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. Author manuscript; available in PMC: 2012 Jun 15.
Published in final edited form as: J Immunol. 2011 May 11;186(12):6771–6778. doi: 10.4049/jimmunol.1100099

IFN-γ Mediates Enhancement of HIV Replication in Astrocytes by Inducing an Antagonist of the β-Catenin Pathway (DKK1) in a STAT 3-Dependent Manner

Wei Li *,1, Lisa J Henderson *, Eugene O Major †, Lena Al-Harthi *
PMCID: PMC3167069  NIHMSID: NIHMS315725  PMID: 21562161

Abstract

Typically, IFN-γ is an antiviral cytokine that inhibits the replication of many viruses, including HIV. However, in the CNS, IFN-γ induces HIV-productive replication in astrocytes. Although astrocytes in vitro are refractory to HIV replication, recent in vivo evidence demonstrated that astrocytes are infected by HIV, and their degree of infection is correlated with proximity to activated macrophages/microglia. The ability of IFN-γ to induce HIV replication in astrocytes suggests that the environmental milieu is critical in regulating the permissiveness of astrocytes to HIV infection. We evaluated the mechanism by which IFN-γ relieves restricted HIV replication in astrocytes. We demonstrate that although astrocytes have robust endogenous β-catenin signaling, a pathway that is a potent inhibitor of HIV replication, IFN-γ diminished β-catenin signaling in astrocytes by 40%, as evaluated by both active β-catenin protein expression and β-catenin-mediated T cell factor/lymphoid enhancer reporter (TOPflash) activity. Further, IFN-γ–mediated inhibition of β-catenin signaling was dependent on its ability to induce an antagonist of the β-catenin signaling pathway, Dickkopf-related protein 1, in a STAT 3-dependent manner. Inhibition of STAT3 and Dickkopf-related protein 1 abrogated the ability of IFN-γ to enhance HIV replication in astrocytes. These data demonstrated that IFN-γ induces HIV replication in astrocytes by antagonizing the β-catenin pathway. To our knowledge, this is the first report to point to an intricate cross-talk between IFN-γ signaling and β-catenin signaling that may have biologic and virologic effects on HIV outcome in the CNS, as well as on broader processes where the two pathways interface.

Interferon-γ (IFN-γ), a type II IFN, is a pleiotropic cytokine involved in antimicrobial and antitumor immunity by enhancing Ag presentation through MHC class I and class II, regulating a variety of genes, and facilitating proapoptotic responses of infected cells (1). Although IFN-γ is predominantly secreted by NK and NK T cells to activate macrophages and by effector CD4+ and CD8+ Ag-specific T cells, it is also secreted by activated astrocytes and microglia in response to mechanical or ischemic injury (2). Further, IFN-γ causes alteration in Ca2+ waves in the astrocytic network, which is a marker of astrocyte activation and may be important in the formation of synapses (3). Although IFN-γ is associated with enhanced anti-HIV immunity in the systemic compartment, in the CNS it is associated with HIV neuroinvasion and severity of neuropathogenesis in the human brain and the brain of SIV-infected macaques (4, 5).

The majority of IFN-γ effects are mediated by signaling through the JAK–STAT pathway (6). IFN-γ signaling through JAK–STAT involves an initial step of IFN-γ binding to its receptor, leading to oligomerization of the IFN-γ receptor subunits (IFNGR1 and IFNGR2), which causes phosphorylation and activation of JAKs. JAK activation leads to phosphorylation and subsequent activation of STAT, which dimerize and translocate to the nucleus, where they bind γ-activated sequences in the promoter of IFN-γ–regulated genes and, with cooperation from other transcriptional factors, such as breast cancer susceptibility gene 1 (BRCA1) and mini-chromosome maintenance protein 5 (MCM5), regulate IFN-γ–responsive genes. Approximately 500 genes are regulated through the IFN-γ–induced JAK–STAT pathway, including IFN-inducible protein 10, GTPase, and suppressor of cytokine signaling I (1, 6). Seven STAT family members have been identified. STAT 3, in particular, is evident in reactive astrocytes and is linked to neuroinflammatory responses in rodent models of ischemia and spinal cord injuries (7, 8). STAT 3 is activated by cytokines (IFN-γ, IL-6, G-CSF) and growth hormones. It induces cell cycle progression, prevents apoptosis, and may be linked to oncogenesis through induction of proto-oncogenes, such as c-myc (9).

