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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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Transcription profiles of epithelia stimulated with RV, UV-RV, and RV in the presence of Type 1 IFN (α/β) antibodiesIntestinal epithelial (HT29) cell monolayers were grown in 6 well plates and infected with RV and UV-RV (MOI 1), and RV (MOI 1) plus Type 1 IFN antibodies (anti-IFN α/β). Control samples received equivalent amounts of trypsin diluted in SFM (Mock). Experiments were performed in biological triplicates. At 24 hpi, cell lysates were extracted for RNA and microarray analyses were performed to assess global transcription of genes. Heat map illustration of genes induced by RV and UV-RV with > 1.3 fold change relative to mock (A). qRT-PCR results used to confirm mRNA synthesis of select anti-viral genes at 24 hpi (B, C, D, E). Data in A shows results of 3 parallel experiments. Data in B, C, D, and E reflects the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).
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Figure 5: Transcription profiles of epithelia stimulated with RV, UV-RV, and RV in the presence of Type 1 IFN (α/β) antibodiesIntestinal epithelial (HT29) cell monolayers were grown in 6 well plates and infected with RV and UV-RV (MOI 1), and RV (MOI 1) plus Type 1 IFN antibodies (anti-IFN α/β). Control samples received equivalent amounts of trypsin diluted in SFM (Mock). Experiments were performed in biological triplicates. At 24 hpi, cell lysates were extracted for RNA and microarray analyses were performed to assess global transcription of genes. Heat map illustration of genes induced by RV and UV-RV with > 1.3 fold change relative to mock (A). qRT-PCR results used to confirm mRNA synthesis of select anti-viral genes at 24 hpi (B, C, D, E). Data in A shows results of 3 parallel experiments. Data in B, C, D, and E reflects the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).

Mentions: We next sought to define the extent to which RV-induced gene expression in general can be mimicked by UV-RV and determine the role of type I IFN in RV-induced changes in IEC gene expression. IEC were mock-infected or exposed to RV, UV-RV, or RV in the presence of neutralizing antibodies to type 1 IFN (anti-IFN α/β) for 24 h, at which time gene expression was assayed by cDNA microarray. The gene chip employed for this purpose permits simultaneous examination of 12 different samples allowing us to assay 4 different experimental conditions in biological triplicates, thus permitting statistical analysis to be performed directly on the microarray data. Such microarray analyses indicated that RV upregulated 1190 genes by at least 1.3 fold relative to mock-treated uninfected cells (cut-off was arbitrarily chosen based on our previous experience with microarray-based studies of IEC (24)). The entire microarray data set is available on-line (posted on GEO) and some of the common means of examining microarray data such as unsupervised clustering analysis are shown in supplemental data (Supplemental Figure 1). In Figure 5A, we sought to display our microarray data in a manner that would most facilitate addressing our central questions. Specifically, we generated a “heat map” that displays gene expression in each of the 12 samples (4 conditions, 3 replicates) relative to the average expression of mock-treated cells. The relative uniformity of the 3 replicates in each condition indicates the high degree of similarity among our biological replicates. Genes were ordered (top to bottom) based on their relative dependence upon type I IFN (ratio of expression upon exposure to RV alone vs. RV in the presence of anti-IFN α/β). The majority of genes that were induced by RV are thus type I IFN-dependent in that their expression was reduced by the neutralizing antibody. The heat map shows that the vast majority of such IFN-dependent gene expression did not appear to require viral replication in that almost of all of these genes were similarly induced by both RV and UV-RV. Such type-I IFN dependent, replication-independent, RV-induced gene expression included a panel of classic anti-viral genes such as IRF7, IFN-β, STAT1, Mx1, OAS-2 and MHC I (Table I). Use of qRT-PCR verified the upregulated mRNA levels of some of these genes (Figure 5B, C, D, E). In contrast to the induction of such classic anti-viral genes, expression of IL-8 was partially dependent upon viral replication (Figure 2C) and independent of type I IFN (Table I). Thus, a large portion of RV-induced gene expression in IEC, particularly upregulation of genes typically associated with viral infection, is independent of viral replication and dependent upon type I IFN.


