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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 partially modulate the IFN-β response and ISG expression in primary Mφ.A. Mφ generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.01 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. B. Whole cell lysates were generated at the indicated times from wild type and DKO Mφ 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. C and D. The induction of (C) IFN-α and (D) IFN-β mRNA in WNV-infected Mφ was analyzed by qRT-PCR as described in Figure 3. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05).
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ppat-1000607-g005: IRF-3 and IRF-7 partially modulate the IFN-β response and ISG expression in primary Mφ.A. Mφ generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.01 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. B. Whole cell lysates were generated at the indicated times from wild type and DKO Mφ 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. C and D. The induction of (C) IFN-α and (D) IFN-β mRNA in WNV-infected Mφ was analyzed by qRT-PCR as described in Figure 3. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05).

Mentions: As previous studies suggested cell type-specific differences in type I IFN induction [21], we evaluated the effect of the combined IRF-3 and IRF-7 deficiency in macrophages (Mφ), a cell type that is permissive to WNV in vivo [26]. Prior experiments established that Mφ lacking either IRF-3 or IRF-7 were more susceptible to WNV infection [21],[22]. Analogously, DKO Mφ supported increased WNV replication (∼35, 250 and 310-fold, P<0.0001 at 24, 48 and 72 h after infection, respectively) compared to wild type or even IRF-3−/− or IRF-7−/− singly deficient cells (Fig 5A and [21],[22]). In contrast to that observed with MEF (see Fig 3), the DKO Mφ produced ∼14-fold less (P<0.05) WNV at 72 h compared to IFN-αβR−/− Mφ infected in parallel. Thus, an absence of both IRF-3 and IRF-7 in Mφ did not recapitulate the replication phenotype of IFN-αβR−/− cells. As such a discrepancy could be related to differences in the type I IFN and/or ISG response, we analyzed these in DKO Mφ after WNV infection. As expected, levels of IFN-α mRNA were completely abolished (Fig 5B), consistent with results from IRF-7−/− Mφ [22]. Whereas IFN-β mRNA levels were reduced at 24 h after infection in DKO Mφ, they accumulated to normal levels by 48 h (Fig 5C). Western blot analysis of ISG expression confirmed this pattern, as the early induction of several ISG was altered at 24 h but not 48 h following WNV infection (Fig 5D). Thus, after WNV infection of Mφ, IRF-3 and IRF-7 coordinately regulate the early but are dispensable for the later IFN-β and ISG responses.


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 partially modulate the IFN-β response and ISG expression in primary Mφ.A. Mφ generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.01 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. B. Whole cell lysates were generated at the indicated times from wild type and DKO Mφ 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. C and D. The induction of (C) IFN-α and (D) IFN-β mRNA in WNV-infected Mφ was analyzed by qRT-PCR as described in Figure 3. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05).
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Related In: Results  -  Collection

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ppat-1000607-g005: IRF-3 and IRF-7 partially modulate the IFN-β response and ISG expression in primary Mφ.A. Mφ generated from wild type, IFN-αβR−/− and DKO mice were infected at an MOI of 0.01 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. B. Whole cell lysates were generated at the indicated times from wild type and DKO Mφ 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. C and D. The induction of (C) IFN-α and (D) IFN-β mRNA in WNV-infected Mφ was analyzed by qRT-PCR as described in Figure 3. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05).
Mentions: As previous studies suggested cell type-specific differences in type I IFN induction [21], we evaluated the effect of the combined IRF-3 and IRF-7 deficiency in macrophages (Mφ), a cell type that is permissive to WNV in vivo [26]. Prior experiments established that Mφ lacking either IRF-3 or IRF-7 were more susceptible to WNV infection [21],[22]. Analogously, DKO Mφ supported increased WNV replication (∼35, 250 and 310-fold, P<0.0001 at 24, 48 and 72 h after infection, respectively) compared to wild type or even IRF-3−/− or IRF-7−/− singly deficient cells (Fig 5A and [21],[22]). In contrast to that observed with MEF (see Fig 3), the DKO Mφ produced ∼14-fold less (P<0.05) WNV at 72 h compared to IFN-αβR−/− Mφ infected in parallel. Thus, an absence of both IRF-3 and IRF-7 in Mφ did not recapitulate the replication phenotype of IFN-αβR−/− cells. As such a discrepancy could be related to differences in the type I IFN and/or ISG response, we analyzed these in DKO Mφ after WNV infection. As expected, levels of IFN-α mRNA were completely abolished (Fig 5B), consistent with results from IRF-7−/− Mφ [22]. Whereas IFN-β mRNA levels were reduced at 24 h after infection in DKO Mφ, they accumulated to normal levels by 48 h (Fig 5C). Western blot analysis of ISG expression confirmed this pattern, as the early induction of several ISG was altered at 24 h but not 48 h following WNV infection (Fig 5D). Thus, after WNV infection of Mφ, IRF-3 and IRF-7 coordinately regulate the early but are dispensable for the later IFN-β and ISG responses.

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