SOCS3 Inhibits Antiviral IFN-β Signaling to Enhance HIV-1 Replication in Macrophages¹

Reagents

HIV-1 Tat_1-72aa was provided by Dr. Avindra Nath (Johns Hopkins University, Baltimore, MD). Recombinant murine IFN-β, IFN-γ, and M-CSF, and recombinant human M-CSF were purchased from R&D Systems (Minneapolis, MN). Recombinant human IFN-β was purchased from PBL Biomedical Laboratories (Piscataway, NJ). BAY 11-7085 was purchased from Alexis Biochemicals (Farmingdale, NY). PMA was purchased from Calbiochem (La Jolla, CA). Antibodies against phospho-STAT1 (Tyr701 and Ser727), phospho-STAT3 (Tyr705 and Ser727), STAT3, and phospho-NF-κB p65 (Ser276) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against SOCS3, NF-κB p65, IκBα, STAT2, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against STAT1 and phospho-STAT2 (Tyr689) were purchased from Upstate Biotechnology (Lake Placid, NY).

Cells

All murine studies were approved by the Animal Studies Committee at the University of Alabama at Birmingham. Primary cells were isolated from C57BL/6J wild-type mice unless otherwise specified. To generate primary murine bone marrow-derived macrophages (BMDM), bone marrow precursor cells were isolated from the femurs of adult mice, and macrophage differentiation induced by incubation with 20 ng/ml M-CSF for 5 days (42). Primary microglia (43), astrocytes (44), and cortical neurons (45) were prepared as previously described. The murine macrophage cell line RAW264.7 was maintained as previously described (46).

To generate primary human peripheral blood-derived macrophages (PBDM), PBMC from healthy donors were isolated by Ficoll Hypaque sedimentation, and macrophage differentiation induced by incubation with 10 ng/ml M-CSF for 7 days (47). To generate primary macaque PBDM, PBMC from healthy adult rhesus macaques were isolated by Ficoll Hypaque sedimentation, and macrophage differentiation induced by incubation with 100 U/ml each of M-CSF and GM-CSF for 3 to 5 days (48).

Human, monocytic THP-GFP reporter cells were derived from THP-1 cells and contain a stably integrated GFP reporter under the control of the HIV-1 NL4-3 LTR. THP-GFP-S3 cells were obtained by retroviral transduction of THP-GFP cells with a SOCS3 expressing retroviral vector, based on the murine stem cell virus (pMSCV-puro; Clontech, Mountain View, CA). All THP-1 derived cell lines were maintained in RPMI 1640 containing 10% FBS.

Ribonuclease protection assay and RT-PCR

Riboprobes for murine SOCS3, SOCS1, and GAPDH were prepared, and ribonuclease protection assay was performed as described previously (42). Briefly, 20 μg of total RNA was hybridized with the riboprobes at 42°C overnight. The hybridized mixture was treated with RNase A/T1 (1/200) and then analyzed by 5% denaturing (8 M urea) PAGE. mRNA expression values were calculated using ImageQuant software.

Alternatively, 1 μg of total RNA was reverse transcribed into cDNA and analyzed by PCR. Endpoint PCR was performed as previously described (49) with primers specific for the genes indicated. Quantitative real-time PCR was performed with Taqman (Applied Biosystems, Foster City, CA) probes according to the manufacturer's instructions. The data were then analyzed using the comparative C_T method to obtain relative quantitation values (fold induction).

Immunoblotting

Cell lysates were separated by electrophoresis on SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with antibodies against the indicated proteins. 25 μg of lysate was sufficient for all proteins except SOCS3 which required 100 μg of lysate. Quantification of band density was performed using Quantity One software.

Luciferase assay

The murine SOCS3 promoter construct was generated as previously described (42), and the IKKβ dominant negative expression vector was provided by Dr. Randolph Noelle (Dartmouth Medical School, Lebanon, NH). Cells were transiently transfected using the Lipofectamine Plus method per manufacturer's instructions. Following treatment, the luciferase activity of each sample was measured using a luminometer and normalized to the protein concentration of each well.

SIV/macaque model and quantitative RT-PCR

Pigtailed macaques were infected with SIV for the times indicated, in accordance with federal guidelines and institutional policies, as previously described (50). Total RNA was collected from macaque basal ganglia, reverse transcribed as described above, and analyzed by quantitative real-time PCR with Taqman probes specific to human SOCS3 (Hs02330328_s1), macaque SOCS1 (Rh02914687_g1), and 18s (Hs99999901_s1).