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)

Transcription profiles of epithelia stimulated with RV, UV-RV, and RV in the presence of Type 1 IFN (α/β) antibodiesIntestinal epithelial (HT29) cell monolayers were grown in 6 well plates and infected with RV and UV-RV (MOI 1), and RV (MOI 1) plus Type 1 IFN antibodies (anti-IFN α/β). Control samples received equivalent amounts of trypsin diluted in SFM (Mock). Experiments were performed in biological triplicates. At 24 hpi, cell lysates were extracted for RNA and microarray analyses were performed to assess global transcription of genes. Heat map illustration of genes induced by RV and UV-RV with > 1.3 fold change relative to mock (A). qRT-PCR results used to confirm mRNA synthesis of select anti-viral genes at 24 hpi (B, C, D, E). Data in A shows results of 3 parallel experiments. Data in B, C, D, and E reflects the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).
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Figure 5: Transcription profiles of epithelia stimulated with RV, UV-RV, and RV in the presence of Type 1 IFN (α/β) antibodiesIntestinal epithelial (HT29) cell monolayers were grown in 6 well plates and infected with RV and UV-RV (MOI 1), and RV (MOI 1) plus Type 1 IFN antibodies (anti-IFN α/β). Control samples received equivalent amounts of trypsin diluted in SFM (Mock). Experiments were performed in biological triplicates. At 24 hpi, cell lysates were extracted for RNA and microarray analyses were performed to assess global transcription of genes. Heat map illustration of genes induced by RV and UV-RV with > 1.3 fold change relative to mock (A). qRT-PCR results used to confirm mRNA synthesis of select anti-viral genes at 24 hpi (B, C, D, E). Data in A shows results of 3 parallel experiments. Data in B, C, D, and E reflects the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).
Mentions: We next sought to define the extent to which RV-induced gene expression in general can be mimicked by UV-RV and determine the role of type I IFN in RV-induced changes in IEC gene expression. IEC were mock-infected or exposed to RV, UV-RV, or RV in the presence of neutralizing antibodies to type 1 IFN (anti-IFN α/β) for 24 h, at which time gene expression was assayed by cDNA microarray. The gene chip employed for this purpose permits simultaneous examination of 12 different samples allowing us to assay 4 different experimental conditions in biological triplicates, thus permitting statistical analysis to be performed directly on the microarray data. Such microarray analyses indicated that RV upregulated 1190 genes by at least 1.3 fold relative to mock-treated uninfected cells (cut-off was arbitrarily chosen based on our previous experience with microarray-based studies of IEC (24)). The entire microarray data set is available on-line (posted on GEO) and some of the common means of examining microarray data such as unsupervised clustering analysis are shown in supplemental data (Supplemental Figure 1). In Figure 5A, we sought to display our microarray data in a manner that would most facilitate addressing our central questions. Specifically, we generated a “heat map” that displays gene expression in each of the 12 samples (4 conditions, 3 replicates) relative to the average expression of mock-treated cells. The relative uniformity of the 3 replicates in each condition indicates the high degree of similarity among our biological replicates. Genes were ordered (top to bottom) based on their relative dependence upon type I IFN (ratio of expression upon exposure to RV alone vs. RV in the presence of anti-IFN α/β). The majority of genes that were induced by RV are thus type I IFN-dependent in that their expression was reduced by the neutralizing antibody. The heat map shows that the vast majority of such IFN-dependent gene expression did not appear to require viral replication in that almost of all of these genes were similarly induced by both RV and UV-RV. Such type-I IFN dependent, replication-independent, RV-induced gene expression included a panel of classic anti-viral genes such as IRF7, IFN-β, STAT1, Mx1, OAS-2 and MHC I (Table I). Use of qRT-PCR verified the upregulated mRNA levels of some of these genes (Figure 5B, C, D, E). In contrast to the induction of such classic anti-viral genes, expression of IL-8 was partially dependent upon viral replication (Figure 2C) and independent of type I IFN (Table I). Thus, a large portion of RV-induced gene expression in IEC, particularly upregulation of genes typically associated with viral infection, is independent of viral replication and dependent upon type I IFN.

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