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. 2010 Aug 15;185(4):2393-404.
doi: 10.4049/jimmunol.0903563. Epub 2010 Jul 14.

Suppressor of cytokine signaling 3 inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages

Lisa Nowoslawski Akhtar  1 , Hongwei Qin, Michelle T Muldowney, Lora L Yanagisawa, Olaf Kutsch, Janice E Clements, Etty N Benveniste
Affiliations

Suppressor of cytokine signaling 3 inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages

Lisa Nowoslawski Akhtar et al. J Immunol. .

Abstract

HIV-1 replication within macrophages of the CNS often results in cognitive and motor impairment, which is known as HIV-associated dementia (HAD) in its most severe form. IFN-beta suppresses viral replication within these cells during early CNS infection, but the effect is transient. HIV-1 eventually overcomes this protective innate immune response to resume replication through an unknown mechanism, initiating the progression toward HAD. In this article, we show that Suppressor of Cytokine Signaling (SOCS)3, a molecular inhibitor of IFN signaling, may allow HIV-1 to evade innate immunity within the CNS. We found that SOCS3 is elevated in an in vivo SIV/macaque model of HAD and that the pattern of expression correlates with recurrence of viral replication and onset of CNS disease. In vitro, the HIV-1 regulatory protein transactivator of transcription induces SOCS3 in human and murine macrophages in a NF-kappaB-dependent manner. SOCS3 expression attenuates the response of macrophages to IFN-beta at proximal levels of pathway activation and downstream antiviral gene expression and consequently overcomes the inhibitory effect of IFN-beta on HIV-1 replication. These studies indicate that SOCS3 expression, induced by stimuli present in the HIV-1-infected brain, such as transactivator of transcription, inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages. This consequence of SOCS3 expression in vitro, supported by a correlation with increased viral load and onset of CNS disease in vivo, suggests that SOCS3 may allow HIV-1 to evade the protective innate immune response within the CNS, allowing the recurrence of viral replication and, ultimately, promoting progression toward HAD.

