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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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IRF-3 and IRF-7 control the IFN-α/β gene induction and ISG expression in MEF.A–D. MEF generated from wild type or DKO mice were infected at an MOI of 0.1 and analyzed for IFN-α/β gene induction. Total RNA from uninfected and WNV-infected MEF was harvested at the indicated times after infection and levels of (A) IFN-α and (C) IFN-β mRNA were measured by qRT-PCR. Data are normalized to 18S rRNA and are expressed as the relative fold increase over normalized RNA from uninfected controls. Accumulation of (B) IFN-α and (D) IFN-β protein in supernatants was evaluated by ELISA. E. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, RIG-I, WNV and tubulin were examined by immunoblot analysis. F. MEF generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated by plaque assay. The data is the average of at least three independent experiments performed in quadruplicate, (***, P<0.0001).
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ppat-1000607-g003: IRF-3 and IRF-7 control the IFN-α/β gene induction and ISG expression in MEF.A–D. MEF generated from wild type or DKO mice were infected at an MOI of 0.1 and analyzed for IFN-α/β gene induction. Total RNA from uninfected and WNV-infected MEF was harvested at the indicated times after infection and levels of (A) IFN-α and (C) IFN-β mRNA were measured by qRT-PCR. Data are normalized to 18S rRNA and are expressed as the relative fold increase over normalized RNA from uninfected controls. Accumulation of (B) IFN-α and (D) IFN-β protein in supernatants was evaluated by ELISA. E. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, RIG-I, WNV and tubulin were examined by immunoblot analysis. F. MEF generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated by plaque assay. The data is the average of at least three independent experiments performed in quadruplicate, (***, P<0.0001).

Mentions: To better understand the net effect of IRF-3 and IRF-7 on WNV infection and induction of a protective IFN response, we infected primary cells from DKO and wild type mice. Because MEF have been studied extensively in virus infection-host immune response assays [16],[29],[30], we initially evaluated the effect of an IRF-3 × IRF-7 deficiency in these cells. Previous experiments had shown a blunted IFN-α response and a normal IFN-β response in IRF-7−/− MEF infected with WNV [22]. In contrast, IRF-3−/− MEF had a diminished IFN-α and -β response early after infection but developed normal levels at later time points (S. Daffis and M. Diamond, unpublished data). In DKO MEF, IFN-α mRNA and protein secretion were completely abolished after WNV infection (Fig 3A and 3B). Similarly, levels of IFN-β mRNA were strongly reduced at 24 h (∼70-fold decrease, P<0.0001) and 48 h after infection (∼130-fold decrease, P<0.0001) but not entirely abolished (Fig 3C). Measurement of secreted IFN-β in the cell supernatants corroborated these findings as a ∼4 and 20-fold reduction (P<0.0001) was observed in DKO MEF at 24 and 48 hours, respectively (Fig 3D). Thus, normal induction of IFN-α and IFN-β in MEF after WNV infection primarily requires transcriptional activation by IRF-3 and IRF-7.


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)

IRF-3 and IRF-7 control the IFN-α/β gene induction and ISG expression in MEF.A–D. MEF generated from wild type or DKO mice were infected at an MOI of 0.1 and analyzed for IFN-α/β gene induction. Total RNA from uninfected and WNV-infected MEF was harvested at the indicated times after infection and levels of (A) IFN-α and (C) IFN-β mRNA were measured by qRT-PCR. Data are normalized to 18S rRNA and are expressed as the relative fold increase over normalized RNA from uninfected controls. Accumulation of (B) IFN-α and (D) IFN-β protein in supernatants was evaluated by ELISA. E. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, RIG-I, WNV and tubulin were examined by immunoblot analysis. F. MEF generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated by plaque assay. The data is the average of at least three independent experiments performed in quadruplicate, (***, P<0.0001).
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ppat-1000607-g003: IRF-3 and IRF-7 control the IFN-α/β gene induction and ISG expression in MEF.A–D. MEF generated from wild type or DKO mice were infected at an MOI of 0.1 and analyzed for IFN-α/β gene induction. Total RNA from uninfected and WNV-infected MEF was harvested at the indicated times after infection and levels of (A) IFN-α and (C) IFN-β mRNA were measured by qRT-PCR. Data are normalized to 18S rRNA and are expressed as the relative fold increase over normalized RNA from uninfected controls. Accumulation of (B) IFN-α and (D) IFN-β protein in supernatants was evaluated by ELISA. E. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, RIG-I, WNV and tubulin were examined by immunoblot analysis. F. MEF generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated by plaque assay. The data is the average of at least three independent experiments performed in quadruplicate, (***, P<0.0001).
Mentions: To better understand the net effect of IRF-3 and IRF-7 on WNV infection and induction of a protective IFN response, we infected primary cells from DKO and wild type mice. Because MEF have been studied extensively in virus infection-host immune response assays [16],[29],[30], we initially evaluated the effect of an IRF-3 × IRF-7 deficiency in these cells. Previous experiments had shown a blunted IFN-α response and a normal IFN-β response in IRF-7−/− MEF infected with WNV [22]. In contrast, IRF-3−/− MEF had a diminished IFN-α and -β response early after infection but developed normal levels at later time points (S. Daffis and M. Diamond, unpublished data). In DKO MEF, IFN-α mRNA and protein secretion were completely abolished after WNV infection (Fig 3A and 3B). Similarly, levels of IFN-β mRNA were strongly reduced at 24 h (∼70-fold decrease, P<0.0001) and 48 h after infection (∼130-fold decrease, P<0.0001) but not entirely abolished (Fig 3C). Measurement of secreted IFN-β in the cell supernatants corroborated these findings as a ∼4 and 20-fold reduction (P<0.0001) was observed in DKO MEF at 24 and 48 hours, respectively (Fig 3D). Thus, normal induction of IFN-α and IFN-β in MEF after WNV infection primarily requires transcriptional activation by IRF-3 and IRF-7.

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