HIV invades the brain early in the course of disease and leads to progressive neurologic impairments. Prior to the era of highly active antiretroviral therapy, HIV led to frank dementia/encephalitis in ~25% of HIV-infected individuals. Today, HIV causes a milder, but much wider, spectrum of neurologic impairments, described as HIV-associated neurocognitive disorders (HAND). HAND symptoms include memory impairment, depression, tremors, psychosis, seizures, and behavioral changes, to name a few. Recent assessments from the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) study (10) indicated that HAND occurs in 53% of HIV-infected individuals. HIV-mediated neuropathogenesis, depending on the severity of disease, includes reactive astrocytosis, myelin pallor, and perturbations in synaptic and dendritic density that may also include selective neuronal loss. The mechanism of HIV-mediated neurologic disorder is not entirely clear, but it is likely driven by both direct (active viral replication) and indirect sequelae to HIV invasion of the brain. Indirect mechanisms include dysregulation of glia, release of viral proteins, and elevation of neurotoxic proteins (TNF-α, IL-6, IL-1β, TGF-β, endothelin, glutamate) from resident brain cells and infiltrating lymphocytes (11).

The primary targets of HIV infection in the CNS are infiltrating monocytes/macrophages and microglia. Astrocytes constitute 40–60% of brain cells and provide vital functions for brain homeostasis, such as regulation of neuronal development, maintenance of the blood–brain barrier, metabolism of neurotransmitters, secretion of neurotrophic factors, and immune surveillance in the brain by secretion of cytokines/chemokines (12–14). Astrocytes are CD4− but may express alternative receptors for HIV entry, including D6, a promiscuous CCR (15), and mannose receptors, which may support HIV entry through endocytosis and subsequent escape from endosomal vesicles (16–18).

Despite the lack of clarity on how HIV enters astrocytes, our group previously demonstrated that astrocytes support productive HIV replication if they are primed with IFN-γ prior to exposure to HIV (19). If IFN-γ is provided to astrocytes post-HIV infection, it does not promote productive HIV replication, and the virus remains latent in astrocytes. Recent studies on postmortem tissue isolated from brains of HIV+ patients with neurocognitive impairment revealed considerable infection of astrocytes in vivo. Interestingly, the severity of HIV-associated dementia (HAD) correlated with the degree of HIV infection of astrocytes and their close proximity to perivascular macrophages (20). These studies suggested that under the appropriate environmental milieu, astrocytes can support productive HIV replication. The mechanism by which signals, such as IFN-γ, prime astrocytes for productive HIV replication is not clear. Astrocytes express robust levels of β-catenin signaling, which causes repression of HIV replication in astrocytes (21, 22) and PBMCs (23, 24). This finding suggests a possible interface between the β-catenin pathway and the IFN-γ–signaling pathway that can impact HIV replication in astrocytes.

The β-catenin pathway is the canonical pathway of Wnt signaling. It is emerging as an important regulator of neurodegenerative diseases (25–28). The β-catenin signal-transduction cascade is multifaceted and is described in detail elsewhere (29). Briefly, the canonical pathway is initiated by the binding of Wnt proteins (a family of 19 soluble secreted glycoproteins) to Frizzled (G-coupled seven transmembrane protein receptor, Fz) and low-density lipoprotein receptor-related protein 5 or 6 coreceptors. This event leads to the inhibition of a multiprotein β-catenin destruction complex (glycogen synthase kinase-3β [GSK3β], axin, adenomatous polyposis coli, casein kinase 1), resulting in accumulation of a stable/hypophosphorylated β-catenin. Active (hypophosphorylated) β-catenin functions as a coactivator for T cell factor/lymphoid enhancer (TCF/LEF) transcription factors and, along with coactivators (CBP and p300), leads to target gene transcription. β-catenin target genes impact cell differentiation, communication, apoptosis/survival, and proliferation (30, 31). Active β-catenin can also bind to cadherins in the cell membrane, along with actin, to provide structural support for adhesion.

In this study, we determined the mechanism by which IFN-γ promotes productive HIV replication in astrocytes. Our study demonstrated a link between IFN-γ signaling and β-catenin signaling that impacts HIV replication in astrocytes and may have a greater biologic impact on mechanisms of viral pathogenesis in the CNS.