Generation of SOCS3^Δ/Δ Mice

Mice homozygous for a floxed (fl, flanked by loxP sites) SOCS3 allele (SOCS3^fl/fl) (51) were serially bred with mice expressing Cre recombinase (Cre) under the control of the LysM promoter, to generate mice in which the conditional SOCS3 allele is excised specifically in macrophages and neutrophils (SOCS3^Δ/Δ). BMDM were then collected as described above.

HIV-1 infection and flow cytometric analysis

THP-GFP or THP-GFP-S3 cells were incubated in the presence of 1 ng/ml PMA for 72 h to induce macrophage differentiation, followed by pretreatment with 0-30 U/ml of IFN-β for 24 h. Medium was removed and the cells were then infected with the molecular HIV-1 clone SG3 or the patient isolate CUCY. Three hours post viral inoculation, RPMI 1640/10% FBS containing the respective concentrations of IFN-β was re-added and cells incubated for an additional 24-72 h prior to collection. The percentage of GFP expressing cells was measured using a Guava EasyCyte flow cytometer.

Statistical analysis

Data was analyzed using GraphPad Prism software. Appropriate statistical tests were chosen for each experiment and are indicated in the respective figure legend.

SOCS3 expression correlates with viral load and CNS disease in a SIV/macaque model of HAD

SOCS proteins are upregulated by a number of pathogens in order to evade protective host IFN responses. Specifically, SOCS1 and SOCS3 have been shown to prevent IFN-mediated inhibition of viral replication (23,24,52). To determine whether either of these molecules are induced in the brain during progression to HAND, we examined SOCS1 and SOCS3 mRNA levels in a SIV/macaque model of HAD. This model closely recapitulates the most severe form of human HAND, known as HAD, although in an accelerated fashion that consistently results in disease (11,14,50). Animals display an increase in CNS viral load shortly following infection, which then rapidly diminishes ∼10 days post-infection in response to an increase in IFN-β expression. Recurrence of viral replication within the brain occurs by 42 to 84 days post-infection, and correlates with onset of disease. We collected mRNA from the basal ganglia of either non-infected animals (C), or animals at a range of timepoints along the course of infection, and analyzed SOCS1 and SOCS3 expression by quantitative real-time PCR. SOCS3 mRNA expression was increased in SIV-infected animals in a pattern that correlated with increases in CNS viral load and onset of disease (Figure 1A). Collectively, 7 out of 9 animals with confirmed CNS disease (Mi, Mo, S) showed increased SOCS3 expression compared to uninfected controls, while animals without disease showed consistently low SOCS3 expression (Figure 1B). Additionally, the 6 most severely affected animals (Mo, S) showed the greatest increases in SOCS3 expression. SOCS1 mRNA expression was also detected in SIV-infected animals, but did not correlate with increases in viral load or CNS disease (data not shown). These data indicate that SOCS1 and SOCS3 mRNA are expressed within the brain during progression to HAD. Because SOCS3 expression correlated with recurrence of viral replication and onset of CNS disease, we concluded that it contributed more significantly to HAD progression and focused subsequent mechanistic studies on this protein.

HIV-1 Tat induces SOCS3 expression in macrophages and microglia

SOCS proteins are expressed in cell types throughout the CNS in a family member- and stimulus-specific manner (19,42,53,54). We hypothesized that SOCS3 must be expressed in macrophages, the most important source of viral replication within the CNS, to have a direct impact on viral replication. To determine whether macrophages could express SOCS3 in response to stimuli present in the HAD brain, we treated cells with HIV-1 Tat, a protein with multiple HAND promoting effects. Primary macaque PBDM were treated with HIV-1 Tat_1-72aa for 0-24 h. A 10 nM concentration, close to the low physiologic concentration of Tat found in the sera of HIV-1-infected individuals (55), was sufficient to produce robust SOCS3 expression between 2 and 8 h, both at the level of mRNA (Figure 2A) and protein (Figure 2B). In order to assess whether Tat treatment of macaque macrophages in vitro also recapitulated the family member specificity seen in our in vivo model, we directly compared SOCS1 and SOCS3 expression by quantitative real-time PCR. Only negligible levels of SOCS1 mRNA expression were detected in comparison to SOCS3 (Supplemental Figure 1), indicating that the ability of Tat to induce SOCS expression is relatively specific for SOCS3 in vitro. Notably, SOCS3 mRNA was not elevated in primary macaque PBDM following a 4 h treatment with SIV alone (Supplemental Figure 1), suggesting that the early stages of virus infection itself are not sufficient to induce SOCS3 expression. To determine whether human macrophages act similarly, we exposed primary human PBDM to HIV-1 Tat_1-72aa. Increased SOCS3 mRNA (Figure 2C) and protein (Figure 2D) levels were detected following Tat exposure, with maximal expression occurring 4 h after treatment. These data indicate that SOCS3 expression is induced by HIV-1 Tat, a stimulus present within the HAD brain, in both human and macaque macrophages.

