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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 RV-infected and UV-RV stimulated epitheliaConfluent intestinal epithelia (HT29) were grown in 6 well plates and treated with RV and UV-RV (MOI 1). Control samples were exposed to trypsin diluted in SFM (Mock), irradiated cellular debris from a mock preparation of UV-RV (Mock Irradiation), or SFM alone (C). Cell lysates and supernatants were collected at various time points (0–48 hpi). Western blot analyses were performed to assess viral protein (VP6) synthesis and protein expression of the indicated anti-viral markers in cell lysates (A, D). ELISA assays were used to measure secretion of IFN-β (B) and IL-8 (C) in supernatants at 48 hpi. Data in A, B and D are results of a single experiment and representative of 3 separate experiments that gave similar results. Data in C is the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).
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Figure 2: Anti-viral protein expression exhibited in RV-infected and UV-RV stimulated epitheliaConfluent intestinal epithelia (HT29) were grown in 6 well plates and treated with RV and UV-RV (MOI 1). Control samples were exposed to trypsin diluted in SFM (Mock), irradiated cellular debris from a mock preparation of UV-RV (Mock Irradiation), or SFM alone (C). Cell lysates and supernatants were collected at various time points (0–48 hpi). Western blot analyses were performed to assess viral protein (VP6) synthesis and protein expression of the indicated anti-viral markers in cell lysates (A, D). ELISA assays were used to measure secretion of IFN-β (B) and IL-8 (C) in supernatants at 48 hpi. Data in A, B and D are results of a single experiment and representative of 3 separate experiments that gave similar results. Data in C is the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).

Mentions: To determine the extent to which RV-induced anti-viral signaling required viral replication or could be mimicked by structural components of RV, we examined the epithelial response to UV-irradiated RV (UV-RV), which is structurally intact but rendered non-replicative (29, 30). The inability of UV-RV to replicate in IEC was verified by monitoring levels of VP6 over time (Figure 2A). In accordance with previous studies, such UV-inactivation of the RV genome substantially reduced induction of IL-8 (Figure 2C). However, in contrast, signaling events typically associated with viral infection including activation of STAT1 and IRF3/7 and induction of IFN-β secretion, were elicited at least as robustly by UV-RV (Figure 2A, B). Similar activation of anti-viral signaling by RV and UV-RV was also observed in polarized epithelia in response to apical stimulation (data not shown). To confirm these events were indeed induced by UV-RV as opposed to UV cross-linked cell debris that might have been present in our virus preparation, we performed a control experiment showing that UV-irradiation of a mock viral preparation, which contained MA104 cell debris but not RV, did not elicit antiviral signaling induced by UV-RV (Figure 2D). Next, we determined if trypsinization, which is known to be required for viral entry, is also required for anti-viral signaling in response to UV-RV. Indeed, robust activation of anti-viral signaling by both RV and UV-RV required the stimulating agonist to be treated with trypsin prior to IEC stimulation (Figure 3). Lastly, we observed that, analogous to the case for RV, activation of innate immune signaling in response to UV-RV was more robust when UV-RV was applied to the apical rather than basolateral surface of polarized epithelia (Figure 4). Together, these results suggest that UV-RV induces anti-viral signaling via a mechanism similar to live virus, and further supports the notion that type I IFN is activated by IEC detection of RV structural components rather than viral replication.


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 RV-infected and UV-RV stimulated epitheliaConfluent intestinal epithelia (HT29) were grown in 6 well plates and treated with RV and UV-RV (MOI 1). Control samples were exposed to trypsin diluted in SFM (Mock), irradiated cellular debris from a mock preparation of UV-RV (Mock Irradiation), or SFM alone (C). Cell lysates and supernatants were collected at various time points (0–48 hpi). Western blot analyses were performed to assess viral protein (VP6) synthesis and protein expression of the indicated anti-viral markers in cell lysates (A, D). ELISA assays were used to measure secretion of IFN-β (B) and IL-8 (C) in supernatants at 48 hpi. Data in A, B and D are results of a single experiment and representative of 3 separate experiments that gave similar results. Data in C is 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 2: Anti-viral protein expression exhibited in RV-infected and UV-RV stimulated epitheliaConfluent intestinal epithelia (HT29) were grown in 6 well plates and treated with RV and UV-RV (MOI 1). Control samples were exposed to trypsin diluted in SFM (Mock), irradiated cellular debris from a mock preparation of UV-RV (Mock Irradiation), or SFM alone (C). Cell lysates and supernatants were collected at various time points (0–48 hpi). Western blot analyses were performed to assess viral protein (VP6) synthesis and protein expression of the indicated anti-viral markers in cell lysates (A, D). ELISA assays were used to measure secretion of IFN-β (B) and IL-8 (C) in supernatants at 48 hpi. Data in A, B and D are results of a single experiment and representative of 3 separate experiments that gave similar results. Data in C is the mean +/− SEM of 3 parallel experiments. Statistically significant differences P< 0.05 are denoted as starred values (*).
Mentions: To determine the extent to which RV-induced anti-viral signaling required viral replication or could be mimicked by structural components of RV, we examined the epithelial response to UV-irradiated RV (UV-RV), which is structurally intact but rendered non-replicative (29, 30). The inability of UV-RV to replicate in IEC was verified by monitoring levels of VP6 over time (Figure 2A). In accordance with previous studies, such UV-inactivation of the RV genome substantially reduced induction of IL-8 (Figure 2C). However, in contrast, signaling events typically associated with viral infection including activation of STAT1 and IRF3/7 and induction of IFN-β secretion, were elicited at least as robustly by UV-RV (Figure 2A, B). Similar activation of anti-viral signaling by RV and UV-RV was also observed in polarized epithelia in response to apical stimulation (data not shown). To confirm these events were indeed induced by UV-RV as opposed to UV cross-linked cell debris that might have been present in our virus preparation, we performed a control experiment showing that UV-irradiation of a mock viral preparation, which contained MA104 cell debris but not RV, did not elicit antiviral signaling induced by UV-RV (Figure 2D). Next, we determined if trypsinization, which is known to be required for viral entry, is also required for anti-viral signaling in response to UV-RV. Indeed, robust activation of anti-viral signaling by both RV and UV-RV required the stimulating agonist to be treated with trypsin prior to IEC stimulation (Figure 3). Lastly, we observed that, analogous to the case for RV, activation of innate immune signaling in response to UV-RV was more robust when UV-RV was applied to the apical rather than basolateral surface of polarized epithelia (Figure 4). Together, these results suggest that UV-RV induces anti-viral signaling via a mechanism similar to live virus, and further supports the notion that type I IFN is activated by IEC detection of RV structural components rather than viral replication.

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