Materials and Methods

Generation of primary human fetal astrocytes and human progenitor-derived astrocytes

Human fetal astrocytes (HFA), isolated at ~20 wk gestation, were purchased from Lonza (BioWhittaker, Walkersville, MD). Progenitor-derived astrocytes (PDA) were generated from neural progenitor cells, as previously described (32). Briefly, progenitor cells were provided by Dr. Eugene Major (National Institute of Neurological Disorders and Stroke, National Institutes of Health [NIH]) and seeded on poly-D-lysine–coated T-75 tissue culture flasks at 2 × 106 cells/flask. Cells were maintained in progenitor medium consisting of neurobasal media (Life Technologies Invitrogen, Carlsbad, CA) supplemented with 0.5% bovine albumin (Sigma, St. Louis, MO), neurosurvival factor (Lonza), N2 components (Life Technologies Invitrogen), 25 ng/ml fibroblast growth factor, 20 ng/ml epidermal growth factor (R&D Systems, Minneapolis MN), 50 μg/ml gentamicin (Lonza) and 2 mM L-glutamine (Life Technologies Invitrogen). To induce differentiation, progenitor medium was replaced with PDA medium containing DMEM (Life Technologies Invitrogen) supplemented with 10% heat-inactivated FBS (Sigma), 2 mM L-glutamine, and 50 μg/ml gentamicin. Cultures were >90% positive for GFAP after 30 d of differentiation. HFA refer to HFA or PDA.

Cell lines and reagents

The astroglioma cell lines U87MG and U251MG were obtained from the NIH AIDS Research and Reference Reagent Program (Frederick, MD) and the American Type Culture Collection (Manassas, VA), respectively. They were propagated in DMEM (Life Technologies Invitrogen) supplemented with 10% heat-inactivated FBS (Sigma) and 1% penicillin-streptomycin (Life Technologies Invitrogen). The cells were used at ~80% confluency. Human rIFN-γ, GSK3β Ab (pY216), pSTAT1 (S727)-AF647 mAb, pSTAT3 (pY705)-AF488, pSTAT4 (pY693)-AF647, pSTAT5 (pTyr694)-AF488, and pSTAT6 (pY641)-PE were purchased from BD Pharmingen (San Jose, CA). pSTAT2 (pTyr690) was purchased from Cell Signaling (Danvers, MA). Allophycocyanin and FITC-conjugated goat anti-mouse Abs and FITC-bovine anti-goat IgG (H+L) Ab were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA). Fludarabine (FLUD) was purchased from Sigma-Aldrich. STAT3 inhibitor V, STATtic, STAT5 inhibitor, and GSK3β inhibitor IX were purchased from CalBiochem/EMD Biosciences (Gibbstown, NJ). hDKK1 (DKK1)-neutralizing Ab was purchased from R&D Systems (Minneapolis, MN), whereas DKK1-detection Ab (clone #569559) was purchased from Abcam (Cambridge, MA). Dkk1 ELISA was purchased from RayBiotech (Norcross, GA) and used as recommended. HIV p24 concentration was measured by conventional ELISA from the AIDS and Cancer Virus Program (Science Applications International Corp., Frederick, MD).

DNA constructs and transfection

U87MG and U251MG astroglioma cell lines and primary human astrocytes were transiently transfected using either the LT-1 DNA transfection reagent (Mirus Bio, Madison, WI) or the Amaxa nucleofection protocol (Amaxa, Gaithersburg, MD), as recommended by the manufacturer. To measure β-catenin–dependent signaling activity, 5 × 106 cells were transfected with 10 μg TOPflash reporter construct (Millipore, Billerica, MA). TOPflash construct consists of two sets of three TCF/LEF-binding sites linked to a luciferase reporter. The cells were also cotransfected with 1 ng Renilla construct (Promega, Madison, WI) to normalize for transfection efficiency and GFP (pMaxGFP; Lonza, Biologics, Portsmouth, NH) to equalize the amount of total DNA used per transfection condition. Firefly and Renilla luciferase activity was measured using dual luciferase assay reporter system (Promega). Where indicated, cells were transfected with TOPflash and Renilla, with or without a constitutively active β-catenin construct (Cara Gottardi, Northwestern University, Chicago, IL) or a dominant-negative (DN) mutant TCF-4 construct (James O’Kelly, University of California, Los Angeles). The constitutively active β-catenin plasmid contains a serine-to-tyrosine mutation at position 33 that protects the protein from proteosomal degradation. DN TCF-4 constructs lack the N-terminal 31 aa required for β-catenin binding.