We then examined whether HIV-1 Tat could enhance SOCS3 expression in murine cells, which are a useful experimental tool for transfection and gene deletion studies. Although Tat is unable to enhance HIV-1 transcription in murine cells (56), it has been shown to successfully regulate host gene expression (57). RAW264.7 murine macrophages, chosen for their ability to induce SOCS3 in response to other stimuli (42), expressed SOCS3 in response to treatment with HIV-1 Tat_1-72aa between 1 and 8 h at both the mRNA (Figure 2E) and protein (Figure 2F) level. Notably, treatment with a mutant form of Tat lacking critical core and basic regions (HIV-1 Tat_Δ31-61aa) did not induce a comparable increase in SOCS3 expression (data not shown). These results indicate that murine macrophages respond specifically to functional Tat by inducing SOCS3 expression, similarly to macrophages of other species. We then examined a range of primary murine CNS-relevant cells for their responsiveness to Tat treatment. Both primary murine BMDM (Figure 2G) and primary murine microglia (Figure 2H) displayed an increase in SOCS3 mRNA for the duration of HIV-1 Tat_1-72aa exposure. In contrast, neither primary murine astrocytes (Figure 2I) nor primary murine cortical neurons (Figure 2J) expressed SOCS3 mRNA in response to HIV-1 Tat_1-72aa treatment. Both of these cell types were capable of expressing SOCS3 in response to Oncostatin M (OSM; Figure 2I) or IFN-γ (Figure 2J) respectively, and have previously been shown to respond functionally to Tat (36,37). These data indicate that HIV-1 Tat-induced SOCS3 expression is restricted to cells of macrophage lineage within the CNS.

HIV-1 Tat-induced SOCS3 expression is transcriptionally regulated

SOCS3 expression can be regulated either at the level of transcription or mRNA stabilization (58,59). To determine how HIV-1 Tat regulates SOCS3 expression in macrophages, we assessed activation of the SOCS3 promoter (Figure 3). RAW264.7 macrophages, chosen for their ease of transfection, were transiently transfected with a murine SOCS3 promoter-driven luciferase reporter construct (42) (Figure 3A). Cells were then treated with HIV-1 Tat_1-72aa for 24 h, which was determined to be the optimal assay point. Tat induced a 17-fold increase in luciferase levels as compared to untreated cells (Figure 3B), a value consistent with Tat-induced increases in SOCS3 mRNA (Figure 2E). These data suggest that HIV-1 Tat regulates SOCS3 expression in macrophages at the level of transcription.

HIV-1 Tat signals through the NF-κB pathway to induce SOCS3 expression

Different signaling pathways contribute to SOCS3 expression in a stimulus- and cell type-specific manner (19-21,23,42). Of these, the NF-κB pathway is frequently utilized by HIV-1 Tat to transcriptionally regulate gene targets (36,38). To determine whether Tat signals through this pathway to induce SOCS3 expression in macrophages, we first examined the kinetics of Tat-induced NF-κB pathway activation. RAW264.7 macrophages were exposed to HIV-1 Tat_1-72aa for 0 to 120 min and analyzed for markers of NF-κB activation (Figure 4A). Phosphorylation of NF-κB p65 (serine 276) was detected at 30 min and remained elevated through 120 min. In addition, total levels of IκBα, a protein that sequesters NF-κB prior to activation, diminished noticeably between 15 and 45 min. HIV-1 Tat_1-72aa treatment of primary murine BMDM showed a similar result, inducing phosphorylation of NF-κB p65 (serine 276) between 30 and 45 min (Figure 4A).

