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Induction of IFN-beta and the innate antiviral response in myeloid cells occurs through an IPS-1-dependent signal that does not require IRF-3 and IRF-7.

Daffis S, Suthar MS, Szretter KJ, Gale M, Diamond MS - PLoS Pathog. (2009)

Bottom Line: Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons.However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells.Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-alpha/beta) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-alpha levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3(-/-)x IRF-7(-/-) double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons. In contrast, the IFN-beta response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-beta response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

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The WNV-induced IFN-β response and ISG expression is primarily IRF-3 and IRF-7-independent in mDC.A. mDC generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments (***, P<0.0001). B–E. Levels of (B) IFN-α and (D) IFN-β mRNA as well as (C) IFN-α and (E) IFN-β protein in WNV-infected mDC were measured by qRT-PCR or ELISA as described in the legend of Figure 3. F. Whole cell lysates were generated at the indicated times from wild type and DKO mDC that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis.
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ppat-1000607-g006: The WNV-induced IFN-β response and ISG expression is primarily IRF-3 and IRF-7-independent in mDC.A. mDC generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments (***, P<0.0001). B–E. Levels of (B) IFN-α and (D) IFN-β mRNA as well as (C) IFN-α and (E) IFN-β protein in WNV-infected mDC were measured by qRT-PCR or ELISA as described in the legend of Figure 3. F. Whole cell lysates were generated at the indicated times from wild type and DKO mDC that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis.

Mentions: Because mDC are likely early targets for WNV infection in animals [31]–[33] and help orchestrate innate and adaptive antiviral immune responses [34], we evaluated IFN and ISG responses in bone marrow-derived mDC. Prior studies showed that mDC lacking either IRF-3 or IRF-7 support enhanced WNV replication, IRF-3−/− mDC developed normal IFN-α/β responses after WNV infection, and IRF-7−/− mDC had reduced IFN-α but relatively intact IFN-β responses after WNV infection (S. Daffis and M. Diamond, unpublished results and [22]). Multi-step growth curve analysis of DKO mDC infected with WNV showed a higher viral burden compared to wild type cells (16 to 95-fold, P<0.0001) and IRF-3−/− or IRF-7−/− mDC (Fig 6A, S. Daffis and M. Diamond, unpublished results, and [22]). The viral titers in DKO mDC were similar to those obtained in IFN-αβR−/− mDC, suggesting a defect in type I IFN signaling in the DKO cells. Surprisingly, whereas levels of IFN-α mRNA and protein were abolished (Fig 6B and 6C), induction of IFN-β gene and protein production was not significantly affected (P>0.2) after WNV infection of DKO mDC (Fig 6D and 6E). Thus, in mDC, IRF-3 and IRF-7 regulate the IFN-α response but are largely dispensable for inducing IFN-β after WNV infection. Western blot analysis of ISG corroborated these findings as similar levels of ISG were observed in wild type and DKO mDC at 24 and 48 h after WNV infection (Fig 6F). These data suggest that the expression of ISG is primarily IFN-β-dependent or that the IFN-α and -β have redundant effects in these cells. Nonetheless, as higher viral replication was sustained in DKO mDC despite a relatively normal IFN-β response and ISG expression profile, it remains possible that IRF-3 directly regulates expression of a key subset of ISG that accounts for anti-WNV activity in this cell type.


Induction of IFN-beta and the innate antiviral response in myeloid cells occurs through an IPS-1-dependent signal that does not require IRF-3 and IRF-7.

Daffis S, Suthar MS, Szretter KJ, Gale M, Diamond MS - PLoS Pathog. (2009)

The WNV-induced IFN-β response and ISG expression is primarily IRF-3 and IRF-7-independent in mDC.A. mDC generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments (***, P<0.0001). B–E. Levels of (B) IFN-α and (D) IFN-β mRNA as well as (C) IFN-α and (E) IFN-β protein in WNV-infected mDC were measured by qRT-PCR or ELISA as described in the legend of Figure 3. F. Whole cell lysates were generated at the indicated times from wild type and DKO mDC that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747008&req=5

ppat-1000607-g006: The WNV-induced IFN-β response and ISG expression is primarily IRF-3 and IRF-7-independent in mDC.A. mDC generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments (***, P<0.0001). B–E. Levels of (B) IFN-α and (D) IFN-β mRNA as well as (C) IFN-α and (E) IFN-β protein in WNV-infected mDC were measured by qRT-PCR or ELISA as described in the legend of Figure 3. F. Whole cell lysates were generated at the indicated times from wild type and DKO mDC that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis.
Mentions: Because mDC are likely early targets for WNV infection in animals [31]–[33] and help orchestrate innate and adaptive antiviral immune responses [34], we evaluated IFN and ISG responses in bone marrow-derived mDC. Prior studies showed that mDC lacking either IRF-3 or IRF-7 support enhanced WNV replication, IRF-3−/− mDC developed normal IFN-α/β responses after WNV infection, and IRF-7−/− mDC had reduced IFN-α but relatively intact IFN-β responses after WNV infection (S. Daffis and M. Diamond, unpublished results and [22]). Multi-step growth curve analysis of DKO mDC infected with WNV showed a higher viral burden compared to wild type cells (16 to 95-fold, P<0.0001) and IRF-3−/− or IRF-7−/− mDC (Fig 6A, S. Daffis and M. Diamond, unpublished results, and [22]). The viral titers in DKO mDC were similar to those obtained in IFN-αβR−/− mDC, suggesting a defect in type I IFN signaling in the DKO cells. Surprisingly, whereas levels of IFN-α mRNA and protein were abolished (Fig 6B and 6C), induction of IFN-β gene and protein production was not significantly affected (P>0.2) after WNV infection of DKO mDC (Fig 6D and 6E). Thus, in mDC, IRF-3 and IRF-7 regulate the IFN-α response but are largely dispensable for inducing IFN-β after WNV infection. Western blot analysis of ISG corroborated these findings as similar levels of ISG were observed in wild type and DKO mDC at 24 and 48 h after WNV infection (Fig 6F). These data suggest that the expression of ISG is primarily IFN-β-dependent or that the IFN-α and -β have redundant effects in these cells. Nonetheless, as higher viral replication was sustained in DKO mDC despite a relatively normal IFN-β response and ISG expression profile, it remains possible that IRF-3 directly regulates expression of a key subset of ISG that accounts for anti-WNV activity in this cell type.

Bottom Line: Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons.However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells.Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-alpha/beta) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-alpha levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3(-/-)x IRF-7(-/-) double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons. In contrast, the IFN-beta response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-beta response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

Show MeSH
Related in: MedlinePlus