IFN-γ treatment and HIV infection

Astrocytes were pretreated with IFN-γ (100 ng/ml) or left untreated for 24 h prior to HIV infection. IFN-γ was maintained postinfection. HIV infection was carried out using IFN-γ–primed astrocytes at 80% confluency and incubating the cells with HIVBal (NIH AIDS Research and Reference Reagents Program, Germantown, MD) at 10 ng HIV p24/1 × 106 cells for 24 h. Postinfection, the cells were washed extensively with 1× PBS and propagated in the presence of IFN-γ (100 ng/ml). At day 7 postinfection, HIV p24 was monitored by conventional ELISA, according to recommendations of the manufacturer (AIDS and Cancer Virus Program, Science Applications International Corp., Frederick, MD).

Immunofluorescence staining and flow cytometry analysis

To detach astrocytes without cleaving surface proteins, they were incubated with 1 mM EDTA for 5 min and then washed and suspended in 1× PBS. Cells were stained with appropriate target Abs and isotype Abs using conventional surface- and/or intracellular-staining methods. When both surface and intracellular staining was desired, cells were first fixed and permeabilized using BD Cytofix/Cytoperm Fixation and Permeabilization Solution (BD Pharmingen), followed by staining for intracellular proteins. Cells were then washed extensively with 1× PBS to remove excess Ab, stained for extracellular targets, and fixed with 2% formaldehyde. Fluorescence was evaluated with a FACSCalibur flow cytometer, and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

STATistical analysis

STATistical analyses were performed using Prism software (GraphPad Prism, San Diego, CA). Untreated and treated (IFN-γ with or without inhibitor) groups were compared using the Student t test when the data were normally distributed. When the data were not normally distributed, the two groups were compared using the nonparametric Mann–Whitney U test. All tests were two-tailed, and a p value <0.05 was considered significant.

Results

IFN-γ–mediated induction of HIV replication in astrocytes is β-catenin–signaling dependent

Active β-catenin signaling inhibits HIV replication in astrocytes and PBMCs (21–24). We evaluated whether IFN-γ downregulates β-catenin in human primary fetal astrocytes (PFA), thereby increasing restricted HIV replication in astrocytes. PFA were cotransfected with a TCF/LEF firefly luciferase construct (TOP-flash) and a control reporter (Renilla luciferase) and then treated or not with IFN-γ. The TOPflash reporter is an indicator of basal and inducible levels of β-catenin–dependent signaling. At 24 h post–IFN-γ treatment, IFN-γ markedly reduced β-catenin signaling by ~38% (Fig. 1A). IFN-γ–mediated inhibition of β-catenin signaling in PFA was also consistent with a reduction in active hypophosphorylated β-catenin, as evaluated by intracellular flow cytometry (Fig. 1B). We also confirmed the ability of IFN-γ to diminish β-catenin signaling in U251MG astroglioma cells, as demonstrated by ~38% decline in TOPflash activity at 24 h postexposure (Fig. 1C). Kinetics of IFN-γ–mediated reduction in the expression of active β-catenin indicated that this process is initiated as early as 1 h posttreatment, and ~45% reduction in active β-catenin expression is achieved by 48 h post–IFN-γ exposure in U251MG cells (Fig. 1D). Specificity of endogenous β-catenin–signaling activity in astrocytes is demonstrated by comparing the activity of the TOPflash construct with a FOPflash construct. FOPflash is a negative control for TOPflash; it consists of the same backbone vector of TOPflash linked to firefly luciferase but with mutated TCF/LEF-binding sites (Fig. 1E). This construct illustrates the expected basal/low activity of backbone vector in these cells (Fig. 1E).

FIGURE 1.

FIGURE 1

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IFN-γ downregulates β-catenin signaling pathway. PFA (A) or U251MG astroglioma cells (C) were left untreated or treated with IFN-γ (100 ng/ml) for 24 h prior to transfection with TOPflash luciferase and Renilla luciferase constructs. After resting for 4 h, the cells were cultured with or without initial treatment of IFN-γ. Dual luciferase activity was measured 24 h later. Data shown are normalized to Renilla activity. Background level of dual luciferase reading is between 0.35–0.13 and is indicated in first two columns of A and C. PFA (B) or U251MG cells (D) were treated with or without IFN-γ for 48 h (B) or for 1, 24, or 48 h (D), and expression of hypophosphorylated/active β-catenin level was measured by conventional intracellular flow cytometry. E, PDA or U87MG cells were transfected with TOPflash or FOPflash with Renilla. Firefly luciferase activity over Renilla activity was measured 24 h posttransfection. Data represent a minimum of three experiments. *p < 0.05.