We used two methods of pathway inhibition to determine whether the NF-κB pathway was necessary for HIV-1 Tat-induced SOCS3 expression. First, RAW264.7 macrophages were assayed for Tat-induced SOCS3 promoter activation in the absence or presence of an IKKβ dominant negative construct. This construct prevents the phosphorylation, and subsequent degradation of IκB, resulting in inhibition of NF-κB activation (60). While HIV-1 Tat_1-72aa stimulation resulted in a nearly 6-fold increase in luciferase expression, inclusion of the IKKβ dominant negative construct reduced this expression by ∼65% (Figure 4B). Next, RAW264.7 macrophages, primary murine BMDM, or primary human PBDM were analyzed for endogenous Tat-induced SOCS3 expression following pre-treatment with a pharmacologic inhibitor of the NF-κB pathway. Cells were pre-treated with 10 μM BAY 11-7085, a concentration determined to prevent phosphorylation of p65, and then exposed to HIV-1 Tat_1-72aa for 1 h. Exposure to BAY 11-7085 inhibited Tat-induced SOCS3 mRNA expression (Figure 4C). These data indicate that activation of the NF-κB signaling pathway is necessary for HIV-1 Tat-induced SOCS3 expression in macrophages.

Activation of the MAPK pathway (ERK, JNK, and p38) and the JAK/STAT pathway (STAT1 and STAT3) were also examined. While all three of the MAPK pathways were activated by HIV-1 Tat_1-72aa with early kinetics (Supplemental Figure 2A), pharmacologic inhibition of these pathways did not prevent Tat-induced SOCS3 expression (Supplemental Figure 2B, C). By contrast, neither STAT1 nor STAT3 activation occurred with early kinetics following Tat treatment (Supplemental Figure 2D). These data suggest that the MAPK and JAK/STAT pathways are not necessary for HIV-1 Tat-induced SOCS3 expression in macrophages.

HIV-1 Tat-induced SOCS3 expression is not mediated by IL-10 or IFN-β expression

SOCS3 can be induced by a variety of stimuli, two of which are readily induced by HIV-1 Tat: IL-10 (61) and IFN-β (62). To determine whether either Tat-induced IL-10 or IFN-β are required intermediates for SOCS3 expression, the kinetics of their expression were first examined. RAW264.7 macrophages and primary murine BMDM were exposed to HIV-1 Tat_1-72aa for the times indicated (Figure 5A). Both IL-10 and IFN-β mRNA expression were induced by Tat treatment, albeit with differential kinetics. To examine whether either of these cytokines were necessary for Tat-induced SOCS3 expression, macrophages deficient for IL-10 (Figure 5B) or the IFNAR (Figure 5C) were compared to wild-type macrophages following HIV-1 Tat_1-72aa treatment. Neither deficiency altered HIV-1 Tat-induced SOCS3 mRNA expression, indicating that neither IL-10 nor IFN-β are intermediates in this process.

HIV-1 Tat inhibits IFN-β-induced STAT activation and antiviral target gene expression

Pathogen-induced SOCS proteins can inhibit signaling downstream of both the type I and type II IFN receptors, thereby diminishing the host immune response (20-25). To determine whether HIV-1 Tat-induced SOCS3 expression is capable of inhibiting IFN signaling, RAW264.7 cells were pretreated with HIV-1 Tat_1-72aa for 4 h to induce SOCS3 protein expression, and then exposed to IFN-β or IFN-γ for 15 min (Figure 6A). Treatment with IFN-β induced tyrosine phosphorylation of STAT1, STAT2, and STAT3 (lane 5), which was abolished by Tat pretreatment (lane 6). This inhibition was somewhat stimulus-specific, as IFN-γ-induced STAT1 and STAT3 tyrosine phosphorylation (lane 3) were affected to a much lesser extent by Tat pretreatment (lane 4). Although serine phosphorylation of STAT1 and STAT3 did not occur to a significant extent at this experimental timepoint, Tat pretreatment was sufficient to inhibit basal serine phosphorylation of STAT3 (lanes 2, 4, 6). Tat-induced inhibition of IFN-β signaling was also observed in primary murine BMDM (Figure 6B). IFN-β induced tyrosine phosphorylation of STAT1, STAT2, and STAT3 (lane 3), while pretreatment with HIV-1 Tat_1-72aa inhibited this activation (lane 4).