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Figures

Figure 1
Figure 1. SOCS3 expression correlates with viral load and CNS disease in a SIV/macaque model of HAD
(A) RNA was collected from the basal ganglia of SIV-infected macaques at the times indicated post-infection (p.i.) and analyzed by quantitative RT-PCR with primers specific for SOCS3. Individual samples were normalized to 18S and displayed as fold increase above the non-infected control (c) with the highest levels of SOCS3 +/- standard error. Disease severity is indicated as mild (Mi), moderate (Mo) or severe (S). CNS viral load is indicated as no infection (0), <100,000 copies (↓), 100,000 to 1 million copies (-), 1 million to 10 million copies (↑), or >10 million copies (*). (B) Infected animals were grouped by disease severity. The number of animals with increased SOCS3 levels (defined as a 3-fold or greater increase above control levels), and average fold induction of SOCS3 expression for each group was determined.
Figure 2
Figure 2. HIV-1 Tat induces SOCS3 expression in macrophages and microglia
Cells were treated with 10 nM HIV-1 Tat1-72aa for the times indicated. mRNA was analyzed by RT-PCR with primers specific for SOCS3 (A, C, G, H, J) or ribonuclease protection assay with a probe specific to SOCS3 (E, I). GAPDH mRNA was examined as a loading control. To calculate induction of SOCS3 mRNA (E), individual samples were normalized to GAPDH and expressed as fold increase above untreated cells. Protein was subjected to immunoblot analysis with antibodies specific for SOCS3 (B, D, F). Blots were stripped and reprobed for actin as a loading control. Data are representative of at least two independent experiments.
Figure 3
Figure 3. HIV-1 Tat-induced SOCS3 expression is transcriptionally regulated
RAW264.7 cells were transiently transfected with 200 ng of a murine SOCS3 promoter-driven luciferase construct (A). Cells were then treated with 10 nM HIV-1 Tat1-72aa for 24 h and analyzed for luciferase production (B). Values are displayed as fold increase above untreated cells +/- standard error and represent three independent experiments performed in triplicate. *p<0.05 versus untreated cells as determined by unpaired t-test.
Figure 4
Figure 4. HIV-1 Tat signals through the NF-κB pathway to induce SOCS3 expression
(A) Cells were treated with 10 nM HIV-1 Tat1-72aa for the times indicated. Protein was subjected to immunoblot analysis with antibodies specific to phosphorylated p65 (serine 276), total p65, and IκBα. Blots were then stripped and reprobed for actin as a loading control. Data are representative of two independent experiments. (B) RAW264.7 cells were transiently transfected with 50 ng of an IKKβ dominant negative (DN) construct or an empty pcDNA3 vector. Cells were also co-transfected with a murine SOCS3 promoter-driven luciferase reporter construct. Following 48 h of recovery, cells were treated with 10 nM HIV-1 Tat1-72aa for 24 h and analyzed for luciferase production. Values are displayed as fold increase above untreated cells +/- standard error. *p<0.001 versus untreated cells and #p<0.05 versus 10 nM Tat treatment alone as determined by one-way ANOVA followed by Bonferroni's multiple comparison test. (C) Cells were pretreated with 10 μM BAY 11-7085 for 2 h and then treated with 10 nM HIV-1 Tat1-72aa for 1 h. mRNA was analyzed by RT-PCR with primers specific for SOCS3. GAPDH mRNA was examined as a loading control.
Figure 5
Figure 5. HIV-1 Tat-induced SOCS3 expression is not mediated by IL-10 or IFN-β expression
(A) RAW264.7 cells or primary murine BMDM were treated with 10 nM HIV-1 Tat1-72aa, or 100 ng/ml LPS as a positive control, for the times indicated. mRNA was analyzed by RT-PCR with primers specific to IL-10 and IFN-β. A vertical line has been inserted to indicate the merge of separate gels. (B) Wild-type (WT) or IL-10 deficient (KO) primary murine macrophages were treated with 10 nM HIV-1 Tat1-72aa, or 100 ng/ml IFN-γ as a positive control, for the times indicated. mRNA was analyzed by RT-PCR with primers specific to IL-10 and SOCS3. (C) WT or IFNAR-deficient primary murine macrophages were treated with 10 nM HIV-1 Tat1-72aa, or 100 U/ml IFN-β as a positive control, for the times indicated. mRNA was analyzed by RT-PCR with primers specific to SOCS3. GAPDH was examined as a loading control in all experiments. Data are representative of two independent experiments.
Figure 6
Figure 6. HIV-1 Tat inhibits IFN-β-induced STAT activation and antiviral target gene expression
RAW264.7 cells (A) or primary murine BMDM (B) were pretreated for 4 h with 10 nM HIV-1 Tat1-72aa or vehicle alone, and then exposed to 100 U/ml murine IFN-β or 100 ng/ml murine IFN-γ for 15 minutes. Protein was subjected to immunoblot analysis with antibodies specific to phosphorylated STAT1 (tyrosine 701 or serine 727), phosphorylated STAT2 (tyrosine 689), phosphorylated STAT3 (tyrosine 705 or serine 727), or total levels of these proteins. Blots were then stripped and reprobed for actin as a loading control. Quantification of band density shown to the right of blots represents pY-STAT levels normalized to total STAT levels, and is displayed as fold decrease below IFN treatment alone. Data are representative of at least two independent experiments. (C) Primary human PBDM were pretreated for 4 h with 10 nM HIV-1 Tat1-72aa or vehicle alone, and then exposed to 100 U/ml human IFN-β for 15 min. Antibodies specific to phosphorylated STAT1 (tyrosine 701), phosphorylated STAT2 (tyrosine 689), and total levels of these proteins were used for immunoblot analysis. Blots were then stripped and reprobed for actin as a loading control. Quantification of band density shown to the right of blots represents pY-STAT levels normalized to total STAT levels, and is displayed as fold decrease below IFN treatment alone. Data are representative of three independent experiments. (D) RAW264.7 cells were pretreated for 4 h with 10 nM HIV-1 Tat1-72aa or vehicle alone, and then exposed to 100 U/ml murine IFN-β for 4 h. mRNA was analyzed by RT-PCR with primers specific to murine ISG20 and PKR. GAPDH was examined as a loading control. Data are representative of two independent experiments.
Figure 7
Figure 7. SOCS3 mediates HIV-1 Tat-induced inhibition of IFN-β signaling
(A) SOCS3fl/fl and SOCS3Δ/Δ primary murine BMDM were treated with 10 nM HIV-1 Tat1-72aa for 2 h, and mRNA analyzed by RT-PCR with primers specific for SOCS3. GAPDH mRNA was examined as a loading control. Data are representative of two independent experiments. (B) Cells were pretreated with 10 nM HIV-1 Tat1-72aa for 4 h, followed by treatment with 100 U/ml murine IFN-β for 30 min. Protein was subjected to immunoblot analysis with antibodies specific for phosphorylated STAT1 (tyrosine 701), phosphorylated STAT2 (tyrosine 689), or total levels of these proteins. Blots were then stripped and reprobed for GAPDH as a loading control. Quantification of band density shown to the right of blots represents pY-STAT levels normalized to total STAT levels, and is displayed as fold decrease below IFN treatment alone. Data are representative of two independent experiments.
Figure 8
Figure 8. SOCS3 prevents the ability of IFN-β to suppress HIV-1 replication
(A) HIV-1 infection in THP-GFP macrophages was monitored by flow cytometric analysis of HIV-1 LTR-driven GFP expression. Background GFP expression levels were defined in uninfected cells (grey line, M1). Positive cells were defined by an increase in GFP expression above background levels (shift of black line into M2). (B) THP-GFP macrophages were infected with HIV-1 CUCY in the absence or presence of 30 U/ml of human IFN-β, and the percentage of GFP positive cells was measured 24-72 h post-infection. Values are displayed as mean +/- standard error. *p<0.001 versus control as determined by two-way ANOVA followed by Bonferroni's multiple comparison test. Data are representative of two independent experiments. (C) Protein from untreated THP-GFP and THP-GFP-S3 cells was subjected to immunoblot analysis with antibodies specific to SOCS3. Blot was stripped and reprobed for actin as a loading control. (D) THP-GFP and THP-GFP-S3 macrophages were treated with 30 U/ml of human IFN-β for 15 min. Protein was subjected to immunoblot analysis with antibodies specific to phosphorylated STAT1 (tyrosine 701), phosphorylated STAT2 (tyrosine 689), or total levels of these proteins. Blots were then stripped and reprobed for actin as a loading control. Quantification of band density shown to the right of blots represents pY-STAT levels normalized to total STAT levels, and is displayed as fold decrease below THP-GFP cells. Data are representative of two independent experiments. (E) THP-GFP and THP-GFP-S3 macrophages were infected with HIV-1 CUCY or HIV-1 SG3 in the presence of 0-30 U/ml of human IFN-β. The percentage of GFP positive cells was measured 72 h post-infection. Values are displayed as a fraction of control and represent an average of two independent experiments performed in triplicate +/- standard error. *p<0.01 versus THP-GFP macrophages as determined by two-way ANOVA followed by Bonferroni's multiple comparison test.
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