To evaluate whether IFN-γ–mediated induction of HIV replication in astrocytes is dependent on downregulation of β-catenin, we used both gain- and loss-of-function studies. For gain-of-function studies, we transfected PFA (Fig. 2A) or U87MG astroglioma cells (Fig. 2B) with a constitutively active construct of β-catenin. For loss-of-function studies, we transfected the cells with a DN construct of TCF-4. Overexpressing β-catenin abrogated the ability of IFN-γ to induce HIV replication in both PFA and U87MG (Fig. 2). These data demonstrated that the ability of IFN-γ to induce HIV replication in astrocytes is dependent on its ability to downregulate β-catenin signaling. Inhibiting β-catenin signaling, through DN TCF-4 expression, had no effect on IFN-γ–mediated induction of HIV replication in both cell types (Fig. 2). This is likely because IFN-γ inhibits β-catenin signaling (Fig. 1), and further inhibition of β-catenin signaling by DN TCF-4 expression did not have additional effects over that already conferred by IFN-γ treatment alone. It is interesting to note that inhibiting endogenous β-catenin activity enhanced HIV replication in untreated cultures (Fig. 2). This observation is consistent with our previous studies demonstrating that β-catenin is an endogenous factor that represses HIV replication and that its inhibition promotes HIV replication in a number of cell types, including astrocytes (21, 23).

FIGURE 2.

FIGURE 2

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Impact of β-catenin and TCF-4 on IFN-γ–mediated induction of HIV in astrocytes. PFA (A) or U87MG astrocytoma cells (B) were transfected with GFP, a constitutively active β-catenin construct (β-catenin pcDNA), a DN construct of TCF-4, or were mock transfected. Transfected cells were treated or not with IFN-γ for 24 h. Subsequently, the cells were infected with HIVBal, and HIV p24 level was measured at day 6 postinfection by conventional ELISA. Data represent a minimum of three experiments. *p < 0.05, compared with untreated samples.

IFN-γ inhibits β-catenin signaling through induction of DKK1, an antagonist of the β-catenin pathway

To determine how IFN-γ downregulates β-catenin–signaling activity, we evaluated the impact of IFN-γ on two prominent antagonists of the β-catenin pathway: DKK1 and GSK3β. DKK1 antagonizes β-catenin signaling by depleting frizzled coreceptors (low-density lipoprotein receptor-related protein), thus inhibiting frizzled activation through Wnt ligands (29). Following casein kinase 1-mediated phosphorylation of β-catenin at serine 45, GSK3β phosphorylates β-catenin at Thr 41, Ser 33, and Ser 37, which tags β-catenin for ubiquitination by βTrCP and proteomic degradation. IFN-γ–treated PFA demonstrated a significant induction in DKK1 expression, as measured by both flow cytometry and ELISA, and this was consistent between PFA and U251MG cells (Fig. 3A–D). Similar results were observed in U87MG (data not shown). Further, addition of neutralizing Ab against DKK1 abrogated the ability of IFN-γ to induce DKK1 and reduced the level of DKK1 from untreated cultures (Fig. 3B, 3D). These data suggested that astrocytes constitutively express DKK1, which is consistent with the knowledge that DKK1 is a target gene of the β-catenin pathway and regulates the expression of this pathway in a feedback-loop mechanism (29). Active GSK3β expression was modestly increased by IFN-γ at 1 h posttreatment, but this induction was transient and returned to background level by 2 h (Fig. 3E).

FIGURE 3.

FIGURE 3

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IFN-γ induces DKK1 expression and has minimal effects on GSK3β. PFA or U251MG cells were treated with or without IFN-γ, and the expression of DKK1 was measured by flow cytometry (A, C) or ELISA (B, D) at 48 h. Endogenous level of DKK1 in untreated PFA is 863 pg/ml and of U251 is 1248 pg/ml. E, Active GSK3β level was measured in U251 cells at 1 h post–IFN-γ exposure, with or without the addition of a GSK3β-specific inhibitor. Data represent a minimum of three experiments. *p < 0.05, compared with untreated samples. MFI, mean fluorescence intensity.

