equivalent amounts of trypsin diluted in SFM (Mock). Cell lysates and supernatants were collected at various time points (0–24 hpi). Western blot analyses were used to detect anti-viral gene expression in cell lysates (A). ELISA assays were performed to measure IFN-β (B) or IL-8 (C) secretion in supernatants at 24 hpi. qRT-PCR analyses were utilized to confirm mRNA synthesis of select anti-viral genes from microarray experiments (see Table III). Data in A are results of a single experiment and representative of 3 separate experiments that gave similar results. Data in B, C, D, and E shows the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).
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Related In: Results  -  Collection

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Figure 6: Anti-viral protein expression exhibited in epithelia treated with UV-RV, RV VLPs and RV RNAConfluent intestinal epithelia (HT29) were grown in 6 well plates and treated with UV-RV (MOI 0.5–1), RV RNA (0.5–5 μg/ml), and VLPs (0.5–5 μg/ml). Control samples received equivalent amounts of trypsin diluted in SFM (Mock). Cell lysates and supernatants were collected at various time points (0–24 hpi). Western blot analyses were used to detect anti-viral gene expression in cell lysates (A). ELISA assays were performed to measure IFN-β (B) or IL-8 (C) secretion in supernatants at 24 hpi. qRT-PCR analyses were utilized to confirm mRNA synthesis of select anti-viral genes from microarray experiments (see Table III). Data in A are results of a single experiment and representative of 3 separate experiments that gave similar results. Data in B, C, D, and E shows the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).
Mentions: Next, we sought to better define the structural determinant of RV that played an important role in activating IEC anti-viral signaling. Specifically, we compared epithelial responses to UV-RV, RV virus-like particles (VLPs), and purified RV RNA. The VLPs used here are protein shells comprised of 4 major RV structural proteins (VP 2/4/6/7) that lack nucleic acid (31). RV VLPs were used at protein concentrations of 0.5–5μg/ml, which is equivalent to the concentration of UV-RV that corresponds to MOIs of 1–10. Purified RV RNA was used at concentrations of 0.5–5 μg/ml, which is approximately 100 times the amount of RNA in UV-RV that corresponds to MOIs of 1–10. In contrast to UV-RV, neither RV VLPs or RNA induced detectable elevations in levels of phospho-STAT1 or IRF7 (Figure 6A). RV VLPs and RNA also failed to recapitulate the induction of IFN-β or IL-8 elicited by UV-RV (Figure 6B, C). To more broadly understand the extent to which these components of UV-RV might recapitulate its ability to activate gene expression in epithelial cells, we measured changes in epithelial gene expression via microarray analysis. A modest concentration of UV-RV (MOI 0.5) induced 401 genes by > 1.3 fold relative to mock-treated control cells including key anti-viral genes such as Mda-5, IFN-β, MHC I (Tables II and III). Only a small portion of the genes upregulated by UV-RV were similarly induced by treatment with RV VLPs or RNA (Table II). In accordance, induction of anti-viral gene expression by RV viral components was also not observed to be comparable to UV-RV (Table III), and these trends were confirmed by measuring mRNA synthesis of select anti-viral genes via qRT-PCR (Figure 6D, E). Thus, neither RV RNA nor VLPs could substantially recapitulate the changes in gene expression induced by inactive but structurally intact RV.

Bottom Line: Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV.In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication.The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Emory University, Atlanta, Georgia, USA.

ABSTRACT
Rotavirus (RV), a leading cause of severe diarrhea, primarily infects intestinal epithelial cells (IECs) causing self-limiting illness. To better understand innate immunity to RV, we sought to define the extent to which IEC activation of anti-viral responses required viral replication or could be recapitulated by inactivated RV or its components. Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV. Use of anti-interferon (IFN) neutralizing antibodies revealed that such replication-independent anti-viral gene expression required type I IFN signaling. In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication. The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC. Together, these results suggest that RV proteins mediate viral entry into epithelial cells leading to intracellular detection of RV RNA that generates an anti-viral response.

Show MeSH
Related in: MedlinePlus