This phenomenon was also observed in primary human PBDM. Human macrophages were pretreated with HIV-1 Tat_1-72aa for 4 h to induce SOCS3 protein expression, and then exposed to IFN-β for 15 min (Figure 6C). IFN-β induced tyrosine phosphorylation of STAT1 and STAT2 (lane 3), but not STAT3, while pretreatment with Tat inhibited this activation (lane 4). Collectively, these data indicate that treatment with HIV-1 Tat, at timepoints sufficient to induce SOCS3 expression, inhibits IFN-β-induced STAT activation in both murine and human macrophages.

We also examined the ability of HIV-1 Tat to inhibit downstream IFN-β-induced antiviral gene expression. RAW264.7 cells were pretreated with HIV-1 Tat_1-72aa for 4 h to induce SOCS3 protein expression, and then exposed to IFN-β for 4 h to induce target gene expression. IFN-β treatment induced mRNA expression of ISG20 and PKR (Figure 6D, lane 3), two important inhibitors of HIV-1 replication, while pretreatment with Tat attenuated this expression (lane 4). These data show that the inhibitory effect of HIV-1 Tat on IFN-β signaling results in decreased antiviral target gene expression.

SOCS3 mediates HIV-1 Tat-induced inhibition of IFN-β signaling

To determine whether the inhibitory activity of HIV-1 Tat is mediated by SOCS3, we evaluated the effect of SOCS3 deficiency on the ability of Tat to dampen IFN-β signaling. Primary murine BMDM were obtained from mice with a conditional SOCS3 allele that was either present and fully functional (SOCS3^fl/fl), or excised specifically in macrophages (SOCS3^Δ/Δ) (51). SOCS3 mRNA expression was abolished in SOCS3^Δ/Δ macrophages in response to HIV-1 Tat_1-72aa, as compared to SOCS3^fl/fl macrophages (Figure 7A). Cells were then pretreated with HIV-1 Tat_1-72aa for 4 h followed by treatment with IFN-β (Figure 7B). While macrophages containing a functional SOCS3 allele exhibited inhibition of IFN-β-induced STAT1 and STAT2 activation in response to Tat (lanes 3 and 4), SOCS3-deletion substantially attenuated this inhibition (lanes 7 and 8). These data indicate that SOCS3 expression mediates HIV-1 Tat-induced inhibition of IFN-β signaling.

SOCS3 prevents the ability of IFN-β to suppress HIV-1 replication

The most important functional consequence of IFN-β in the HIV-1-infected brain is inhibition of viral replication in macrophages. To determine whether the inhibitory effect of SOCS3 observed on upstream IFN-β signaling correlates with an inhibition of downstream antiviral function, we examined the effect of SOCS3 on HIV-1 replication. PMA differentiated THP-1 cells containing a stably integrated HIV-1 LTR-driven reporter construct (THP-GFP macrophages) were used to perform these experiments. In these cells, GFP expression is induced upon productive HIV-1 infection (Figure 8A), and can therefore be used to monitor viral replication. THP-GFP macrophages were infected with HIV-1 CUCY (R5/macrophage preference) in the absence or presence of IFN-β, and then incubated for 24-72 h (Figure 8B). A significant decrease in HIV-1 LTR-driven GFP expression was observed in the presence of IFN-β, indicating an inhibitory effect on HIV-1 replication in these cells. To evaluate the effect of SOCS3 on this process, THP-GFP macrophages containing an integrated SOCS3 expression construct (THP-GFP-S3, Figure 8C) were utilized rather than inducing SOCS3 with HIV-1 Tat, in order to avoid Tat's broader replication enhancing effects. In these cells, SOCS3 overexpression was sufficient to substantially diminish the robust STAT1 and STAT2 activation induced by IFN-β in parental THP-GFP macrophages (Figure 8D, lanes 3 and 4). THP-GFP and THP-GFP-S3 macrophages were then infected with HIV-1 CUCY or HIV-1 SG3 (X4/lymphocyte preference) in the presence of increasing amounts of IFN-β and incubated for 72 h prior to collection (Figure 8E). HIV-1 LTR-driven GFP expression generated in response to infection with either HIV-1 strain was suppressed ∼60% by the highest dose of IFN-β tested in THP-GFP macrophages, while IFN-β had no inhibitory effect in SOCS3 overexpressing THP-GFP macrophages. These results indicate that SOCS3 expression is sufficient to overcome the inhibitory effect of IFN-β and thereby enhance HIV-1 replication in macrophages.