To determine whether IFN-γ inhibition of β-catenin signaling is mediated by its induction of DKK1, PFA were treated with IFN-γ in the presence or absence of a DKK1-specific inhibitor. The ability of IFN-γ to inhibit the β-catenin pathway was abrogated in the presence of the DKK1 inhibitor, as measured by TOPflash activity (Fig. 4). The DKK-1 inhibitor alone, in the absence of IFN-γ treatment, enhanced TOPflash activity, suggesting that astrocytes express endogenous DKK1 to regulate β-catenin–mediated signaling. Similar results were observed in U251MG cells (data not shown).

FIGURE 4.

FIGURE 4

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IFN-γ inhibition of β-catenin signaling is through its induction of DKK-1. PFA were left untreated or were treated with IFN-γ, anti–DKK-1, or both IFN-γ and anti–DKK-1 for 24 h prior to transfection with TOPflash and Renilla constructs. Posttransfection, the cells were maintained in their initial treatment for 24 h. Dual luciferase activity was measured, and TOPflash luciferase was normalized to Renilla firefly activity. Data represent a minimum of three independent experiments. *p < 0.05, compared with untreated samples.

IFN-γ induction of HIV replication in astrocytes is dependent on its ability to induce DKK1 and STAT3

We determined whether the ability of IFN-γ to increase DKK1 and, to a lesser extent, GSK3β may play a role in the mechanism by which IFN-γ overcomes restricted HIV replication in astrocytes. Primary astrocytes were pretreated with IFN-γ, with or without neutralizing Abs against DKK-1 (αDKK-1) or a GSK3β inhibitor (G3I). The cells were then infected with HIVBal, and HIV p24 level was measured 6 d postinfection. IFN-γ pretreatment induced HIV replication in primary astrocytes by 4-fold (Fig. 5A). Inhibiting DKK-1 reduced the ability of IFN-γ to induce HIV replication by 50% (Fig. 5A). Using G3I (Fig. 3E) had no statistically significant effect on the IFN-γ–mediated induction of HIV replication. This observation was also consistent in U87MG cells (Fig. 5B).

FIGURE 5.

FIGURE 5

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IFN-γ induces HIV replication in astrocytes via upregulation of DKK1. PFA (A) or U87MG cells (B) were pretreated or not with IFN-γ in the presence or absence of anti–DKK-1 or G3I for 24 h, infected with HIVBal, and cultured with initial treatment. At day 6 postinfection, HIV p24 was measured by ELISA. Data represent a minimum of three independent experiments. *p < 0.05, compared with IFN-γ–treated cultures alone.

Because inhibiting DKK-1 did not completely abrogate the ability of IFN-γ to promote HIV-productive replication in astrocytes (Fig. 5), we investigated the contribution of classical IFN-γ signaling on its ability to enhance HIV replication in astrocytes. Within 30 min of exposure, IFN-γ activated STAT 1 and STAT 3 and had no effect on STAT 2, 4, 5, or 6 in PFA, U251, and U87MG cells (data not shown). STAT3 inhibitor (S3I) reduced IFN-γ–mediated induction of HIV replication by ~32%, whereas inhibiting STAT1 by FLUD had no effect (Fig. 6). Further, combining inhibitors of STAT3 and DKK1 abrogated the effect of IFN-γ on increased HIV replication in astrocytes (Fig. 6). These data demonstrated that the ability of IFN-γ to induce productive HIV replication in astrocytes is mediated by STAT 3 and DKK1.

FIGURE 6.

FIGURE 6

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IFN-γ enhancement of HIV replication in astrocytes is DKK1 and STAT 3 dependent. PFA were treated or not with IFN-γ in the presence of S3I, STAT1 inhibitor (FLUD), or S3I, FLUD and anti–DKK-1 for 48 h prior to HIVBal infection. At day 6 postinfection, HIV p24 was measured by ELISA. Data represent a minimum of three independent experiments. *p < 0.05, compared with IFN-γ–treated cultures alone.

Given that IFN-γ induction of DKK1 is a prominent pathway by which it downregulates β-catenin signaling and subsequently enhances HIV replication in astrocytes, we evaluated whether IFN-γ induction of DKK1 and inhibition of β-catenin are STAT 3 dependent. Inhibition of STAT3 abrogated the ability of IFN-γ to downregulate β-catenin (Fig. 7A) and induce DKK-1 (Fig. 7B). STAT1 had no effect on IFN-γ induction of DKK1 and inhibition of β-catenin (data not shown). These data demonstrated that IFN-γ–mediated inhibition of β-catenin and induction of DKK-1 are also STAT3 dependent. Collectively, these findings demonstrated an interaction between two prominent signaling pathways, β-catenin and IFN-γ signaling, that interface with each other to impact the outcome of HIV in the CNS.

FIGURE 7.

FIGURE 7

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IFN-γ activation of STAT3 induces DKK1 expression and inhibits β-catenin. U87MG cells and PFA were treated or not with IFN-γ in the presence of S3I for 48 h. Subsequently, active β-catenin level was measured by flow cytometry (A), and DKK1 level was measured by ELISA (B). Data represent a minimum of three independent experiments. *p < 0.05, compared with IFN-γ–treated cultures alone.

Discussion

Using sophisticated assessment of HIV infection of postmortem tissue, Churchill et al. (20) recently demonstrated that ≤19% of GFAP+ astrocytes are infected by HIV. The level of HIV infection of astrocytes was highest among those in close proximity to macrophages/microglia. Although a disconnect existed between in vitro and in vivo data with regard to whether astrocytes are infected by HIV, these postmortem data demonstrated that astrocytes are productively infected in vivo and require biologic signals to promote productive HIV replication, which may be lacking in an in vitro model system. The nature of the biologic signals promoting HIV permissiveness in astrocytes is not completely clear. We demonstrated that IFN-γ may be such a signal that primes HIV productive infection in vitro (19). IFN-γ levels are elevated in neuroAIDS and may drive higher levels of HIV replication in astrocytes in vivo (5). Further, IFN-γ is secreted by activated macrophages/microglia, which may explain the recent findings of higher levels of HIV infection in astrocytes that are in close proximity to macrophages/microglia (20). Astrocytes themselves secrete IFN-γ, which may function in an autocrine fashion to enhance HIV infection in these cells.

Astrocytes have robust β-catenin signaling (21), which is inversely correlated with HIV replication in a number of cell types, including astrocytes (21, 23). Specifically, inhibiting β-catenin signaling in astrocytes through the use of a DN construct of β-catenin or TCF-4 promoted HIV productive replication in astrocytes. Because IFN-γ inhibits β-catenin, which is a negative regulator of HIV replication, we evaluated whether IFN-γ promotes HIV replication in astrocytes by inhibiting β-catenin and determined the mechanism by which it does so. In this study, we demonstrated that the ability of IFN-γ to mediate productive HIV replication in astrocytes occurs through inhibition of the β-catenin–signaling pathway in a STAT3-dependent manner. Further, IFN-γ–mediated STAT3 activation induces an antagonist of the β-catenin pathway, DKK-1. Both IFN-γ induction of STAT3 and DKK-1 are critical in its ability to promote HIV replication in astrocytes. This finding is especially intriguing because it points to interplay between β-catenin and IFN-γ signaling leading to enhanced HIV replication. Our data also add to the body of evidence pointing to STAT1-independent mechanisms of IFN-γ signaling events that lead to IFN-γ–dependent effects and gene expression (6).

IFN-γ inhibition of β-catenin signaling demonstrates a significant cross-talk between the IFN-γ and β-catenin pathways. Although, classically, the presence or absence of Wnt ligands dictates whether the β-catenin pathway is engaged, there is a growing appreciation for Wnt ligand-independent regulation of β-catenin. Extensive cross-talk exists between the β-catenin pathway and other signal-transduction cascades, including the PI3K/Akt and p38 MAPK pathways, which interact with the canonical pathway by converging on a third signaling partner: GSK3β. This study adds to the growing number of pathways that can regulate β-catenin signaling, independent of Wnt ligand engagements. Further, β-catenin is tightly regulated to avoid aberrant activation. A number of proteins in the β-catenin–signaling pathway are regulated at the gene level by β-catenin/TCF transcriptional regulation via the presence of LEF/TCF-binding sites in these genes. DKK1 is one of these genes and is positively regulated by β-catenin/TCF-mediated gene regulation. Because DKK1 is an antagonist of the β-catenin pathway, this may be a mechanism by which β-catenin is self-regulated to avoid over-activation that can lead to uncontrolled cell survival/proliferation and, ultimately, oncogenesis. Although IFN-γ inhibited active β-catenin expression and signaling, it induced DKK1 protein. This finding suggested that, in addition to β-catenin/TCF gene regulation of DKK1, IFN-γ may be engaging another pathway by which it leads to induction of DKK1, whether via an alternative mechanism of DKK1 gene regulation or posttranscriptional regulatory events. Greater understanding of the interplay between β-catenin and other signaling pathways, including the IFN-γ pathway, may provide tools for enhancing function and survival of neurons and glia, as well as manipulating HIV replication in the CNS reservoir.

Astrocytes make up 40–60% of brain cells and play vital functions in maintaining brain homeostasis, which suggests that any level of HIV replication from astrocytes in vivo may have dramatic consequences in the brain. Indeed, dysregulation of astrocytes is associated with a number of neurodegenerative diseases, including HAD. In HAD, astrogliosis, characterized by hypertrophy, increased GFAP immunoreactivity, enhanced proliferation, and apoptosis, is one of the hallmarks of HIV infiltration in the brain and the severity of encephalopathy (33). However, the exact mechanism by which astrocytes contribute to HIV neuropathogenesis is not clear. Astrocytes may contribute to the release of neurotoxins (gp120, Tat) and inflammatory cytokines/chemokines, such as TNF-α, IFN-γ, and MCP-1, that cause dysregulation of neurons. Astrocytes themselves may be dysregulated by HIV [e.g., their inability to scavenge for glutamate post-HIV infection and/or exposure (34)]. We propose that IFN-γ may also contribute to the dysregulation of astrocytes in the context of HIV by downregulating an important prosurvival-signaling pathway. Wnt/β-catenin plays a key role in axonal remodeling and regulation of synaptic connectivity in the CNS (35). Activation of Wnt signaling by exogenous molecules, such as LiCl or Wnt-3a, protects cells from a number of toxic insults, such as glutamate, N-methyl-D-aspartate, calcium, and β-amyloid and deprivation of KCl, serum, and nerve growth factor (36, 37). In a mouse model of neuroAIDS, LiCl was able to restore loss of microtubule-associated protein-2+ neurites and synaptic density that is often observed with HIV invasion of the CNS (38). Lithium remarkably improved neurocognition in HIV+ patients in a small clinical trial (39). Our previous studies defined an antiviral effect of LiCl that is β-catenin pathway dependent (39–41). Many of the neuroprotective effects of lithium in the context of neuroAIDS seem to be driven by its effects on β-catenin (38). As such, there seems to be an intricate balance between downregulation of β-catenin signaling in astrocytes to promote productive HIV replication and negative effects that may ensue as a result of this downregulation. Wnt signaling regulates hippocampal neurogenesis in the adult brain (42) and is a well-established prosurvival pathway. These findings suggest that signals that downregulate β-catenin signaling, including IFN-γ, may have negative effects on neurogenesis and cell survival while enhancing HIV replication. Conversely, β-catenin signaling may be manipulated to favor neurogenesis and neuroprotection against toxic insults in a number of neurodegenerative diseases, including HAD and/or HAND.

The link that we demonstrated in this study between IFN-γ and β-catenin–signaling pathway suggests that IFN-γ may have broader roles than was previously appreciated. Understanding the interactions between IFN-γ and β-catenin signaling will have a broader impact on viral infection, as well as on understanding the normal biology of immune or brain cells at the interface of IFN-γ and β-catenin–dependent pathways.

Acknowledgments

This work was supported by Grants R01 NS060632 (to L.A.-H.) and F31 NS071999 (to L.J.H.) from the National Institutes of Health. It was also supported by the Chicago Developmental Center for AIDS Research (P30 AI 082151), a National Institutes of Health-funded program supported by the National Institute of Allergy and Infectious Diseases; the National Cancer Institute; the National Institute of Mental Health; the National Institute of Drug Abuse; the National Institute of Child Health and Development; the National Heart, Lung, and Blood Institute; and the National Center for Complementary and Alternative Medicine.

We thank Dr. Cara Gottardi (Northwestern University, Chicago, IL) for providing a constitutively active β-catenin construct and Dr. James O’Kelly (University of California, Los Angeles) for providing a DN-mutant construct of TCF-4.

Abbreviations used in this article

DN

dominant negative

FLUD

fludarabine

G3I

glycogen synthase kinase-3β inhibitor

GSK3β

glycogen synthase kinase-3β

HAD

HIV-associated dementia

HAND

HIV-associated neurocognitive disorders

HFA

human fetal astrocyte

LEF

lymphoid enhancer

NIH

National Institutes of Health

PDA

progenitor-derived astrocyte

PFA

primal fetal astrocyte

S3I

STAT3 inhibitor

TCF

T cell factor

Footnotes

Disclosures

The authors have no financial conflicts of interest.

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